PDTM Vienna, Austria, March 29 to April 2, 2017: Abstracts.

Neurodegener Dis 2017;17(suppl 1): 1-7- Page 1

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Plenary Lecture

ADPD7-0168 GENE SILENCING THERAPY FOR HUMAN NEURODEGENERATIVE DISEASE 1 D. Cleveland 1 University of California at San Diego, Ludwig Institute, La Jolla, USA


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The genes whose mutation causes human neurodegenerative disease are widely expressed within neurons and non-neurons of the nervous system, producing damage not only within the most vulnerable neurons but also within their partner neurons, glia, and endothelia. Sustained gene silencing or altered pre-mRNA splicing broadly within neurons and non-neurons throughout the nervous system has been achieved using a clinically feasible antisense oligonucleotide (ASO) injection into the nervous system. Beginning with the founding example for inherited ALS caused by mutation in superoxide dismutase, single doses of this “designer DNA-based” drug approach have been shown to produce sustained, catalytic (RNase H-dependent) RNA degradation of a target gene, thereby producing slowing of disease progression (for ALS-like disease in rodents) or sustained partial disease reversal for Huntington’s-like disease from single dose injection. Therapy with ASO injection is now in trial for ALS, Huntington’s disease, and myotonic dystrophy. An additional trial using an ASO that corrects the splicing of the SMN2 gene has demonstrated efficacy in spinal muscular atrophy (SMA). A trial is anticipated to initiate in 2017 for the most frequent cause (hexanucleotide expansion in the C9orf72 gene) of both ALS and frontal temporal dementia. An extension of this approach is development of synthetic CRISPR RNAs to induce transient activation of Cas9 nuclease to cleave and permanently inactivate a selected target gene.

ADPD7-1446 TRANSMISSION OF Α-SYNUCLEIN STRAINS IN SYNUCLEINOPATHIES 1 V.M.Y. Lee 1 , Philadelphia, USA Aims The accumulation of pathological α-synuclein (α-syn) as Lewy bodies (LBs) in neurons is found in a number of neurodegenerative diseases including Parkinson’s disease without (PD) and with dementia (PDD) and dementia with Lewy bodies (DLB). However, in multi-system atrophy (MSA) misfolded α-synuclein accumulates as glial cytoplasmic inclusions (GCIs) in oligodendrocytes suggesting that they could represent a different unique strain of synucleinopathies. Method Here we found that pathological α-syn in GCIs and LBs (GCI-α-syn and LB-α-syn, respectively) are conformationally and biologically distinct. GCI-α-syn forms more compact structures and is ~1,000-fold more potent than LB-α-syn in seeding α-syn aggregation, consistent with the highly aggressive nature of MSA. Results Surprisingly, the seeding properties of GCI-α-syn and LB-α-syn show no cell type preference, raising the question of why they demonstrate different cell type distribution in diseased brains. Strikingly, we found that oligodendrocytes but not neurons could transform misfolded α-syn into GCI-like strain, highlighting that distinct αsyn strains could be generated by different intracellular milieus. Finally, GCI-α-syn maintains its high activity when propagated in neurons.

Conclusion


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Thus, the properties of pathological α-syn are determined by both misfolded seeds and intracellular environments.

ADPD7-0008 APOE, SYNAPSES, AND ALZHEIMER’S DISEASE 1 T. Südhof 1 , USA

APOE, SYNAPSES, AND ALZHEIMER’S DISEASE Thomas C. Südhof Humans express three genetic isoforms of the gene encoding the apolipoprotein ApoE, ApoE2, ApoE3, and ApoE4, that differ by two residues. Of these genetic variants, ApoE4 represents the most important risk factor for Alzheimer’s disease, whereas ApoE2 protects against Alzheimer’s disease. In addition to serving as an apolipoprotein for LDL and VLDL particles in blood, ApoE is abundantly produced in brain by astrocytes, but its function in brain and the relation of that function to the role of ApoE in Alzheimer’s disease has remained unclear. In my talk, I will discuss our studies on human neurons that investigate a possible signaling function of ApoE, and try to relate this function to Alzheimer’s disease.


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Thomas Südhof has received several recognitions for his work on neurotransmitter release, including the Wilhelm Feldberg Award, the U.S. National Academy Award in Molecular Biology (shared with Richard Scheller), the Bernhard Katz Award, Biophysical Society (shared with Reinhard Jahn), the Passano Foundation Award, the Kavli Award (shared with James Rothman and Richard Scheller), the Lasker-deBakey Award in Basic Medical Research (shared with Richard Scheller) and the Nobel Prize in Physiology or Medicine (shared with James Rothman and Randy Schekman).

ADPD7-0063 CURRENT DEVELOPMENTS IN THE TREATMENT OF PARKINSON’S DISEASE - FROM DISEASE MODIFICATION TO PREVENTION? 1 W. Poewe 1 Innsbruck Medical University, Neurology, Innsbruck, Austria

Current developments in the treatment of Parkinson’s disease – from disease modification to prevention ? Werner Poewe, Medical University Innsbruck, Department of Neurology Over the past 50 years, the treatment of Parkinson’s Disease has seen dramatic advances. Ever since the discovery of striatal dopamine deficiency in the late 1950s substituting striatal dopamine has remained the gold standard approach. Levodopa has remained the gold standard of efficacy to reduce the cardinal motor symptoms of PD, but long-term efficacy is limited by the evolution of motor complications. Ever since the 1980s until today, drug delivery efforts concentrate on optimizing levodopa pharmacokinetics and delivery and progress continues to evolve with current development programs of novel oral extended-release formulations, novel COMT-inhibitors, as well as formulations for novel delivery routes including intrapulmonary and subcutaneous levodopa delivery. Alternative approaches at restoring striatal dopamine via cell-based therapies were pioneered in the 1980s, but have remained challenging with important concerns raised by observations of Lewy-body formation in grafted neurons. Delivery of dopamine synthesizing enzymes via gene therapy is under current investigation with recent encouraging findings with a tri-cistronic vector encoding for TH, CH1 and AADC. Drug therapy beyond the dopaminergic system has received much attention and effort in the past 10 years, but so far with limited success. In contrast, multiple randomized controlled trials have established superiority of DBS versus best medical management in patients with levodopa-related motor complications and efforts continue to improve stimulation protocols, hardware and targets.


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The holy grail of PD therapy remains elusive, however. While neuroprotection moved into the focus with the DATATOP trial in the late 1990s, the history of disease modification trials has remained largely disappointing. There is new hope with the identification of pathological synuclein processing and trafficking as a novel target and the first alpha-synuclein-devoted immunotherapy trials in PD are underway. The urgency of this development is also driven by important progress in identifying prodromal stages of PD opening up perspectives for preventive therapies for subjects at risk to develop PD.

ADPD7-1667 STAGING AD PATHOLOGY WITH PET 1 K. Johnson 1 , USA


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Amyloid-beta and tau protein fibril depositions, the defining pathologies of Alzheimer’s disease, can now be detected during life with molecular imaging using positron emission tomography. Quantitative, anatomically localized measures of amyloid-beta and tau deposits can disclose the presence and evolution of Alzheimer’s pathology in preclinical as well as clinical populations. Recent studies suggest that molecular measures relate to clinical diagnosis, cognitive performance, and likelihood of progression. Amyloid-beta measures are widely employed to determine eligibility for anti-amyloid therapeutic trials, but of perhaps greatest interest is recently available evidence indicating that the increase in tau pathologic burden occurs more rapidly than disease changes reflected in more established Alzheimer's biomarkers. If these early indications from natural history studies are confirmed, efforts to develop therapies aimed at modifying Alzheimer’s pathology could be substantially more efficient.

ADPD7-0102 BLOOD BRAIN BARRIER DYSFUNCTION, NEURODEGENERATION AND ALZHEIMER’S DISEASE 1 B. Zlokovic 1 , USA

BLOOD BRAIN BARRIER DYSFUNCTION, NEURODEGENERATION AND ALZHEIMER’S DISEASE Objectives: Blood vessels in the brain are organized with surprising precision, patterned in parallel with the major brain circuits tasked with sensation, memory and motion. This tight interrelationship may reflect key functional roles in neuronal normal function, brain aging and diseases such as Alzheimer’s disease (AD). Methods: I will examine the cellular and molecular composition of the blood-brain barrier (BBB) and the role of neurovascular unit in neurodegeneration in rare human monogenic disorders, and complex neurological diseases such as AD. We will make comparison with animal transgenic models. We use multiparametric magnetic resonance imaging (MRI) in the living human brain and animal brain, CSF biomarkers and tissue analysis to evaluate how changes in brain microcirculation relate to brain connectivity and cognition. Results: I will show i) cellular and molecular findings in blood vessels and signaling in pericytes, endothelium and astrocytes that can lead to neurodegeneration in humans and animal models; ii) the effects of AD risk genes (e.g., APOE4, PICALM, CLU) on blood vessels and BBB; iii) new BBB imaging and molecular biomarkers in the living human brain in relation to cognitive impairment; iv) targets and treatments directed at the BBB that have advanced to Phase 3 and Phase 2 clinical studies in AD and stroke patients, respectively, based on our preclinical findings.


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Conclusions: It is probably good time for us to begin thinking how to update our model of the AD pathophysiological cascade by including vascular dysfunction as a major contributor and/or a possible driver of disease pathogenesis.

Neurodegener Dis 2017;17(suppl 1):8-590 – Page 8

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Symposium & Short Orals

SYMPOSIUM 01 - THE ALZHEIMER’S DISEASE SEQUENCING PROJECT ADPD7-1672 LARGE-SCALE DNA SEQUENCE ANALYSIS AND ALZHEIMER’S DISEASE GENETICS 1 2 3 4 4 1 2 G. Schellenberg , C. Xia , J. Malamon , J. Farrell , L. Farrer , L.S. Wang , Z. Nancy 1 University of Pennsylvania, Pathology and Laboratory Medicine, Philadelphia, USA 2 University of Pennsylvania, Biostatistics, Philadelphia, USA 3 University of Pennsylvania, Pathology and Laboboratory Medicine, Philadelphia, USA 4 Boston University, Epidemiology & Biostatistics, Boston, USA


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A substantial amount of the heritability of Alzheimer’s disease (AD) remains to be explained. Early family studies lead to identification of rare mutations in 3 genes (APP, PSEN1 and PSEN2), and common variants in APOE. Subsequent work using high-density genotyping arrays identified over 30 common variants loci. The advent of low-cost high-throughput DNA sequencing makes it possible to identify additional rare single nucleotide variants (SNVs) in risk and protective genes. Study designs include studies of multiplex late-onset AD kindreds and larger case-control samples. The Alzheimer’s Disease Sequence Project (ADSP) generated whole genome sequence (WGS) from 1,000 family members and exome sequence data from 6,000 AD cases and 5,000 elderly normal controls. Other efforts are generating additional WGS and WES data relevant to AD. While analyses of these large data sets may yield AD risk genes, to identify rare variants of modest effect size will require much larger data sets. While WGS/WES costs are declining, obtaining genetic data from very large samples will require use of high-density imputation panels to follow up candidates from sequencing experiments. In addition to SNVs, it is now possible to derive high-quality structural variants (SVs; indels. Insertions, deletions, copy number variation, and chromosome-level alterations) from sequence data. SVs, particularly short indels and variants 10 points) (P < 0.001). Conclusion


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Both cognitive and functional assessment scales can be used to predict survival of AD patients after the start of ChEI therapy.

SYMPOSIUM 03 - CHARACTERIZATION OF EARLY DISEASE STAGE ADPD7-1109 THE IMPACT OF OBESITY AND WEIGHT LOSS ON BRAIN STRUCTURE: A 2-YEARS PROSPECTIVE STUDY 1 2 1 1 1 1 2 1 J. Pegueroles , A. Jimenez , E. Vilaplana , V. Montal , D. Alcolea , M. Carmona-Iragui , A. Andreu , J. Clarimon , 1 1 1 R. Blesa , A. Lleó , J. Fortea 1 Hospital de la Santa Creu i Sant Pau, Neurology, Barcelona, Spain 2 Hospital Clínic, Medicine, Barcelona, Spain Aims Obesity is a risk factor for AD and weight loss has been proposed as an early sign of pre-clinical AD. We aimed to assess the relationship between obesity and brain structure taking into account the potential confounding effect of weight loss in the association. Method We included 139 subjects (104 with 2-years follow-up) with available 3T MRI scan from the ADNI cohort. We compared the cross-sectional and the longitudinal changes in cortical thickness (CTh) in relation to the weight trajectories and to the BMI categories (normal-weight, overweight or obesity). CTh and symmetrized percent change (spc) between baseline and the 2-year scan were extracted using Freesurfer. Results Subjects with significant weight loss during the follow-up presented decreased CTh already at baseline and an accelerated 2-years cortical thinning. Overweight subjects showed increased baseline CTh and an attenuation in the 2-year cortical thinning as compared with normal-weight and obese subjects. When subjects with significant weight loss were excluded from the analyses, no significant differences in CTh or spc were found between normal weight and overweight subjects whereas obese subjects showed extensive clusters of decreased CTh in both hemispheres and accelerated 2-year atrophy rates as compared with normal-weight and overweight subjects. Conclusion


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Late-life unintentional weight loss is related to cortical thinning and longitudinal brain atrophy. Late life obesity is associated with cortical thinning and with accelerated atrophy rates although weight loss negatively confounds this association.

SYMPOSIUM 03 - CHARACTERIZATION OF EARLY DISEASE STAGE ADPD7-0519 BODY WEIGHT DEFICITS AND ALTERATIONS IN PLASMA ADIPOKINE LEVELS IN THE PRECLINICAL STAGE OF ALZHEIMER’S DISEASE 1 1 M. Ishii , C. Iadecola 1 Weill Cornell Medical College, Feil Family Brain and Mind Research Institute, New York, USA Aims Weight loss is an early manifestation of Alzheimer’s disease (AD) that often precedes the cognitive decline (Cell Metabolism 22:761,2015). We previously found that transgenic mice with amyloid-beta (Aβ) accumulation have body weight/adiposity deficits and low plasma leptin levels prior to plaque formation, suggesting that early Aβ pathology can cause metabolic deficits (J Neuroscience 34:9096,2014). In this study, we sought to verify our animal studies by investigating whether body mass index (BMI) and plasma levels of adipocyte-derived hormones or adipokines are altered in human preclinical AD subjects. Method Cognitively intact (CDR=0) volunteers from the Healthy Aging & Senile Dementia and Adult Children Study (Missouri, USA) with BMI forebrain > cerebellum > WT). In human brain synaptosomes the levels of pathological tau were correlated with the patient's Braak stage. Importantly, we demonstrated that hyper-phosphorylated and aggregated tau is released by exocytosis in a calcium-dependent manner, and is seeding competent. Conclusion


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All together these data build on previous evidence of pathological tau presence in presynaptic terminals and demonstrate its calcium-dependent release. This could underlie the spreading of tau in AD brain. Therapeutic interventions aimed at blocking tau release could be beneficial in reducing tau propagation and disease progression in AD.

SYMPOSIUM 11 - SEEDING, SPREADING, AND PROTEIN AGGREGATION MECHANISMS ADPD7-1403 PERIPHERAL AMYLOID-BETA SEEDS AS EFFICIENT INDUCERS OF BRAIN AMYLOIDOSIS AND CEREBRAL AMYLOID ANGIOPATHY 1 2 2 2 2 2 R. Morales , J. Bravo-Alegria , C. Kramm , C. Duran-Aniotz , I. Moreno-González , C. Soto 1 University of Texas Medical School at Houston, Neurology, Houston, USA 2 The University of Texas Health Science Center at Houston, Neurology, Houston, USA Aims To assess the effect of peripherally administered amyloid-beta (Abeta) seeds from different sources (brain, blood and recombinant) over brain amyloidosis. Method Three experimental paradigms were used: i) administration of brain extracts from old tg2576 mice or Alzheimer’s Disease (AD) patients into young transgenic mice by different routes including intra-cerebral, intra-peritoneal, intra-muscular, eye-drops and oral-gavages; ii) intra-venous administration of recombinant/purified Abeta structures (monomers, oligomers and dimers) in APP/PS1 mice; and iii) blood transfusions from old and young transgenic mice to young individuals. Treated animals were sacrificed at times when amyloid deposition was minimal in untreated mice. Results Our results show that Abeta seeds administered intra-peritonealy, intra-muscularly and by eye drops were able to accelerate pathological changes in the brains of transgenic mice. Surprisingly, the eye drop route was the most efficient among all the ones tested. Importantly, large quantities of seeds administered orally were unsuccessful to seed into the brain. Administration of recombinant/purified Abeta seeds into the circulation also increased brain amyloidogenesis, being oligomers more efficient seeds compared to fibrils. Blood transfusions also increased the levels of brain amyloidosis, albeit in a smaller degree. Peripheral Abeta seeding to the brain promoted the appearance of large quantities of vascular aggregates. Interestingly, different types of aggregates were generated depending of the administration route. Conclusion


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Our data indicates that peripherally circulating Abeta aggregates may contribute importantly to AD brain pathology. These results may help understanding the mechanisms related to the origin and spread of Abeta pathology and be of importance to design novel therapeutic and diagnostic strategies.

SYMPOSIUM 11 - SEEDING, SPREADING, AND PROTEIN AGGREGATION MECHANISMS ADPD7-0125 ΑLPHA-SYNUCLEIN PRIONOIDS NEUROINVADE THE CNS AFTER PERIPHERAL INJECTION 1 1 1 2 2 1 E.G. Tamgüney , M.E. Bernis , J.T. Babila , M.C. Garza , H. Wille , S. Breid 1 German Center for Neurodegenerative Diseases DZNE, AG Tamgüney, Bonn, Germany 2 University of Alberta, Centre for Prions and Protein Folding Diseases & Department of Biochemistry, Edmonton, Canada Aims α-Synuclein forms pathologic protein deposits such as glial cytoplasmic inclusions in oligodendrocytes of patients with multiple system atrophy, or Lewy bodies and Lewy neurites in neurons of patients with Parkinson’s disease. Also, misfolded α-synuclein shows prion-like propagation along synaptic connections after intracerebral injection into transgenic and wild-type mice. Here we investigated the potential of α-synuclein fibrils to induce neurodegeneration after intraperitoneal or intraglossal injection. Method We injected wild-type α-synuclein fibrils or phosphate-buffered saline as control into the peritoneum or tongue of +/+/bigenic Tg(M83 :Gfap-luc ) mice that hemizygously express the A53T-mutant of human α-synuclein and firefly +/luciferase. We also intraglossally injected monogenic Tg(Gfap-luc ) mice that only express endogenous wild-type mouse α-synuclein. Results All PBS-injected mice remained healthy during the 420 d course of the experiment. In contrast, injection of αsynuclein fibrils into the peritoneum caused neurologic disease with kyphosis and paralysis in 80% of the +/+/Tg(M83 :Gfap-luc ) mice with a median incubation time of 229 ± 17 d. Diseased mice accumulated aggregates of phosphorylated and sarkosyl-insoluble α-synuclein in brain and spinal cord, which colocalized with p62 and ubiquitin and were accompanied by gliosis as detected by immunohistochemistry and bioluminescence imaging. +/+/Injection of alpha-synuclein fibrils into the tongue of Tg(M83 :Gfap-luc ) mice caused neurodegeneration in only +/20% of the mice in 285 d. In contrast, intraglossal injections of Tg(Gfap-luc ) mice did not cause pathology. Conclusion +/-

+/-


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We show here that similar to prions, α-synuclein prionoids can neuroinvade the CNS of Tg(M83 :Gfap-luc ) mice after intraperitoneal or intraglossal injection and cause neurodegeneration and disease.

SYMPOSIUM 11 - SEEDING, SPREADING, AND PROTEIN AGGREGATION MECHANISMS ADPD7-0740 A UNIVERSAL AGGREGATION MECHANISM FOR FAMILIAL ALPHA-SYNUCLEIN MUTATIONS IN PRIONLIKE SPREAD 1 1 1 1 1 J. Sanderson , N. Kim , E. Grignaschi , U. Dettmer , T. Bartels 1 Harvard Medical School - Ann Romney Center for Neurologic Diseases, Neurology, Boston, USA Aims Parkinson’s disease (PD) is the most common movement disorder, characterized by severe cell loss in the substantia nigra and neuronal/neuritic accumulation of aggregated α-Synuclein (αSyn). We discovered that αSyn normally exists in cells principally as an α-helically folded tetramer that resist aggregation. We have recently established an invariant tetramer-destabilizing effect of fPD mutations, leading to a shift of the tetramer/monomer ratio towards the latter, aggregation-prone form. Recent studies suggest a seeded propagation of aggregated αSyn in in vivo models. We are trying to address experimentally here how tetramer destabilization influences the susceptibility of neuronal cells to prion-like spread. Method We use a variety of molecular biology techniques (ThT fluorescence, SDS-PAGE/Western Blot, in vivo crosslinking) to quantify the structural changes and aggregation propensity of different αSyn variants in vitro and neuronal cells. Results The data we are presenting here indicate that the native, tetrameric form is not only resistant to time-dependent aggregation but also shows greater resistance (vs. unfolded monomers) to αSyn misfolding initiated by small amounts of fibrillar material - so-called “seeded aggregation”. Tetramer-destabilizing factors like fPD mutations make physiological αSyn susceptible to conversion into pathological self-templating oligomers in our experiments, analogous to transthyretin-based amyloidosis or prion diseases in which a destabilization event is necessary to initiate pathologic aggregation. Conclusion


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Our data tie the discovery of the soluble physiological tetramer with the recently discussed “prion-like” aggregation model thus clarifying some of the earliest events that need to happen before the spread of amyloid in the brain is possible.

SYMPOSIUM 11 - SEEDING, SPREADING, AND PROTEIN AGGREGATION MECHANISMS ADPD7-0692 ASSESSMENT OF ALPHA-SYNUCLEIN AGGREGATION AND TRANSMISSION IN VIVO 1 V. Prasad 1 Uniklinik RWTH Aachen, Neurology, Aachen, Germany Aims The pathological hallmark of synucleinopathies like Parkinson’s disease (PD) and multiple system atrophy is the presence of aggregated alpha-synuclein. Although a clear correlation of alpha-synuclein aggregation and disease is evident, in vivo data on the cellular mechanisms promoting alpha-synuclein aggregation is largely missing.In addition, our system allows monitoring of neuron-to-neuron transfer of alpha-synuclein. In humans, a detailed analysis revealed that alpha-synuclein aggregates appear first in the peripheral nervous system from where they seem to spread via connected neuronal populations throughout the brain, eventually reaching the dopaminergic neurons in substantia nigra. Method We apply bimolecular fluorescence complementation to visualize alpha-synuclein aggregation and spreading in vivo . Combined with the powerful genetics of Drosophila as a model system, we show that soluble alphasynuclein oligomers are degraded by the chaperone/proteasome degradation system, while the insoluble alphasynuclein is degraded via the authophagic/lysosomal pathway. In addition, we analyzed factors implicated to cause PD (e.g. rotenone, metal ions) and presumably protective agents like resveratrol on aggregation properties of alpha-synuclein. Results With the exception of ferric ion and rotenone, other insults did not, or only mildly enhance the aggregation of alpha-synuclein. Interestingly, resveratrol provided protection towards alpha-synuclein dependent toxicity but had no impact on aggregation. However, in our in vivo model we show that neuron-to-neuron transfer is facilitated via synaptic release of alpha-synuclein. Conclusion


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We conclude that the extraneous insults have no dramatic effect on alpha-synuclein aggregation. With regard to spreading, our in vivo data suggests that neuronal activity is required for alpha-synuclein aggregation and neuronto-neuron transfer.

SYMPOSIUM 12 - UPDATE ON PD ETIOLOGY AND MANIFESTATIONS ADPD7-0133 IS PARKINSON REALLY A PRION DISEASE? 1 A. Korczyn 1 , Tel-Aviv, Israel

Is Parkinson's a prion disease? Amos D. Korczyn Sackler School of Medicine Tel-Aviv University, Ramat-Aviv 69978, Israel The hallmarks of prion diseases consist of four basic elements: An initial spontaneous stochastic conversion of a normal endogenous protein into a misfolded form. This misfolded form serves as a template encouraging conversion of further normal molecules into misfolded isoforms; the resultant isoform is toxic to cells; and the neurons then release the abnormal isoform into the neuropil, causing damage to neighboring cells, resulting in "status spongiosus".


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The recent designation of Parkinson's disease (PD) as a prion disease does not conform to any of these basic elements. The presumed prion-like molecule, α-synuclein, can of course be misfolded like all cellular proteins. This can occur in all neurons throughout the CNS, not just the structures damaged in PD. But there is no evidence that this process is self-replicating. Neither is there substantial evidence that α-synuclein, in its native or misfolded isoforms, is toxic to cells. And finally, there is no data to support the assumption that neighboring neurons are affected through the release of misfolded α-synuclein from dying cells in PD. The sequential degeneration of neurons, which occurs in PD, is unparalleled by lesions in prion diseases.

SYMPOSIUM 12 - UPDATE ON PD ETIOLOGY AND MANIFESTATIONS ADPD7-0098 HALLUCINATIONS IN PARKINSONS DISEASE 1 2 3 3 3 J. Kulisevsky , J. Pagonabarraga , S. Martínez-Horta , E. Bejr-Kasem , J. Marín-Lahoz 1 Hospital Sant Pau- Universitat Autónoma de Barcelona- Universitat Oberta de Catalunya UOC and CIBERNED, Neurology, Barcelona, Spain 2 Sant Pau Hospital- IIB Sant Pau, Neurology Department, Barcelona, Spain 3 Research Institute- Sant Pau Hospital IIB Sant Pau, Neurology, Barcelona, Spain

Visual Hallucinations in PD: from premotor phases to dementia. The development of visual hallucinations (VH) is a frequent complication of Parkinson's disease (PD). Presence of hallucinations is one of the main risk factors associated with dementia, and severity progression of VH mainly contributes to impaired quality of life in PD. The clinical spectrum of VH in PD goes from minor hallucinations (presence, passage hallucinations) to well-formed VH with loss of insight. The neuropsychological features associated with severity progression of VH are only partially known and might help to detect patients at risk of a more severe outcome. Previous work has shown that patients with minor VH did not differ from patients without VH in any cognitive domain. PD patients with major VH and insight retained performed significantly worse on the action verbal fluency task, and patients with VH and loss of insight showed a greater impairment on posterior cortical tasks such as denomination and copying. A double dissociation was found in the neuropsychological profile of patients with VH with and without loss of insight. While the presence of major VH with insight retained appeared related to a predominant frontal-striatal impairment, loss of insight was characterized by further impairment of cognitive functions related to posterior cortical areas. A comprehensible continuum pattern of clinical relationships emerged among VH and cognitive functioning in PD. Structural and functional abnormalities within the primary visual system and visual association areas, including ventral and dorsal pathways, have been reported. Structural MRI studies have shown that non-demented PD patients with VH present grey matter reduction in parieto-occipital areas and the hippocampal head. unlike PD patients without VH, PD patients with VH frequently develop dementia associated with progressive atrophy in limbic, paralimbic and neocortical areas. Functional MRI (fMRI) studies have revealed altered activation in occipito-temporal and frontal areas in response to simple and complex visual stimuli in PD patients with VH, suggesting a marked impairment in bottom-up visual processing, as well as an attentional deficit in the pathophysiology of VH in PD. Although minor hallucinations are generally not disturbing, they may progress to well-structured hallucinations with loss of insight and a great impact on quality of life. Compared to controls, PD with minor hallucinations (PDmH) show reduced gray matter volume bilaterally in different areas of the dorsal visual stream, and in functionally related midbrain and cerebellar structures. Additionally, bilateral gray matter volume increases were observed in the PD-mH group in limbic and paralimbic regions. Thus, existint data support a major role of the dorsal visual stream in the genesis of minor hallucinations in PD, reinforcing the importance of posterior cortical regions for the development of cognitive and psychiatric complications in PD.


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Because early detection of minor hallucinations may be useful for screening patients with more severe endophenotypes, we prospectively assessed 50 drug-naive, "de novo" PD patients and 100 controls using the MDS-UPDRS Scale-Part I and a structured interview covering all types of psychotic phenomena reported in PD. Minor hallucinations were experienced in 42% (21 of 50) PD patients and 5% controls (P E3>E2. There may be other mechanisms that link APOE to both risk and progression of AD as well as other neurodegenerative diseases. We sought to ask whether ApoE isoforms influences tauopathy and tau-mediated neurodegeneration in a mouse model. We utilized a mouse model of tauopathy (P301S Tau transgenic mice) in which a form of human tau is overexpressed in the brain and asked whether the presence or absence of human ApoE isoforms influenced tauopathy and tau-mediated neurodegeneration relative to murine apoE and no apoE. By 9 months of age, P301S/ApoE4 mice had strikingly more brain atrophy and neuronal loss as well as increased insoluble tau accumulation than P301S mice on the other ApoE backgrounds. In addition, both microglial and astrocyte gene expression patterns consistent with neurodegeneration were seen in P301S mice expressing different forms of ApoE. This neuroinflammatory pattern of gene expression was markedly reduced in P301S/ApoE knockout mice. These results show that ApoE4 can drive increased tau-mediated neurodegeneration. This may result from its ability to increase tau aggregation independent of Aβ together with the effects of ApoE on neuroinflammation.

SYMPOSIUM 15 - APOE: MECHANISMS AND APOE-BASED TREATMENT STRATEGIES ADPD7-0039 CENTRAL AND PERIPHERAL APOE IN COGNITION AND AD 1 1 G. Bu , C.C. Liu 1 Mayo Clinic, Neuroscience, Jacksonville, USA


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The ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). In the central nervous system (CNS), apoE4 inhibits the clearance and promotes the aggregation of amyloid-β (Aβ), and has several Aβ-independent effects including age-dependent inferior functions in transporting lipids, supporting synapses, and controlling neuroinflammation. ApoE is also abundantly expressed in peripheral tissues including liver where it mediates plasma lipid metabolism. APOE4 carriers have increased risk for hypercholesterolemia and atherosclerosis. To address apoE isoform-dependent functions in CNS and periphery, we developed cell type-specific and inducible apoE mouse models. Expression in the CNS was driven by astrocyte-specific GFAP-Cre and in the periphery by liver-specific Alb-Cre. These mice were further bred to the background of amyloid model APPSWE/PS1ΔE9 mice. When expressed by astrocytes, we found that expression of apoE3 but not apoE4 led to improved synaptic functions and memory performance, whereas expression of apoE4 but not apoE3 inhibited Aβ clearance and accelerated amyloid deposition. More interesting, expression of apoE4 prior to amyloid deposition had a greater impact on amyloid pathology than that during the rapid growth period. When apoE isoforms were expressed exclusively in the periphery in the Apoe-knockout background, apoE4 inhibited synaptic functions, impaired cognition, and was associated with compromised blood-brain barrier integrity and vascular functions. These results provide mechanistic insights as to how apoE4 increases the risk for AD and how we can develop apoE isoform-specific strategies for AD prevention and therapy.

SYMPOSIUM 15 - APOE: MECHANISMS AND APOE-BASED TREATMENT STRATEGIES ADPD7-1580 INTERRELATIONSHIP OF BETA AMYLOID RATIO 42/40 AND TTAU AND ITS DEPENDENCY ON APOE GENOTYPE 1 1 1 2 O. Peters , K. Luther , S. Röhling , J. Wiltfang 1 Charité - Campus Benjamin Franklin, Klinik für Psychiatrie und Psychotherapie, Berlin, Germany 2 Universitätsmedizin Göttingen, Klinik für Psychiatrie und Psychotherapie, Göttingen, Germany Aims Levels of amyloid- and TAU-species in the cerebrospinal fluid (CSF) are considered to mirror neurodegeneration in AD. Yet their interdependence remains partly unclear. Here we investigated the Aß-1-42/Aß-1-40-ratio (Aßratio), tTAU and APOE genotype in parallel to elucidate the interdependencies of the most common biomarkers. Method A total of 593 patients with objectified cognitive deficits (age > 50; MMSE ≥ 21) were included. CSF Aß-1-40, Aß1-42 and tTAU were quantified using established ELISAs. For pairwise correlations between amyloid species and tTAU Pearson correlation coefficients were calculated. ROC curves were determined for single Aß-species and Aß-ratio to classify the cohort into patients with high and low tTAU levels (threshold 350 pg/ml). Subgroup analyses were conducted for APOE ε4 carriers and non-carriers. Results The Aß-ratio correlates to a higher degree with tTAU levels compared to Aß-1-42 alone (rAß-ratio&tTAU= -0.61, p= -62 -23 6.8*10 ; rAß-1-42&tTAU= -0.39, p= 3.7*10 ) in all subjects. Aß-ratio showed a significant higher classification -14 performance than Aβ-1-42 alone (AUCAß-ratio= 0.83, p= 2.1*10 , AUCAß-1-42 = 0.71). Patients with none or a single ApoE-4 allele were best classified by Aß-ratio, whereas only in patients with two ApoE-4 alleles the ratio did not outperform single markers. Conclusion


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The CSF Aß-ratio exceeded Aß-1-42 alone in segregating the cohort into patients with high and low tTAU. These results indicate an association between the TAU and amyloid metabolism, influenced by ApoE genotype, whereby considering the Aß-ratio instead of Aß-1-42 alone is of advantage.

SYMPOSIUM 15 - APOE: MECHANISMS AND APOE-BASED TREATMENT STRATEGIES ADPD7-0540 EFFECT OF APOE GENOTYPE ON PATHOLOGICAL CEREBROSPINAL FLUID CUT-POINTS IN MILD COGNITIVE IMPAIRMENT 1 1 2 3 4 5 3 6 7 M. Marizzoni , C. Ferrari , J. Jovicich , D. Albani , C. Babiloni , M. Didic , G. Forloni , J.L. Molinuevo , F. Nobili , 8 9 10 11 12,13 14 15 L. Parnetti , P. Payoux , P.M. Rossini , P. Schönknecht , A. Soricelli , M. Tsolaki , P.J. Visser , 16,17 18 19 1,20 J. Wiltfang , R. Bordet , O. Blin , G.B. Frisoni 1 IRCCS Centro San Giovanni di Dio Fatebenefratelli, Laboratory of Neuroimaging and Alzheimer's Epidemiology, Brescia, Italy 2 University of Trento, Center for Mind/Brain Sciences, Trento, Italy 3 IRCCS Istituto di Ricerche Farmacologiche "Mario Negri", Neuroscience Department, Milano, Italy 4 Sapienza University of Rome, Department of Physiology and Pharmacology, Rome, Italy 5 Aix-Marseille Université and Hôpital Timone Adultes, INSERM and Service de Neurologie et Neuropsychologie, Marseille, France 6 Hospital Clínic de Barcelona, Alzheimer's Disease Unit and Other Cognitive Disorders Unit, Barcelona, Spain 7 University of Genoa- Genoa- Italy-, Department of Neuroscience- Ophthalmology- Genetics and Mother– Child Health DINOGMI, Genoa, Italy 8 University of Perugia, Section of Neurology- Centre for Memory Disturbances, Perugia, Italy 9 Université de Toulouse, Toulouse NeuroImaging Centre, Toulouse, France 10 Catholic University, Department of Gerontology- Neurosciences & Orthopedics, Rome, Italy 11 University Hospital, Department of Mental Health- Psychiatry and Psychotherapy, Leipzig, Germany 12 IRCCS SDN, Neuroimaging, Naples, Italy 13 University of Naples Parthenope, University of Naples Parthenope, Naples, Italy 14 Aristotle University of Thessaloniki, 3rd Department of Neurology-, Thessaloniki, Greece 15 VU University Medical Center, Alzheimer Center and Department of Neurology, Amsterdam, The Netherlands 16 University of Duisburg-Essen, Department of Psychiatry and Psychotherapy, Essen, Germany 17 University Medical Center UMG- Georg-August-University, Department of Psychiatry and Psychotherapy, Goettingen, Germany 18 University of Lille Nord de France, Department of Pharmacology, Lille, France 19 Aix-Marseille University-CNRS, Pharmacology- Assistance Publique-Hôpitaux de Marseille, Marseille, France 20 University Hospitals of Geneva, Laboratory of Neuroimaging of Aging, Geneva, Switzerland Aims To investigate the effect of APOE genotype on cerebrospinal fluid (CSF) biomarkers distribution and on cut-points to identify prodromal AD. Method WP5 of PharmaCog (E-ADNI) harmonized and collected data from 13 European centres. 147 mild cognitive impairment (MCI) patients were followed up for at least 2 years or until they progressed to clinical dementia. Baseline analysis. T-test to compare APOEε4 carriers and non-carriers features; Mixture Model Analysis, adjusted or not for APOE genotype, for pathological CSF cut-points determination. Longitudinal analysis. Linear Mixed Model for repeated measures to study the association between CSF status and time on clinical and biomarker values. Results


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At baseline, APOEε4 carriers reported lower CSF levels of Aβ42 and Aβ42/p-tau, and higher levels of t-tau and ptau relative to non-carriers (p19 it was clear that A-Beta deposition had the earliest onset, whereas in non-e4 carriers and poly-Tlength≤19 it was hippocampal volume. A 40% risk of having abnormal Aβ deposition occurred 22 years earlier in e4 vs none4 carriers, and 18 years earlier in poly-Tlength>19 vs poly-Tlength≤19, no significant differences were observed for gender or BDNF Met carriage. Conclusion


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The order of onset of abnormal levels of disease markers as measured here appears to align with other findings APOE e4 carriage and TOMM40 poly-Tlength>19 appear to be associated with earlier onset.

SYMPOSIUM 19 - BIOMARKERS 2: AD ADPD7-1251 KALLIKREIN-RELATED PEPTIDASES IN ALZHEIMER’S DISEASE – SUITABLE BIOMARKERS FOR AMYLOID PATHOLOGY? 1 1 1 1 2 3 O. Goldhardt , T. Grimmer , H. Förstl , P. Alexopoulos , V. Magdolen , E. Diamandis 1 Klinikum rechts der Isar der Technischen Universität München, Department of Psychiatry and Psychotherapie, Munich, Germany 2 Klinikum rechts der Isar der Technischen Universität München, Department of Gynaecology, Munich, Germany 3 Mount Sinai Hospital of University of Toronto, Department of Laboratory Medicine and Pathophysiology, Toronto, Canada Aims Alterations in the expression of human kallikrein-related peptidases (KLKs) have been described in patients with Alzheimer’s disease (AD) compared to cognitively normal controls (NC): KLK10 was increased in cerebrospinal fluid (CSF) in AD. In mice, KLK-8 inhibition impeded amyloidogenic amyloid-precursor-protein processing and facilitated amyloid β (Aβ) clearance. KLK6 was highly expressed in the nervous system, positively associated with age, increased in brain tissue and in CSF and associated with pTau levels. In this study, we aimed at elucidating the suitability of KLK6, 8, and 10, to distinguish between NC and patients with probable AD and explored associations with AD biomarkers. Method We used ELISA to measure KLK6, 8, 10 levels in CSF of AD patients (n=33, 71.4 ± 4.64 years, CDR global=0.65 ± 0.318) with evidence for amyloid pathology in amyloid PET and/or CSF, and compared to NC (n=23, 64.7 ± 9.16, CDR global=0) with physiological levels of amyloid and tau in CSF. Results KLK6 and KLK8 were increased in patients (KLK6 p97%) involve genetic susceptibility factors, e.g. APOE, and lifestyle, e.g. diet, exercise, sleep, intellectual and social engagement, stress levels, and brain trauma. Most recently we have found that low-grade infections, e.g. bacterial, fungal, viral, in the brain may also play a role by rapidly nucleating beta-amyloid deposition as an antimicrobial protection response of the brain's innate immune system. Genetic susceptibility factors have been elucidated over the past decade using genome-wide association studies (GWAS) and more recently by follow up with whole genome sequencing (WGS) and whole exome sequencing (WES). We are now carrying out GWAS using approximately 50 million single nucleotide variants (SNV) from WGS and WES (whole genome sequencing association studies; WGSAS). As AD-linked/associated functional SNVs are identified in these studies, they are being tested in our 3D human stem cell-derived neural culture models of AD, in which we have shown betaamyloid directly drives tangle formation. Many of the more recently identified AD genes are involved in innate immunity, e.g. CD33, which we first reported in our family-based GWAS in 2008 (along with ADAM10 and ATXN1). To study CD33 and other innate immune-related AD genes, we have incorporated microglia into our 3D neural cultures while also utilizing classic transgenic mouse models. Aspects of all these studies will be covered.

SYMPOSIUM 35 - GENETICS 2 - EPIGENETIC AND GENETIC MECHANISMS ADPD7-0067 PROGRESS IN UNRAVELLING THE GENETICS OF EARLY-ONSET DEMENTIA 1 C. Van Broeckhoven 1 VIB, Department of Molecular Genetics, Antwerp, Belgium


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The majority of genes involved in early-onset neurodegenerative dementia have been identified by molecular genetic studies in extended families with autosomal dominant disease. This approach has successfully discovered several causal genes for the major clinical subtypes i.e. Alzheimer disease and frontotemporal dementia. Knowledge of these genes has provided fundamental insights in the disease mechanisms that are underlying neurodegenerative dementia that became cornerstones in potential pharmaceutical interventions. However, mutations in the known genes associated with early-onset dementia (EOD) explain only a minor fraction of the patients and families. Continued interest in EOD genetics is warranted since recent studies using massive parallel sequencing, demonstrated that rare genetic variants might contribute to our further understanding of the molecular disease mechanisms behind EOD. Clinical studies have indicated that EOD is highly heterogeneous with a substantial fraction of patients in whom the disease is unrelated to previous identified biological pathways. Further, many EOD patients have been excluded from genetic studies since their clinical phenotype was atypical and did not fit the de strict diagnostic entities. Nonetheless, several of those atypical EOD patients have a positive family history of dementia and some belong to large families. We showed that mutations in known causal genes can be found in patients with diagnostic unclassified EOD. Also, mutations are found in genes that were associated with late-onset dementia (e.g. Sorl1, ABCA7, TBK1, etc.). In extended families, we used exome sequencing and identified rare genetic variants in genes that had not yet been associated with EOD.

SYMPOSIUM 35 - GENETICS 2 - EPIGENETIC AND GENETIC MECHANISMS ADPD7-0042 REGULATION OF GENE EXPRESSION: THE GENETIC MECHANISMS UNDERLYING NEURODEGENERATIVE DISEASES 1,2 1,2 1 1,3 O. Chiba-Falek , L. Tagliafierro , M. Lutz , A. Roses 1 Duke University Medical Center, Neurology, Durham, USA 2 Duke University Medical Center, Center for Genomic and Computational Biology, Durham, USA 3 Zinfandel, Pharmaceuticals, Chapel Hill, USA


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In the post genome-wide association studies (GWAS) era we are shifting gears toward translation of genetic disease loci to molecular mechanisms of pathogenesis and pinpointing the causal genetic factors and their functional effects. It has been suggested that changes, even subtle, in the expression levels of wild-type genes in the brain can, over years, lead to neurodegenerative diseases. Moreover, differences in gene expression profiles between brain tissues from neurodegenerative disease patients compared to healthy controls have been reported. We have been studying the expression regulation of key genes implicated in Alzheimer’s (AD) and Parkinson’s (PD) diseases and a wide-spectrum of related disorders. In particular, we focus on noncoding variants and their cis-regulatory effects on gene expression using a comprehensive strategy that combines multiple-level approaches. (1) in silico: we have developed a new bioinformatics tool for prioritizing candidate functional/causal short structural variants (SSVs). The tool is a searchable, annotated database of SSVs, with associated customizable scoring software that is designed to evaluate and prioritize SSVs that are most likely to have significant biological effects and impact on disease risk. (2) in vitro: we have established induced Pluripotent Stem Cell (iPSC)-derived neurons, dopaminergic and cholinergic, from a normal subject and PD patients. We are currently using this model system to determine how cis-variants modulate expression of disease risk genes via their interactions with the trans-acting factors, by applying CRISPR/Cas9 – mediated genome editing. (3) in vivo: we have quantified cell-type specific gene expression in neurons, astrocytes, and microglia by single-cell gene expression assays using cells isolated from frozen human brain samples via LCM. We have implemented this strategy in studies of genomic loci implicated in Lewy body and AD diseases, specifically SNCA gene and the TOMM40-APOE cluster. Collectively, the innovative approaches we developed represent a cohesive strategy for discovering potentially functional and causal variants within candidate risk-genes associated with AD-PD spectrum disorders.

SYMPOSIUM 35 - GENETICS 2 - EPIGENETIC AND GENETIC MECHANISMS ADPD7-1694 REPEAT ASSOCIATED NON-ATG (RAN) TRANSLATION IN NEUROLOGIC DISEASE: LESSONS FROM C9ORF72 ALS/FTD BAC MICE 1 1 1 1 1 1 2 1 T. Zu , A. Pattamatta , L. Nguyen , Y. Liu , B. Perez , O. Bardhi , A.T. Yachnis , L. Ranum 1 University of Florida Center for NeuroGenetics, Dept. MolecularGeneticsandMicrobiology and Genetics, Gainesville, USA 2 University of Florida Center for NeuroGenetics, Dept. of Pathology Immunology and Laboratory Medicine, Gainesville, USA

Microsatellite expansion mutations cause more than 30 different neurologic diseases. In 2011, we discovered that in the absence of an AUG initiation codon, expanded CAG and CUG repeats can express homopolymeric proteins from all three reading frames. We showed this repeat-associated non-ATG (RAN) translation occurs in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1). Since this initial discovery, we and others have demonstrated that RAN translation occurs in C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD), Fragile X tremor ataxia syndrome (FXTAS) and Huntington’s disease (HD). An emerging theme is that coding and non-coding expansion mutations are bidirectionally expressed, producing two mutant RNAs and up to six mutant RAN proteins. These findings impact our fundamental understanding of gene expression and protein translation and should now be considered for a broad category of neurological disorders.


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Efforts to understand the mechanisms of RAN translation and the roles that RNA gain-of-function and RAN proteins play in C9ORF72 ALS/FTD will be discussed. We have recently developed a BAC transgenic model of C9ORF72 ALS/FTD that develops key phenotypic and molecular features of the disease. These C9-BAC mice show decreased survival, paralysis, muscle denervation, motor-neuron loss, anxiety-like behavior and cortical and hippocampal neurodegeneration in a repeat-length and expression dependent manner. These mice express C9ORF72 sense and antisense transcripts and RNA foci are found throughout the CNS. RAN protein accumulation increases with age and severity of disease and TDP-43 inclusions are found in degenerating brain regions in end-stage animals. Insights into RNA vs. RAN mechanisms in C9ORF72 from our BAC transgenic mouse model will be discussed along with efforts to reverse the disease in our mice.

SYMPOSIUM 35 - GENETICS 2 - EPIGENETIC AND GENETIC MECHANISMS ADPD7-0085 MICRORNA (MIRNA) SIGNALING IN ALZHEIMER'S DISEASE (AD) 1 W. Lukiw 1 , USA

microRNA (miRNA) signaling in Alzheimer's disease (AD) and other inflammatory neurodegenerations of the CNS


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MicroRNAs (miRNAs) are small, non-coding single-stranded RNA molecules 20-26 nucleotides (nt) in length that are regulators of gene expression in the CNS in neurodevelopment, aging, health and disease. There are about 2650 human miRNAs (only ~35 of which are human brain abundant) and 27,000 human messenger RNAs (mRNAs) so far identified; the major mode of miRNA action and the down-regulation of gene expression is that an up-regulated miRNA typically recognizes (via base pair complementarity) and binds to the mRNA-3’ untranslated region (mRNA-3’-UTR) of selective mRNA targets and in doing so downregulates expression from that mRNA. Certain up-regulated miRNAs contribute to chronic inflammation, innate-immune and synaptic disturbances in various brain cell types (such neurons, astroglia, microglia, endothelial cells), thereby leading to progression of neurodegenerative diseases like sporadic Alzheimer's disease (AD), prion disease (PrD) and age-related macular degeneration (AMD). Our current studies involve the extraction, quantitation and characterization of miRNA populations from (i) short post-mortem brain tissues and biopsies of sporadic AD, PrD, AMD and other agerelated neurological diseases; (ii) human brain cells in primary culture that have been stressed with AD-relevant ROS- and/or NF-kB-inducing stressors including IL-1β, TNFα, Aβ42 peptides, hypoxia, neurotoxic metals (Al, Hg) and viruses (HSV-1); and (iii) aging, amyloid-overexpressing transgenic AD (Tg-AD) murine models focusing on the 5xFAD (C57BL/6J background) mouse. Our ongoing DNA-array-, miRNA array-, LED-Northern- and RNAsequencing-based analyses of ~160 human brain tissues indicate the significant up-regulation of a small family of inducible, NF-kB-regulated miRNAs including miRNA-7, miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a and miRNA-155; these target and down-regulate the expression of a family of at least 9 mRNAs including 15-LOX, CFH, IkBKG, IRAK-1 (with a compensatory surge of IRAK-2), TREM2, TSPAN12, SYN1/2, UBE2A, VDR; these induced changes can explain much of AD neuropathology including impaired amyloid clearance and amyloidogenesis, inflammation, tau pathology and deficits in neurotropism and synaptogenesis. Several of these more well-studied miRNA-mediated pathogenic circuitries will be discussed in this lecture. Lastly, our ideas on miRNA-mRNA coupling and their regulatory mechanism in the developing and aging CNS continue to evolve, and technological advancement, refinement and recent discoveries continue to contribute to our understanding of the pathogenic mechanisms that characterize these age-related neurodegenerative and neuropsychiatric disorders of the human CNS.

SYMPOSIUM 35 - GENETICS 2 - EPIGENETIC AND GENETIC MECHANISMS ADPD7-1165 TAU BUT NOT AMYLOID PATHOLOGY IS ASSOCIATED WITH LARGE-SCALE EPIGENOMIC CHANGES IN THE HUMAN BRAIN 1 1 2 P.L. De Jager , H.U. Klein , D.A. Bennett 1 Brigham and Women's Hospital and Harvard Medical School, Neurology, Boston, USA 2 Rush University Medical Center, Rush Alzheimer’s Disease Center, Chicago, USA Aims Epigenetic mechanisms are likely to play a key role in the pathogenesis of Alzheimer's disease (AD). We conducted an epigenome-wide association study to characterize the interplay between the brain epigenome and the pathologic AD hallmarks Tau and amyloid-β. Method ChIP-seq against H3K9Ac was conducted in 669 post-mortem human brain samples. Genome-wide DNA methylation and transcription profiles were available for a large subset of these samples. Tau and amyloid-β loads were quantified using immunohistochemnistry. ATAC-seq was applied to induced forebrain neurons to measure chromatin accessibility. Results We identified 26,384 H3K9Ac domains which primarily occurred at promoters (15,225) and enhancers (8,071). H3K9Ac levels at promoters were positively correlated with transcription. Tau protein loads were significantly associated with H3K9Ac levels in 5,980 domains and had a much broader impact than amyloid-β (610 domains). Domains positively associated with Tau showed a strong enrichment (p= 0.5 and 10 popular network inference algorithms. We prioritize genes within each study’s ensemble network based on each gene’s network distance to known genetic drivers of Alzheimer’s disease from the IGAP study. Results We demonstrate highly ranked genes from the integration of networks and genetic evidence across studies are enriched for known Parkinson’s Disease, AD, and Huntington’s signatures. Furthermore, we find evidence that transcriptomic networks mediate the effect of genetic loci via mitochondrial function. Previous evidence suggests a role of mitochondrial dysfunction in early stage AD pathophysiology. Conclusion


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Transcriptional network inference holds great promise for elucidating molecular pathways that cause or modulate Alzheimer’s disease, specifically when integrated with known genetic drivers of disease, and our method prioritize specific genes for further functional follow-up.

SYMPOSIUM 46 - GENETICS, TRANSCRIPTOMICS AND EPIDEMIOLOGY ADPD7-0265 WHOLE EXOME SEQUENCING IN 522 EARLY-ONSET ALZHEIMER PATIENTS REVEALS MAPT DUPLICATIONS AS A CAUSE OF PROMINENT TAU-RELATED DEMENTIA WITH INCREASED MAPT EXPRESSION 1 2 2 3 4 5 5 A. Rovelet-Lecrux , K. Le Guennec , O. Quenez , G. Nicolas , D. Wallon , J. Alexander , P. Paschou , 6 7 8 9 9 10 11 12 J.F. Deleuze , M. Lathrop , O. Kalev , S. Mehrabian , L. Traykov , T. Ströbel , I. Le Ber , T. Jonveaux , 13 10 4 2 C. FREX , G. Kovacs , D. Hannequin , D. Campion 1 Rouen University, Inserm U1079, Rouen, France 2 Inserm, U1079, Rouen, France 3 Rouen University Hospital, Department of Genetics, Rouen, France 4 Rouen University Hospital, Department of Neurology, Rouen, France 5 Democritus University of Thrace, Department of Molecular Biology and Genetics, Alexandroupoli, Greece 6 CEA, Centre National de Génotypage, Evry, France 7 McGill University, Génome Québec Innovation Centre, Montréal, Canada 8 Landes-Nervenklinik Wagner-Jauregg, Institute of Pathology and Neuropathology, Linz, Austria 9 UH “Alexandrovska”, Department of Neurology, Sofia, Bulgaria 10 Medical University Vienna, Institute of Neurology, Vienna, Austria 11 University Hospital Pitié-Salpêtrière, CRICM- IM2A- UMR-S975 AP-HP, Paris, France 12 Nancy University hospital, Department of Neurology, Nancy, France 13 CHU Brest, Inserm- UMR1078, Brest, France Aims Copy-number variants (CNVs), such as duplications of the APP locus or deletions of the exon 9 of PSEN1, are known causes of Early-Onset Alzheimer Disease (EOAD). To further decipher the role of CNVs in EOAD, we conducted a case-control study using whole-exome sequencing (WES) data from 522 EOAD cases and 584 controls of French origin. Method WES data were screened for rare (1.4 and 1.9, n= 89]. AB groups were compared on rates of disease 2 clinical progression using X and on rates of cognitive decline using ANCOVA. Statistical models were adjusted for clinical or demographic factors that also differed between the groups. Results ANCOVA models showed that rates of disease progression were lower in the AB+ [10%] than in AB++ group [5.6 %; OR 5.1 95%CI 3.4 -6.7]. ANCOVA showed that the rate of decline on the ADCS-PACC was lower in the AB+ group than in the AB++ group: with [d = 0.89] or without [d = 0.65] progressors included in the analyses. Conclusion


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This data provides a basis for the extent to which a change in amyloid levels, measured by amyloid plaque burden, could manifest in terms of change in cognition or clinical disease progression.

SYMPOSIUM 59 - IMAGING, BIOMARKERS, AND TRANSLATIONAL ASPECTS ADPD7-1690 APOE*E4 IMPACT ON THE CORTICAL THICKNESS BRAIN NETWORKS TOPOLOGY IN MILD COGNITIVE IMPAIRMENT 1 1 1 1 G. Sanabria Diaz , B. Draganski , L. Melie Garcia , F. Kherif 1 Centre Hospitalier Universitaire Vaudois CHUV, Laboratoire de recherche en neuroimagerie LRENDépartement des neurosciences cliniques, Lausanne, Switzerland Aims The apolipoprotein E ε4 allele (APOE*E4) has been consistently associated with a higher risk of developing Alzheimer’s Disease (AD). In the present study, we investigated how APOE*E4 status modulates the topological organization of structural brain networks in Mild Cognitive Impairment (MCI) depending on their progression into AD. Method We used graph theory and cortical thickness obtained from T1-weighted structural magnetic images to study brain network co-variance properties (NPs) in a longitudinal sample comprised 100 MCI who convert into AD and 100 non-converters. The groups were divided into 50 APOE*E4-positive (‛carriers’) and 50 APOE*E4-negative (‛non-carriers’). All subjects were selected from the ADNI database. Results The co-variation patterns among anatomical regions showed the double of differences over time between carriers and non-carriers for MCI who progressed into AD. Large co-variations patterns between carries and non-carriers involved structures like superior and inferior parietal cortex, precuneus and middle frontal regions described previously as a core of highly connected brain structures. The MCI carriers in this group showed a significant increase over time in the characteristic path length, clustering index, local efficiency, global connectivity and a global efficiency decrease. Interestingly, this was not the case for MCI non-converters in which the same patterns of differences between groups were preserved over time. Conclusion


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This study demonstrates an APOE*E4-related disruption of the brain topological network organization in MCI. Our findings suggest a differential genetic modulation associated with the disease progression. The APOE*E4 effect over whole-brain topological attributes could be a potential biomarker to predict conversion from MCI to AD.

SYMPOSIUM 59 - IMAGING, BIOMARKERS, AND TRANSLATIONAL ASPECTS ADPD7-1879 SUSTAINED ATTENTION AND MEMORY TASKS WITH CONCURRENT EEG PROVIDE POTENTIAL BIOMARKERS FOR MILD COGNITIVE IMPAIRMENT 1 1 1 1 2 3 S. Waninger , C. Berka , M.S. Karic , A. Meghdadi , D. Salat , A. Verma 1 Advanced Brain Monitoring, Neurotechnology, Carlsbad, USA 2 MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Charlestown, USA 3 Biogen, Neurodegenerative Drug Development, Cambridge, USA Aims There is a critical unmet need for reliable, cost effective and noninvasive biomarkers to quantify cognitive defects associated with dementia at early stages and facilitate development of efficacious, disease modifying therapeutics. Neurophysiological metrics including quantitative EEG and event-related potentials (ERPs) reliably measure the physiology associated with cognition and may provide sensitive metrics for early diagnosis, tracking disease progression and assessing efficacy of novel interventions for dementia. Method 3-Choice Vigilance (3CVT) and Image Recognition Tasks (IR) were administered with concurrent EEG acquisition to elicit ERPs in MCI and healthy cohorts. EEG was filtered and independent component analysis applied (EEGLAB) to clean the data. F-Measure, a combined measure of processing speed and accuracy, was used to evaluate performance. Results There were significant decreases observed in the ERP waveform late positive component (LPC) in the MCI group compared to the healthy controls during 3CVT, predominately in occipital and right temporal-parietal region. MCI patients evidenced larger and more widespread decreases in the LPC during IR with significant decreases over central, temporal, parietal and occipital regions. Significant differences were observed in performance during IR; F-measure in the healthy group (0.77 ± 0.1) are higher than MCI (0.70 ± 0.1) (p = 0.038). Conclusion


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The data suggest that cognitive ERPs obtained during tasks that activate the neural circuits involved in sustained attention and recognition memory may provide a powerful tool for assessing MCI and have strong potential as sensitive and robust biomarker for tracking disease progression and evaluating response to investigative therapeutics.

SYMPOSIUM 59 - IMAGING, BIOMARKERS, AND TRANSLATIONAL ASPECTS ADPD7-1778 COMPARISON OF BIOMARKERS BETWEEN A PHASE 0 STUDY IN ALZHEIMER'S DISEASE AND THE ALZHEIMER’S DISEASE NEUROIMAGING INITIATIVE LONGITUDINAL COHORT 1 2 3 3 3 3 3 4 4 N. Mendonca , Z. Sun , H.K. Lon , R. Li , Z. Jin , C. Locke , S. Goss , D. Wang , W. Robieson , B. Rendenbach1 Mueller 1 AbbVie Deutschland GmbH & Co. KG, Neuroscience, Ludwigshafen, Germany 2 University of Pittsburgh, Graduate School of Public Health, Pittsburgh, USA 3 AbbVie Inc., Clinical Pharmacology and Pharmacometrics, North Chicago, USA 4 AbbVie Inc., Statistics, North Chicago, USA Aims A Phase 0 (Ph0) exploratory biomarker study was conducted to confirm that the Alzheimer’s Disease Neuroimaging Initiative (ADNI) data-derived conclusions on biomarkers apply to the Alzheimer’s disease (AD) population of planned clinical trials and to better characterize the biomarkers relevant for AD. Method In the multicenter Ph0 (non-treatment) study, 26 AD subjects, 52 mild cognitive impairment (MCI) subjects, and 26 healthy subjects of similar age were enrolled. Subjects underwent magnetic resonance imaging (MRI) and amyloid positron-emission tomography (PET) imaging as well as 2 lumbar punctures to collect cerebrospinal fluid (CSF). The 2 studies (ADNI and the Ph0 study) were similar with respect to inclusion and exclusion criteria for each of the three diagnostic groups. Results Comparison between the Ph0 study and the ADNI cohort showed that within each diagnosis group, the 2 study samples were comparable in most aspects including: (1) demographic composition and genetic risk profile; (2) classic CSF biomarkers (abeta1-42, tau and phosphorylated-tau); (3) brain MRI (volumetric MRI); (4) amyloid PET standard uptake value ratio; and (5) cognitive assessments. Overall, the patient selection criteria used in our Ph0 study resulted in preservation of key findings on CSF and neuroimaging biomarkers from ADNI, even with a relatively modest sample size. Conclusion


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These results increase confidence in the disease-progression assumptions that were derived through ADNI data analyses and in clinical trial decisions such as those on patient selection criteria that may be taken in our clinical development programs based on the results from the Ph0 study.

SYMPOSIUM 59 - IMAGING, BIOMARKERS, AND TRANSLATIONAL ASPECTS ADPD7-1893 [18F]T807/AV-1451 (FLORTAUCIPIR) IMAGING IN ATHLETES WITH POSTCONCUSSIVE SYNDROMES INCLUDING CLINICALLY PROBABLE CTE: PROMINENCE OF PSYCHIATRIC CLINICAL SYMPTOMS AND IMPLICATIONS FOR EXPERIMENTAL THERAPY 1 2 3 4 4 5 6 6 D. Dickstein , M. Pullman , J. Short , L. Kostakoglu , K. Knesaurek , B. Jordan , W. Gordon , K. Dams-O'connor , 3 3 7 8 9 10 3 2 3 C. Tang , E. Wong , S. DeKosky , J. Stone , G. Farmer , E. Peskind , M. Sano , H. Patrick , S. Gandy 1 Uniformed Services University of Health Sciences, Pathology, Bethesda, USA 2 Icahn School of Medicine at Mount Sinai, Neuroscience, New York, USA 3 Icahn School of Medicine at Mount Sinai, Psychiatry, New York, USA 4 Icahn School of Medicine at Mount Sinai, Nuclear Medicine, New York, USA 5 Burke Rehabilitation Hospital, Neurorehabilitation, New York, USA 6 Icahn School of Medicine at Mount Sinai, Rehabilitation Medicine, New York, USA 7 University of Florida, Neurology, Gainesville, USA 8 University of Virginia, Radiology and Neurosurgery, Charlottesville, USA 9 Cortice Bioscience, NA, New York, USA 10 University of Washington School of Medicine, Psychiatry and Behavioral Sciences, Seattle, USA Aims Chronic post-concussive syndromes are a major source of morbidity and mortality and candevelop into neurodegenerative disorders such as chronic traumatic encephalopathy (CTE). Methods ofassessment and prediction of outcome are major areas of research focus, in particular molecular neuroimagingwith positron emission tomography (PET) using ligands for neuropathological lesions such as plaques andtangles. CTE is characterized pathologically by the presence of neurofibrillary tau deposits. In living persons,advances in diagnosis have been made through positron emission tomography (PET) using tracers binding toaggregated tau. Here we examined [ 18 F]T807 uptake in athletes with mTBI resulting from multiple concussions. Method Twenty-four athletes and four control subjects underwent PET for tau ([18F]T807/AV-1451) andamyloid ([18F]florbetapir). All athletes had experienced multiple concussions and presented with symptoms ofCTE (i.e. memory complaints, anxiety, depression, aggression). Results Eight athletes had abnormal [ 18 F]T807 ligand retention. None retained [ 18 F]florbetapir. While theamount of ligand retention varied, the pattern resembles postmortem CTE, with tau distribution at the whitematter-grey matter junction. Our data also show a high frequency of positive tau scans in clinically probableCTE while mostly negative in less severe post-concussive states. Conclusion


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Our study of in vivo imaging of tau deposition in the brains of retired athletes can provide insightinto the pathogenesis, diagnosis, and potential treatment of tauopathies such as CTE. Whether tauopathyimaging can provide useful diagnostic or prognostic screening information and/or clinically important outcomemeasures for anti-tauopathy therapies, such as tauopathy-reducing interventions, are major questions for thefuture.

SYMPOSIUM 59 - IMAGING, BIOMARKERS, AND TRANSLATIONAL ASPECTS ADPD7-0252 TREATING ACUTE AND CHRONIC NEURODEGENERATION BY INHIBITING NEUROTOXIC AGGREGATING PROTEINS 1 2 3 4 5 6 7 M. Maccecchini , O. Arancio , M.F. Chesselet , P. Davies , J. Sundstrom , R. Vasquez , K. Yien-Ming , 8 9 10 M. Salim , B. Mobley , H. Feldman 1 QR Pharma- Inc., Neuroscience, Berwyn, USA 2 Columbia University, Taub Institute of Neuroscience, New York, USA 3 University of California- Los Angeles, Neurology, Los Angeles, USA 4 Hofstra North Shore - LIJ University, Neuropathology and Neuroscience, Hempstead, USA 5 Hershey Medical Center, Ophthalmology, Hershey, USA 6 Mount Sinai Hospital, Molecular Modeling and Imaging, New York, USA 7 University of California San Francisco, Parkinson's and Movement Disorders, San Francisco, USA 8 Temple University, Pharmacy, Philadelphia, USA 9 University of California- San Diego, Neuroscience, San Diego, USA 10 University of California- San Diego, ADCS, San Diego, USA Aims Neurodegenerative disorders associated with accumulation of neurotoxic aggregating proteins are chronic like Alzheimer’s (AD) and Parkinson’s disease (PD) or acute like traumatic brain injury (TBI). Posiphen® is a small, hydrophobic, orally available molecule that readily enters the brain and inhibits translation of more than one neurotoxic aggregating protein. 1. show that by inhibiting more than one neurotoxic aggregating proteins, Posiphen may be effective in more than one disorder 2. elucidate the underlying mechanism of this inhibition 3. transition Posiphen into human phase II studies - AD and TBIMethod Animal Studies: the appropriate models were run and treated vs. untreated animals were compared as to symptomatology and pathology. Mechanism of Action: we ran RNA pulldown inhibition, thermophoresis binding, proteomics, protein inhibition, molecular modeling. Human Data: the phase IIa study in early AD is a double-blind, placebo-controlled, dose-escalating study, with state of the art stable isotope labeling, inhibition of markers, safety and cognition. Results Posiphen was found to be effective in APP/PS1 AD mice, in alpha-synuclein PD mice, in TBI rats and in acute glaucoma rats. The underpinning mode of action: neurotoxic aggregating proteins have a homologous 5’UTR region in their mRNA and a preserved translational regulatory mechanism. We will further report on the clinical study in early AD and the planned phase II/III study in TBI. Conclusion


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Posiphen inhibits more than one neurotoxic aggregating protein and shows efficacy in more than one model of neurodegeneration. While the animal data and preliminary human data look very encouraging, the final endpoint is efficacy in humans.

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SYMPOSIUM 59 - IMAGING, BIOMARKERS, AND TRANSLATIONAL ASPECTS ADPD7-0373 AUTOLOGOUS TRANSPLANTATION OF ENTERIC GLIA IMPROVES NEUROPATHOLOGY AND RESTORES COGNITIVE DEFICITS IN Β AMYLOID-INDUCED NEURODEGENERATION. 1 1 1 2 G. Esposito , S. Gigli , L. Seguella , G. Sarnelli 1 Sapienza University of Rome, Physiology and pharmacology "V. Erspamer", Rome Italy, Italy 2 University Federico II Naples, Experimental medicine and surgery, Naples, Italy Aims Progressive inability of astrocytes in removing amyloid plaques is a key hallmark featuring Alzheimer's disease (AD) brain. Enteric glial cells (EGC) are the intestine corresponding CNS astrocytes and might be replaced in the injured AD-brain as a "brand-new astroglia" population capable of restart normal astrocytic disrupted by amyloid accumulation. We evaluated the efficacy of a gut-brain autotransplantation of EGC in a rat model of Aβ-induced AD. Method Rats received Aβ 1–42 peptide (n = 120) or its vehicle infusion (n = 60) by artificial sterile cerebrospinal fluid (CSF). Aβ was infused into the lateral ventricle the 20 days following its implantation, and left in place for further transplantation. After stereotaxic surgery, Aβ-infused animals underwent appendectomy through laparotomy and EGC cells were isolated and tracked with PKH26GL nontoxic marker. At different time points (2-4-8 weeks post transplantation), immunofluorescence, immunoblot, behavioral and neuroimaging analysis were performed to evaluate: 1. engraftment of EGC and their impact on amyloid plaques burden formation, neurofibrillary tangles density and reactive gliosis. 2. neurogenesis rescue and behavioral improvement in Aβ-injuried rats. Results EGC successfully and stably engrafted in the CNS and caused: 1) considerable reduction of amyloid plaque burdens and size; 2) significant reduction of neurofibrillary tangles density; 3) marked improvement of cognitive functions and 4) forthright rescue of neurogenesis rate in Aβ-injuried rats. Conclusion


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The overall result was the amelioration of the pathological hallmarks and the cognitive deficits typical of Aβassociated disease. Autologous EGC transplantation could represent a novel alternative in cell-replacement therapies to treat neurodegeneration in AD.

SYMPOSIUM 59 - IMAGING, BIOMARKERS, AND TRANSLATIONAL ASPECTS ADPD7-0346 THE ANTI-INFLAMMATORY ANNEXIN A1 AMELIORATES BLOOD BRAIN BARRIER DISRUPTION IN A MOUSE MODEL OF ALZHEIMER’S DISEASE 1 1 2 3 1 M. Ries , M. Yanez Lopez , N. Baxan , E. Solito , M. Sastre 1 Imperial College London, Division of Brain Sciences, LONDON, United Kingdom 2 Imperial College London, Medicine, LONDON, United Kingdom 3 Queen Mary University London, William Harvey Research Institute, London, United Kingdom Aims Annexin A1 (ANXA1) is an anti-inflammatory protein, which in the brain appears to be released by microglial cells, but is also expressed in neurons and in the blood brain barrier (BBB) endothelium, where it has an important role in regulating BBB tightness. Because Alzheimer’s disease (AD) patients show increased BBB permeability, the aim of this study was to elucidate the extent of BBB leakage in the 5xFAD model of AD and whether ANXA1 could reverse this effect. Method BBB permeability was determined in male wild type and 5xFAD mice, and in 5xFAD mice following an acute intravenous treatment with 0.67 μg/kg human recombinant ANXA1 at 12-13 weeks of age using an Evans blue dye assay, as well as MRI using the contrast agent dimeglumine gadopentetate. Amyloid pathology was investigated using immunofluorescence staining. Results Our data show an increased BBB leakage in 5xFAD mice compared to wild type littermates in both the frontal cortex (+82.8%) and hippocampus (+63.7%) using the Evans blue dye assay. This was significantly reduced in 5xFAD mice treated with human recombinant ANXA1 (frontal cortex: -42.4%, hippocampus: -25.9 %). Furthermore, a positive correlation between BBB leakage in 5xFAD mice and Aβ load was seen. Our preliminary results indicate the same pattern of BBB leakage using dimeglumine gadopentetate, which coincided with abnormalities in the vascular architecture observed by MRI scan. Conclusion


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Our results show that human recombinant ANXA1 rescues BBB deficits in a mouse model of AD, and suggest that targeting the BBB is a promising avenue for therapeutic interventions in AD.

SYMPOSIUM 60 - MICROGLIA AND INFLAMMATION ADPD7-1425 STUDY OF CD33: AN ALZHEIMER’S DISEASE SUSCEPTIBILITY LOCUS IN NEURODEGENERATION 1 1 1 1 1 2 1 1 M. Taga , M. Olah , M. Cimpean , S. Connor , X. Ying , D.A. Bennett , E.M. Bradshaw , P.L. De Jager 1 Brigham and Women's hospital, Neurology, Boston, USA 2 RUSH University, Rush Alzheimer's Disease Center, Chicago, USA Aims Many genes identified in AD GWAS are implicated in inflammation, including CD33. Our group has reported an association of the CD33 risk allele with increased cell surface expression of CD33 and decreased Aβ1-42 internalization. Our group also identified an intronic variant in the myeloid specific gene, TREM1 that is associated with an increased burden of neuritic plaques, and leads to a decreased expression of TREM1. TREM1 has been associated with protection from apoptosis. Objectives: To explore the distribution of CD33 in microglia in the human brain and to evaluate the effects of Aβ1-42 on cell phenotype in vitro in subjects with the risk allele compared with the protective allele. Method Using human post-mortem brain tissue from the ROS/MAP cohorts, we performed immunohistochemistry staining for CD33 and Aβ1-42 and co-stained for cell-specific markers for microglia (such as Iba1). Primary monocytes from genotyped subjects were used to evaluate whether the risk allele presents a greater sensitivity to apoptosis after treatment with Aβ1-42 using flow cytometry, and TREM1 expression. Results CD33 is expressed by microglia, however only a subset of Iba1+ microglia express this protein. Furthermore, a specific association between CD33 and neuritic plaques was observed. In vitro, lower apoptosis and higher TREM1 expression was observed in subjects with the CD33 protective allele post Aβ1-42 treatment. Conclusion


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Evidence suggests that CD33 expression on microglia is related to neuritic plaques, and CD33 may regulate the myeloid cell response to Aβ1-42 in a TREM1 dependent manner.

SYMPOSIUM 60 - MICROGLIA AND INFLAMMATION ADPD7-1551 AN ALZHEIMER’S DISEASE MOUSE MODEL LACKING FUNCTIONAL ADAPTIVE IMMUNE CELLS SHOWS REDUCED AMYLOID PATHOLOGY, ALTERED MICROGLIAL RESPONSES AND REDUCED SURVIVAL 1,2,3 3,4 1,3 1,3 1 1,3 1,3 1,3 C. Späni , T. Suter , R. Derungs , M.T. Ferretti , T. Welt , F. Wirth , C. Gericke , R.M. Nitsch , 1,3,5 L. Kulic 1 University of Zurich, Institute for Regenerative Medicine IREM, Zurich, Switzerland 2 University of Kentucky, Sanders Brown Center on Aging, Lexington- KY, USA 3 University of Zurich, Neuroscience Center Zurich ZNZ, Zurich, Switzerland 4 University Hospital Zurich, Department of Neurology- Section of Neuroimmunology and MS Research, Zurich, Switzerland 5 University of Zurich, Zurich Center for Integrative Human Physiologie ZIHP, Zurich, Switzerland Aims Accumulation and deposition of amyloid peptides is a hallmark of Alzheimer’s disease, which is accompanied by complex immune responses including both beneficial and detrimental properties. The contribution of the adaptive arm of the immune system in these responses is currently poorly understood. This study was designed to elucidate whether adaptive immune cells have an influence on amyloid pathology in a mouse model of Alzheimer’s disease. Method We analyzed PSAPP transgenic mice crossbred with recombination-activating gene-2 knockout (Rag2ko) mice lacking functional T and B cells. In a second experimental paradigm PSAPP mice reconstituted with bone marrow from Rag2ko or wildtype (WT) control mice were investigated. Results PSAPP transgenic mice lacking functional T and B cells showed significantly reduced amyloid pathology and decreased brain amyloid beta-peptide levels. This reduction was paralleled by elevated microgliosis and increased phagocytosis of amyloid beta-peptide. qPCR analysis showed a shift from pro- towards antiinflammatory microglia responses, in detail a rescue of reduced Ym1 expression levels and reduced TNFalpha expression levels. In contrast to these beneficial effects, reduced survival rates could be observed in PSAPP mice crossed with Rag2ko animals compared to WT, PSAPP or Rag2 ko mice. Conclusion


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The results of this study show that the adaptive immune system has an impact on cerebral amyloid-beta pathology by regulating microglia-mediated clearance. However, reduced survival rates show that a total adaptive immune ablation in combination with Alzheimer’s disease-like pathology can be detrimental, indicating that further studies are needed to identify the specific lymphocytic subpopulation(s) causing the observed effects.

SYMPOSIUM 60 - MICROGLIA AND INFLAMMATION ADPD7-1277 IMPACT OF MICROGLIA ON THE HIPPOCAMPUS DETERIORATION IN ALZHEIMER’S DISEASE 1 1 1 2 3 1 1 D.S. Bouvier , L. Salamanca , P. Antony , N. Mechawar , K.K. Murai , R. Balling , A. Skupin 1 University of Luxembourg, Luxembourg Centre for Systems Biomedicine, Belvaux, Luxembourg 2 McGill University, Douglas Mental Health University Institute, Montreal, Canada 3 The Research Institute of the McGill University Health Centre, Centre for Research in Neuroscience, Montreal, Canada Aims Memory loss is one of the first signs detected in Alzheimer’s Disease (AD) patients. Indeed, a rapid deterioration of the hippocampus leads to early memory deficits. The CA1 subfield is particularly affected by a severe neuronal loss. But the causes of the hippocampus vulnerability to AD still need to be deciphered. Interestingly, hippocampal microglia might engage strong maladaptive responses in AD that could promote neurodegeneration. Here, we aim to characterize how microglial structural remodeling impacts the hippocampus in AD and if microglia adopt phenotypic signatures that could mediate neuronal loss. Method We have combined imaging and automated computational methods to analyze microglial populations in postmortem samples from AD patients and controls. Briefly, hippocampal sections were immunostained for microglial morphological markers. Representative volumes of hippocampal subfields were acquired with high-resolution confocal microscopy. Next, we have implemented algorithms to extract geometrical and graph-based features of each microglia within these volumes. We have performed a subsequent cluster analysis to identify the various microglial subgroups. Results Human hippocampal microglia appear as a heterogeneous population of morphologically distinct cells in both healthy and disease conditions. In AD, microglial remodeling is particularly severe in the CA1 subfield compared to the CA3. Furthermore, we report a high occurrence of group of microglia actively engulfing CA1 pyramidal neurons carrying neurofibrillary tangles in AD. Surprisingly, CA1 microglia can regroup in super-clusters to phagocyte the full morphology of neurons affected by Tau aggregation. Conclusion


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Microglia of the CA1 show stronger responses to AD pathology than CA3. Microglia promote CA1 atrophy in AD.

SYMPOSIUM 60 - MICROGLIA AND INFLAMMATION ADPD7-1442 MICROGLIA IDENTITY IN THE AGED HUMAN BRAIN 1 2 2 1 1 2 2 3 3 M. Olah , E. Patrick , A.C. Villani , J. Xu , M. Cimpean , C. McCabe , P. Nejad , D.A. Bennett , J.A. Schneider , 1 1 1 L.B. Chibnik , P.L. De Jager , E.M. Bradshaw 1 Brigham and Women's Hospital- Harvard Medical School, Neurology, Boston, USA 2 The Broad Institute, The Broad Institute, Cambridge- MA, USA 3 Rush University Medical Center, Neurology, Chicago, USA Aims Recent genome wide association studies (GWAS) have implicated the innate immune system in the pathogenesis of late onset Alzheimer’s disease (LOAD). Next to the genetic predisposition, aging is the other major risk factor for developing LOAD. One of the most profound changes occurring in the aging human brain is the activation of the brain’s resident innate immune cells, called microglia. Since this observation is primarily based on the changes in microglia morphology, the exact nature of this activation state, or phenotype, is not well understood yet. Here we set out to empirically establish what constitutes the aged human microglia phenotype. Method We established a new pipeline for the isolation of microglia from aged human autopsy brain specimens. Furthermore, we utilized a novel library construction protocol for RNA-Sequencing that has been specifically optimized for low cell numbers. Results We identified the microglia specific genes in the aged human brain and established which neurodegenerative disease GWAS hits were allocated to the microglia compartment. Furthermore, we explored the microglial transcriptomic response to the aging brain environment. Conclusion


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Our findings reinforce earlier notions, that within the aged human brain, a significant amount of the genetic load that influences the onset of neurodegenerative disease exerts its function through microglia and that immunosenescence in the brain may exhibit itself by conveying a downregulated immunophenotype on microglia, rather than resulting in an overt activation. Our study will serve as an imporant resource for the LOAD community in particular and to the microglia field in general.

SYMPOSIUM 60 - MICROGLIA AND INFLAMMATION ADPD7-1509 HUMAN IPSC-DERIVED MICROGLIA-LIKE CELLS TO STUDY ALZHEIMER’S DISEASE 1 2 3 4 3 3 5 5 6 E. Abud , R. Ramirez , E. Martinez , L. Healy , C. Nguyen , S. Marsh , V. Scarfone , S. Newman , Y. Andriy , 7 8 6 4 9 2 3 1 R. Kayed , K. Gylys , M. Cahalan , J. Antel , M. Carson , A. Mortazavi , W. Poon , M. Blurton-Jones 1 University of California - Irvine, Neurobiology and Behavior, Irvine, USA 2 University of California - Irvine, Developmental and Cell Biology, Irvine, USA 3 University of California - Irvine, Institute for Memory Impairments and Neurological Disorders, Irvine, USA 4 McGill University- Montreal Neurological Institute, Neuroimmunology Unit, Montreal, Canada 5 University of California- Irvine, Sue and Bill Gross Stem Cell Research Center, Irvine, USA 6 University of California- Irvine, Department of Physiology & Biophysics, Irvine, USA 7 University of Texas Medical Branch at Galveston, Department of Neurology, Galveston, USA 8 University of California- Los Angeles, Brain Research Insitute, Los Angeles, USA 9 University of California- Riverside, Biomedical Sciences, Riverside, USA Aims Microglia play important roles in development and homeostatic brain function and profoundly influence the development and progression of many neurological disorders including Alzheimer’s disease. Identification of microglial-genes with AD-associated risk variants have further highlighted the need to study microglia in AD. To begin investigating the role of microglia in AD, a renewable and consistent source of microglia must be developed. We have recently developed a protocol that allows for the generation of microglia-like cells (iMGLs) from human iPSCs. Method We generated hematopoietic progenitors (iHPCs) from iPSCs using fully defined serum free conditions. iHPCs are exposed to cytokines and growth factors implicated in the development of microglia. Analysis of iMGLs reveals that they develop from CD45+/ CX3CR1+/- progenitors and mature to CX3CR1+ /CD11b+ /CD45-lo cells when compared to macrophages. Results Whole-transcriptome and protein expression analysis reveals high expression of microglia-rich markers including P2RY12/13, OLFML3, GPR34, and TREM2 in iMGLs. Functional assays demonstrate that iMGLs phagocytose and secrete cytokines in response to amyloid, human brain-derived tau oligomers, and human synaptosomes. Here, we show that iMGLs exposed to fibrillar amyloid and tau have differential mRNA expression of AD-risk associated genes. Finally, we show that iMGLs successfully integrate, tile in volume, and project processes in human 3D brain cultures. Conclusion


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Collectively, our data shows that human iMGLs can be efficiently derived from human iPSCs using a two step fully-defined protocol. RNA, protein, and functional assays highlight that iMGLs can be used as surrogates to study microglia in the context of human health and neurological diseases including AD.

SYMPOSIUM 60 - MICROGLIA AND INFLAMMATION ADPD7-1358 SHARED POLYGENIC CONTRIBUTIONS TO NEURODEGENERATIVE AND IMMUNE-RELATED TRAITS REVEALS EFFECTS OF SUSCEPTIBILITY LOCI ON MICROGLIAL ACTIVATION 1 1 1 2 2 1 P. De Jager , D. Felsky , N. Pastopoulos , J. Schneider , D. Bennett , E.M. Bradshaw 1 Brigham and Women's Hospital- Harvard Medical School, Neurology, Boston, USA 2 Rush Alzheimer's Disease Center, Neurology and Neuropathology, Chicago, USA Aims Large-scale genome-wide studies suggest pleiotropic mechanisms of neurodegenerative disease. We tested the polygenic basis of multiple neurodegenerative, inflammatory, and vascular traits against late-life cognition and postmortem neuropathology. Method Antemortem cognition and postmortem amyloid and tau were measured in 819 subjects. Regional postmortem counts of active microglia were available for 162 samples. Polygenic scores (PS) for Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), schizophrenia (SCZ), rheumatoid arthritis (RA), coronary artery disease (CAD), and telomere length (TL) were calculated. PS showing significant associations with pathology or cognition were deconstructed in tests of individual SNPs. To parse pleiotropy, the MHC region was imputed and examined separately, and loci overlapping between PS were checked for gene regulatory effects in GTEx. Results The AD PS was associated with all cognitive domains, and the SCZ PS influenced episodic memory only (p=0.0067). For neuropathology, the AD PS was associated with amyloid and tau, whereas the RA and CAD PS -4 were associated with microglial counts (min p=2.3x10 ). Independent effects of MHC variation on microglial count localized to known eQTLs in PSORCS1 and HLA-DQB2 5’ UTR. The non-MHC variant driving association of RA PS with microglial count was in proximity to an MS variant on chromosome 2; rs13414105 (MS) is a cis-eQTL for -6 -4 LBH in heart (p=3x10 ) and brain (p=3.3x10 ), while rs10175798 (RA) has no effects. Conclusion


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We have shown that polygenic contributions to disease impact cognitive decline and neuropathology in elderly. Further, implicated genetic variants both within and outside the MHC region differentially affect microglial activity and transcriptomic regulation.

SYMPOSIUM 60 - MICROGLIA AND INFLAMMATION ADPD7-0617 EARLY MICROGLIAL ACTIVATION BEYOND THE SUBSTANTIA NIGRA IN PARKINSON’S DISEASE 1 1 2 1 A.M. Van Dam , K. Doorn , P.J. Lucassen , B. Drukarch 1 VU University Medical Center, Anatomy and Neurosciences, Amsterdam, The Netherlands 2 Swammerdam Institute for Life Sciences- University of Amsterdam, Center for Neuroscience, Amsterdam, The Netherlands Aims Brain microglial cells contribute to brain homeostasis which upon disturbance can change their phenotype with concomitant altered function. This is seen under various neurodegenerative conditions, including Parkinson’s disease (PD). Until now most attention has been focused on microglia in the substantia nigra in PD. It has become clear that various other brain regions are affected and express pathological alpha-synuclein which relates to non-motor symptoms in PD. In our human post-mortem studies, we questioned if different microglial phenotypes are present in brain regions beyond the substantia nigra, i.e. hippocampus and olfactory bulb. Method Immunohistochemistry has been performed on brain material of PD patients (Braak 4-6), incidental Lewy body disease patients (iLBD; Braak 1-3) and control subjects (Braak 0). Results Our data show that microglial activation (CD68) occurs in substantia nigra, hippocampus and olfactory bulb of PD patients. Moreover, cell proliferation processes (MCM2) in the hippocampus of PD patients are mostly accounted for by microglial cells. Moreover, phenotypical changes in microglia occur already in iLBD patients which coincide with elevated expression of toll-like receptor 2 (TLR2). However, in the substantia nigra TLR2 remains present during PD, whereas in the hippocampus, TLR2 is then reduced till control level immunoreactivity. Conclusion


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These studies indicate that microglial cells are involved in the early PD pathological process. Moreover, the differential responsiveness of hippocampal versus nigral microglial cells suggest that they are not a uniform cell population throughout the brain but respond differentially to pathological situations depending on the local environment, and stage of the disease process.

SYMPOSIUM 60 - MICROGLIA AND INFLAMMATION ADPD7-1295 THE PSYCHOSIS PHENOTYPE OF ALZHEIMER’S DISEASE IS ASSOCIATED WITH INCREASED TAU BURDEN AND SHIFT IN MICROGLIAL ACTIVATION 1 2 3 J. Kofler , O.L. Lopez , R.A. Sweet 1 University of Pittsburgh, Pathology, Pittsburgh, USA 2 University of Pittsburgh, Neurology, Pittsburgh, USA 3 University of Pittsburgh, Psychiatry, Pittsburgh, USA Aims A more severe phenotype of Alzheimer disease is identified by the occurrence of psychosis (AD+P). However, the underlying neurobiology of psychosis in AD is poorly understood. We aimed to compare quantitative AD disease burden, comorbid disease pathologies and inflammatory markers between AD+P and AD-P subjects without endstage neuropathology. Method 140 autopsy-confirmed AD cases with known psychosis status and Braak stage III-V were identified in the University of Pittsburgh Alzheimer’s disease research center. Immunohistochemical burden of phospho-tau and microglial markers Iba1 and HLA-DR were assessed in dorsal prefrontal cortex using a whole slide imaging approach and automated measurement of immunoreactive area fraction. Results The cohort included 81 AD+P and 59 AD-P cases with no significant differences between groups in age, sex, postmortem interval, presence of Lewy body pathology and Braak stage. The AD+P group had significantly higher tau burden and mean tau signal intensity than the AD-P group. Tau burden showed a positive correlation with HLA-DR but not Iba1 area fraction. In a regression analysis, tau burden but not age, sex, age of onset, disease duration, ApoE, Iba1 or HLA-DR burden were associated with presence of psychosis. In a subset of cases with last evaluation within 30 months of death, tau burden remained associated with the presence of psychosis, and an additional association of the HLA-DR:Iba1 ratio with psychosis was found. Conclusion


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Our study confirms previous findings of an association between psychosis phenotype in AD and tau burden and shows a novel but weaker association with microglial activation markers.

Neurodegener Dis 2017;17(suppl 1):591-1890 - Page 591

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Posters

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0320 RELATIONSHIP OF PKCΕ GENE EXPRESSION BETWEEN HIPPOCAMPUS AND PLATELET IN ALZHEIMER DISEASE 1 2 1 S. Amiri , K. Azadmanesh , N. Naghdi 1 Pasteur Institute of Iran, Physiology and Pharmacology, Tehran, Iran 2 Pasteur Institute of Iran, Virology, Tehran, Iran Aims Alzheimer’s disease (AD) is one of the most common neurodegenerative disease and accounts for more than 80% of dementia cases worldwide in elderly people. Alzheimer is characterized by loss of memory and cognitive decline. Protein kinase C (PKC) is a family of serine/threonine protein kinases which is involved in signal transduction in the central nervous system. Abnormalities in the levels and activities of protein kinase C isozymes in brain and other tissues have been reported. Isozymes of this family are activated in different disease and among them; PKCε is the isoform that most effectively suppresses Aβ production and therefore synaptic loss that is important in AD pathophysiolology. So the aim of this study was to investigate the PKCε gene expression changes in the platelet and hippocampus of Rat model of Alzheimer’s disease.

Method Amyloid beta (Aβ) fragment 1–42 was injected bilaterally into the CA1 region of adult male rats hippocampus and spatial performance was assessed by Morris water maze task (MWM). Hipocampus and Platelets (from whole blood) were isolated in 0,5,10 and 30 days after injection from AD and control groups. PKCε gene expression was assessed by RT-qPCR. Results Our results showed that injected Aβ in CA1 region of hippocampus could impair learning and memory and it also showed decreases in PKCε gene expression as compared to the control groups at hippocampus and platelets. Conclusion


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The findings are consistent with altered PKCε in neurodegenerative disease and showed relationship between PKCε gene expression in hippocampus and platelets.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1437 CHARACTERIZATION OF PATHOLOGICAL CASCADES IN A SINGLE-CLONAL 3D CELL CULTURE MODEL OF ALZHEIMER’S DISEASE 1 1 1 1 2 3 1 3 J. Aronson , K. Washicosky , C. D'Avanzo , E. Bylykbashi , S. Hartmann , K.C. Roet , S.H. Choi , C.J. Woolf , 1 1 R.E. Tanzi , D.Y. Kim 1 Massachusetts General Hospital/ Harvard Medical School, Neurology, Charlestown, USA 2 University of Erlangen-Nuremberg, Institute of Physiology and Pathophysiology, Erlangen, Germany 3 Boston Children's Hospital/Harvard Stem Cell Institute, FM Kirby Neurobiology Center, Boston, USA Aims Recently we developed a 3D human neural stem cell culture model recapitulating both key hallmarks of AD: betaamyloid(Abeta) plaques and Neurofibrillary tangles (NFTs). However, the heterogeneity of these cells hampered detailed characterization of downstream AD pathologies. We improved the model by creating stable single-clonal cell lines. Here, we report applications of single-clonal human neural stem cell lines for understanding neuronal deficits, cell death, and other Amyloid-triggered pathogenic cascades.

Method Single-clonal cells were plated in 3D with Matrigel as described (Choi et al., 2014). Using FACS-assisted cell sorting, we established single-clonal lines consistently expressing GCaMP6, a calcium reporter protein with fastkinetic response. After 3-8 weeks differentiation, LDH release assays, active caspase-3 staining and DAPI staining were performed, while laser confocal microscopy was used to identify Abeta and tau aggregates, as well as dystrophic neurites. GCaMP6 calcium reporter protein technology and other techniques identified altered neural activity. Results Single-clonal cell lines secreted higher levels of pathogenic Abeta42 than mixed parental cells. Accordingly, we observed more robust ThioflavineS-positive Abeta plaques and phospho-tau accumulation in cell bodies and neurites in 6-7 weeks, with dystrophic neurites co-staining with phospho-tau in cells with highest Abeta42 levels. Furthermore, we observed abnormal calcium influx and hyperexcitability, suggesting functional deficits in AD cells. Increased LDH release and caspase-3 activity combined with decreased ATP levels and DAPI-positive AD cells demonstrate increased cell stress and neurodegeneration.

Conclusion


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Our single-clonal AD models can be used to study not only amyloid and tau tangle pathology, but also functional deficits, neuronal injury, cell death, and their relationships.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0919 HALLMARKS OF ALZHEIMER'S DISEASE IN STEM CELL-DERIVED HUMAN NEURONS TRANSPLANTED INTO MOUSE BRAIN 1 1 1 1 1 1 2 2 A. Arranz , I. Espuny-Camacho , M. Fiers , A. Snellinx , S. Munck , E. Radaeli , K. Roussel-Ando , K. Leroy , 2 2 1 J.P. Brion , P. Vanderhaeghen , B. De Strooper 1 KU Leuven - VIB, Center for Human Genetics, Leuven, Belgium 2 ULB Neuroscience Insitute UNI, Université Libre de Bruxelles- Faculty of Medicine, Brussels, Belgium Aims Alzheimer’s disease (AD) is the most common neurodegenerative disorder of the elderly. The recurrent pathological hallmarks of AD found in patients are the presence of amyloid-beta (Aβ) plaques, the formation of neurofibrillary Tau tangles, chronic neuroinflammation and neuronal injury and loss. The use of stem cell technology is revealing important mechanisms associated to AD and providing essential clues for the identification of candidate treatments. Human pluripotent stem cells (PSC) have a great potential in AD research because they can be differentiated into neurons and allow the investigation of human-specific phenotypes and mechanisms. Our main goal is to use the stem cell technology for the generation of a new chimeric model of AD that recapitulates more accurately the cascade of pathogenic events leading to neurodegeneration and will allow a more complete study of human AD pathology. Method To generate a chimeric model of AD we transplanted human PSC-derived neuronal precursors into the brain of a murine AD model. Human PSC were first differentiated into cortical progenitors in vitro and about 100,000 cells were injected. Results Human neurons integrate efficiently into the mouse brain, show Aβ pathology, neuroinflammation, Tau pathology and undergo neurodegeneration. By GWAS we observed up-regulation of genes involved in myelination and down-regulation of genes related to memory and cognition, synaptic transmission and neuron projection. Conclusion


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This novel chimeric model for AD displays human-specific pathological features, allows the analysis of different genetic backgrounds and mutations during the course of the disease and can be useful to unveil molecular pathways involved in AD pathogenesis.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0843 NON NATURAL PEPTIDES THAT MODULATE COPPER (II)-INDUCED AMYLOIDOGENICITY AND REDOX ACTIVITY OF AMYLOID BETA PEPTIDE AS POTENTIAL DRUGS FOR ALZHEIMER DISEASE 1 1 1 2 2 3 1 F. Attanasio , I. Monaco , M.F.M. Sciacca , I. Naletova , V.G. Nicoletti , C. Satriano , M.F. Tomasello 1 Institute of Biostructure and Bioimaging, Department of Biomedical Science- Research National Council, Catania, Italy 2 Biochemistry Institute, Department of Biomedical Science- University of Catania, Catania, Italy 3 Physical-Chemical Institute, Department of Chemical Science- University of Catania, Catania, Italy Aims There is a general consensus that the copper ions participate in two processes related to AD pathology: accelerating amyloid-β (Aβ) aggregation and catalyzing the production of neurotoxic reactive oxygen species (ROS). Here we report the inhibition of copper catalyzed oxidation of Aβ peptide and the modulation of the copper- induced Aβ aggregation by Semax, a synthetic analog of ACTH4-10, and two new designed bifunctional peptides, that combine the known beta-breaker properties of the oligomerization inhibitors KLVFF and LPFFD with the chelating and redox silencing ability of Semax. Method The inhibitory effect of Semax and its derivatives on Aβ aggregation, ROS production and cytotoxicity with and without copper was assessed by thioflavin T (ThT) fluorescence assay, Atomic Force Microscopy, coumarin-3carboxilic acid (CCA) fluorescence assay for cell free experiments. For in cell experiments, human neuroblastoma SH-SY5Y were exposed to stress generated by a mixture of copper(II) and ascorbic acid, Aβ-Cu(II) and ascorbic acid, Aβ, Aβ and copper(II) and the inhibitory effect of the peptides were assessed by MTT and flow cytometry assay. Results Semax and its bifunctional derivative peptides were able to quench ROS generation mediated by Cu(II)/ascorbate and Aβ-Cu(II)/ascorbate either in the cell-free system or in cultured neuroblastoma cell line. Furthermore, the bifunctional peptides had nearly identical efficacy to inhibit copper-induced Aβ aggregation, ROS production and cytotoxicity. Conclusion


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Semax and its bifunctional derivative peptides modulate copper-induced Aβ cytotoxicity rescue SH-SY5Y cells by limiting ROS production and inhibiting aggregation. Overall, these molecules may be a promising therapeutic drug for AD.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1538 XANTHURENIC ACID SYNTHESIS IN THE HUMAN BRAIN AND ITS BLOCKADE BY D-CYCLOSERINE – IN AN IN VITRO STUDY 1 2 H. Baran , B. Kepplinger 1 Karl Landsteiner Research Institute Mauer, Neurochemistry and Neuropharmacology, Mauer-Amstetten, Austria 2 Karl Landsteiner Research Institute Mauer, Rehabilitation and Pain Treatment, Mauer-Amstetten, Austria Aims D-Cycloserine, known as a partial agonist at the glycine modulatory site of the glutamatergic N-methyl-Daspartate receptor improves cognitive function and is able to block formation of kynurenic acid synthesis. In present study we investigated xanthurenic acid synthesis from 3-hydroxykynurenine in human brain tissue. Subsequently, we evaluated the action of D-cycloserine with respect to the biosynthetic machinery of xanthurenic acid synthesis e.g. the activity of enzymes synthesizing kynurenic acid as well as xanthurenic acid, kynurenine aminotransferases I, II and III (KAT I, KAT II and KAT III) in human brain in the presence of the anti-mycobacterial drug D-cycloserine, in an in vitro study. Method Xanthurenic acid was determined using HPLC method. KATs activities were determined as described (Eur. Neuropsychopharmacol. 2009; 19, 161-168). The number of human brain samples used was N=6. The study was carried out according to the ethical regulations of Austria. Results We found that KATs of human brain are able to synthesize xanthurenic acid using 3-hydroxykynurenine as a substrate. Furthermore, D-cycloserine at various µmolar concentrations in the incubation mixture blocked dosedependently and significantly KATs activities and respectively the formation of xanthurenic acid. Conclusion


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Due to D-cycloserine action lowering of xanthurenic acid might play a role in the lowering of plaque formation, while lowering of kynurenic acid might activate the NMDA and nicotine cholinergic receptors. We propose that this mechanism(s) can be involved in the improvement of symptoms like dementia, cognition and/or delusion in Schizophrenia, Alzheimer’s, Parkinson’s patients or in HIV-1 dementia. Study supported by SeneCura Austria.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1560 CONFORMATIONALLY DISTINCT AMYLOID-Β VARIANTS IN HUMAN ALZHEIMER’S DISEASE BRAIN 1,2 1,2 1,2 3 4 5,6 5,6 N. Beschorner , J. Mahler , J. Rasmussen , K.P.R. Nilsson , L.C. Walker , A.L. Oblak , B. Ghettii , 1,2 M. Jucker 1 Hertie Institut for Clinical Brain Research, Cellular Neurology, Tuebingen, Germany 2 German Center for Neurodegenerative Diseases, DZNE, Tübingen, Germany 3 IFM Linköping University, Department of Chemistry, Linköping, Sweden 4 Emory University, Department of Neurology and Yerkes National Primate Research Center, Atlanta, USA 5 Indiana University School of Medicine, Indiana Alzheimer Disease Center, Indianapolis, USA 6 Indiana University School of Medicine, Department of Pathology and Laboratory Medicine, Indianapolis, USA Aims Alzheimer's disease (AD) pathogenesis is widely believed to be driven by the pathological aggregation of the amyloid-β peptide (Aβ). Evidence suggests that Aβ can adopt distinct conformations that propagate through the brain by templated protein misfolding. Recently, structural differences in Aβ were identified by NMR spectroscopy on seeded fibrils from two patients’ brain tissue, with a single Aβ fibril structure detected in each case. However, the diversity of structural characteristics in the broader population of AD patients and their relation to the clinical phenotype remains obscure. Method In our study, we approached this question by analyzing post-mortem brain tissue from more than thirty familial and sporadic AD patients using conformation-sensitive methods including biochemical analysis as Aβ quantification, Aβ 42/40 ratio and protease-resistance as well as the use of luminescent conjugated oligothiophenes (LCOs) that spectrally discriminate among various amyloid structures. Results Whereas biochemical analysis revealed slight differences among the AD cases, the use of LCOs showed significant and remarkable differences in plaque conformation among the familial and sporadic cohorts. Based on these results, we have initiated experiments to further analyze the impact of the plaque microenvironment and the capability of the human Aβ aggregates to pass on their conformational properties in mouse models. Conclusion


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Our results provide evidence for the structural variety of Aβ conformers among AD cases, a diversity that is not directly attributable to a single biochemical characteristic. In the future, the observed conformational variety might be correlated to clinical data of the patients and finally facilitate the development of novel diagnostics.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1677 THE DETERMINANTS OF AMYLOID-Β FIBRIL FORMATION 1 1 2 1 K. Brännström , T. Islam , L. Sandblad , A. Olofsson 1 Umeå University, Medical chemistry, Umeå, Sweden 2 Umeå University, Molecular Biology, Umeå, Sweden Aims Self-assembly of the amyloid-β peptide (Aβ) into amyloid fibrils is associated with the development of Alzheimer’s disease. Several different lengths of Aβ are found in vivo, but Aβ1-40, Aβ1-42, and N-terminally truncated variants thereof, are the dominant forms. The architectures of Aβ1-40 and Aβ1-42 fibrils differ, and Aβ1-42 assemblies generally have higher cytotoxicity. We want to investigate the polymerisation between Aβ1-42 fibrils and Aβ1-40 monomers and vice versa under physiological like conditions. Method

We have used surface plasmon resonance to study polymerisation between Aβ1-42 fibrils and Aβ1-40 monomers and vice versa. Results We performed cross-templating experiments using surface plasmon resonance and discovered that monomeric Aβ1-42 can be readily incorporated onto the ends of immobilized Aβ1-40 fibrils, but that monomeric Aβ1-40 is poorly incorporated onto immobilized Aβ1-42 fibrils. Intriguingly, the most N-terminal residue, Asp1, controls this barrier. We further showed that the monomers that are incorporated onto the fibrillar ends also adopt the properties of the templating fibril. Conclusion


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This consequently implies an important role in the mechanism behind the formation and architecture of nuclei from which a fibril subsequently propagates. These results demonstrate a complicated interplay between different Aβ variants where the ability to incorporate monomers control the properties and formation of the amyloid fibrils.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0296 A NOVEL ASSAY FOR QUANTITATIVE ANALYSIS OF THE ABETA OLIGOMER ELIMINATION BY DRUG CANDIDATES 1 2 1 2 O. Brener , L. Gremer , L. Nagel-Steger , D. Willbold 1 Heinrich-Heine-University- Duesseldorf, Physikalische Biologie, Duesseldorf, Germany 2 Forschungszentrum Jülich, ICS 6, Jülich, Germany Aims Several lines of research provide strong evidence for a central role of amyloid-beta (Abeta) oligomers in the pathogenesis of Alzheimer’s disease (AD). Any screening for oligomer eliminating compounds requires a well characterized target. Therefore, new methods for the preparation, purification and quantification of specific Abeta oligomers, which are representative for the toxic oligomers involved in AD pathogenesis, combined with a reliable in vitro assay to quantify the removal of Abeta oligomers by any potential drug are urgently needed in AD drug development. Method We have designed an assay for the quantitative determination of interference with Abeta aggregate size distribution (QIAD). It is a fast, reliable and robust in vitro assay that is able to quantify the Abeta oligomer eliminating potential of any compound. Results With the help of QIAD we could demonstrate the influence of different potential drugs against AD on the Abeta oligomer distribution. The QIAD outcomes correlate with the treatment effects in different AD animal models. Conclusion


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The hereby described QIAD assay enables quantitative, fast and reliable in vitro screening of the Abeta oligomer removal by potential drugs and allows a comparison between them. Furthermore, the observed relation between Abeta oligomer elimination potential and in vivo efficacy strengthens the role of Abeta oligomers in AD pathology.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0845 NEGATIVELY CHARGED GOLD NANOPARTICLES INHIBIT ALZHEIMER’S AMYLOID-Β FIBRILLIZATION, INDUCE FIBRIL DISSOCIATION, AND MITIGATE NEUROTOXICITY 1 2 Y.C. Chang , Y.R. Chen 1 Academia Sinica, Research Center for Applied Sciences, Taipei, Taiwan R.O.C. 2 Academia Sinica, Genomic Research Center, Taipei, Taiwan R.O.C. Aims 1. Amyloids are the major pathogenic hallmarks in many neurodegenerative diseases where amyloid-β (Aβ) aggregation is considered the pathogenesis of Alzheimer’s disease (AD). 2. The effects of gold nanoparticles (AuNPs) on amyloids (Aβ40) are studied. Method 1. Synthetic Aβ40 peptide was synthesized using Fmoc (N-(9-fluorenyl) methoxycarbonyl) chemistry and purified by reversed phase high performance liquid chromatography. 2. The molecular mass was identified by MALDI-TOF mass spectrometry 3. The aggregation of Amyloid is detected by Thioflavin T assay 4. The size of Amyloid fibril is measured by Dynamic light scattering method 5. The aggregation of fibril with AuNPs is imaged by Transmission electron microscopy 6. We employed MTT (3-[4,5-Dimethy- lthiazol-2-yl]-2,5-diphenyl-tetrazdium bromide) assay to examine cytotoxicity. Results 1. We found that bare AuNPs inhibited Aβ fibrillization in a dose-dependent manner and redirected Aβ forming fragmented fibrils and spherical oligomers. 2. When adding bare AuNPs to preformed fibrils, smaller and ragged Aβ species were observed. 3. The bare AuNPs bound preferentially to Aβ fibrils but not amorphous aggregates. 4. The phenomenon results from the negative surface potential of AuNPs as evidenced from observing similar effects from carboxyl- but not amine-conjugated AuNPs. 5. The neurotoxicity assays showed reduced Aβ toxicity after incubation with negatively charged AuNPs during Aβ fibrillization. Conclusion


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We demonstrate that AuNPs possessing negative surface potential serve as nano-chaperones to inhibit and redirect Aβ fibrillization, which could contribute to potential applications for AD. Furthermore, we developed a sensitive assay using functionalized AuNPs conjugated with peptides for Aβ oligomer detections.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1686 PEPTIDE DRUG AMELIORATES THE COGNITIVE DECLINE OF ALZHEIMER TRANSGENIC MICE VIA INTRANASAL ROUTE 1 R. Chen 1 Academia Sinica, Institute of Biological Chemistry, Taipei, Taiwan R.O.C. Aims Imbalance between the production and clearance of amyloid β (Aβ) peptides is considered the primary cause for the pathogenesis of Alzheimer's disease (AD). We designed a peptide drug which can be used to prevent or delay the onset of Alzheimer's disease by inhibiting Aβ aggregation in the brain. Method The peptide contains a segment for target recognition and a segment for inhibiting Aβ aggregation. The peptide was dissolved in buffer and was given to the AD transgenic mice via intranasal route. Results After treating the AD transgenic mice with our peptide (2 nmole/mouse/day) for 4 months, the Aβ40 and Aβ42 levels in the hippocampus and cortex significantly decreased. The peptide treatment also reduced the amyloid deposition in the brain and ameliorated the memory deficits of the transgenic mice. The brain penetration efficiency of the peptide is ~17%. The amount of peptide reached maximum in the brain at 6 hours after dosing and more than half of the peptide remained in the brain after 12 hours. Conclusion


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Intranasal delivery is a non-invasive drug delivery method and suitable for disease prevention. Our peptide therapy is a cheap, effective, and user-friendly therapy to delay the disease progression of AD.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1533 BIO-INSPIRED MOLECULES TO PREVENT ALZHEIMER'S DISEASE 1 2 2 3 3 3 3 1 D. Galante , S. Tomaselli , K. Pagano , C. Lambruschini , L. Moni , R. Riva , L. Banfi , C. D'Arrigo 1 Institute for Macromolecular Studies, National Research Council, Genova, Italy 2 Institute for Macromolecular Studies, National Research Council, Milano, Italy 3 Università degli studi di Genova, Dipartimento di chimica e di chimica industriale, Genova, Italy Aims Recently many natural substances, as curcumin, flavonoids etc., have proved effective in preventing amyloid aggregation. The limits of these substances are represented by their poor bioavailability and their reduced ability to pass the blood-brain barrier. For this reason we synthesized new polyphenolic substances to improve the bioavailability of these compounds and make them more effective in the activity antiplatelet towards β amyloid Method We studied amyloid-β (Aβ) aggregation, using the fluorescence of some probes (ANS and ThT), the precipitation of the peptides by means of turbidity measurements, the morphology by electron microscopy and the interactions between Aβ and synthesized polyphenolic compounds by NMR. Results We transformed simple, monocyclic, natural (poly)phenols into suitable building blocks. In a second time we joined these building blocks in a combinatorial manner (thanks to a multicomponent reaction, MCR), in order to generate a series of new bio-based polyphenols. The antiplatelet efficacy of the new compounds was tested on the Aβ1-42 and AβpE3-42 peptides. Some of the bio-based polyphenols reduced the aggregation process of these peptides. The aggregates were smaller and fewer in comparison to the Aβ samples alone. But the antiplatelet efficacy dipends from the new polyphenols and from the type of peptide studied. Conclusion


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We found some bio-based polyphenols as good candidates out to develop compounds more skilled to inhibit the fibrillization. The new compounds will be tested also in vivo to check after their antiplatelet power also their antitoxic ability

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1535 AMYLOID BETA INTERACTIONS WITH THE PRION PROTEIN 1 2 2 3 4 4 4 5 D. Galante , S. Tomaselli , K. Pagano , E. Gatta , A. Corsaro , M. Nizzari , T. Florio , C. D'Arrigo 1 Institute for Macromolecular Studies, National Research Council, Genova, Italy 2 Institute for Macromolecular Studies, National Research Council, Milano, Italy 3 University of Genoa, Department of Physics, Genova, Italy 4 Section of Pharmacology- University of Genova, Department of Internal Medicine and Centre of Excellence for Biomedical Research CEBR, Genova, Italy 5 Institute for Macromolecular Studies, National Research Council CNR, Genova, Italy Aims C

In the last years, a new molecular actor has appeared in AD pathophysiology: the cellular prion protein (PrP ). The role of this protein is still not completely disclosed, but its involvement in the complex pathway connecting amyloid beta (Aβ) to Fyn/Tau neuronal toxicity is now recognized. In this work we exploit the interactions between C c Aβ peptides and N-terminal truncated PrP . In particular we study the effect of PrP (90-231) on aggregation of Aβ1-42, AβpE3-42 and of their mixtures. Method To reach this goal we investigated the different interactions by NMR and we characterized the intermediate species by turbidimetry, circular dichroism, transmission electron microscopy, ThT and ANS fluorescence. To C determine if PrP is a mediator of Aβ-induced neurotoxicity, we studied the physiological response of cells to Aβ C oligomers, and the dysregulation of calcium homeostasis, both on glioblastoma cells where PrP expression was inhibited or not. Results c

We found that PrP (90-231) strongly inhibits aggregation of Aβ1-42 while it has a weak effect on that of AβpE3C 42. Also the size of the fibrils and the content of β-sheets decrease in the presence of PrP . Specific interactions C of Aβ1-42 with PrP are detected at the level of the N-terminal tail and in the turn. We evaluated A1 neuron C survival and we found that PrP is a mediator of AβpE342-induced neurotoxicity. Conclusion c


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From these preliminary data, Aβ1-42 and AβpE3-42 interact with PrP and it is a mediator of AβpE3-42 toxicity.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1890 PURIFICATION OF NEW POWERFUL INHIBITORS OF AMYLOID-Β AGGREGATION AND NEURONAL TOXICITY 1 Z. Dhouafli 1 Faculty of Sciences of Tunis, Biology, Tunis, Tunisia Aims The aggregation and deposition of misfolded β-amyloid peptide (Aβ)42 is a consistent pathological hallmark of Alzheimer’s disease (AD) and contribute to the progressive central nervous system decline. Presently, new treatment strategies based on medicinal plants have been the subject of current focus. Therefore, bioactive compounds derived from natural products that inhibit or even reverse Aβ aggregation might be useful for treatment or prevention of AD. Method Accordingly, in this study we targeted to purify the bioactive compound from Lawonia inermis extract and to test its ability to counteract amyloid aggregation, cytotoxicity and its effect on Aß/GM1 interaction. In particular, by using a panel of different spectroscopic, imaging techniques and cell viability assays we provide a detailed description of the Aβ42 structural modifications arising in the presence of the inhibitor and the resulting effects on peptide aggregation and cytotoxicity. Results Thus, we report that the new molecule hinders Aβ42 aggregation and reduce its cytotoxicity, ROS level and decrease the co-localization of Aß with GM1 Conclusion


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These results suggested that the new molecule act as a neuroprotective and therapeutic factors against toxic Aβ oligomers and it’s of great potential as neuroprotective agent that could be a good candidate for future drug of AD

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1587 PROTECTIVE EFFECTS OF GINGEROL ON STREPTOZOTOCIN-INDUCED SPORADIC ALZHEIMER’S DISEASE; EMPHASIS ON INHIBITION OF BETA-AMYLOID, COX-2 AND SECRETASES 1 2 2 N. El Sayed , A. El Halawany , H. Abdallah 1 Faculty of pharmacy- cairo university, Pharmacology & Toxicology, Cairo, Egypt 2 Faculty of pharmacy- cairo university, Pharmacognosy, Cairo, Egypt Aims Gingerol is a major dietary compound occurs in several plants belonging to family Zingiberaceae. In the current study, the protective effect of gingerol on STZ-induced sporadic Alzheimer’s disease (AD) was determined. Method Gingerol was isolated from the seeds of Aframomum melegueta K. Schum. and tested in a adose of (10 and 20 mg/kg.bwt.) for its possible effect on STZ-induced sporadic AD model in mice using celecoxib (30mg/Kg.bwt.) as a reference standard. The curative effects of gingerol were assessed through measurement of beta-amyloid, alpha, beta, gamma-secretases and COX-2 levels. In addition the improvement of cognitive deficit in mice after treatment were confirmed using the water maze and Y-maze with intramaze cues. Results Gingerol, improved the cognitive and behavioral impairment and AD-like pathology in streptozotocin model in mice. Conclusion


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These beneficial effects occur with an increase in α-secretase activity and a decrease in: cerebral amyloid beta 42, beta-and gamma secretase activities, and COX2 linked neuro-inflammation.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0129 THE ROLE OF Β -AMYLOIDOPATHY IN THE PATHOGENESIS OF AGE-RELATED MACULAR DEGENERATION IN CORRELATION WITH ALZHEIMER’S DISEASE 1 V. Ermilov 1 Dvinskaya 8-85, 400087, Volgograd, Russia Aims Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in the elderly. The concord appearance of β-amyloid in eye and brain tissues makes it possible to see in a new light the problem of eye amyloidosis. The commonality of pathological processes in brain in Alzheimer’s disease (AD) and in the eye for AMD has encouraged the authors to study the clinical and morphological features of amyloidogenesis in AMD as a manifestation of proteinopathy - β-amyloidopathy. Method With the help of electron microscopic study, selective methods for amyloid detecting its proteins 111 eyes with morphological signs of “dry” and “wet” forms of AMD and the brain taken from 56 cadaver were examined. Results β-amyloidopathy was identified in 39% of eyes with "dry" form of AMD, while "wet" form - in 80 % of cases. The combined accumulation of β-amyloid deposits in the structures of the eye and the brain was found in 50.6 % of cases. Amyloid- β deposits was specific to drusen from eyes with AMD and to senile plaques in brain tissue in the patients with Alzheimer disease (AD). Conclusion


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The keynote of AMD and AD pathogenesis is β-amyloidopathy that is a manifestation of proteinopathy leading to cytotoxicity, neurodegeneration and the development of pathological apoptosis activated by the formation of intracellular Aβ. This view on the problem creates the preconditions for the development of new strategies for creating of ophthalmogeriatric and neuroprotective drugs affecting the pathogenesis and including all stages of Aβ formation and pathological aggregation.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0717 SYSTEMIC ADMINISTRATION OF EPOTHYLONE-D RECUES MEMORY AND AMELIORATES ALZHEIMER’S DISEASE-LIKE PATHOLOGY IN APP/PS1 MICE 1 1 2 3 3 1 J.J. Fernandez-Valenzuela , R. Sanchez-Varo , V. De Castro , F.J. Moyano , M.I. Vizuete , J.C. Davila , 3 1 J. Vitorica , A. Gutierrez 1 Faculty of Science - University of Malaga/ CIBERNED/IBIMA, Department of Cell BiologyGenetics and Physiology NeuroAD, Málaga, Spain 2 Faculty of Science - University of Malaga/CIBERNED/IBIMA, Department of Cell BiologyGenetics and Physiology NeuroAD, Málaga, Spain 3 Faculty of Pharmacy - University of Seville/CIBERNED/IBIS, Department of Biochemistry and Molecular Biology, Seville, Spain Aims Cognitive and memory decline in Alzheimer's disease (AD) patients is highly related to synaptic dysfunction and neuronal loss. Tau hyperphosphorylation destabilizes microtubules leading to axonal transport failure, accumulation of autophagy/vesicular material and the generation of dystrophic neurites, thus contributing to axonal/synaptic dysfunction. In this study, we analyzed the effect of a microtubule-stabilizing drug in the progression of the disease in an APP751SL/PS1M146L transgenic model.

Method APP/PS1 mice (3 month-old) were weekly treated with 2 mg/kg intraperitoneal injections of Epothilone-D (Epo-D) for 3 months. Vehicle-injected animals were used as controls. For memory performance, animals were tested on the object-recognition tasks, Y-maze and Morris water maze. Levels of Abeta, ubiquitin, AT8 and synaptic markers were analyzed by Western-blot. Hippocampal plaque burden, dystrophic and synaptic loadings were quantified after immunostaining by image analysis.

Results Epo-D treated mice showed a significant improvement in the performance of hippocampus-associated cognitive tests compared to controls. This memory recovery correlated with a significant reduction in the AD-like hippocampal pathology. Abeta, APP and ubiquitin levels were significantly reduced in treated animals, and a decrease in both the plaque loading and the axonal pathology was also found. Finally, synaptic levels were significantly preserved in treated animals in comparison with controls.

Conclusion Epo-D treatment promotes synaptic and cognitive improvement, reduces the accumulation of extracellular Abeta and the associated neuritic pathology in the hippocampus of APP/PS1 model. Therefore, microtubule stabilizing drugs could be considered therapeutical candidates to slow down AD progression.


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Supported by FIS-PI15/00796 (AG), FIS-PI15/00957(JV) and co-financed by FEDER funds from European Union.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1433 AGGREGATION AND TOXIC BEHAVIOUR OF N-TERMINAL TRUNCATED AMYLOID-BETA PEPTIDES 1 1 1 S. Hossain , M. Zhang , G. Multhaup 1 McGill University, Pharmacology and Therapeutics, Montreal, Canada Aims Amyloid-β (Aβ) peptides are produced from the cleavage of the amyloid precursor protein (APP) by β- and γsecretases. Of the three most commonly detected Aβ species (Aβ38, Aβ40 and Aβ42), Aβ40 is usually non-toxic while Aβ42 is the most toxic. Aβ peptides tend to aggregate in solution: monomers form neurotoxic soluble oligomers which spontaneously aggregate into non-toxic proto-fibrils, fibrils and lastly Aβ plaque deposits. Nterminally truncated (5-X) Aβ peptides were detected in cells expressing the FAD K16N mutation affecting the αsecretase cleavage site of APP (Kaden et al, 2012). The resulting truncated Aβ likely triggers AD pathology. Here, we studied the aggregation and toxic behaviour of N-terminally truncated Aβ peptides containing the APP K16N FAD mutation. Method Oligomer formation was assessed by size exclusion chromatography and fibril formation of N-terminally truncated Aβ peptides was analyzed by the Thioflavin T assay and electron microscopy. Cytotoxicity in neurons and glial cells was studied by mitochondrial reduction and cell membrane permeability assays. Results N-terminal truncated Aβ 5-40 and 5-42 K16N peptides exhibited increased aggregation profiles and a pronounced fibril formation compared to its wild-type (wt) truncated counterparts. N-terminal truncated K16N species were more toxic than its wt counterparts, but most notably, truncation toxified the widely non-toxic Aβ40 wild-type peptide. Conclusion


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Overall, our findings suggest that the K16N mutation altered APP processing, producing more toxic Aβ species to likely contribute to the disease pathogenesis.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0839 THE ARCHITECHTURE OF THE AMYLOID FIBRIL CONTROLS THE ABILITY TO INDUCE FIBRILATALYZED SECONDARY NUCLEATION 1 1 1 T. Islam , K. Brännström , A. Olofsson 1 Umeå University, Medical Biochemistry and Biophysics, Umeå, Sweden Aims Self-assembly of the amyloid-beta peptide (Abeta) into amyloid fibrils is associated with the development of Alzheimer’s disease. In vivo Abeta1-40 and Abeta1-42 are the dominant forms. The architectures of Abeta1-40 and Abeta1-42 fibrils differ, and Abeta1-42 assemblies generally have higher cytotoxicity. Abeta fibril formation follow a nucleation dependent polymerization process and it is known that new initiation sites, from which the incorporation of monomers can propagate into a fibril, can be generated through at least three different routes, these include; primary nucleation where nuclei spontaneously form in solution, the breaking of fibrils to form new fibrillar ends, and fibril-catalyzed secondary nucleation where formation of new nuclei is catalyzed laterally along already formed fibrils .The overall rate of conversion into amyloid is consequently dependent on the number of initiation sites from which the template-dependent polymerization can start. Under stagnant conditions fibrilcatalyzed secondary nucleation dominates. Method Using Thioflavin-T assay we have performed cross-seeding studies of Abeta1-42 by monomeric Abeta1-40. Results Our results show that fibrils of Abeta1-42 are impaired to induce fibril-catalyzed secondary nucleation of Abeta140 while the elongation process is unaffected. Intriguingly the formed Abeta fibril acquire the same properties as the Abeta1-42 and is, in contrast to spontaneously formed fibrils of Abeta1-40, impaired to induce fibril-catalyzed secondary nucleation of monomeric Abeta1-40. Conclusion


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The results suggest that the incorporated Abeta1-40 acquired the structure of Abeta1-42 fibrils and that the architecture of the Abeta fibrils rather than the sequence controls the ability to induce fibril-catalyzed secondary nucleation.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1003 AΒ34-BASED CHARACTERIZATION OF HUMAN POST-MORTEM BRAIN SAMPLES 1 2 1 1 2 1 T. Kirabali , F. Liebsch , C. Hock , R.M. Nitsch , G. Multhaup , L. Kulic 1 University of Zurich, Institute for Regenerative Medicine, Schlieren, Switzerland 2 McGill University, Department of Pharmacology and Therapeutics, Montreal, Canada Aims Enzymatic degradation of amyloid β-peptide (Aβ) leads to formation of shorter, non-toxic Aβ fragments that are less prone to aggregate formation. The C-terminally truncated Aβ isoform, Aβ34, has been identified as an important intermediate product of γ- and β-secretase-mediated Aβ40 and Aβ42 degradation. Since Aβ34 is detectable both in CSF and cell culture media, it has the potential to be used as a pharmacodynamic fingerprint and a possible biomarker of enzyme-mediated Aβ clearance activity. In this study, we investigated Aβ34 immunoreactivity in different neuropathological AD stages. Method Human post-mortem cortex and hippocampus samples categorized according to Braak stage, CERAD scores, ApoE genotype and disease status (AD or not-AD) were further assessed for Aβ34 immunoreactivity using a recently developed highly specific anti-Aβ34 monoclonal antibody. Immunohistochemical analysis additionally included various other AD-related markers (e.g. anti-amyloid and vascular markers, among others.) Results Aβ34 immunoreactivity was observed mostly in the earlier Braak stages and showed vessel-associated localization. It was not detected in amyloid plaques and did not colocalize with other amyloid species in earlier stages. On the other hand, most of the CAA-affected vessels in the Braak stage VI samples were found to be positive for both Aβ34 and anti-amyloid antibody. Preliminary analyses revealed an inverse relationship between Aβ34 and total amyloid load in human post mortem samples. Conclusion


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Our results point to an early involvement of Aβ34 in AD pathology and are thus in line with its proposed potential as a promising novel biomarker of early AD.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1348 NANO-SCALE INFRARED IMAGING OF Β-SHEET STRUCTURES IN SYNAPTIC JUNCTIONS OF PRIMARY NEURONS FROM TRANSGENIC MOUSE MODELS OF ALZHEIMER´S DISEASE 1 2 3 4 1 5 6 1 O. Klementieva , J. Mathurin , A. Engdahl , R. de Oliveira Freitas , K. Willén , P. Uvdal , L. Miller , G. Gouras 1 Lund University, Department of Experimental Medical Science, Lund, Sweden 2 Université Paris-Sud Orsay, Laboratoire de Chimie Physique, Paris, France 3 Lund University, MAXIV, Lund, Sweden 4 Brazilian Center for Research in Energy and Materials - CNPEM, Brazilian Synchrotron Light Laboratory - LNLS, Campinas, Brazil 5 Lund University, Department of Physical and Theoretical Chemistry, Lund, Sweden 6 Brookhaven National Laboratory, NSLS-II, Upton, USA Aims Amyloid-β (Aβ) comprises a class of aggregation-prone peptides, which may misfold into stable, β-sheet rich fibrils. Aβ is linked to the development of synaptic pathology in Alzheimer’s disease (AD). However, a main question is how Aβ contributes to AD neuropathology. Our aim is to study the distribution of β-sheet structures in AD transgenic neurons in order to uncover sub-cellular mechanism(s) by which amyloid β-sheet structures are involved in AD pathology. Method We use two atomic force microscopy (AFM)-based spectroscopic approaches: AFM-infrared spectroscopy (AFMIR) and AFM-near-field scanning optical microspectrosopy (s-SNOM). AFM provides information on neuronal morphology, while AFM-IR and s-SNOM characterise conformational changes in protein structures inside and on the surface of neurons, respectively. Experiments were done at SOLEIL (France), Lawrence Berkeley National Laboratory (USA) and Brazilian Synchrotron Light Laboratory. Results For the first time, the hyperspectral maps of protein structures in AD transgenic neurons were performed and the beta-sheet distributions were mapped in AD transgeinc neurons at nano-scale spatial resolution (~ 40 nm). Conclusion


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Our results show that β-sheet structures are distributed within AD neurites of AD transgenic compared to wildtype neurons. The presence of β-sheet structures in synaptic junctions in AD transgenic neurons supports the conclusion that synapses are the sites where Aβ accumulates and aggregates, thereby mediating synapse dysfunction in AD brain. However, further experiments are required to understand the spread of β-sheet structures, and nano-scale AFM-based infrared spectroscopies are useful tools for this purpose.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0625 PRION-LIKE INDUCTION OF BETA-AMYLOID DEPOSITION IN THE ABSENCE OF APP OVEREXPRESSION 1,2 1,2 1,2 3,4 1,2 H. Lau , E. Stuart , Z. Wang , G. Schmitt-Ulms , J. Watts 1 Tanz Centre for Research in Neurodegenerative Diseases, Biochemistry, Toronto, Canada 2 University of Toronto, Biochemistry, Toronto, Canada 3 Tanz Centre for Research in Neurodegenerative Diseases, Lab Medicine and Pathobiology, Toronto, Canada 4 University of Toronto, Lab Medicine and Pathobiology, Toronto, Canada Aims The beta-amyloid (Abeta) peptide is a proteolytic cleavage product of the Amyloid Precursor Protein (APP). The aggregation and deposition of Abeta is hypothesized to be an initiating event in the pathogenesis of Alzheimer’s disease (AD). Mounting evidence suggests that the formation and spread of self-propagating, prion-like Abeta aggregates during AD may contribute critically to disease progression. Indeed, a number of groups have shown that intracerebrally inoculating transgenic mice that overexpress wild-type or mutant APP with Abeta aggregates results in the induction of cerebral Abeta deposition. Our goal was to assess whether Abeta deposition can also be induced when physiological APP levels are present in the brain. Method NL-F

The APP mouse is a knock-in model of AD that overproduces wild-type human Abeta peptide while avoiding NL-F potential artifacts associated with APP overexpression. We inoculated APP mice with partially purified brainderived Abeta aggregates and then monitored the induction of cerebral Abeta deposition. Results Induced Abeta deposition was observed in the corpus callosum of inoculated mice at 5 months post-inoculation, whereas uninoculated and buffer-inoculated animals did not exhibit spontaneous Abeta deposits at this age. Induced Abeta deposits were also observed within meningeal blood vessels. Interestingly, the induced Abeta deposits were composed almost exclusively of Abeta-42 peptide. Conclusion


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These results demonstrate that APP overexpression is not a prerequisite for the prion-like induction of cerebral Abeta deposition. Thus, spreading of Abeta deposition may contribute to disease progression in AD patients.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0776 EFFECT OF SIMVASTATIN ON INFLAMMATION IN CLINICAL AD PATIENTS AND ANIMAL MODEL OF AD 1 Z. Liandong 1 The second hospital of Huai'an, Neurology, Huai'an, China Aims Explore the effect of simvastatin on inflammation was investigated and the underlying mechanisms. Method First, we tested the effect of simvastatin on AD in clinical research. The fasting venous blood was collected in order to evaluate the levels of IL-6, IL-1β, ACT and TNF-α, which were measured with the enzyme-linked immunosorbent assay (ELISA) kits. Amyloid-β (Aβ), amyloid-β precursor protein(APP) and β-site APP-cleaving enzyme 1(BACE1) were tested by western blotting. Second, we used an APPswe/PS1E9 (APP/PS1) double transgenic mouse to evulate the amelioration ability of simvastatin against the in vivo memory impairment. Spatial learning and memory of mice were investigated by the Morris water maze test. The mRNA of inﬂammatory cytokines were measured using real-time PCR. Third, the phospho-proteome profile of SH-SY5Y human neuroblastoma cells treated with simvastatin was used to investigate the possible mechanisms. Results The results showed that simvastatin ameliorated the memory deﬁcits both in clinical AD patients and animal model of AD. Simvastatin could reduce the mRNA expression of inflammatory cytokines and mediators, suppress the apoptosis of neural stem cells and improve the survival rate of neurons. Conclusion


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In conclusion, these results suggest that simvastatin could be of benefit in preventing the progression of AD and expected to be potentially used as a lead drug for further anti-AD treatment.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1098 HUMAN AMYLIN BUT NOT RAT AMYLIN PROMOTES INTRACELLULAR AMYLOID-BETA AGGREGATION VIA OXIDATIVE STRESS. 1 1 1 Y.A. Lim , W.T. Lee , C. Chen 1 National University of Singapore, Pharmacology, Singapore, Singapore Aims Epidemiological studies have shown that patients with type 2 diabetes (T2DM) have a significantly greater risk of developing Alzheimer's disease (AD). We have previously shown that the amyloidogenic peptides amyloid-beta and HA induce oxidative stress in neurons. HA has previously been shown to co-localise within amyloid-beta plaques in the AD brain, but the underlying mechanisms remains unclear since studies in diabetic AD mice could not recapitulate the findings in humans. Presumably, this is because rodent amylin is non-amyloidogenic, but this remains to be formally investigated. This study aims to assess whether amyloidogenic HA exacerbates amyloidbeta burden in a neuronal cell culture model of AD. Method SYSY5Y neuroblastoma cell line were stably-transfected with the human amyloid precursor protein harbouring the Swedish mutation (APPSw), causing an overproduction of amyloid-beta, and treated with either amyloidogenic HA or the non-amyloidogenic RA with various concentrations and time points, prior to biochemical analyses. The role of oxidative stress in promoting intracellular amyloid-beta aggregation was also assessed. Results We found that the amyloidogenic HA, but not the non-amyloidogenic RA, induced a significant increase in intracellular amyloid-beta aggregation in APPSw cells in a time and dose-dependent manner via oxidative stress. Furthermore, HA-induced intracellular amyloid-beta aggregation is in part, mediated via oxidative stress. Conclusion


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Together, our results show that the amyloidogenic HA, but not the non-amyloidogenic RA, promotes amyloid-beta aggregation and is mediated via oxidative stress. Our results provide support to further assess the role of HA on promoting amyloid-beta pathology as an important event underlying the link between T2DM and AD.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1307 THE RATIO BETWEEN BETA-AMYLOID (AΒ) 42 AND AΒ40 IN CSF IS A BETTER PREDICTOR OF STRUCTURAL BRAIN CHANGES THAN AΒ42 ALONE IN COGNITIVELY NORMAL PEOPLE 1 2 1 3 4 5 6 1 O. Lindberg , E. Stomrud , X. Li , M. Schöll , D. van Westen , K. Blennow , H. Zetterberg , L.O. Wahlund , 7 O. Hansson 1 Karolinska Institutet, Department of Neurobiology- Care Sciences and Society, Stockholm, Sweden 2 privat, Clinical Memory Research Unit- Department of Clinical Sciences- Malmö- Lund University, Malmö, Sweden 3 University of Gothenburg, MedTech West and the Division of Clinical Neuroscience- University of GothenburgGothenburg- Clinical Memory Research Unit- Department of Clinical Sciences- Malmö- Lund University Sweden, Gothenburg, Sweden 4 Lund university, Department of Clinical Sciences- Diagnostic Radiology- Lund University- LundSweden and Imaging and Function- Skåne University Health Care- Lund- Sweden, Lund, Sweden 5 University of Gothenburg, Institute of Neuroscience and PhysiologyDepartment of Psychiatry and Neurochemistry- the Sahlgrenska Academy at the University of GothenburgMölndal- Sweden- Clinical Neurochemistry Laboratory- Sahlgrenska University Hospital- Mölndal, Gothenburg, Sweden 6 University of Gothenburg, - Institute of Neuroscience and PhysiologyDepartment of Psychiatry and Neurochemistry- the Sahlgrenska Academy at the University of GothenburgMölndal- Sweden- Clinical Neurochemistry Laboratory- Sahlgrenska University Hospital- Mölndal- SwedenDepartment of Molecular Neuroscience- UCL Institute of Neurology- Queen Square- London- UK, Gothenburg, Sweden 7 Lund university, Clinical Memory Research Unit- Department of Clinical Sciences- Malmö, Lund, Sweden Aims Decreased levels of the amyloid β peptide 42 (Aβ42) in cerebrospinal fluid (CSF) is a validated biomarker for Alzheimer´s disease (AD). However, recent evidence suggests that the ratio between Aβ 42 and Aβ40 may improve the possibility to detect cortical Aβ fibrils at an earlier stage. It is not clear whether this ratio also is superior at detecting neurodegeneration in preclinical AD. We investigated whether cerebrospinal fluid (CSF) Aβ42/Aβ40 ratio is a better predictor of brain grey and white damage and of amyloid fibrillary load than CSF Aβ 42 levels alone in 303 cognitively normal subjects between 65-87 years of age. Method Quantification of Aβ and P-tau was done using ELISA, brain atrophy and white matter abnormalities using MRI, Aβ fibrils using PET. Results Low Aβ42/Aβ40 ratio, but not CSF Ab42 alone, was associated with atrophy of frontoparietal cortex and with white matter abnormalities. The Aβ42/Ab40 ratio was more strongly correlated with Aβ fibrillary load than Aβ42 alone. Further, CSF P-tau was only associated with medial temporal atrophy in cases with a low Aβ 42/Aβ40 ratio, but not low Aβ42. Conclusion


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CSF Aβ42/Aβ40 ratio is a better predictor of amyloid deposition and structural grey and white-matter changes in the brain of cognitively normal subjects than Aβ42 alone. In preclinical AD Aβ accumulation might be associated with subtle frontoparietal cortical atrophy, while tau pathology is associated with cortical thinning of the medial temporal lobe.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1655 INHIBITOR DESIGN OF AMYLOID TOXICITY BASED ON ATOMIC STRUCTURES OF AMYLOID 1 2 3 2 1 D. Li , L. Jiang , J. Nowick , D. Eisenberg , C. Liu 1 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China 2 UCLA-DOE Institute for Genomics and Proteomics, and Department of Chemistry and Biochemistry, University of California, Los Angeles, USA 3 Department of Chemistry, University of California, Irvine, USA The abnormal aggregation of amyloid-beta (Aβ) into plaques in brain is the hallmark of Alzheimer disease (AD)， one of the major forms of neurodegenerative disorders. Over 50 million patients are suffering from AD worldwide1,2. Currently, the most widely used strategy in search of drug candidates for AD is to prevent the formation of pathogenic Aβaggregates3,4. However, no effective drug has been successful developed so far. In this study, we developed two different strategies of structure-based inhibitor design targeting Aβ amyloid aggregation and cytotoxicity. The first one is to design peptide-based beta-sheet mimics to target the nucleus of Aβ aggregates. By blocking the nucleation process, beta-sheet mimics is capable of inhibiting Aβ aggregation and reduced its cytotoxicity in mammalian cells. The second one is to design small molecules capable of stabilizing the final aggregation product -Aβ fibrils. From structure-based rational design, several compounds were identified as tight fibril binders. Importantly, the compounds were able to detoxify Aβ by shifting the equilibrium of Aβ from oligomers to fibril. Structure-activity relationship studies further revealed the pharmacorphore of Aβ fibril which is essential for optimization of more potent amyloid fibril binder.


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1. Chiti, F. & Dobson, C. M (2006) Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333– 366. 2. Eisenberg D, Jucker M (2012) The amyloid state of proteins in human diseases. Cell 148(6):1188–1203. 3. Aguzzi, A. & O’Connor, T (2010) Protein aggregation diseases: pathogenicity and therapeutic perspectives. Nature Rev. Drug. Discov. 9, 237–248. 4. Härd T, Lendel C (2012) Inhibition of amyloid formation. J Mol Biol 421(4–5):441–465.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0576 THE ROLE OF INSOLUBLE OFF-PATHWAY AGGREGATES AS CROWDING AGENTS DURING PROTEIN FIBRILLATION 1 2 2 1 3 1 R. Crespo , E. Villar-Alvarez , P. Taboada , F. Rocha , A. Damas , P.M. Martins 1 University of Porto, LEPABEB FEUP, Porto, Portugal 2 Universidade Santiago de Compostela, Física de la Materia Condensada, Santiago de Compostela, Spain 3 University of Porto, ICBAS, Porto, Portugal Aims The screening of drugs candidates for the treatment of amyloidosis and neurodegenerative diseases frequently involves in vitro measurements of amyloid fibril formation. Macromolecular crowding and off-pathway aggregation (OPA) are, by different reasons, two important phenomena affecting the scalability of amyloid inhibitors and their successful application in vivo. On the one hand, the cellular milieu is crowded with macromolecules that drastically increase the effective (thermodynamic) concentration of the amyloidogenic protein. On the other, offpathway aggregates, rather than amyloid fibrils, are increasingly appointed as the causative agents of toxicity. Method Protein depletion and thioflavin-T fluorescence progress curves indicate that OPA rebirth is not accompanied by additional amyloid fibril formation. The crystallization-like model [1] extended to account for OPA [2] and timedependent activity coefficients is able to fit multiple kinetic results using a single set of three parameters describing amyloid nucleation, autocatalytic growth and off-pathway nucleation. The list of fitted results notably includes all types of progress curves measured for different HEWL concentrations. Results Our results reveal that insoluble off-pathway aggregates of hen egg-white lysozyme are a peculiar type of crowding agents that, unlike classical macromolecular crowders, decrease the thermodynamic concentration of protein. Illustrating this effect, OPA is shown to resume after lowering the fraction of insoluble aggregates at constant protein concentration. Conclusion


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The quantitative challenges posed by macromolecular crowding and OPA find here a unified response with broader implications for the efficient design of on- and off-pathway inhibitors. [1]- Crespo et al. (2012) JBC, 287(36): 30585-30594. [2]- Crespo et al. (2016) JBC, 291(4): 2018-2032.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0853 PREGNANCY AFFECTS ABETA PATHOLOGY AND ADULT NEUROGENESIS IN 5XFAD MICE 1 1 1 1 1 1 2 2 3 S. Waldkirch , K. Marksteiner , L. Neudel , D. Eiler , V. Sturn , I. Opper , B. Nuscher , C. Haass , M. Datta , 3 1 M. Prinz , M. Meyer-Luehmann 1 Neurocenter, Neurology, Freiburg, Germany 2 BioMedical Center BMC, Biochemistry, Munich, Germany 3 Neurocenter, Neuropathology, Freiburg, Germany Aims Several studies suggest that women have a higher risk to develop Alzheimer’s disease (AD) than men. In particular, the number of pregnancies was shown to be a risk factor for AD and women with several pregnancies on average had an earlier onset of the disease, thus making childbearing a risk factor. However, the impact of being pregnant on Abeta plaque pathology and adult neurogenesis still remains elusive. Method 3 months old pregnant 5xFAD transgenic mice were analyzed at either gestation day 5 (G5), G10, G18 or at P20 by using immunohistochemical stainings. We quantified Ki67 positive cells as a marker for proliferation, counted the number of doublecortin (DCX) positive cells as a marker for neurogenesis and measured the Abeta content with an antibody against Abeta. Results Postmortem analysis at different time points revealed that pregnant 5xFAD transgenic mice had significantly more Abeta plaques in the hippocampus from G10 on. Interestingly, 5 months old 5xFAD transgenic mice that also nursed their offsprings for 4 weeks had a similar Abeta plaque load than merely pregnant mice, indicating that pregnancy alone is sufficient to elevate Abeta plaque levels. In both groups, the number of Ki67- and DCXpositive cells was dramatically decreased in the postpartum period compared to 5xFAD mice. Conclusion


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Our results suggest that pregnancy alters Abeta plaque load in 5xFAD transgenic mice and diminishes the generation of newborn neurons. We conclude that pregnancy alone is sufficient to induce this phenotype as lactation has no influence on this pathology.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1359 POTENTIAL ROLE OF SORLA IN ALZHEIMER’S DISEASE PATHOGENESIS 1 1 1 1 M. Micsenyi , R. Pandit , M. Wittmann , T. Bussiere 1 Biogen, Neurology, Cambridge, USA Aims SORL1 variants are implicated in both sporadic and familial forms of Alzheimer’s disease (AD). SORL1 encodes the sorting receptor SorLA, which is reported to function in APP trafficking. Published studies suggest SorLA protein levels might be reduced in sporadic AD patient brains, and this reduction is potentially cellspecific. Additionally, it has been reported that reduced SorLA might alter APP trafficking and be associated with increased beta-amyloid generation. As such, we intend to refine our understanding of the role of SorLA in APP trafficking and amyloidogenesis in AD. Method Expression of SorLA protein in human brains is assessed by biochemistry and IHC with the aim of validating the reported reduction of SorLA in AD. Cell-specific expression of SorLA is evaluated in neurons versus glia using IHC and ISH. The implications for modulating SorLA levels on beta-amyloid generation is being assessed in cell lines and primary cultures. Further studies are focused on defining the subcellular interaction of SorLA and APP, and how this interaction may modulate beta-amyloid levels. Results The reported reduction in SorLA protein in AD brains has not been observed in our samples, however these results need to be confirmed with a larger cohort. Additionally, cell-specific SorLA expression is being currently evaluated. Transient siRNA knock-down of SorLA in several cell lines does not affect beta-amyloid levels. Similar experiments using primary cultures are being conducted. Conclusion


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These studies will offer further insight for the role of SorLA in amyloidogenesis and AD pathogenesis, and define its potential as a druggable target.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1495 TOWARDS GENETIC DISSECTION OF SEVERITY AND ONSET MODULATING GENES FOR ALZHEIMER’S PATHOLOGY IN DOWN SYNDROME USING CELLULAR SYSTEMS 1 D. Nizetic 1 Lee Kong Chian School of Medicine, Mechanisms of Ageing and Disease, Singapore, Singapore Aims Trisomy of APP seems obviously responsible for extremely high incidence and early onset of AD pathology in Down syndrome (DS) brains. However, other genes on human chr21 likely modulate the age of onset, severity and modality of the clinical picture, as DS individuals have later or absent onset of clinical AD, and less intracerebral haemorrhage pathology, than euploid individuals with dupAPP. Our aim is to identify such modulator genes on chr21 using cellular models. Method Neurons derived from isogenic hiPSCs we generated from a mosaic DS individual, unpublished iPSCs from segmental trisomy 21 DS and non-DS individuals, as well as from DS individuals with extremely early or late dementia onset are used in 2D, 3D and cerebral organoid paradigms. Optogenetic stimulation of Channelrhodopsin-2 engineered hiPSCs is used to determine the neuronal activity-dependent cellular phenotype modulation. CRISPR/Cas9 mediated reduction from trisomy to disomy for individual selected candidate genes on chr21 are performed on isogenic T21 iPSCs.

Results We detect T21-caused neurodevelopmental delay, increased β-amyloid and phospho-Tau presence, complex mitochondrial dysfunction, accelerated DNA damage and abnormal endosomes, and some of these phenotypes exacerbate in 3D and cerebral organoid paradigms, and/or can be reproduced in primary human neuroectodermal cells expanded and differentiated in vitro. Conclusion


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Cellular phenotypes relevant for AD pathology caused by trisomy21 can be reproduced in iPSC-derived and primary NSC-derived neurons and differences sharpened by the use of cerebral organoid technology. Isogenic iPSCs allow detection of subtle changes in phenotypes and evaluation of single gene’s trisomy contribution using CRISPR/Cas9 editing.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0612 AMYLOID-PLAQUES PROPERTIES CHANGE WITH THE PROGRESSION OF ALZHEIMER’S DISEASE 1 1 1 1 1 1 C. Nuñez-Diaz , E. Sanchez-Mejias , R. Sanchez-Varo , L. Trujillo-Estrada , A. Gomez-Arboledas , J.C. Davila , 2 1 J. Vitorica , A. Gutierrez 1 University of Malaga. CIBERNED/IBIMA, Cell Biology. Faculty of Science., Málaga, Spain 2 University of Seville. CIBERNED/IBIS, Biochemistry & Molecular Biology. Faculty of Farmacy., Seville, Spain Aims Accumulation of beta-amyloid peptides (Abeta) exacerbates during the progression of Alzheimer’s disease (AD). These Abeta peptides aggregate forming toxic oligomers, fibrillar structures and amyloid plaques. The latter may release soluble oligomers causing synaptic and neuronal damage. Here we analyzed amyloid pathology progression and plaque toxicity in the hippocampus of an APP/PS1 model and AD patients. Method Immunohistochemistry was performed in hippocampal samples from 4 to 18 month-old APP751SL/PS1M146L mice and in post mortem human AD samples along Braak stages (II-VI). Quantifications were done by densitometry and stereology. Results In APP/PS1, the accumulation of Abeta had a specific spatiotemporal pattern, beginning at 3-4 months in the subiculum and the hippocampus proper. On the contrary, AD patients were affected by Abeta deposition in more advanced stages, reaching the hippocampus in Braak V-VI stages. In the model, plaques were principally fibrillar and neuritic. Their compaction decreased with the pathology progression, whereas the ring of oligomeric Abeta surrounding the plaque core enlarged. However, human samples displayed a clear regional and interindividual variability, developing different types of deposits (fibrillar, classic and diffuse). Plaques had a toxic effect on synapses as a function of distance from the plaque center and its size. Conclusion We propose that amyloid plaques change their properties with the disease progression, releasing toxic Abetaoligomer species to the brain parenchyma and promoting neurodegeneration. Moreover, our results suggest a different origin or progression pattern for the extracellular amyloid deposits between AD mice and human samples.


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Supported by FIS-PI15/00796(AG), FIS-PI15/00957(JV) and co-financed by FEDER funds from European Union.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1051 A NOVEL METALLOPORPHYRIN-PEPTIDE CONJUGATE PREVENTS AMYLOID-Β FIBRILLATION AND OLIGOMERS’ CYTOTOXICITY 1 1 1 2 1 3 3 G. Pappalardo , R. Tosto , G. Di Natale , A. Sinopoli , M.F. Tomasello , V. Villari , N. Micali 1 National Research Council CNR, Institute of Biostructures and Bioimaging, Catania, Italy 2 International PhD Program in Translational Biomedicine, University of Catania, Catania, Italy 3 National Research Council CNR, Institute for chemical and physical processes, Messina, Italy Aims Molecules that inhibit Aβ mis-folding have received considerable attention in view of their potential use in prevention or treatment of Alzheimer’s disease (AD). The construction of a di-functional system capable to exploit the metal coordination ability of the Aβ N-terminus and the hydrophobic interaction between Aβ chains as anchoring sites would provide new devices to look inside the Aβ’s fibrillation process in addition to promising therapeutic approaches. We designed a peptide derivative that incorporates two recognition sites. Such a system consists of a Zn-porphyrin conjugated to the well-known Aβ-recognizing KLVFF aminoacid sequence. Method The interaction between the peptide conjugate and Aβ was studied by using an array of different biophysical techniques including Dynamic Light Scattering (DLS), far-UV Circular Dichroism (CD), Fluorescence spectroscopy, MALDI-TOF-MS and NMR. Furthermore Th-T kinetics and CD data shown that the Zn-porphyrin conjugate abolish the fibrillogenic process of Aβ42. Results Here we show that the novel Zn-porphyrin-peptide conjugate is a potent inhibitor of Aβ42 amyloid formation and elicits the formation of large-size amorphous aggregates. This evidence is accompanied by a significant reduction of the toxicity of Aβ-oligomers toward primary neuronal cell cultures. Conclusion


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A hypothetical working mechanism of the inhibitory effects of the conjugate can be proposed based on the experimental observations.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0851 CSF BIOMARKERS IN PRODROMAL ALZHEIMER’S DISEASE 1,2 2,3 4 2,5 2 2,5 2,6 2,6 1,2 B.C. Kim , K.H. Lee , J.E. Park , K.Y. Choi , J.J. Lee , Y.Y. Choi , H. Kim , J.M. Ha , S.M. Choi , 1,2 2,3,4 M.K. Song , J.S. Lee 1 Chonnam National University Medical School, Departments of Neurology, Gwangju, Republic of Korea 2 Chosun University, National Research Center for Dementia, Gwangju, Republic of Korea 3 Chosun University, Department of Biomedical Science, Gwangju, Republic of Korea 4 Chosun University, BK21-plus Research Team for Bioactive Control Technology, Gwangju, Republic of Korea 5 Chosun University, Premedics, Gwangju, Republic of Korea 6 Chosun university, School of Medicine, Gwangju, Republic of Korea Aims Disease-modifying therapy for Alzheimer’s disease has led to a need for biomarkers to identify prodromal Alzheimer’s disease and very early stage of Alzheimer’s dementia. Here, we are aimed to identify the cut-off values of CSF biomarkers for detecting prodromal Alzheimer’s disease in patients with mild cognitive impairment (MCI), who have positive uptake of amyloid PET scan. Method We assessed 12 patients with MCI who underwent successful lumbar puncture. Additionally, 21 healthy elderly and 5 patients with Alzheimer’s dementia served as controls. To detect human brain amyloid-beta (Aβ) deposition, PET scan was conducted using florbetaben as a probe. We analyzed protein concentration of beta 181 amyloid (Abeta1-42), total tau (t-tau), and phosphorylated tau (p-tau ) in CSF using INNOTEST ELISA (Fujirebio, Belgium). Results 181

The cut-off values of CSF Abeta1-42, t-tau, and p-tau are 735 pg/ml, 370 pg/ml, and 51 pg/ml for detection of prodromal Alzheimer’s disease in patients with MCI, respectively. The combination ratios (t-tau/Abeta1-42 and ptau/Abeta1-42) have also been recently considered as biological diagnostic criterion of Alzheimer’s disease. The ttau/Abeta1-42 and p-tau/Abeta1-42 ratios defined cut-off values at 0.57 and 0.075 to discriminate prodromal Alzheimer’s disease in patients with MCI, respectively. Conclusion


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CSF biomarkers are very useful tools for diagnosis of the prodromal Alzheimer’s disease in patients with mild cognitive impairment.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1480 PHOTO-INDUCED DAMAGE OF AMYLOID BETA BY THE CHLORIN E6 PHOTOSENSITIZER 1 M. Richman 1 Bar Ilan university, Chemistry, Ramat gan, Israel Aims Inhibition of amyloid beta (Aβ) aggregation and destabilization of toxic Aβ oligomers are promising approaches for the prevention and therapy of Alzheimer's disease (AD). Compounds block Aβ aggregation have been identified in many studies, for example, peptides containing a recognition element KLVFF (residues16-20 in Aβ) were developed to bind Aβ and disrupt its aggregation, thus protecting neurons from Aβ-associated toxicity. Herein, we utilize the high affinity of the porphyrin, Chlorin e6 (Ce6), to Ab and its well-known photosensitizing property to selectively damage Ab upon illumination with visible light. The ThT assay as well as the transmission electron microscopy (TEM) studies demonstrated that Ce6 exhibits potent antiamyloidogenic activity in dark, however, brief illumination of the Ab-Ce6 mixture dramatically increases its activity. Ce6 also protected rat pheochromocytoma PC-12 cells from Ab toxicity in dark, however illumination with visible light significantly increased this activity. Method Using various immunoassays, circular dichroism spectroscopy, photoinduced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying Ce6 antiamyloidogenic activity. Results NMR spectroscopy indicated that Ce6 selectively binds the His residues in positions 13 and 14 of Ab. A significant loss of His residues was observed by NMR as well as amino acid analysis upon irradiation of the AbCe6 mixture with light. Overall, the NMR and the amino acid analysis together with the PICUP results demonstrated that. Conclusion


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irradiation of Ce6 in the presence of Aβ induces intra-molecular cross-links and also modifies His residues in Aβ sequence, which can be responsible for its photo-induced antiamyloidogenic activity

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0303 ALPHA SYNUCLEIN SPECIES STRONGLY AFFECT THE AMYLOID FORMATION MECHANISM OF MEMBRANE-BOUND PROTEIN 1 1 1 S. Rocha , J. Kiskis , P. Wittung-Stafshede 1 Chalmers University of Technology, Biology and Biological Engineering, Gothenburg, Sweden Aims The aggregation of alpha-synuclein, from monomers to amyloid fibrils, is related to degeneration of dopaminergic neurons in Parkinson’s disease. The protein proposed function involves synaptic vesicle regulation. Alphasynuclein binds to negatively charged vesicles in vitro, undergoing a transition from unordered to partially alphahelical structure. Here, we describe the effect of the chemical structure of lipid head groups on the membraneinduced aggregation of alpha-synuclein. Method Using circular dichroism spectroscopy, thioflavin T fluorescence and atomic force microscopy, we studied the binding of alpha-synuclein to DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) and DOPG (1,2-dioleoyl-snglycero-3-phospho-(1'-rac-glycerol) vesicles. Results In order to have the same α-helix content, higher Lipid:Protein (L:P) ratios are needed in the case of DOPS vesicles as compared to DOPG. We found that the number of lipid molecules associated with one protein in the DOPS model membranes is 5x higher than that in the DOPG model. At low L:P ratios, when part of the protein molecules is bound to vesicles and part is free in solution, alpha-synuclein forms amyloid fibrils at a faster rate for DOPG than for DOPS. We discovered also that the protein aggregation mechanism depends on the homogeneity of the starting protein sample. When only monomers are present, the vesicles stimulate primary nucleation. However, if a small fraction of oligomers is present along with the monomers, elongation of thin fibrils dominates the aggregation reaction. Conclusion


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This study reveals a key role of the lipid head group for the interaction of alpha-synuclein with membranes and demonstrates that the presence of oligomers alters the mechanism of membrane-bound protein aggregation.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1351 AMYLOID BETA OLIGOMERIZATION NEGATIVELY INFLUENCES BRAIN CLEARANCE MECHANISMS 1 2 2 2 3 2 A. Rostagno , P. Giannoni , F. McIntee , E. Cabrera , T. Neubert , J. Ghiso 1 New York University School of Medicine, New York, USA 2 New York University School of Medicine, Pathology, New York, USA 3 New York University School of Medicine, Skirball Institute, New York, USA Aims Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to progressive accumulation of neurotoxic amyloid beta (Abeta) oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Abeta influencing the delicate balance between rate of synthesis, dynamics of aggregation and clearance kinetics. Emerging new data suggest that reduced Abeta clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Method We have used a combination of stereotaxic injection into the hippocampal region of C57BL/6 wild-type mice with biochemical and mass spectrometric analyses of CSF to evaluate the brain clearance and catabolism of welldefined monomeric and low molecular mass Abeta oligomeric assemblies. Results Abeta physiologic removal from the brain is extremely fast, involves local proteolytic degradation with generation of heterogeneous C-terminally cleaved proteolytic products, and is negatively influenced by oligomerization. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Abeta. Clearance from brain interstitial fluid follows local and systemic paths; in addition to the BBB, local enzymatic degradation and transport through the choroid plexus into the CSF play significant roles. Conclusion


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Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0486 FACTORS CONTROLLING THE FORMATION OF SELF-PROPAGATING AΒ AGGREGATES 1 2 A. Ruiz-Riquelme , J. Watts 1 Tanz Centre for Research in Neurodegenerative Diseases, -, Toronto, Canada 2 Tanz Centre for Research in Neurodegenerative Diseases, Biochemistry, Toronto, Canada Aims The amyloid cascade hypothesis proposes that aggregation of beta-amyloid (Aβ) peptide triggers a cascade of events that leads to the development of Alzheimer’s disease. Prions are proteins that in their misfolded form selfpropagate, becoming infectious and causing prionopathies. Recent studies support the idea that Aβ aggregates may exhibit prion-like characteristics, including the ability to self-propagate. In prion disease, proteinase K (PK)resistant aggregates are a major component of the most infectious strains. Recent evidence suggests that PKresistance may also be a crucial feature of self-propagating Aβ aggregates. Our aim was to characterize the relative PK resistance of Aβ aggregates formed under different conditions. Method A variety of Aβ peptides of distinct lengths and of different origins (synthetic and recombinant) were tested. Hexafluoroisopropanol (HFIP) or ammonium hydroxide (NH4OH) pre-treatment was employed. Aβ aggregation was achieved by protein misfolding cyclic amplification (PMCA) or shaking. Biochemical features of Aβ aggregates were assessed by PK digestion, conformational stability assay, and fluorescence spectroscopy. Results HFIP pre-treated Aβ peptides formed PK-resistant Aβ aggregates whereas NH4OH pre-treated peptides only formed PK-sensitive aggregates. Conformational differences between synthetic and recombinant Aβ aggregates were also observed, potentially indicative of distinct “strains”. The relative seeding capacity of the various preparations is currently being assessed in mice. Conclusion


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HFIP pre-treatment appears to be key for obtaining PK-resistant Aβ aggregates. Seeding conditions might be also important. These findings will lead to a better understanding of the factors influencing the generation of selfpropagating Aβ aggregates.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0887 INCREASED LEVELS OF TOXIC ABETA PROTOFIBRILS IN ALZHEIMER´S DISEASE BRAIN SUPPORT THE IMPORTANCE OF ABETA PROTOFIBRILS IN ALZHEIMER´S DISEASE PATHOGENESIS 1 1 1 1 2 2 3 1 C. Sahlin , A. Sandegren , J. Fälting , L. Lannfelt , B. Winblad , C. Graff , M. Ingelsson , L. Söderberg 1 BioArctic AB, Warfvinges väg 35, Stockholm, Sweden 2 Karolinska Institutet, Department of Neurobiology- Care Sciences and Society, Stockholm, Sweden 3 Uppsala University, Department of Public Health/Geriatrics- Rudbeck Laboratory, Uppsala, Sweden Aims Soluble Abeta oligomers and protofibrils have been implicated to mediate neurotoxicity and alter synaptic function in the brain. Increasing evidence suggests that these Abeta species may better correlate with disease severity. The aim of the present study was to analyze levels and composition of Abeta protofibrils in brain extracts of normal and Alzheimer’s disease (AD) patients. Moreover, the presence of N-terminally truncated Abeta was investigated in different detergent soluble brain fractions. Method Abeta from human brain tissue (n=39) was biochemically fractionated in presence or absence of SDS. Levels of Abeta protofibrils were analyzed by immunoprecipitation using the protofibril selective murine antibody mAb158 combined with Abeta analysis using the Meso Scale Discovery (MSD) platform. Total levels of Abeta and the extent of N-terminally truncation were studied using MSD Abeta triplex for measuring Abeta 38, 40 and 42 using both 6E10 and 4G8 as detection antibodies. Results Immunoprecipitation analysis revealed significantly increased levels of soluble Abeta protofibrils in AD brains compared to control brains. Also, an increase of total Abeta in both soluble and insoluble brain fractions were observed in AD compared to controls. Characterization of brain samples demonstrates high levels of N-terminally truncated forms of Abeta in human brain, both in AD and control brains suggesting that N-terminal truncations are normal events in the adult human brain. Conclusion


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The findings in this study provide strong evidence to support the contention that elevated soluble protofibrils are involved in the pathogenesis of AD.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1842 PURIFICATION OF AMYLOID PEPTIDES USING PREPARATIVE SDS-PAGE 1 1 2 1 1 1 C. Spahn , R. Eichentopf , G. Hause , D. Schlenzig , S. Schilling , M. Wermann 1 Fraunhofer Institute for Cell Therapy and Immunology, Department for Drug Design and Target Validation IZIMWT, Halle Saale, Germany 2 Martin-Luther University Halle-Wittenberg, Biocenter, Halle Saale, Germany Aims To investigate the molecular basis of aggregation and toxicity of Aβ1-42 and pGlu-Aβ3-42, recombinantly produced peptides gained considerable interest. However, the isolation of recombinant human Aβ is hampered by inefficient separation of the Aβ peptide from the fusion protein used for heterologous expression. The aim was to purify Aβ1-42 and pGlu-Aβ3-42 applying preparative SDS-PAGE and to characterize and compare the peptide preparations. Method Aβ1-42 and pGlu-Aβ3-42 were produced as NANP-fusion peptides in E. coli BL21 (DE3). The fusion protein was isolated using affinity chromatography and subjected to TEV protease cleavage. We performed preparative SDSPAGE (Model 491 Prep Cell, BioRad) under reducing conditions. SDS was removed by a combination of RPchromatography and peptide precipitation. Aβ1-42 and pGlu-Aβ3-42 were characterized by analytical SDSPAGE, the Thioflavin T aggregation test ( ThT-test), electron microscopy and cell toxicity analysis. Results We successfully isolated 8 mg of Aβ1-42 and 4.7 mg of pGlu-Aβ3-42 from 40 mg and 20 mg of fusion peptide, respectively. The peptide preparations were devoid of any impurities. The characterization revealed faster aggregation and higher toxicity of pGlu-Aβ3-42 versus Aβ1-42. Conclusion


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The application of preparative SDS-PAGE represents the key step to isolate ultrapure recombinant Aβ versions. The method might be of interest for purification of other amyloid peptides.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0365 EVALUATION OF BLOOD-BRAIN BARRIER TRANSLOCATION OF THE NEW ANTI-AMYLOID BRI2BRICHOS AFTER INTRAVENOUS INJECTION. 1 1 1 2 1 1 S. Tambaro , L. Galán-Acosta , A. Leppert , G. Hultqvist , J. Johansson , J. Presto 1 Karolinska Institutet, Department of Neurobiology- Care Sciences and Society NVS- H1, Huddinge, Sweden 2 Uppsala University, Department of Public Health and Caring Sciences, Uppsala, Sweden Aims Aggregation of the amyloid–beta peptide (Aβ) into toxic oligomers and amyloid fibrils is linked to the development of Alzheimer´s disease (AD). Recently, we have shown that the chaperone-like protein domain BRICHOS, present in the dementia related Bri2 /Itm2b, efficiently delays Aβ42 fibril formation and toxicity in vitro and in vivo. Based on these studies, we want to investigate if BRICHOS could be developed as a future AD drug, given peripherally and pass the blood brain barrier (BBB). We analyzed the biological half-life in mouse sera, permeability and distribution of the Bri2-BRICHOS domain trough the BBB. Method The Bri2-BRICHOS (10-20mg/kg) were injected intravenously in adult wild type mice (C57BL/6) and blood samples were collected at different time points. Brains were perfused and collected after 1, 2 and 6 hours from the injections. Furthermore, Bri2-BRICHOS was fused to a single-chain antibody (scFv8D3) targeted for the transferrin receptor in order to increase the BBB penetrance of BRICHOS. Results At the dose of 20mg/kg i.v. and 1 hour after injection, the Bri2-BRICHOS was detected by immunohistochemistry and western blot in the mice brain. Furthermore, Bri2-BRICHOS was detected in the blood for more than 2 hours from the injection. Conclusion


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Our study supports that Bri2-BRICHOS reaches the brain after systemic injection and provides the bases for exploring it as a therapeutic tool for AD. The new fusion construct is currently under investigation and will be analysed for ability to inhibit Ab aggregation in vitro and BBB translocation in wild-type and AD mouse models.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1524 BINDING PROPERTIES OF AMYLOID OLIGOMER-BINDING DNA APTAMERS AGAINST AMYLOID BETA OLIGOMERS 1 1 1 1 K. Tsukakoshi , C. Hosoi , K. Sode , K. Ikebukuro 1 Tokyo University of Agriculture and Technology, Department of Biotechnology and Life Science, Koganei, Japan Aims Alzheimer’s disease (AD) and Parkinson’s disease (PD) are common neurodegenerative disorders and are believed that protein aggregates, especially highly toxic amyloid oligomers cause neuronal degeneration. We succeeded in obtaining DNA aptamers that bind to α-synuclein (α-syn) oligomer related to onset of PD and amyloid β (Aβ) oligomer that causes onset of AD (Tsukakoshi et al., Anal Chem 2012). In this study, we investigated binding properties of amyloid oligomer-binding DNA aptamer for Aβ oligomers. Method Aβ oligomers were prepared by in vitro experiment. Aliquots were arbitrarily removed from the incubated sample, flash frozen in liquid nitrogen, and stored at −80 °C. The time course of Aβ fibrillization was determined by TfT fluorescence assay. We detected Aβ oligomers by the biotinylated DNA aptamers. Results We found the DNA aptamers appeared to bind to high-molecular-weight oligomers but not to small oligomers. This result corresponded that the bivalent DNA aptamers showed greater binding signals than that of the monovalent aptamer (Tsukakoshi et al., Chem Commun in press). Conclusion


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We believe this investigation of the binding properties of the DNA aptamers would provide information for the development of new diagnostic and therapeutic tools of AD.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0974 IDENTIFICATION THROUGH HIGH CONTENT SCREENING OF CALCIUM CHANNELS ANTAGONIST AS NOVEL NEUROPROTECTIVE INHIBITORS 1 1 1 1 1 1 M. VALCARCEL , R.M. Mella , P. Villace , M. Roura , D. Kortazar , C. Salado 1 Innoprot SL, Innoprot, Derio, Spain Aims Production and deposition of β-amyloid protein is an important key player involved in mechanism in ischemic neurodegeneration as well as Alzheimer´s disease. To date, efficient therapeutics drugs are still insufficient, making necessary to find relevant compounds amongst masses of potential candidates. Method A first cell-based screening model for secretase inhibitors using MCDK cells followed by a second assay in cortical neurons where cell parameters associated with membrane damage, cell viability and neurite outgrowth were measured using HCS analysis. Results In the primary screen we identified 57 inhibitors of β-secretase in the 1200 compounds of Prestwich library. These initial hits were further analyzed for their capacity to protect primary cortical neurons against the neurotoxicity induced by oxygen glucose deprivation (OGD). In this neuron-screen, just only 4 and of these 57 compound (metergoline, bepridil, carbetapentane and raloxifene), all antagonist of calcium channels, displayed as highly neuroprotective in a pre-treatment toxicity assay but only metergoline compound remained active when tested in a post-treatment protocol administered six hours post OGD damage. Conclusion


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Our work stablishes the feasibility of identifying molecule inhibitors of neuronal damage using both screening models and provides novel insights into the mechanisms of neuroprotection by calcium channels antagonists.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0299 N-ACETYLASPARTATE SUPPRESSES PROTEIN AGGREGATION: IMPLICATIONS FOR THE TREATMENT OF ALZHEIMER’S DISEASE 1 1 2 M. Warepam , M.H. Rahaman , R. Laishram 1 Manipur University, Biotechnology, Imphal, India 2 University of Delhi, Dr B R Ambedkar Center for Biomedical Research, Delhi, India Aims Protein aggregation or amyloid formation is a common hallmark of Alzheimer’s Disease (AD). N-Acetylaspartate, NAA is one of the most concentrated (upto 10 mM) intracellular solute in neuronal cells whose level got altered in many neurodegenerative diseases which make it one of the widely accepted neuronal biomarkers in several human brain disorders (especially in Alzheimer’s Disease). Our primary focus is to examine the role of NAA in suppressing protein aggregation or amyloid formation. Method Aggregation was monitored by observing the change in the light scattering intensity at 500 nm using UV visible spectrophotometer followed by TEM images. Protein stability was measured by fluorescence spectrophotometer. Results We have studied the aggregation behaviour of carbonic anhydrase, catalase and amyloid β (Aβ) 1-42 in the presence and absence of NAA. We observed that NAA decreases the aggregation propensity of all the three proteins. Mechanistically the reduction in aggregation or amyloid formation is due to its effect on the nucleation phase and exponential or growth rate of the aggregation process. We speculate that NAA helps to prevent the generation of aggregation prone intermediates. Conclusion


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The study indicates the importance of NAA level in the brain cells of AD patients to control the deposition of protein aggregates.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1150 GENERATION OF SINGLE-CLONAL HUMAN NEURAL STEM CELL LINES FOR A 3D CULTURE MODEL OF ALZHEIMER’S DISEASE 1 1 1 1 1 1 1 1 K.J. Washicosky , J.L. Aronson , S.H. Choi , C. D'Avanzo , E. Bylykbashi , I. Kim , R.E. Tanzi , D.Y. Kim 1 Massachusetts General Hospital/ Harvard Medical School, Neurology, Charlestown, USA Aims We previously reported the recapitulation of Alzheimer’s disease (AD) pathologies using genetically engineered human neural progenitor cells (hNPCs) expressing the familial Alzheimer’s disease (FAD) gene APP with or without presenilin in a 3D culturing environment (Choi et al., Nature 2014). Although we successfully recapitulated the two pathological hallmarks of AD, amyloid plaques and neurofibrillary tangles, these engineered hNPC populations are heterogeneous and have variable FAD gene expression that limit their use for drug screening and basic mechanistic studies. Here we describe a protocol for generating single-clonal hNPCs expressing uniform levels of FAD genes with multiple mutations. Method ReNcell VM hNPCs transfected with GFP and/or mCherry polycistronic CSCW lentiviral vectors containing FAD genes with multiple mutations (FAD ReN cells) were enriched as previously described (Choi et al., Nature 2014). These heterogeneous cell populations were then subject to FACS-assisted 96-well single-clonal cell sorting and cultured until individual colonies formed. Each single-clonal cell line was expanded, differentiated, and screened for pathogenic beta-amyloid (Abeta) levels using western blot and ELISA. FAD gene expression was also monitored by fluorescence microscopy. Results We successfully engineered single-clonal FAD ReN cell lines that each exhibit homogenous FAD gene expression. These single-clonal FAD lines are stable across multiple passages, differentiate into neurons and glial cells under 2D and 3D conditions, and in some cases express higher Abeta levels (~10 fold) compared to their heterogeneous parental cells. Conclusion


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Single-clonal FAD ReN cells when combined with our 3D culture technology can provide an improved platform for AD drug discovery and basic mechanistic studies.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0368 PYROGLUTAMATE-MODIFIED AMYLOID-BETA(3-42) ACCELERATES AGGREGATION KINETICS OF AMYLOID-BETA(1-42) BY AFFECTING PRIMARY AND SECONDARY PATHWAYS 1 2 1 1,2 C. Dammers , A. Buell , M. Schwarten , D. Willbold 1 Forschungszentrum Jülich, Institute of Complex Systems Structural Biochemistry, Jülich, Germany 2 Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, Düsseldorf, Germany Aims A variety of amyloid-β (Aβ) peptides differing in length have been discovered with pyroglutamate-modified Aβ (pEAβ) as a significant proportion. pEAβ has a dramatically enhanced aggregation propensity compared to the non-modified Aβ demonstrating it to be a potential seeding species of Aβ accumulation. Here, we analyze the coaggregation mechanism of Aβ(1-42) with the more toxic pEAβ(3-42). Method Highly pure recombinant pEAβ(3-42) and Aβ(1-42) were used for kinetic studies elucidating the interaction of both Aβ variants on a molecular level. The cross-seeding effects of both species were analysed in the presence of homo-type and hetero-type monomers. Results Aggregation kinetics of pEAβ(3-42) are accelerated and the concentrations needed for fibrillation were drastically decreased by one order of magnitude when compared to Aβ(1-42). Mixtures of both peptides in concentrations where they did not aggregate alone showed co-aggregation. pEAβ(3-42) monomers shortened the aggregation half-time of Aβ(1-42). Cross-seeding assays showed that pEAβ(3-42) fibrils catalysed Aβ(1-42) aggregation. In contrast, pEAβ(3-42) aggregation kinetics were slowed down in the presence of Aβ(1-42) monomers and fibrils. Conclusion


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We elucidated the mechanism of co-aggregation of Aβ(1-42) with pEAβ(3-42) on a molecular level. pEAβ(3-42) monomers increase the primary nucleation of Aβ(1-42) and pEAβ(3-42) fibrils serve as a highly catalytic surface for secondary nucleation and elongation of Aβ(1-42). Aβ(1-42) monomers drastically decelerate pEAβ(3-42) primary and secondary nucleation but do not influence pEAβ(3-42) elongation rate. Actually, high concentrations of Aβ(1-42) fibrils prevent pEAβ(3-42) aggregation due non-reactive surface binding. Thus, pEAβ(3-42) catalyses aggregation of Aβ(1-42) affecting all reaction processes while Aβ(1-42) dramatically slows down pEAβ(3-42) aggregation.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0370 PYROGLUTAMATE AMYLOID-Β(3-42) FORMS TRANSIENT Α-HELICES PRIOR TO AMYLOID FORMATION 1 1 2 1 1,2 C. Dammers , K. Reiss , L. Gremer , M. Schwarten , D. Willbold 1 Forschungszentrum Jülich, Institute of Complex Systems Structural Biochemistry, Jülich, Germany 2 Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, Düsseldorf, Germany Aims Amyloid-β (Aβ) peptides are highly heterogeneous in size and modification, however N-terminally truncated pyroglutamate-modified Aβ (pEAβ) has been described to be a dominant isoform of senile plaque. Compared to non-truncated Aβ species, pEAβ is more resistant to degradation, shows higher toxicity and has increased aggregation propensity. To date, there is only little insight about the structure of pEAβ and its difference to the non-truncated isoform of Aβ. Method Circular dichroism and heteronuclear multi-dimensional nuclear magnetic resonance spectroscopy (NMR) in solution state were used for analysis and secondary structure determination of pEAβ monomers. Aggregation kinetics were monitored by thioflavin-T assays. Results Soluble pEAβ(3-42) had increased tendency to form β-sheet-rich structures leading to fibrillation under solution conditions, where Aβ(1-42) formed α-helices. The formation to pEAβ(3-42) affected the NMR chemical shifts of 30 % of the total amino acid residues and some peak intensities are drastically decreased when compared to Aβ(1-42). Chemical shift analysis indicated that monomeric pEAβ(3-42) contained two helical regions connected by a linker. These α-helical pEAβ(3-42) monomers were instable due to intermolecular interactions ultimately leading to fibrillation. Conclusion


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We obtained insights into the driving force of pEAβ to form oligomers and fibrils more rapidly than Aβ. pEAβ forms transient α-helices as an intermediate to β-sheet formation and fibrillation. The N-terminal modification towards pEAβ has a significant effect on secondary structure elements resulting in a severe tendency to form β-sheet-rich structures extending to fibrils via transient α-helices. These transient intermediates seem to be on-pathway of the highly helical monomeric conformation and the amyloidogenic fibrils.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0196 PEN-2 AND PRESENILIN ARE SUFFICIENT TO CATALYZE NOTCH PROCESSING 1 1 1 X. xu , C. Hu , M.Z. Cui 1 The University of Tennessee, Biomedical & Diagnostic Sciences, Knoxville, USA Aims Presenilin-1 (PS1) or presenilin-2 (PS2), nicastrin (NCT), anterior pharynx-defective 1 (Aph-1), and presenilin enhancer-2 (Pen-2) have been considered the minimal essential subunits required to form an active gammasecretase complex. Besides PS, which has been widely believed to function as the catalytic subunit, the functional roles of the other subunits in the gamma-secretase complex remain debatable. In the current study, we set out to determine the role of Pen-2 in gamma-secretase activity. Method Using knockout cells in combination with siRNA and immunoprecipitation approaches. Results To this end, our results revealed that Pen-2 together with presenilin are sufficient to form a functionally active enzyme capable of processing Notch. Specifically, our data demonstrated that Pen-2 plays a crucial role in substrate binding, a mechanism by which Pen-2 contributes directly to the catalytic mechanism of gammasecretase activity. Our data also suggested that there may be different requirements for components to process APP and Notch. These information would be important for therapeutic strategy aimed at inhibition or modulation of gamma-secretase activity. Conclusion


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Pen2 plays a critical role in substrate binding to Presenilin.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0218 IS ANGIOTENSIN CONVERTING ENZYME GENE TO ALZHEIMER’S DISEASE INDEPENDENTLY FROM HYPERTENSION? 1,2,3 Y.H. Yang 1 Kaohsiung Municipal Ta-Tung Hospital, Neurology, Kaohsiung City, Taiwan R.O.C. 2 Kaohsiung Medical University Hospital, Neurology, Kaohsiung, Taiwan R.O.C. 3 Kaohsiung Medical University, Neurology, Kaohsiung City, Taiwan R.O.C. Aims Angiotensin converting enzyme gene insertion/ deletion (ACE I/D) polymorphisms were considered as a biomarker of late-onset Alzheimer’s disease (AD) with inconsistent results. ACE protein can directly degrade the beta-amyloid that will decrease the risk of having AD. Contrarily, it can activate the renin–angiotensin– aldosterone system to have hypertension or cardiovascular events that will increase the risk of having AD. The exact mechanisms of ACE gene to AD still have to be determined for these two proposed hypotheses. Method We have examined the bidirectional hypotheses of ACE gene to AD by controlling one of the effects of ACE gene, hypertension. Clinically diagnosed AD patients with history of hypertension have recruited and identified from the department of neurology, Kaohsiung Municipal Ta-Tung Hospital. The diagnosis of AD was made by the NINCDS-ADRDA criteria with referring to a series of comprehensive neuropsychological tests, laboratory tests, and neuroimaging to exclude other possible causes to the diagnosis of AD. Results In total, 983 clinically diagnosed AD patients were recruited and have completed the ACE I/D genotyping. ACE I/D genotype (p=0.355 for I/I vs D/D; p=0.888 for I/D vs D/D) and I allele (p=0.895) were not at significantly different distribution between AD patients with or without hypertension. Conclusion


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ACE gene contributing to having the diagnosis of AD is less likely to be associated with hypertension or related cardiovascular effects. The detailed mechanisms of ACE to AD could be resulted from other undetermined mechanisms.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1557 ANALYSIS OF PYROGLUTAMATE-AMYLOID AND TAU EXPRESSION IN HUMAN CASES OF ALZHEIMER’S DISEASE: COMPARISON WITH MOUSE MODELS OF AD 1 1 1 1 1 V. Niederkofler , K. Beutl , J. Neddens , S. Flunkert , B. Hutter-Paier 1 QPS Austria GmbH, Neuropharmacology, Grambach, Austria Aims Alzheimer´s disease (AD) is associated with the formation of neuritic plaques composed of beta-amyloid (Abeta) and the formation of neurofibrillary tangles composed of hyperphosphorylated tau. Neuritic plaques often show high levels of N-terminally truncated forms of beta-amyloid, namely 3-glutamate-beta-amyloid whose leading residue is modified to pyroglutamate (pE) by glutaminyl cyclase. The current study is designed to analyze features of AD-related pathological changes in human brain that are spatially associated with pE-Abeta immunoreactive structures, and to verify whether similar changes are present in different animal models of AD. Method Brain sections of human AD cases and controls are labeled by multichannel immunofluorescence to analyze the spatial relations of different proteins. Cryosections from different animal models of AD will also be labeled to compare the results with the human samples. For immunohistochemical analyses, uniform systematic random sets of cryosections will be labeled and quantitatively analyzed to evaluate the occurrence and location of ADrelated markers (pE-Abeta, Tau, phosphoTau), and indicators of inflammation (IBA1, GFAP). Additional markers may be used. Results We will show data on AD-associated pathology and compare the patterns between humans and animal models. Conclusion


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A major focus during interpretation of the data will be to find common features of pathology.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-0629 BIOPHYSICAL CHARACTERIZATION OF THE SELF-ASSOCIATION OF AMYLOID BETA PEPTIDES IN THE PRESENCE OF ZINC IONS 1,2 1,2 1,2 T. Zhang , L. Nagel-Steger , D. Willbold 1 Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, Düsseldorf, Germany 2 Forschungszentrum Jülich, Institute of Complex Systems- Structural Biochemistry ICS-6, Jülich, Germany Aims Abnormal aggregation of Amyloid β (Aβ) in the brain is one of the major neuropathological changes in Alzheimer’s disease. Zinc dyshomeostasis has been demonstrated to be closely associated with Aβ deposition in the brain. However, the impact of zinc ions on the self-association of Aβ and the underlying mechanisms are still incompletely resolved. Method A combination of complementary approaches, including circular dichroism (CD) spectroscopy, thioflavin T (ThT) assay, analytical ultracentrifugation (AUC) and atomic force microscopy (AFM), was used to analyze the aggregation process of Aβ42 in the presence or absence of zinc ions. Results Aβ42 aggregates found in the presence of zinc ions were highly heterogeneous in size, non fibrillar and showed less β-sheet structures than aggregates formed without zinc ions. Besides, there remained more monomers in Aβ42 sample treated with zinc. Co-incubation with zinc chelator EDTA totally abolished the inhibitory effect of zinc ions on Aβ42 fibrillization. Conclusion


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Treatment with zinc ions suppressed Aβ42 fibrillogenesis by shifting toward a heterogeneous aggregation process. In addition, AUC and AFM analysis could complement each other with respect to the evaluation of particle size distributions.

A01.a. Disease Mechanisms, Pathophysiology: Abeta aggregation, protein misfolding ADPD7-1067 CYCLODEXTRIN-BEARING PORPHYRINS AS INHIBITORS OF ASS OLIGOMERIC TOXICITY 1 2 1 1 2 S. Zimbone , V. Oliveri , M.L. Giuffrida , M.F. Tomasello , G. Vecchio 1 National Council of Research CNR, Institute of Biostructure and Bioimaging IBB, Catania, Italy 2 University of Catania, Department of Chemical Sciences, Catania, Italy Aims Aggregation of Amyloid-beta (Aß) is one of the crucial events occurring during Alzheimer’s disease progression, clearly linked to the pathology. Abeta oligomers (oAß) have been identified as primary mediators of Aß toxicity, leading to the development of strategies aimed at interfering with the early stage of Aß aggregation. Here we show the activity of a cyclodextrin compound bearing a porphyrin moiety and its zinc complex in suppressing Aß toxicity. Method 5[4-(6-O-β-cyclodextrin)-phenyl],10,15,20-tri(4-hydroxyphenyl)-porphyrin (CDTHPP) and its zinc complex were synthesized and characterized by mass spectrometry and NMR. To test the ability of CDTHPP and its derivative to affect Aß toxicity, we treated differentiated neuroblastoma cell, SH-SY5Y, with oligomeric preparation of Aß incubated in the presence or absence of the two different compounds. The parent compounds, ß-cyclodextrin (CD) and 5,10,15,20-tetra(4-hydroxyphenyl)porphyrin (THPP), were also studied to demonstrate that CDTHPP activity, could arise from the synergy of THPP and CD properties. Dot Blot analysis were performed to confirm that the absence of toxicity observed in the presence of CDTHPP and its zinc complex, were correlated with the lack of immunoreactivity with the anti-oligomeric conformation-specific antibody, A11. Finally, we took advantage of the intrinsic fluorescent properties of the derivatives to verify the cell internalization of these systems

Results The synthetized porphyrin-cyclodextrin conjugate CDTHPP and its zinc complex were able to inhibit Aβ toxicity and oligomers formation. Conclusion


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Our results provide a basis for the development of novel molecule with potential applications in AD.

A01.b. Disease Mechanisms, Pathophysiology: Cell to cell transmission, spreading of pathology, prionlike ADPD7-0952 AΒ–TAU INTERACTION: A QUANTITATIVE ANALYSIS OF THE BRAAK CROSS-SECTIONAL POSTMORTEM DATA OFFERS SUPPORT FOR THE ACCELERATION HYPOTHESIS 1 2 1 2 J. Kemp , T. Jacobs , M. Mercken , T. Van de Casteele 1 Janssen Research & Development, Neuroscience Discovery, Beerse, Belgium 2 Janssen Research & Development, Statistics and Decision Sciences, Beerse, Belgium Aims Alzheimer’s disease (AD) is defined by the presence of the two neuropathological hallmarks, comprised of extracellular β-amyloid plaques and intracellular neurofibrillary tangles (NFT) composed of aggregated, hyperphosphorylated tau. The emergence of causative mutations of familiar AD indicate a key role of amyloid in the disease. Nevertheless, these familial AD patients still have the usual NFT pathology. Braak and colleagues have characterized and staged the amyloid and tau pathology present in a collection of 2,366 human brains spanning the ages of 1 – 100. We have performed a quantitative statistical analysis of these data to evaluate the hypothesis that the progression of amyloidosis accelerates tau pathology. Method Tauopathy was considered an ordinal response variable (stages 0 to 8). Dependency of tauopathy on candidate predictors was analyzed with a proportional odds cumulative logit model. Several candidate predictors for tauopathy were evaluated, including β-amyloid level, age, gender and their interactions. Results Results show that the stage of tauopathy is best described by a model including age, β-amyloid level and an interaction between age and β-amyloid stage (P < 0.0001). For low β-amyloid stages (0,1 or 2), parameter estimates indicate that the frequency of higher tau stages is positively associated with age but is not associated with β-amyloid level. For high β-amyloid stages, parameter estimates indicate that the frequency of high tauopathy stages is not associated with age, but is positively associated with β-amyloid level. Conclusion


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These findings are consistent with the hypothesis that the presence of β-amyloid plaques increases the likelihood of later stage tau pathology.

A01.b. Disease Mechanisms, Pathophysiology: Cell to cell transmission, spreading of pathology, prionlike ADPD7-0537 CELL CULTURE MODEL FOR THE ANALYSIS OF THE SEEDING-ACTIVE AMYLOID SPECIES IN ALZHEIMER'S BRAIN TISSUE 1 1 D. Markx , M. Fändrich 1 University Ulm, Protein Biochemistry, Ulm, Germany Aims Alzheimer’s disease (AD) is the classical example of a neurodegenerative disease that is not a transmissible 1,2 spongiform encephalopathy but that shows prion-like characteristics . However, the responsible factor(s) for this prion-like mechanism still have to be elucidated. We here aim to develop a novel cell culture-based model to efficiently screen the seeding-activity in a large number of biochemical fractions from AD brain homogenates. Method THP-1 cell culture, Biochemical fractionation This study was conducted under the approval of the Ethics Committee of Ulm University. Results We could demonstrate that the cell culture model can successfully be used to monitor the seeding-activity of AD brain homogenates. We could reproduce the seeding effect for three cases of AD brain tissue while none of the tested age-matched, non-demented control brain homogenates or buffer alone produced discernible seedingactivity. In vitro aggregation assays, in which chemically pure Aβ peptide was incubated in presence of nondemented and AD brain homogenate or phosphate-buffered saline, did not suffice to demonstrate the seedingactivity of AD brain homogenates. Based on the cell culture model we could test several hundred different fractions to reveal the molecular characteristics of the seeding-active components. Conclusion These data demonstrate the power of the cell culture model for characterizing the seeding-active species present in AD brain homogenates.


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References: 1. Meyer-Luehmann, M., et al., Science 2006, 313, 1781-1784 2. Stöhr, J., et al., Proc Natl Acad Sci USA 2012, 109, 11025-11030

A01.b. Disease Mechanisms, Pathophysiology: Cell to cell transmission, spreading of pathology, prionlike ADPD7-1182 CATTLE BRAIN CONTAINING AMYLOID PLAQUES ACCELERATES ALZHEIMER’S PATHOLOGY WHEN INTRACEREBRALLY INOCULATED IN MICE 1 1 1 1 2 2 1 I. Moreno-Gonzalez , G. Edwards III , R. Morales , C. Duran-Aniotz , M. Marquez , M. Pumarola , C. Soto 1 University of Texas Health Science Center, Neurology, Houston, USA 2 Animal Tissue Bank of Catalunya BTAC- Universidad Autonoma de Barcelona, Animal Medicine and Surgery Department, Bellaterra Cerdanyola del Valles, Spain Aims Amyloid beta (Ab) and hyperphosphorylated tau are the proteins undergoing misfolding in Alzheimer’s disease (AD). Recent studies have shown that brain homogenates rich in amyloid aggregates are able to seed the misfolding and aggregation of amyloidogenic proteins inducing an earlier onset of the disease in mouse models of AD. This seeding behavior is analogous to the disease transmission by propagation of prion protein misfolding observed in prion diseases. Prion diseases can be transmitted across species by inoculation of the misfolded prion protein from one specie into an appropriate host. For example, material from cattle affected by bovine spongiform encephalopathy can be propagate in humans inducing variant Creutzfeldt-Jakob disease. Method In this study, we analyzed the presence of AD-related protein aggregates in the brain of old cows and investigated whether these aggregates are capable to induce pathology in animal models of AD. Results We observed that many of the typical hallmarks detected in human AD brains, including Ab aggregates, were present in cow brains. When cattle tissue containing Ab aggregates was intracerebrally inoculated into APP/PS1 mice, we observed an acceleration of brain Ab deposition and faster cognitive impairment compared to controls. However, when this material was orally inoculated, no effect was observed. Conclusion


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These findings may contribute to understand the prion-like behavior of Ab aggregates and the factors modulating the initiation and spreading of protein aggregates. Although our results indicate that cattle material may promote AD pathology under experimental conditions, the fact that no transmission was observed by oral route is reassuring.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0107 ISO-Α-ACIDS, BITTER COMPONENTS OF BEER, PREVENT CEREBRAL INFLAMMATION AND COGNITIVE DECLINE IN MOUSE MODEL OF ALZHEIMER’S DISEASE 1 2 2 3 2 Y. Ano , A. Dohata , K. Uchida , T. Akihiko , H. Nakayama 1 Kirin Company- Limited, Reaserach and Development Division, Kanagawa, Japan 2 The University of Tokyo, Department of Veterinary Pathology, Tokyo, Japan 3 Gakushyuin University, Department of Life Sciences, Tokyo, Japan Aims Epidemiological studies suggest that moderate consumption of alcoholic beverages is beneficial for preventing dementia, but there are few reports about the effective constituents except for resveratrol in red wines. In the present study, we evaluated the effects of iso-α-acids, hop-derived bitter compounds in beer on the development of Alzheimer’s disease. Method To evaluate the effects of iso-α-acids on microglia, primary CD11b-positive microglia were treated with iso-αacids, and then with lipopolysaccharide for anti-inflammatory activity or with Aβ-FAM for phagocytic activity. Next, 3-month-old 5xFAD mice, a model for Alzheimer’s disease, were fed with a diet containing 0 or 0.05% (w/w) isoα-acids for 2.5 months. The amount of Aβ1-42, cytokines, neurotrophic factors, and synaptophysin in the brain were quantified by ELISA. The expressions of surface markers of microglia were analyzed with a flow cytometer. Results Iso-α-acids treatments enhanced microglial phagocytosis of Aβ and suppressed the production of TNF-α. The treatment also changed microglial phenotype to CD206-positive anti-inflammatory one and enhanced the expression of CD36. In the 5xFAD mice, the intake of iso-α-acid significantly suppressed soluble and insoluble Aβ accumulations and inflammatory cytokine productions such as MIP-1α, TNF-α and IL-1β in the brain. The amount of neurotrophic factors such as BDNF and synaptophysin were also significantly increased in the mice fed with iso-α-acid. The novel object recognition test showed that the intakes of iso-α-acid improved episodic memory in 5xFAD mice. Conclusion


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The present study first reported that iso-α-acids, bitter ingredients of beer, might have preventive effects on the development of Alzheimer’s disease via the regulation of microglia.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1819 BAICALEIN ATTENUATES INTRACEREBROVENTRICULAR STREPTOZOTOCIN INDUCED LEARNING AND MEMORY DEFICIT AND OXIDATIVE STRESS IN RATS 1 1 R. Arora , R. Deshmukh 1 ISF College of Pharmacy- Moga, Pharmacology, Moga, India Aims The present study has been designed to evaluate the therapeutic potential of baicalein against intracerebroventricular streptozotocin (ICV-STZ) induced learning and memory deficit and oxidative stress in rats Method STZ was infused icv bilaterally (3 mg/kg) to induce experimental sporadic dementia of Alzheimer's type in rats. Rats were treated with baicalein –a flavone (1, 2 and 4 mg/kg i.p) for 21 days following 1st STZ infusion Rats were sacrificed on day 22 and hippocampal brain regions were isolated and used to assess memory deficits and oxidative stress [Malondialdehyde (MDA), nitrite, protein carbonyls and reduced glutathione levels (GSH)] and cholinergic functions acetylcholinesterase activity Morris water maze and passive avoidance task were used to assess learning and memory deficits in rats Results STZ infused rats showed significant learning and memory deficits which can be due to the increase in neuronal cell death Further, STZ infused rats showed increase in oxidative stress, compromised antioxidant defense (GSH) and cholinergic hypofunction as evidenced by increased AChE activity Chronic treatment with baicalein at different doses significantly attenuated STZ-induced cognitive deficits, oxidative stress and restored hippocampal cholinergic functions in rats an>and passive avoidance task were used to assess learning and memory deficits in rats Conclusion


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The outcomes of the current study suggest that baicalein improve cognitive functions in STZ-infused rats which could be due to its antioxidant effect and its ability to restore cholinergic functions

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0753 SOLUBLE TREM2 LEVELS IN ALZHEIMER’S DISEASE 1 L. Bekris 1 Cleveland Clinic Lerner Research Institute, Genomic Medicine Institute, Cleveland, USA Aims Triggering receptor expressed on myeloid cells 2 (TREM2) genetic variants are associated with increased lateonset Alzheimer’s disease (AD) risk. TREM2 is a microglial receptor involved in innate immunity. Soluble TREM2 (sTREM2) is a cleavage product of TREM2 and is present in cerebrospinal fluid (CSF) and plasma. Increased CSF sTREM2 levels have been associated with CSF biomarkers of neurodegeneration. Genetic analyses have found associations between variants at the TREM2 locus and sTREM2 levels in biofluids from neurodegenerative disease patients. C-reactive protein (CRP) levels in healthy African American and Hispanic American women are associated with a TREM locus variant. The hypothesis of this investigation is that not only are biomarkers of neurodegeneration associated with sTREM2 in neurodegenerative disorders, but also markers of inflammation are associated with sTREM2 in neurodegenerative disorders. The aim of this investigation is to demonstrate a correlation between sTREM2, CRP and inflammation markers, in neurodegenerative disorders, such as AD. Method CSF CRP, inflammation markers and sTREM2 from cognitively normal (CN), mildly cognitively impaired (MCI), AD and Parkinson’s disease (PD) were measured. Correlation analyses were performed to determine whether inflammation markers correlated with sTREM2 levels from CN compared to MCI, AD or PD. Results We found that inflammation markers are indeed associated with sTREM2 levels disease status. In addition, sTREM2 correlates with AD biomarkers as a function of disease status. Conclusion


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These results suggest that sTREM2 is linked to the inflammatory processes in neurodegenerative disease. Therefore, CSF sTREM2 may be an important biomarker for inflammatory activity in neurodegenerative disease.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0478 THE MODULATION OF INTERLEUKIN-1Β BY INTRA AND EXTRACELLULAR AMYLOID BETA IN THE PROGRESSION OF ALZHEIMER’S DISEASE 1 1 1 1 1 1 1 A. Cadete Martini , S. Forner , D. Baglietto-Vargas , L. Trujillo-Estrada , C. Da Cunha , J. Ha , R. Ager , 2 1 R. Medeiros , F. LaFerla 1 University of California - Irvine, Institute for Memory Impairments and Neurological Disorders, Irvine, USA 2 The University of Queensland, Queensland Brain Institute, Brisbane, Australia Aims Inflammation is a key pathological hallmark of Alzheimer’s disease, although its impact on disease progression and neurodegeneration remains an area of active investigation. Interestingly, previous studies from our lab have demonstrated that interleukin-1β (IL-1β)-mediated chronic inflammation represents a key mechanism by which Aβ drives the development of tau pathology and cognitive decline in AD. Increased IL-1β levels and IL-1R1 expression have been detected in close association with the buildup of intraneuronal Aβ in the 3xTg-AD mouse model, which places IL-1β as one of the earliest mediators in the neurodegenerative process induced by Aβ. Here, we further investigated how Aβ triggers IL-1β signaling and whether intra- or extracellular Aβ is responsible for this. Method We used adeno-associated virus (AAV) vectors and biochemical and molecular assays to evaluate the molecular mechanisms through which intra- or extracellular Aβ trigger an inflammatory response in mice. Results Our study reveals that both intra- and extracellular Aß trigger a pro-inflammatory response. Interestingly, important changes in IL-1β pathway are observed with both AAVs. Conclusion


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Overall, our study indicates that IL-1β plays a critical pathogenic role in the development of AD. A more in-depth understanding on the molecular effects of Aβ on IL-1β transcription and release will lead to the development of more effective disease modifying therapeutics.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1656 THE EXTRACELLULAR CHAPERONE HAPTOGLOBIN BINDS COMPLEMENT RECEPTOR 1 (CR1) AND INFLUENCE ITS ACTIVATION IN MICROGLIA 1 1 1 2 L. Cigliano , L. Iannotta , V. Farina , M.S. Spagnuolo 1 Dipartimento di Biologia, Università degli Studi di Napoli Federico II, Napoli, Italia 2 ISPAAM, Consiglio Nazionale delle Ricerche, via Argine 1085, 80147 Napoli, Italia Objectives: Genome wide association studies highlighted the importance of the complement cascade in the pathogenesis of Alzheimer’s disease (AD). The association between complement receptor 1 (CR1) loci and agerelated cognitive decline and plaque burden was suggested to be linked to impaired clearance of beta amyloid. Microglial CR1 activation exerts neurotoxic effects, correlated with enhanced cytokine and superoxide production. The ligand of CR1 is C3b complement protein, which shows amino acid sequences that are homologous to those of Haptoglobin (Hpt). We previously showed that Hpt is an extracellular chaperone with critical roles in brain by modulating beta amyloid and cholesterol homeostasis. The aim of this study was to evaluate whether Hpt binds CR1, and whether this interaction modulates CR1-mediated effects in microglia. Methods: Co-immunoprecipitation, ELISA, and Western blotting with purified proteins or human hippocampal homogenates from AD patients were performed in this study. BV-2 microglia cells were used in cell-based ELISA and FACS analyses. Results: We report here, for the first time, that Hpt binds CR1 and this binding increases in conditions of inflammation. Hpt interaction with CR1 was confirmed by co-immunoprecipitation experiments in hippocampal homogenates from AD and healthy samples. Also we found that Hpt, by binding CR1, may influence microglia physiology as it interferes with the production of both superoxide and pro-inflammatory cytokines induced by LPS.


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Conclusions: The interaction between Hpt and CR1 might represent a mechanism by which the extracellular chaperone Hpt might counteract or limit pro-oxidative and pro-inflammatory effects induced by CR1 activation on microglia.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1840 REDUCED BETA-AMYLOID SENSITIVITY FOR PLATELET-MONOCYTE AGGREGATES IN EDTA BLOOD OF ALZHEIMER PATIENTS 1 2 1 M. Defrancesco , J. Marksteiner , H. Christian 1 University Hospital, Department of Psychiatry- Psychotherapy and Psychosomatics-, Innsbruck, Austria 2 LKH Hall, Psychiatry, Hall, Austria Aims Objectives: Alzheimer´s disease (AD) is a severe neurodegenerative brain disorder characterized by beta-amyloid (Aβ) plaques, Tau pathology, inflammation, neurodegeneration and cerebrovascular dysfunction. Besides that, alterations in monocytes and platelets have been reported in the blood of AD patients. The aim of the present study was to explore the interaction of platelets and monocytes in whole blood before and after a stimulation by Aβ peptides. Method Methods: We analyzed whole EDTA blood from 46 healthy controls and 40 AD patients and measured CD14, CD62P and CD42a using fluorescence-activated cell scanning (FACS) analysis. Results Results: Our data show that the Platelet-Monocyte-Aggregates were not different between controls and AD patients before stimulation. Incubation of whole blood with 10 µg/ml Aβ40, Aβ42, or pyroglutamate-Aβ for 1 hour increased the platelet-monocyte aggregation in healthy controls, but not AD patients. Conclusion


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Conclusion: Our data conclude that the sensitivity of Aβ to the Platelet-Monocyte aggregation is lowered in AD patients due to chronic overactivation during the disease progression.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1568 REGULATION OF AMYLOID BETA-INDUCED LIPOCALIN 2 EXPRESSION IN PRIMARY BRAIN CELLS 1,2 1,2 1,3,4 2,5 D. Dekens , P. Naudé , P. De Deyn , U. Eisel 1 University of Groningen- University Medical Center Groningen, Alzheimer Research Center GroningenNeurology, Groningen, The Netherlands 2 University of Groningen- Groningen Institute for Life Sciences, Department of Neurobiology, Groningen, The Netherlands 3 Hospital Network Antwerp, Department of Neurology and Memory Clinic, Antwerp, Belgium 4 Institute Born-Bunge- University of Antwerp, Laboratory of Neurochemistry and Behavior, Antwerp, Belgium 5 University of Groningen- University Medical Center Groningen, University Center of Psychiatry & Interdisciplinary Center of Psychopathology of Emotion Regulation, Groningen, The Netherlands Aims Lipocalin 2 (Lcn2) is a pro-inflammatory mediator and regulator of iron homeostasis, known to play a role in the pathophysiology of many diseases. We hypothesize that Lcn2 may also significantly contribute to neurodegenerative processes in Alzheimer’s disease (AD). Lcn2 levels are increased in brain regions affected by AD pathology, and in vitro studies showed that Lcn2 promotes pro-inflammatory signaling, impairs neuroprotective signaling and aggravates amyloid beta-induced neurotoxicity in primary brain cell cultures. Normalization of Lcn2 expression in AD may therefore be of great therapeutic value. The upregulation of Lcn2 in AD appears to in part be induced by amyloid beta, but the signaling pathways underlying this process remain unclear. Therefore, we aim to elucidate the mechanisms via which amyloid beta stimulates Lcn2 expression, and identify inhibitors of amyloid beta-induced Lcn2 expression. Method Primary murine astrocytes are incubated with a range of compounds known to modulate specific signaling pathways, followed by administration of amyloid beta. Western blot analysis is performed to determine changes in Lcn2 protein levels, demonstrating what signaling pathways are involved in amyloid beta-induced Lcn2 production. Studied pathways include amongst others STAT1/STAT3 and MAPK signaling. Results Studies are still ongoing. Results so far show that incubation with an iron chelator can significantly reduce amyloid beta-induced Lcn2 production in primary astrocytes. Conclusion


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Results suggest that iron chelation may interfere with amyloid beta-induced Lcn2 production. More results are currently being collected, and will be presented during the AD/PD conference.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1407 EFFECTS OF NATURAL ANTIBODIES DELIVERED TO THE BRAIN USING FOCUSED ULTRASOUND ON THE NEUROINFLAMMATORY ENVIRONMENT IN A MOUSE MODEL OF AMYLOIDOSIS 1 1 2 3 1 S. Dubey , B. Kim , D.R. Branch , K. Hynynen , I. Aubert 1 Sunnybrook Research Institute, Biological Sciences, Toronto, Canada 2 University of Toronto, Laboratory Medicine & Pathobiology, Toronto, Canada 3 Sunnybrook Research Institute, Physical Sciences, Toronto, Canada Aims Intravenous immunoglobulins (IVIg) are natural antibodies collected from the plasma of thousands of healthy blood donors. We have previously shown that the delivery of IVIg to the brain is increased by transcranial focused ultrasound (FUS). Treatments with FUS, IVIg and combined FUS and IVIg lead to significant reduction in plaque load. Combined FUS and IVIg treatments had the highest impact on promoting neurogenesis. Here, we investigate inflammatory responses, which could contribute to the observed therapeutic effects. Method Using an amyloidosis mouse model, we administered IVIg intravenously, with or without application of FUS. FUS was performed once a week, for two consecutive weeks, and was targeted bilaterally to the hippocampus. After treatment, blood and tissue were collected for biochemical analyses of mRNA and protein expression using qPCR and western blotting. Results Our results indicate that treatments with IVIg, FUS, and combined IVIg and FUS altered cytokines and chemokines levels in the brain compared to saline treated animals. In particular, one cytokine, which is involved in the JAK/STAT pathway regulating amyloid pathology and neurogenesis, was found to be three times lower in the combined FUS and IVIg treatment group. The mRNA levels of two pro-inflammatory cytokines decreased, antiinflammatory levels of two cytokines increased while levels of eight cytokine mRNA (such as PPARG and IL-10) remained unchanged. Further work evaluating protein expression of markers downstream of this and other cytokines is underway. Conclusion


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These results suggest an anti-inflammatory mechanism via which therapy with IVIg, FUS, and combined treatments contribute to decreased plaque pathology and enhanced neurogenesis.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0349 OBESITY AND NEUROINFLAMMATORY PHENOTYPE IN MICE LACKING ENDOTHELIAL MEGALIN 1 1 2 1 2 3 4 F. Bartolome , D. Antequera , E. Tavares , C. Pascual , R. Maldonado , C. Antonio , C. Eva 1 Instituto de Investigacion Hospital 12 de Octubre, Neuroscience, Madrid, Spain 2 Valme University Hospital, Clinical and Experimental Pharmacology Research Unit, Sevilla, Spain 3 Universitat de Barcelona, Unitat de Farmacologia i Farmacognòsia, Barcelona, Spain 4 Instituto de Investigacion Hospital 12 de Octubre, Madrid, Spain Aims The multiligand receptor megalin controls the brain uptake of a number of ligands, including insulin and leptin. Despite the role of megalin in the transport of these metabolically relevant hormones, the role of megalin at the blood-brain-barrier (BBB) has not yet been explored in the context of metabolic regulation. Method Here we investigate the role of brain endothelial megalin in energy metabolism and leptin signalling using an endothelial cell-specific megalin deficient (EMD) mouse model. Results We found megalin is important to protect mice from developing obesity and metabolic syndrome when mice are fed a normal chow diet. EMD mice developed neuroinflammation, by triggering several pro-inflammatory cytokines, displayed reduced neurogenesis and mitochondrial deregulation. Conclusion


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These results implicate brain endothelial megalin expression in obesity-related metabolic changes through the leptin signalling pathway proposing a potential link between obesity and neurodegeneration.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1706 SYSTEMIC AUTOIMMUNITY: A SEX-SPECIFIC PROTECTIVE MECHANISM AT THE ONSET OF AD-LIKE DISEASE 1 2 1 3 1 B. Sakic , P. Forsythe , M. Kapadia , D. Ma , M. Fahnestock 1 McMaster University, Psychiatry & Behavioural Neurosciences, Hamilton, Canada 2 McMaster University, Medicine, Hamilton, Canada 3 McMaster University, Pathology and Molecular Medicine, Hamilton, Canada Aims The 3xTg-AD mouse is a widely used animal model of Alzheimer’s disease (AD). Recently, we reported that 1year-old 3xTg-AD males no longer show plaque/tangle pathology but exhibit anxiety-like behavior, learning/memory deficits and signs of systemic autoimmunity as early as two months of age. This study was designed to better understand the nature and course of the systemic autoimmunity. Method 3xTg-AD and non-transgenic mice of both sexes were administered the immunosuppressant cyclophosphamide 2 days/week in drinking water from 4 weeks to 6 months of age. Results Chronic immunosuppression abolished autoimmune manifestations and reduced soluble Aβ levels in 3xTg-AD mice but failed to normalize spatial learning capacity and worsened performance in anxiety-related tasks. Immunosuppression accelerated graying of fur in 3xTg-AD mice only, showing different skin patterns between sexes. Systemic autoimmunity (e.g., prevalence of CD4 CD8 T-splenocytes, splenomegaly, low hematocrit and increased anti-dsDNA autoantibody levels) were more profound in untreated 3xTg-AD males, suggesting sex as a key factor in determining the magnitude of immunological perturbations. Conclusion


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This study reveals multisystem involvement in the etiology of disease in 3xTg-AD mice. It also suggests an adaptive, genetically-driven autoimmune process that is more profound in males. Consistent with “protective autoimmunity”, our study supports the hypothesis of an early, sex-dependent immune activation that interferes with plaque/tangle formation in brains of 3xTg-AD males. If confirmed across species, this discovery may shed new light on the increased prevalence of AD in females and the relatively low number of patients with mild cognitive impairment converting to dementia of the Alzheimer’s type.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0667 EARLY AND LATE NEUROINFLAMMATORY PROCESSES IN DOWN SYNDROME: A LINK WITH ALZHEIMER’S DISEASE? 1 2 3 4 5 L. Flores Aguilar , M.F. Iulita , T. Wisniewski , J. Busciglio , A.C. Cuello 1 McGill University, Anatomy and Cell Biology, Montreal, Canada 2 Universite de Montreal, Neurosciences, Montreal, Canada 3 New York University, Neurology- Pathology- Psychiatry, New York City, USA 4 University of California Irvine, Neurobiology and Behavior, Irvine, USA 5 McGill University, Pharmacology and Therapeutics- Neurology and Neurosurgery- Anatomy and Cell Biology, Montreal, Canada Aims Down syndrome (DS) individuals exhibit a gradual intraneuronal-amyloid-beta (AB) deposition, which progresses to overt-Alzheimer’s disease(AD)-pathology (Busciglio,2002). We have reported an early neuroinflammatory process concurrent with intraneuronal-AB in transgenic models of amyloid pathology (Hanzel,2014, Ferretti,2012). Moreover, we have shown that DS individuals exhibit upregulation of inflammatory molecules in plasma before dementia onset (Iulita,2016). We propose that an early neuroinflammatory process will occur in preclinical stages of AD in DS. Given that recent investigations implicate inflammasome activation in AD pathogenesis; we hypothesize that this should occur early in DS. Our main objective is to characterize the evolution of the neuroinflammatory process and inflammasome activation throughout AD progression in DS. Method Classical inflammatory and inflammasome-related genes were analyzed via a gene expression array (Qiagen) and by qPCR in frontal cortex tissue (UCI-NYU brain banks) of DS (3 weeks-9 months-old) and non-DS-infants (22 days- 13 months-old), non-DS-AD (44-68 years-old) and DS-AD-adults (43-63 years-old). Results DS-infants show upregulation of IL-1B, IL-33, IL-6, IL-12a, IL12b, MCP-1, TNFSF11, TNFSF14, TNFSF4, CD40lg and downregulation of IFN-B. Regarding inflammasome components, DS-infants show upregulation of caspases1 and 5, NLRP3, NLRP4, NLRP6, CARD6 and downregulation of CARD18. While DS-adults with dementia also show upregulation of some of these molecules, the difference between DS and non-DS cases is more substantial in DS-infants. Moreover, DS-adults show upregulation of IFN-B and CARD18 contrary to the downregulation observed in DS-infants. Conclusion


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Preliminary gene expression analysis revealed differential levels of inflammatory molecules and inflammasome components in the silent (DS-infants) and clinical (DS-adults with dementia) stages of AD in DS.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1368 ACTIVATED MICROGLIA IN CORTICAL WHITE MATTER OF COGNITIVELY INTACT ELDERLY 1 1 1 1 1,2 1 1,3 1,4 T. Gefen , G. Kim , A. Geoly , K. Bolbolan , S. Weintraub , E. Rogalski , E.H. Bigio , M.M. Mesulam , 1 C. Geula 1 Northwestern University- Feinberg School of Medicine, Cognitive Neurology and Alzheimer's Disease Center, Chicago, USA 2 Northwestern University- Feinberg School of Medicine, Psychiatry and Behavioral Sciences, Chicago, USA 3 Northwestern University- Feinberg School of Medicine, Department of Pathology, Chicago, USA 4 Northwestern University- Feinberg School of Medicine, Department of Neurology, Chicago, USA Aims Microglial activation is a major indicator of neuroinflammation in a number of age-related neurodegenerative disorders, such as Alzheimer's disease. We recently found that activated microglia are extremely abundant throughout white matter—at levels that surpass grey matter densities—in brains of individuals who show postmortem Alzheimer’s disease or TDP-43 pathology. In this study, we evaluated microglial densities in brains of cognitively normal elderly (over age 65, N=4) and cognitive “SuperAgers”, individuals over age 80 who show outstanding memory capacity (N=3). One cognitively normal young case (45 years) was included for preliminary age-related comparisons. Method Specimens were cut into whole-hemisphere sections and immunohistochemically stained to visualize HLA-DRpositive activated microglia. Five cortical regions (inferior frontal, anterior cingulate, inferior parietal, superior temporal, and entorhinal) were examined for microglial activation in both grey and white matter. Densities were ranked based on a 0-5 scale, with 0 denoting absence of activated microglia. Results Preliminary rankings revealed higher microglial activation in white matter compared to adjacent grey matter in all cases. Greatest white matter microglial activation was observed in entorhinal cortex compared to other regions. Examination of the one young case showed substantially less white matter microglia than elderly post-mortem cases. Conclusion


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Patterns of white matter microglia were prominent in brains of cognitively intact elderly, at levels that exceeded grey matter densities. Future directions will investigate age-related white matter microglial differences in larger groups of both young and old specimens, and will examine the contribution of white matter microglia to cognitive performance.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1564 IKAROS AS A MICROGLIAL AND ALZHEIMER’S DISEASE MODULATOR 1 1 1 S. Georgopoulos , L. Velentza-Albany , E. Paouri 1 Biomedical Research Foundation of the Academy of Athens, Cell biology, ATHENS, Greece Aims Although beta-amyloid (Ab) aggregation is traditionally accepted to be the primary initiator of Alzheimer’s disease (AD), neuroinflammation has also a causal role in AD. Increasing evidence suggests that AD pathogenesis is not restricted to the neuronal compartment, but includes interactions with immunological mechanisms outside the brain. The role of the innate immune system in AD has been recently in the focus of research, yet the role of adaptive immunity remains largely unknown. Clinical and mouse studies have suggested that T and B lymphocytes are involved in AD pathogenesis.

Method To further elucidate the role of adaptive immunity in AD we have deleted T and B lymphocytes and NK cells on an AD mouse that develops amyloid deposits and neuroinflammation, by using Ikaros (Ik) null mice. Ik is a transcription factor that regulates lymphoid deferentiation and Ik-/- mice do not have T, B and NK cells. We have employed an Ik promoter-GFP reporter mouse to examine expression of Ikaros in the brain and an Ik conditional Knock-out mouse for further studies. Results Our preliminary analysis revealed expression of Ikaros in microglia. Deletion of Ikaros in the brain of Ic-/- mice resulted in an altered, unspecified microglial phenotype. 5XFAD mice on the Ik-/- background (5XFAD/Ik-/-) have an immense decrease in amyloid deposition in the brain. Conclusion


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These results suggest a novel role for Ikaros in microglial cells and AD pathogenesis.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1300 BETA-AMYLOID PATHOLOGY IMPAIRS BRAIN IMMUNE SURVEILLANCE IN TRANSGENIC MICE 1 2 3 4 3 1 1 1 C. Gericke , C. Spaeni , N. Schweizer , M. Merlini , T. Suter , L. Kulic , M.T. Ferretti , R. Nitsch 1 University of Zurich, Institute for Regenerative Medicine, Schlieren, Switzerland 2 University of Kentucky, Sanders Brown Center on Aging, Lexington, USA 3 University Hospital Zurich, Department of Neurology- Section of Neuroimmunology and MS Research, Zurich, Switzerland 4 University of Zurich, Center for Molecular Cardiology, Schlieren, Switzerland Aims Chronic neuroinflammation accompanies brain beta-amyloid pathology in Alzheimer’s Disease (AD). This study was designed to test the hypothesis that pathologic forms of amyloid beta peptide (Abeta) can escape brain immune surveillance by actively inhibiting it. Method We analyzed brain-derived antigen-presenting cells (APCs) and T cells from aged APP-transgenic (tg) mouse models with beta-amyloid pathology by flow-cytometry and compared results with non-tg, age-matched littermates. Effects of Abeta on major histocompatibility class II (MHC-II)-dependent antigen presentation were studied ex vivo using ovalbumin (OVA)-inducible OT-II T cells and bone-marrow-derived APCs. T cell cytokine production and expression of the proliferation marker Ki67 following OVA-presentation were analyzed by using flow-cytometry. Results MHC-II surface expression on APCs was significantly reduced in APP-tg brains, along with intracellularly accumulated MHC-II, suggesting low antigen-presentation capability. Intracellular staining revealed that cerebral CD4+ and CD8+ T cells from APP-tg mice had low levels of IFNgamma and TNFalpha cytokines. No such decreases were present in spleen or cerebellum with lacking amyloid pathology, indicating a cerebral, betaamyloid-related effect. Oligomeric recombinant Abeta1-42 reduced IFNgamma and Ki67 levels in OT-II CD4+ T cells following OVA-presentation ex vivo, indicating impaired antigen-presentation. Conclusion


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Beta-amyloid pathology in APP-tg mice is accompanied by both impaired MHC-II surface expression and low T cell effector-cytokine production in vivo. Ex vivo results further support the idea that Abeta oligomers inhibit MHCII-dependent antigen-presentation. Together, these results suggest that beta-amyloid pathology is associated with impaired brain immune surveillance. If confirmed in human brains, compromised brain immune surveillance could play a role during the development of AD.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1055 A HIGH-THROUGHPUT SCREEN FOR INHIBITORS OF PU.1-INDUCED INFLAMMATION IN NEURODEGENERATION 1 1 1 1 S. Godin , G. Elizabeta , G. Tyler , T. Li-Huei 1 Massachusetts Institute of Technology, Picower Institute of Memory and Learning, Cambridge, USA Aims Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by amyloid-beta plaque formation, neurofibrillary tangles, and cognitive impairment in older individuals. During AD, as well as numerous other neurodegenerative diseases, the brain exhibits symptoms of inflammation and reactive microgliosis. The importance of reactive microglia and the resulting inflammatory response in driving disease progression has been highlighted by several studies that found that inhibiting microglia has clear benefits in ameliorating many disease phenotypes in mice models of AD. Recent work from our laboratory underscored the significance of the immune response in the AD brain during an evaluation of the transcriptional and chromatin alterations that occur during disease progression. In this study we found a consistent and significant activation of immune response genes, largely regulated by the immune-lineage specific transcription factor PU.1. Method To attenuate the inflammatory response, we have generated a high-throughput drug screen to test tens of thousands of compounds in microglia for their ability to inhibit PU.1-dependent transcription. Results This work has identified numerous compounds as potential options for PU.1 inhibition. Moreover, the targets of these compounds have uncovered several previously unreported signaling pathways that act to regulate PU.1dependent transcription in the cell. Conclusion


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Together, the results of the screen will help to elucidate the regulation of PU.1 and identify pharmacological agents to inhibit its activity.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0686 ELUCIDATING REACTIVE ASTROGLIA FUNCTION IN APP/PS1 MOUSE MODEL AND HUMAN ALZHEIMER’S DISEASE BRAIN 1 1 1 2 2 3 A. Gomez-Arboledas , R. Sanchez-Varo , E. Sanchez-Mejias , V. Navarro , M.L. Vizuete , J.X. Comella , 4 4 2 1 1 R. Masgrau , E. Galea , J. Vitorica , J.C. Davila , A. Gutierrez 1 University of Malaga, Department of Cell Biology- Physiology and Genetics NeuroAD NeuroAD, Malaga, Spain 2 University of Seville, Dept of Biochemistry and Molecular Biology- Faculty of Pharmacy- IBIS. CIBERNED, Seville, Spain 3 Autonomic University of Barcelona UAB, Institut de NeurocienciesDepartment of Biochemistry and Molecular Biology- Faculty of Medicine. VHIR- CIBERNED., Barcelona, Spain 4 Autonomic University of Barcelona UAB, Institut de Neurociencies and BiochemistryFaculty of Medicine. Institucio Catalana de Recerca i Estudis Avancats., Barcelona, Spain Aims Reactive astrocytes are a hallmark of Alzheimer’s disease (AD), however whether these cells actively contribute to the neurodegenerative processes or, by contrast, play a neuroprotective role during disease progression it is still unknown Method Immunohistochemistry at light and electron microscopy was performed in the hippocampus of APP 751SL/PS1M146L transgenic mice. Post mortem human AD brains were also studied at light microscopy. The viability of astroglial cells to soluble toxic agents (Abeta and/or phospho-tau) from S1 fractions derived from APP/PS1 and AD brains were tested by in vitro assays. Results The hippocampus of APP/PS1 mice and Braak V-VI samples was characterized by an intense astroglial response. In vitro assays revealed that soluble S1 fractions from both models and patients were not lethal for cultured astroglia. Reactive astrocytes located in the vicinity of amyloid plaques displayed numerous thick, GFAPpositive, primary processes. Ultrastructural analysis in APP/PS1 mice clearly showed that the astrocyte processes, positive for AQP4, GS or EAAT, extended and contacted Abeta fibers. Moreover, thick astrocyte processes located near plaques were surrounding or even engulfing dystrophic neurites, suggesting an active role of these glial cells in the removal of abnormal axonal/presynaptic structures. Conclusion Astrocyte survival rate is not affected by brain-derived soluble Abeta or phospho-tau species. Reactive astrocytes might act as a protective barrier around Abeta plaques and, more interestingly, they are actively involved in the stripping of neuronal dystrophies. Therefore, astrocytes could be a potential therapeutical target for AD.


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Supported: FIS PI15/00796(AG),PI15/00957(JV), CIBERNED, Fundacion La Marato TV3 and co-financed by FEDER funds from European Union

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0333 EFFECTS OF BRD2 INHIBITION ON ASTROCYTE-DEPENDENT NEUROPROTECTION VIA MODULATION OF TPA/PAI-1 ACTIVITY 1 1 2 2 3 4 S.H. Han , K. Kyoung Ja , K. Ryoung En , E. Pyeong Hwa , K. Hee Jin , S. Chan Young 1 Konkuk University Medical Center, Neurology, Seoul, Republic of Korea 2 Konkuk University School of Medicine, Neuroscience, Seoul, Republic of Korea 3 Hanyang University Medical Center, Neurology, Seoul, Republic of Korea 4 Konkuk University School of Medicine, Pharmacology, Seoul, Republic of Korea Aims Neuro-inflammation is one of the pathological hallmarks of neurodegenerative disease such as Alzheimer’s disease and Parkinson’s disease. The bromodomain and extra terminal domain (BET) protein family are a group of epigenetic readers, which is a critical regulator of transcription in many cell types including neurons. We tested whether the inhibition of BET protein inhibit neuronal death and regulate the neurite outgrowth using a rat primary cortical neurons. We also investigated whether a potent inhibitor of BET protein could prevent the amyloid beta aggregation and cognitive decline in an animal model of AD. Method We investigated the effects of BET inhibition on neuronal death on neuronal cell death in primary cortical neurons, astrocytes and mixed neurons: 1) cell viability and ROS generation; 2) enzyme activity of tPA/PAI-1; 3) the signal pathway including BDNF/CREB and MAPKs using Western blotting; 4) astrocyte-dependent neurite outgrowth using co-culture system. Results JQ1 significantly decreased the neuronal death in concentration-dependent manner in mixed cortical neuronal but not in pure neuronal cultures. JQ1 decreased the ROS generation, and decreased cleaved caspase-3 expression. JQ1 concentration-dependently increased PSD95 and nNOS expression in mixed cortical neurons and organotypic hippocampal slice cultures. JQ1 decreased the H2O2-induced p38 and JNK activation in mixed cortical neurons. Brd2 inhibition by transfection of Brd2 siRNA reduced Aβ aggregation. Conclusion


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Our experimental results show Brd2 inhibition by JQ1 could prevent the neuronal death and neuroinflammation, and increase astrocyte-dependent neurite outgrowth through regulation of tPA/PAI-1 system. These findings indicate that BET inhibition may have a therapeutic potential of AD.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1410 TREM2 DEFICIENCY IMPACTS MYELOID CELL FUNCTION AND AMYLOID PATHOLOGY IN A DISEASESTAGE DEPENDENT MANNER IN MOUSE MODELS OF ALZHEIMER’S DISEASE 1 1 1 1 2 3 4 5 T. Jay , V. von Saucken , A. Hirsch , M. Broihier , C. Miller , R. Ransohoff , B. Lamb , G. Landreth 1 Case Western Reserve University, Neurosciences, Cleveland, USA 2 Cleveland Clinic Lerner Research Institute, Neurosciences, Cleveland, USA 3 Biogen, Neuroimmunology/Discovery Biology, Cambridge, USA 4 Indiana University, Stark Neurosciences Research Institute, Cleveland, USA 5 Case Western Reserve University, Neuroscience, Cleveland, USA Aims The importance of neuroinflammation in Alzheimer’s disease (AD) has been supported by the identification of variants in immune-related genes that alter risk for developing AD. Variants in Trem2, a receptor expressed exclusively on myeloid cells in the brain, are among the strongest risk factors for AD. Understanding how these Trem2 variants modify myeloid cell function and impact AD pathology promises to provide important insight into the role of neuroinflammation in AD. Method In order to examine the role of Trem2 in AD, we examined myeloid cell proliferation, survival and internalization of amyloid at different stages of disease progression in APPPS1 and 5XFAD Trem2 deficient mice. Results We found that Trem2 deficient AD mouse models had reduced proliferation and increased expression of apoptotic markers specifically late in disease progression. Amyloid internalization was decreased in plaque-associated myeloid cells both early and late in the development of pathology. Late in pathology, these changes in myeloid cell function led to a significant increase in amyloid plaque area. In contrast, early in pathology, Trem2 deficient mice had reduced amyloid accumulation. How these changes early in pathology lead to decreased amyloid pathology is currently under further investigation. Conclusion


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Together, these stage specific changes in Trem2 function suggest a dynamic role of the innate immune response in AD and provide insight into how these changes in myeloid cell function affect pathology throughout disease progression.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0849 HEPARAN SULFATES ARE INVOLVED IN AMYLOID-BETA CLEARANCE IN APP-TRANSGENIC MOUSE MODELS OF ALZHEIMER DISEASE 1 2 2 3 1 2 C. Jendresen , A. Digre , H. Cui , X. Zhang , L. Nilsson , J.P. Li 1 University of Oslo, Department of Pharmacology, Oslo, Norway 2 Uppsala University, Department of Medical Biochemistry and Microbiology, Uppsala, Sweden 3 Uppsala University, Department of Neuroscience, Uppsala, Sweden Aims Heparan sulfate (HS) is ubiquitously present on cell surfaces regulating basic cell functions. In Alzheimer disease (AD) and animal models thereof, HS is associated with amyloid-beta (Aβ) deposits in the brain. Here, we investigated whether a systemic lipopolysaccharide (LPS)-injection could reduce Aβ-burden in amyloid-beta precursor protein (AβPP)-transgenic mice and if HS is involved in LPS-induced Aβ-clearance. We also investigated the neuroinflammatory profiles after LPS-injection of AβPP-transgenic mice compared to wildtype and heparanase-overexpressing AβPP-mice with fragmented HS. Method 13-months-old mice of three genotypes: 1) wildtype mice, 2) AβPP-transgenic mice, and 3) heparanase/AβPP double-transgenic mice was given a single intraperitoneal injection of LPS (1.7 µg/g mouse) or PBS-vehicle. 1-1.5 months after injections the mice were euthanized, and brains were examined histologically and biochemically for amyloid burden and neuroinflammation. Results Heparanase-overexpression leads to reduced amyloid burden in AβPP-mice. LPS-treatment reduced amyloid burden in single-transgenic AβPP-mice, but not in double heparanase/AβPP-transgenic mice. Interestingly, LPStreated single-transgenic mice had a higher ratio of soluble-to-insoluble Aβ compared to PBS-treated singletransgenic mice. The double-transgenic mice generally had lower cytokine/chemokine levels than singletransgenic AβPP-mice and wildtype mice. Conclusion


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LPS-induced neuroinflammation solubilizes Aβ-deposits in AβPP-transgenic mice and this effect depends on HS. Furthermore, amyloid burden was lower in LPS-treated AβPP-mice, while there is no significant difference in the HS-reduced mice. This indicates that 1) systemically induced LPS-neuroinflammation solubilizes cerebral Aβ and this likely facilitates clearance, and 2) HS is involved in this Aβ-clearance process.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1712 INTERLEUKIN-33 MODULATES THE IN VITRO PHAGOCYTOSIS OF AMYLOID-BETA BY MACROPHAGES 1 1 1 1 1,2 F. La Rosa , M. Saresella , I. Marventano , F. Piancone , M. Clerici 1 Don Carlo Gnocchi Foundation, Molecular and Biotechnology, Milan, Italy 2 University of Milan, Pathophysiology medical and transplantation, Segrate, Italy Aims Alzheimer’s disease (AD) is a neurodegenerative disease associated with the deposition of extracellular (Aβ) plaques within the brain and neuroinflammation. The NLRP3 inflammasome, in particular, is activated in AD. This results in the downstream production of the proinflammatory cytokines IL1β and IL18, a reduced generation of the antiinflammatory IL33 cytokine, and the impairment of Aβ-phagocytosis. Herein we analyzed possible correlations between inflammasome activation and Aβ-phagocytosis in an in vitro system. Method Existing flow cytometry methods that lack image-based analysis capabilities fail to fully discriminate between adherent or cells-internalized Ab; the AMNIS technology can perform simultaneous detection of Aβ-phagocytosis in different in vitro conditions. THP-1 cells differentiated into macrophages by PMA were stimulated with LPS (1ug/ml for 24 hours)+ Aβ(1ug/ml for 1 hour), or with LPS+ Aβ+ the NLRP3 activators Nigericin (5uM for 1 hour) or with Aβ (1ug/ml for 1 hour) in the presence/absence of IL33 (10ng/ml for 18 hours). Phagocitosis was measured as the ratio between intra and extra cellular FITC-coniugated Aβ; cytokine production was measured by ELISA. Results A significant increase in IL1β production (p=0.001) and a drastic reduction of Aβ-phagocytosis (p=0.0001) were seen when cells stimulated by LPS+ Aβ+Nigericin were compare to those stimulated by LPS+Aβ or Aβ alone. Notably, IL1β production was drastically reduced and Aβ-phagocytosis was significantly increased (both p=0.001) in LPS+ Aβ+Nigericin-stimulated cells in the presence of IL33. Conclusion


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IL33 reduces inflammasome-associated inflammation and improves macrophage-mediated Aβ-phagocytosis. These results allows the speculation that this cytokine could be beneficial in the therapy of AD.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1192 BETA-AMYLOID AND NEUROINFLAMMATION IN LEWY BODY DEMENTIA 1 1 2 3,4 1,5 1 J.H. Lee , Y. Chai , P.T. Francis , D. Aarsland , C.P. Chen , M.K. Lai 1 National University of Singapore, Department of Pharmacology- Yong Loo Lin School of Medicine, Singapore, Singapore 2 King's College London, Wolfson Centre for Age-Related Diseases, London, United Kingdom 3 Karolinska Institutet, Department Neurobiology- Care Sciences and Society- Center for Alzheimer ResearchDivision of Clinical Geriatrics, Stockholm, Sweden 4 King’s College London, Department of Old Age Psychiatry, London, United Kingdom 5 National University Health System, Memory- Aging and Cognition Centre, Singapore, Singapore Aims Alzheimer’s disease (AD) is the most common form of dementia, characterised by beta-amyloid (A-beta) plaques, neurofibrillary tangles and neuroinflammation. However, the state of neuroinflammatory involvement in Parkinson’s disease dementia (PDD) and Dementia with Lewy body (DLB) is less well characterised. As A-beta is known to induce inflammation, we aimed to assess the burden of AD-related pathology as well as to measure the expression of key neuroinflammatory markers in PDD and DLB. We hypothesize that neuroinflammatory activation will be lower in PDD compared with DLB in line with lower PDD cortical A-beta burden. Method Postmortem frontal cortex of PDD, DLB, and controls from longitudinally assessed cohorts were used to assay for total A-beta and tau using ELISA and inflammatory markers including interleukin-1alpha(IL-1a), IL-10, IL-13, transforming growth factor-alpha (TGF-a), interferon-gamma (IFN-g)and granulocyte-macrophage colonystimulating factor using Luminex technology. Results As expected, total A-beta load was significantly increased in DLB but not in PDD compared to controls. Tau burden was not significantly increased in PDD or DLB. Interestingly, inflammatory cytokines were not significantly increased in PDD or DLB, while TGF-a was significantly reduced in both. Conclusion


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The lack of marked inflammation in PDD and DLB predicts limited efficacy of anti-inflammation therapy in these conditions, and that specific A-beta species, rather than total amyloid burden, may be the determinants of inflammatory activation. Further work is required to investigate the mechanism underlying the relatively mild neuroinflammation in DLB despite the high amyloid load.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0260 RELATIONSHIP BETWEEN THE HS-CRP AS NON-SPECIFIC BIOMARKER AND ALZHEIMER’S DISEASE ACCORDING TO AGING PROCESS 1 1 J.Y. Lee , S. In-Uk 1 Incheon St. Mary’s Hospital- The Catholic University of Korea, Neurology, Incheon, Republic of Korea Aims Microglia are involved in immune surveillance in intact brains and become activated in response to inflammation and neurodegeneration. Microglia have different functions, neuroprotective or neurotoxic, according to aging in patients with PD. The clinical effect of microglia in patients with Alzheimer’s disease (AD) is poorly defined. This prospective study was conducted to investigate the clinical effects of microglia according to the aging process in newly diagnosed AD. Method We examined 532 patients with newly diagnosed AD and 119 healthy controls, and the differences in hs-CRP between these groups were investigated. The patients with AD were classified into 3 subgroups according to age of newly diagnosed AD to investigate the relationship between hs-CRP and the aging process in newly diagnosed AD. Results There was significantly higher serum high-sensitivity C-reactive protein (hs-CRP), levels in patients with AD compared with healthy controls. A post-hoc analysis of the 3 AD subgroups showed no significant differences in serum hs-CRP level between each group. Conclusion


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We assumed that neuroinflammation play a role in the pathogenesis of AD, but found no clinical evidence that microglia senescence underlies the microglia switch from neuroprotective in young brains to neurotoxic in aged brains. To clarify the role of microglia and aging in the pathogenesis of AD, future longitudinal studies involving a large cohort are required.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0557 HIGH SERUM HAPTOGLOBIN LEVELS IN ASSOCIATION WITH THE PATHOGENESIS OF ALZHEIMER'S DISEASE 1 2 Y.H. Lee , I.U. Song 1 Incheon St.Mary's Hostpital, Neurology, Seoul, Republic of Korea 2 Incheon St.Mary's Hospital, Neurology, Incheon, Republic of Korea Aims Haptoglobin is known to have several functional properties, including antioxidant and anti-inflammatory activities. In addition, it has been shown that the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease (AD), involves inflammation as well as oxidative stress. However, evidence suggesting an association between the serum haptoglobin level and AD is lacking. Therefore, we conducted this study in order to investigate whether serum haptoglobin is associated with AD. Method We compared the serum haptoglobin levels of 121 pateitns with newly diagnosed AD, 58 patients with Parkinson's disease (PD) and 43 healthy controls. We also evaluated the relationship between the severity of cognitive impairment in patients with AD and the serum haptoglobin level. Results The mean serum haptoglobin level of the patients with AD was significantly higher than that of the healthy controls (p=0.042), although it was not significant different from that observed in the PD group (p=0.613). We also found a significant positive association between the serum haptoglobin level and the severity of cognitive impairment, as measured using several neuropsychological tests, in the patients with AD. The odds ratio (95% confidence interval) of the patients with AD grouped according to the haptoglobin level was 2.417 (95% confidence interval=1.134-5.149). Conclusion


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We observed a significantly higher mean serum haptoglobin level among the patients with AD compared to the healthy controls. These results support the hypothesis that oxidative stress and neuroinflammatory reactions play a role in the pathogenesis of AD.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0987 TREM2 COORDINATES ANTI-INFLAMMATORY RESPONSE AND GENE EXPRESSION ASSOCIATED WITH NEUROPROTECTION IN PRIMARY MICROGLIAL CULTURE 1 1 2 1 1 W. Liu , D.M. Cummings , P. Garcia-Reitboeck , F.A. Edwards , D.A. Salih 1 Univeristy College London, Neuroscience- Physiology and Pharmacology, LONDON, United Kingdom 2 Univeristy College London, Institute of Neurology, LONDON, United Kingdom Aims Neuroinflammation is one of the major pathological features in Alzheimer’s disease (AD), and SNPs in immune genes like TREM2 and CD33 are found to be risk factors for AD by human GWAS studies. Here we use siRNA to knockdown Trem2 in primary microglia and investigate changes in microglial gene expression and function. Method Primary microglia, generated from 1-3 day old wild type mice, were transfected with Trem2 siRNA. Trem2 knockdown level and expression levels of immune genes were assessed by qRT-PCR. Phagocytosis was assayed using pHrodo E. coli bioparticles and analysed with fluorescence-activated cell sorting. Pro- or antiinflammatory activation was induced by lipopolysaccharide (LPS) or interleukin-4 (IL4) treatment. All experiments in accordance with Animals (Scientific Procedures) Act 1986 Results In primary microglia, expression of Trem2 was substantially depressed by LPS treatment, while preliminary data with IL4 treatment suggested little change or increased expression. At 72-hour after siRNA transfection, ~70% knockdown of Trem2 was obtained. Cd68, Csf1r, and Igf1 (normalised to Aif1) were found to be significantly down-regulated after Trem2 knockdown, and phagocytosis was significantly impaired. Additionally, there was a decrease in microglial number after Trem2 knockdown. The Cd68, Csf1r, and Igf1 down-regulation was also observed with LPS treatment. Conclusion Trem2 expression is largely down-regulated in primary microglia with LPS, suggesting pro-inflammatory stimuli depress TREM2-related functions of microglia. TREM2 is involved in microglial anti-inflammatory response and maintaining normal phagocytic function.


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Decreased function of TREM2, due to loss-of-function variations in the gene, may alter microglia-neuron communication by decreased production of microglia-derived neuronal growth factors such as IGF1.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1581 INFLAMMATORY MARKERS AS POTENTIAL BIOMARKERS OF DEMENTIA IN AGED 1 2 3 2 N.M. Mikhaylova , L. androsova , S. gavrilova , T. klushnik 1 Mental Health Research Center of the Russian Academy of Medical Sciences, Alzheimer's disease department, Moscow, Russia 2 Mental Health Research Center of the Russian Academy of Medical Sciences, laboratory of immunology, Moscow, Russia 3 Mental Health Research Center of the Russian Academy of Medical Sciences, department of geriatric psychiatry, Moscow, Russia Aims The aim was to study some immunological characteristics in main forms of neurocognitive disorders in aged. There are some evidences of an important role of neuroinflammation in the pathogenesis of dementia. Method A study made up 79 cases with Alzheimer’s disease (AD), 52 cases of mixed dementia (MD), 34 cases of Vascular Dementia (VaD), 17 cases of Frontotemporal dementia (FTD) and 57 patients with amnestic mild cognitive impairment (aMCI). Activity of leukocyte elastase (LE) and alpha1-proteinase inhibitor (α1-PI), the level of IL-6 and CRP were determined in patients’ blood plasma. Results Cases of AD and MD characterized by significant decrease of LE activity and significant increase of α1-PI and IL-6 level compared to control. Indicators of innate immunity in the groups of AD and MD did not differ from each other. CRP concentration was higher in MD as well as in VaD but this difference did not reach the level of significance. FTD characterized by significant increase of LE and α1-PI activity and CRP level depending on the different variants of this disease (behavioral FTD and primary progressive aphasia). One third of aMCI cases showed similar changes of inflammatory markers as in AD. This group may have a high risk of AD and needs long-term follow-up clinical and immunological examinati Conclusion


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Obtained data suggest the involvement of inflammatory reactions in the pathway of dementia in aged. Immune indices of inflammation in patient blood plasma may be useful in discovery of biomarkers for early clinical differential diagnostics of cognitive impairment and dementia in aged.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1154 PERIPHERAL TUMOR NECROSIS FACTOR-ALPHA (TNF-Α) MODULATES AMYLOID PATHOLOGY BY REGULATING BLOOD-DERIVED IMMUNE CELLS AND GLIAL RESPONSE IN THE BRAIN OF AD MICE WITH TNF-INDUCED ARTHRITIS 1 1 1 1 1 E. Paouri , O. Tzara , G.I. Kartalou , S. Zenelak , S. Georgopoulos 1 Biomedical Research Foundation of the Academy of Athens, Center of Basic Research, ATHENS, Greece Aims TNF-α is a pro-inflammatory cytokine involved in systemic inflammatory and neurodegenerative diseases, including rheumatoid arthritis (RA) and Alzheimer’s disease (AD). Evidence has suggested that anti-TNF-α therapy or RA itself can modulate AD pathology although the underlying mechanism is unclear. To investigate the role of TNF-α as a common mediator between AD and RA, we employed the 5XFAD AD mice and the Tg197 human TNF-α (huTNF-α) transgenic mice that develop arthritis. Method For the purposes of our study, we generated double transgenic 5XFAD/Tg197 mice. We evaluated the effect of: (a) huTNF-α expression and (b) peripheral huTNF-α inhibition by infliximab huTNF-α neutralizing antibody, in the amyloid and brain inflammatory phenotype of 5XFAD/Tg197 mice, using histological and biochemical techniques. Results We show that 5XFAD/Tg197 mice express huTNF-α both in the brain and the periphery resulting in decreased amyloid deposition, robust brain inflammation characterized by diffused gliosis and highly elevated numbers of perivascular macrophages and infiltrating leukocytes, and synaptic degeneration. To evaluate whether this phenotype can be attributed to peripheral or brain huTNF-α, we treated 5XFAD/Tg197 mice systemically with infliximab anti-huTNF-α antibody that does not penetrate the blood-brain-barrier and prevents arthritis. Peripheral huTNF-α inhibition significantly lowers peripheral but not brain huTNF-α and reverses the amyloid and brain inflammatory phenotype, leading to increased amyloid deposition, decreased gliosis and peripherally-derived immune cells and restored synaptic integrity. Conclusion


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Our study shows for the first time that manipulation of peripheral TNF-α modulates the amyloid phenotype by regulating peripherally-derived immune cell trafficking and glial response in the mouse brain.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1113 LFA-1 INTEGRIN MEDIATES NEUTROPHIL TRAFFICKING IN THE BRAIN IN MOUSE MODELS OF ALZHEIMER’S DISEASE AND CONTRIBUTES TO DISEASE PATHOLOGY 1 1 1 1 1 1 1 1 E. Pietronigro , V. Della Bianca , E. Zenaro , G. Piacentino , T. Carlucci , S. Dusi , R. Nagarajan , G. Iannoto , 1 1 1 M. Bonani , T. Saatchi , G. Constantin 1 University of Verona, Department of Medicine, Verona, Italy Aims We have previously demonstrated that neutrophils infiltrate the brain in animal models of mice Alzheimer's disease (AD), contributing to the induction of cognitive deficits and neuropathological changes. Our goal was to study the effect of LFA-1 integrin blockade on neutrophil trafficking and disease pathogenesis in transgenic animals with AD-like disease. Method We performed two-photon laser-scanning microscopy (TPLSM) experiments in the brain to determine the effect of LFA-1 integrin blockade on neutrophil adhesion and extravasation in the brain. To better evaluate LFA-1 integrin blockade on disease, we also generated 3xTg-ADxItgal-/- mice lacking functional LFA-1 integrin, by crossing 3xTg-AD mice and LFA-1-deficient Itgal-/- strain. Mice were examined in behavioral tests and neuropathological studies. Results TPLSM experiments showed that LFA-1 integrin blockade inhibits neutrophil intravascular adhesion and cerebral extravasation, and also impairs intraparenchymal motility. Notably, the LFA-1 integrin blockade during the early stages of disease improves memory in 3xTg-AD mice and also has long-term benefits on cognition. In addition, -/we found that the 3xTg-ADxItgal mice show improved memory in behavioral tests compared to control 3xTg-AD animals during early and late phases of disease. These findings were supported by neuropathological data showing a reduction in Abeta deposition, microgliosis and tau hyperphosphorylation compared to aged-matched control animals. Conclusion


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Our data demonstrate that LFA-1 integrin has a key role in the development of cognitive dysfunction and neuropathological changes in animal models of AD, suggesting that anti-adhesion therapy may represent a new therapeutic strategy to interfere with the development of this disease.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1622 INNATE IMMUNITY IN NEURODEGENERATIVE DISEASES: STUDIES OF NLRP3 INFLAMMASOME ACTIVATION AND IL-1BETA SECRETION IN HUMAN MICROGLIA 1 1 1 A. Pike , M. Herrebout , R. Veerhuis 1 VUMC, Clinical Chemistry, Amserdam, The Netherlands Aims Activation of the NLRP3 inflammasome, a multimolecular scaffolding complex that amplifies proinflammatory signals through cytokine processing and secretion in response to an unusually wide array of stimuli, is associated with neurodegenerative diseases (NDs) including Alzheimer’s disease, Parkinson’s disease, and frontotemporal dementia. NLRP3 inflammasome signaling requires activated caspase-1, which has been detected in brain tissue of patients with these diseases and is expressed along with other inflammasome components by microglia. Thus, we study NLRP3 inflammasome activation and inhibition mechanisms in human microglia with the aim of clarifying the inflammasome’s roles in these diseases and the potential for their modulation. Method Using primary human microglia and several THP-1 cell lines, we are characterizing in vitro the mechanisms and extent of NLRP3 inflammasome activation and IL-1beta production in ND conditions with an array of activators, including disease-relevant proteins, and inhibitors. Results Primary human microglia and THP-1 cells secrete IL-1beta and IL-18 in response to inflammasome priming and activation. However, in agreement with previously published observations in non-human primate microglia, we find that the IL-1beta secreted by human microglia appears to be only partially caspase-1-dependent and indeed not entirely inflammasome-dependent. We are currently studying contributions to IL-1beta production under ND conditions by other caspases and by entirely different mechanisms. Conclusion


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NLRP3 inflammasome inhibitors are under development for therapeutic use in selectively blocking IL-1beta signaling, which is a known contributor to neurodegeneration. However, if this inflammasome is only partially responsible for IL-1beta production in human microglia in ND, other mechanism-specific interventions may be needed.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0607 SOLUBLE PHOSPHO-TAU FROM ALZHEIMER’S DISEASE HIPPOCAMPUS DRIVES MICROGLIAL DEGENERATION 1 2 1 2 1 1 E. Sanchez-Mejias , V. Navarro , C. Nuñez-Diaz , S. Jimenez , R. Sanchez-Varo , L. Trujillo-Estrada , 2 2 1 2 1 M.V. Sanchez-Mico , M.L. Vizuete , J.C. Davila , J. Vitorica , A. Gutierrez 1 University of Malaga. CIBERNED. IBIMA., Cell Biology, MALAGA, Spain 2 University of Seville. CIBERNED. IBIS, Biochemistry and Molecular Biology, Seville, Spain Aims The role of neuroinflammation in the development/progression of Alzheimer’s disease (AD) is still unknown. The actual view based on the findings in transgenic models gives a cytotoxic/proinflammatory role to activated microglia. In this sense, we have previously reported a massive microglial activation process in parallel with the amyloid pathology in a APP/PS1 mouse model. Here, we studied the microglial response in human AD hippocampus in order to compare with that observed in mice. Method In vitro studies, RT-PCR, western blots and immunostainings were performed in post-mortem mild (Braak II) to severe (Braak V-VI) AD cases obtained from Neurological Tissue Banks of Spain. Results The hippocampus of Braak V-VI AD patients exhibited limited microglial activation associated with amyloid plaques. Most relevant, microglia suffered a degenerative process that was most prominent in the dentate gyrus followed by the CA3, CA1 and parahippocampal gyrus and included fragmented and dystrophic prolongations with spheroids formation, reduced numerical density and a significant decrease in the area of surveillance (microglial domain). Moreover, in vitro studies revealed that the phosphorylated tau, and not Abeta, from soluble S1 fractions of human AD hippocampus, was detrimental for microglial cells. Conclusion


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These data reveal a significant decline in the area covered by microglia, which may compromise the immune protection and, therefore, neuronal survival. This microglial vulnerability provides new insights on immunological mechanisms underlying disease progression and supports therapeutic efforts through the modulation of the innate immune system. Supported by FIS-PI15/00796(to AG) and FIS-PI15/00957(to JV) and co-financed by FEDER funds from European Union.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0608 IMMUNOSUPPRESSION EXACERBATES ALZHEIMER’S DISEASE PATHOLOGY IN APP/PS1 TRANSGENIC MICE 1 1 2 2 2 1 2 R. Sanchez-Varo , R. Gomez-Gutierrez , S. Jimenez , V. Navarro , M.L. Vizuete , J.C. Davila , J. Vitorica , 1 A. Gutierrez 1 Faculty of Science - University of Malaga/ CIBERNED/IBIMA, Cell Biology, Malaga, Spain 2 Faculty of Pharmacy- University of Seville. CIBERNED. IBIS., Biochemistry and Molecular Biology Department, Seville, Spain Aims Previous works have demonstrated that neuroinflammatory response plays a key role in Alzheimer’s disease (AD) progression. Microglia, the main cell type of the innate immune system in the brain, exhibit activation in both patients and animal models; however, it is still unknown whether it is a cause or a consequence of AD. An inefficient immunologic response could trigger an increase of amyloid-beta levels with disease progression, so we aimed to investigate the effect of immunosuppression over the pathological progression in the hippocampus of APP/PS1dE9 transgenic mouse model. Method Cyclosporine (15 mg/kg) and prednisone (20 mg/kg) were intraperitoneally administered to 9 month-old APP/PS1dE9 mice for 3 months. Untreated mice were used as controls. Hippocampal amyloid burden, microglia and astroglia, cytokines production, tau phosphorylation and neurodegeneration were assessed by immunohistochemistry and molecular techniques (Western-blot and RT-PCR). Results The microglial marker Iba1 and some proinflammatory cytokines were found to be decreased by RT-PCR in the immunosuppressed mice compared to controls. Conversely, a significant increase in Abeta levels and the astroglial marker GFAP was detected. Moreover, the treatment negatively affected SOM and NPY subpopulations. Conclusion Immunosuppression treatment downregulated microglial population activity and accelerated not only amyloid pathology but also the neuronal degeneration in this APP/PS1 model. Deficiencies in the innate immune system with age could promote AD pathology and cognitive decline in patients. Therefore, regulating microglial activation signalling pathways might be considered as a therapeutical target for AD.


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Supported by FIS-PI15/00796 (AG), PI15/00957 (JV), and co-financed by FEDER funds from European Union.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0948 AMYLOID-BETA PLAQUE ASSOCIATED CELLULAR PLASTICITY IN ALZHEIMER'S DISEASE 1 1 1 2 2 3 4 M.S. Unger , J. Marschallinger , B. Klein , M. Johnson , A. Khundakar , M. Heneka , S. Couillard-Despres , 5 2 1 E. Masliah , J. Attems , L. Aigner 1 PMU Salzburg, Molecular Regenerative Medicine, Salzburg, Austria 2 Institute of Neuroscience Newcastle University, Ageing Research Laboratories, Newcastle upon Tyne, United Kingdom 3 University Hospital Bonn, Clinic and Polyclinic for Neurology, Bonn, Germany 4 PMU Salzburg, Institute of Experimental Neuroregeneration, Salzburg, Austria 5 University of California San Diego School of Medicine, Department of Neuroscience, San Diego, USA Aims Alzheimer´s disease (AD) is the most prevalent neurodegenerative disease in the Western world characterized by a progressive loss of cognitive functions leading to dementia. Besides others, AD is characterized by the formation of amyloid-beta plaques, which often co-localize with increased neuroinflammation, i.e. activation of brain resident glial cells, in particular microglia, as well as infiltrating peripheral immune cells. The function and interaction of peripheral immune cells with the brains microglia are so far not fully understood. By serendipity, we observed doublecortin (DCX; generally used as a marker for young immature neurons) positive cells located at sites of amyloid-beta plaques in various transgenic amyloid mouse models and in human AD specimen. Method Immunohistochemical fluorescence (IHC) staining was performed on human AD samples and free floating brain slices from transgenic amyloid mouse models. Sections were analysed by confocal laser scanning microscopy and APP-PS1 brain sections were immunogold stained for ultrastructure analysis by electron microscopy. Results Detailed IHC analysis of these cells in the APP-PS1 mouse model demonstrated that a fraction of the plaque+ + + associated DCX cells showed co-expression of markers for microglia (Iba1 ), while the Iba1 negative DCX cells + + were highly positive for the pan-leukocyte marker CD45. These DCX /CD45 cells express the classical T-cell + marker CD3 and ultrastructure analysis revealed a close association of CD3 cells with the brains resident microglia. Conclusion


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It will be essential to uncover the functional interaction between these two cell types, as peripheral derived immune cells and their interaction with microglia might be a target for future therapeutic approaches in AD.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0055 THROMBIN ENHANCED MATRIX METALLOPROTEINASE-9 EXPRESSION AND MIGRATION OF SK-N-SH CELLS VIA ACTIVATION OF PROTEIN TYROSINE KINASES, ERK1/2 AND AP-1 1 2 C.M. Yang , C.C. Yang 1 Chang Gung University, Pharmacology, Taoyuan, Taiwan 2 Chang Gung Memorial Hospital at Lin-Kou, Department of Traditional Chinese Medicine, Tao-Yuan, Taiwan Aims Neuroinflammation is a hallmark of neurodegenerative disorders in the CNS. Thrombin has been shown to be a regulator of matrix metalloproteinase (MMPs) expression leading to cell migration, neuroinflammation, and neuronal death. The elevated expression of MMP-9 has been observed in various brain diseases, which may contribute to neuroinflammatory and neurodegenerative diseases. However, the mechanisms underlying thrombin-induced MMP-9 expression in SK-N-SH cells were not completely understood. Method We used gelatin zymography, Western blot, real-time PCR, promoter activity assay, and cell migration assay to demonstrate that thrombin induced the expression of pro-form MMP-9 protein and messenger RNA (mRNA), and promoter activity in SK-N-SH cells, coupled by using the selective pharmacological inhibitors and transfection with siRNAs. Results We demonstrated that the expression of thrombin-induced pro-form MMP-9 protein and mRNA, and promoter activity in SK-N-SH cells were attenuated by pretreatment with the pharmacological inhibitor of protease-activated receptor-1 (PAR-1, SCH79797), Gi-coupled receptor (GPA2), c-Src (PP1), Pyk2 (PF431396), EGFR (AG1478), PI3K (LY294002), Akt (SH-5), MEK1/2 (U0126), or AP-1 (Tanshinone IIA) and transfection with small interfering RNA (siRNA) of PAR-1, Gi, c-Src, Pyk2, EGFR, Akt, p44, p42, or c-Jun. Moreover, thrombin-stimulated c-Src, Pyk2, EGFR, Akt, p42/p44 MAPK, or c-Jun phosphorylation was attenuated by their respective inhibitor of PP1, PF431396, AG1478, SH-5, U0126, or Tanshinone IIA. Finally, pretreatment with these inhibitors also blocked thrombin-induced SK-N-SH cell migration. Conclusion


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Our results concluded that thrombin binding to PAR-1 receptor activated Gi-protein/cSrc/Pyk2/EGFR/PI3K/Akt/p42/p44 MAPK cascade, which in turn elicited AP-1 activation and ultimately evoked MMP-9 expression and cell migration in SK-N-SH cells.

A01.c. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1114 BLOCKADE OF ALPHA4 INTEGRINS AMELIORATES COGNITIVE DYSFUNCTION AND NEUROPATHOLOGICAL CHANGES IN TRANSGENIC ANIMALS WITH ALZHEIMER'S-LIKE DISEASE 1 1 1 1 1 1 1 1 E. Zenaro , G. Piacentino , E. Pietronigro , V. Della Bianca , T. Carlucci , S. Dusi , R. Nagarajan , G. Iannoto , 1 1 1 M. Bonani , T. Saatchi , G. Constantin 1 Università di Verona, Medicine, Verona, Italy Aims Our recent data have shown that neutrophils infiltrate the brain of subjects with Alzheimer’s disease (AD) and that neutrophils contribute to the induction of cognitive deficits and neuropathological changes in transgenic mouse models of AD. Leukocyte trafficking mechanisms during AD are largely unknown and our goal was to determine the role of alpha4 integrins in disease pathogenesis. Method We performed two-photon laser scanning microscopy (TLPSM) studies to visualize and analyze leukocytevascular interactions in the brain of 3xTg-AD mice. Contextual fear conditioning and Y maze tests and neuropathological studies were performed to analyze the effect of alpha blockade on disease development. Results Using TLPSM we observed that blockade of apha4 integrins prevents neutrophil and Th1 cell adhesion in cortical venules and extravasation in the brain of 3xTg-AD mice. Treating 3xTg-AD mice with an anti-alpha4 integrin antibody improved memory compared to control mice in both Y maze and contextual fear conditioning tests. Notably, restoration of cognitive function in mice with temporary anti-alpha4 treatment during early disease was maintained also at later time points in aged animals, suggesting a long-term beneficial effect on cognition in mice with alpha4 blockade. Furthermore, neuropathological studies showed a reduction of amyloid beta deposition, tau hyperphosphorylation and microglial activation in mice with alpha4 blockade compared to animals treated with a control antibody. Conclusion


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Antibodies anti-alpha4 integrin are currently used for the treatment of patients with autoimmune diseases and our data suggest that blockade of alpha4 integrins may represent a novel therapeutic approach in AD.

A01.d. Disease Mechanisms, Pathophysiology: Synaptic plastcity & synapse pathology ADPD7-0722 ALTERATIONS OF SYNAPTIC MORPHOLOGY AND PLASTICITY IN A MOUSE MODEL OF ALZHEIMER'S DISEASE 1 2 3 3 1 1 1 4 A. Androuin , B. Potier , U.V. Nägerl , D. Cattaert , I. Youssef , C. Duyckaerts , K.H. El Hachimi , A. Triller , 2 1 1 P. Dutar , B. Delatour , S. Marty 1 Institut du Cerveau et de la Moelle épinière, INSERM U1127- CNRS UMR7225- Université Pierre et Marie Curie, Paris, France 2 Centre de Psychiatrie et Neurosciences, INSERM UMR 894- Université Paris Descartes- Sorbonne Paris Cité, Paris, France 3 Centre de Neurosciences Intégratives et Cognitives, CNRS UMR 5228, Bordeaux, France 4 Ecole Normale Supérieure- Institut de Biologie de l'ENS, INSERM- CNRS- PSL Research University, Paris, France Aims A loss of synapses, correlated with cognitive decline, is observed from the early stages of Alzheimer’s Disease (AD). The mechanism of this loss remains unknown. In a mouse model of AD (APPxPS1-Ki) accumulating betaamyloïd, we observed a loss of synapses in the CA1 stratum radiatum at 4 months that precedes a loss of neurons at 6 months. However, a deficit in spatial memory was detected before the onset of synapse loss. The aim of this study was to analyze changes in the morphology and function of synapses that occur before synapse loss, and could be responsible for the early memory deficit. Method We performed serial section electron microscopy, electrophysiology and modeling study to characterize early synaptic alterations. Results As early as 3 months of age, we showed in the CA1 stratum radiatum modifications of dendritic spine morphology. An increased proportion of spines without head (sessile spines), associated with a decrease in their length and an enlargement of their neck, was observed. Similar alterations of spine morphology were observed in AD biopsies presenting a loss of synapses, with an increased fraction of sessile spines and a widening of spine necks. Despite maintenance of basal parameters of synaptic transmission, alterations of spine morphology in APPxPS1-Ki were associated with deficits in the induction of long-term potentiation. Conclusion


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The change in the morphology of dendritic spines indicates an alteration of the actin cytoskeleton, which could be responsible for the deficit in LTP.

A01.d. Disease Mechanisms, Pathophysiology: Synaptic plastcity & synapse pathology ADPD7-0923 SENSORY EXPERIENCE RESTRICTS SYNAPSE NUMBER THROUGH NEURONAL INDUCTION OF FN14 1 2 1 1 1 3 1 L. Cheadle , L. Burkly , C. Tzeng , B. Kalish , S. Rivera , C. Chen , M. Greenberg 1 Harvard Medical School, Neurobiology, Boston, USA 2 Biogen, Neuro/Immunology, Cambridge, USA 3 Boston Children's Hospital, Neurology, Boston, USA Aims Sensory experience drives the refinement of neural circuits during critical periods of postnatal brain development. Disruption of this process contributes to neurodevelopmental disorders, yet molecular mechanisms linking experience with synaptic pruning are largely unknown. Inappropriate activation of synapse pruning may contribute to cognitive impairment in neurodegenerative diseases, such as Alzheimer’s disease. Thus we sought to identify molecular regulators of synaptic refinement. Method The retinogeniculate circuit was used to study refinement because the visual experience-dependent critical window is well characterized, and the level of visual activity easily and non-invasively manipulated in vivo by dark rearing. Whole transcriptome RNA-sequencing was performed on the lateral geniculate nucleus (LGN) upon visual stimulation following dark rearing during the critical period. Results Visual experience induced immediate early genes such as c-Fos, Npas4, and Egr1 within one hour of stimulation, as expected. Gene programs induced at later time points were enriched for intercellular signaling modules, innate immune genes, and genes associated with neurodevelopmental disorders. Among these candidate regulators of synaptic refinement, we focused on the tumor necrosis factor receptor superfamily member Fn14 based on its high degree of induction following visual stimulation, its selective expression in excitatory relay neurons, and its known roles in brain diseases such as neuropsychiatric lupus and multiple sclerosis. Analysis of Fn14 knockout mice revealed an increase in the number of retinal synapses maintained at the end of the critical period, consistent with a deficit in synaptic pruning. Conclusion


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These ongoing studies indicate a novel Fn14-mediated signaling mechanism of synaptic pruning.

A01.d. Disease Mechanisms, Pathophysiology: Synaptic plastcity & synapse pathology ADPD7-1798 INTERACTION OF TRKA WITH APP IN NGF-TARGET NEURONS IS MODULATED DURING CELL DEATH 1 2 2 2 2 3 4 2 2 N. Canu , I. Pagano , L.R. LaRosa , M. Pellegrino , D. Mercanti , V. Triaca , V. Sposato , C. Petrella , F. Moretti , 2 5 2 4 M.T. Ciotti , I. Maruyama , A. Levi , P. Calissano 1 Univeristy of Rome Tor Vergata, Department of System Medicine- Section of Physiology, Rome, Italy 2 Consiglio Nazionale delle Ricerche, Institute of Cell Biology and Neurobiology, Rome, Italy 3 EBRI Foundation, European Brain Institute, Rome, Italy 4 EBRI Foundation, European Brain Research Institute, Rome, Italy 5 Okinawa Institute of Science and Technology Graduate University, Information Processing Biology Unit-, Okinawa, Japan Aims The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in normal but not in Alzheimer’s disease (AD) brain tissue. It has not been reported whether the two proteins interact directly, and if so, which domains are involved. Clarifying these points will increase our understanding of the role and regulation of the TrkA/APP interaction in normal brain functioning as well as in AD

Method HEK293 cells expressing full length and mutant constructs of APP and TrkA were assessed for mapping the domains involved in TrkA/APP association by co-immunoprecipitation and Western blot analysis. Bimolecular fluorescence complementation was used for direct visualization of APP/TrkA complex and for evaluating the involvement of shared partners in favoring APP/TrkA complex. Proximity ligation assay was used to detect the complex and its modulation by several agents in primary septal neurons Results Exogenously expressed APP and TrkA associate through their juxtamembrane/transmembrane domains to form a complex, insensitive to cation chelation, that localizes to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Formation of the complex does not require p75NTR, ShcC or Mint-2. The association between endogenous APP and TrkA in primary septal neurons was modified by NGF, by drugs that either inhibit ER-toGolgi transport or perturb microtubules and microfilaments. Interestingly, several agents that induce cell death albeit via different mechanisms, all caused dissociation of APP/TrkA complexes, formation of p75NTR/APP complex and increased production of β‐CTF APP fragment

Conclusion


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Our findings open new perspectives for investigating the interplay between APP and TrkA during neurodegeneration and AD

A01.d. Disease Mechanisms, Pathophysiology: Synaptic plastcity & synapse pathology ADPD7-1693 A PROTEIN ENRICHED IN UMBILICAL CORD PLASMA IS NECESSARY AND SUFFICIENT TO REVITALIZE HIPPOCAMPAL FUNCTION IN AGED MICE 1 1 1 1 2 1 1 3 3 J. Castellano , K. Mosher , R. Abbey , A. McBride , M. James , D. Berdnik , J. Shen , B. Zou , X.S. Xie , 4 1 4 1 M. Tingle , I. Hinkson , M. Angst , T. Wyss-Coray 1 Stanford University, Neurology and Neurological Sciences, Palo Alto, USA 2 Stanford University, Radiology, Palo Alto, USA 3 AfaSci Research Labs, N/A, Redwood City, USA 4 Stanford University, Anesthesiology, Palo Alto, USA Aims Aging drives changes in neuronal and cognitive function, the decline of which is a major feature of Alzheimer’s disease. Exposure to young blood counteracts key aspects of age-related decline, yet the identity of cognitionpromoting factors or whether they exist in human plasma remains unknown. We hypothesized that umbilical cord plasma represent a reservoir of “rejuvenating” factors that revitalizes synaptic function. Method We provide the first characterization of a human plasma transfer model using immunodeficient mice as a tool to screen blood-borne rejuvenating activities. Conservation between mouse and human plasma candidates was achieved using protein microarrays on hundreds of plasma proteins, which we followed with functional assays in vivo. Results Cord plasma improved synaptic and cognitive function compared to plasma treatment with older human donors. Coupling these analyses with array-identified candidates in vivo uncovered blood-borne plasticity-promoting factors, including CSF2, previously shown to improve cognitive function in a mouse model of beta-amyloidosis. We further reveal that systemic TIMP2 is sufficient to enhance synaptic plasticity and cognitive performance in aged mice and is necessary in young mice for normal hippocampal-dependent memory. Depletion experiments reveal that TIMP2 is necessary for the hippocampal-dependent cognitive improvements conferred by cord plasma. We further demonstrate a loss of TIMP2 expression in the blood of APP-transgenic mice, reminiscent of its systemic deficiency we observed in normal aging. Conclusion


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Together, our results suggest that blood-borne cues modulate the brain’s response to aging and its susceptibility to synaptic failure, perhaps providing novel targets for the development of therapies targeting neurodegeneration associated with AD.

A01.d. Disease Mechanisms, Pathophysiology: Synaptic plastcity & synapse pathology ADPD7-0259 ASTROCYTES ARE INVOLVED IN AΒ-INDUCED ALTERATIONS OF SYNAPTIC AND EXTRASYNAPTIC PTYR1472-GLUN2B IN PRIMARY HIPPOCAMPAL CELLS 1 1 2 1 1 L. Chang , Y. Zhang , J. Liu , Y. Song , Y. Wu 1 Capital Medical University, anatomy, Beijing, China 2 Tsinghua University, School of Medicine, Beijing, China Aims Astrocytes are the most abundant glia cell type in the brain. They are viewed as key elements in synaptic transmission, memory formation and neuronal communication. Astroglial failure may aggravate cognitive decline in Alzheimer’s disease (AD). Synaptic dysfunction during early stages of AD is triggered by soluble amyloid-b (Aβ) oligomers that interact with NMDA receptors. Our previous study had found Aβ exerts complex and distinct regulatory effects on the trafficking and phosphorylation of GluN2A and GluN2B, as well as on their localization within synaptic and non-synaptic sites. What specific roles astrocytes could play in this process are still unclear. Method In the present study, the immunofluorescence staining was applied. Results We revealed that Aβ induced the similar decreases in dendritic or extrasynaptic pTyr1325-GluN2A puncta densities, but quite different alterations of synaptic and extrasynaptic pTyr1472-GluN2B, between pure primary hippocampal neuron culture (NE-S, mainly containing neurons) and mixed culture system (MIX-S, mainly containing neurons and astrocytes). In MIX-S, after Aβ treatment for 1h or 24h, the puncta densities of synaptic pTyr1472-GluN2B decreased significantly, while that of extrasynaptic pTyr1472-GluN2B did not change; in NE-S, synaptic pTyr1472-GluN2B increased with 24h Aβ treatment, but extrasynaptic pTyr1472-GluN2B decreased significantly after Aβ treatment for either 1h or 24h. Conclusion


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This Aβ-induced distinct alterations of NMDA receptor subunits between NE-S and MIX-S suggest astrocytes play a critical role in neuronal communication through regulating phosphorylation of GluN2, especially GluN2B. The precise mechanisms need further investigation.

A01.d. Disease Mechanisms, Pathophysiology: Synaptic plastcity & synapse pathology ADPD7-0942 THE PERIPHERAL PENTRAXIN SERUM AMYLOID P COMPONENT CAN ENTER THE ALZHEIMER’S BRAIN AND INTERACT WITH NEURONAL PENTRAXINS, MODULATING GLUTAMATE RELEASE 1 1 1 1 1 2 3 D.M. Cummings , T.A. Benway , H. Ho , A. Tedoldi , M.M. Fernandes Freitas , L. Shahab , C.E. Murray , 3 1 1 T. Lashley , D.A. Salih , F.A. Edwards 1 Univeristy College London, Neuroscience- Physiology & Pharmacology, LONDON, United Kingdom 2 Univeristy College London, Department of Epidemiology and Public Health, LONDON, United Kingdom 3 Univeristy College London Institute of Neurology, The Queen Square Brain Bank for Neurological DisordersDepartment of Molecular Neuroscience, LONDON, United Kingdom Aims Neuronal pentraxin 1 (NPTX1), NPTX2 and NPTX receptor influence central glutamatergic synapses by clustering AMPA receptors. The liver also secretes pentraxins, including serum amyloid P component (SAP), possibly entering the brain under conditions that compromise the blood-brain barrier, including Alzheimer’s disease. Here we observe additional presynaptic effects of pentraxins in the hippocampus and interactions between SAP and NPTX1. Method Patch clamp recordings in hippocampal organotypic slice cultures from wild type mice. Immunohistochemistry: antibodies against neuronal pentraxins and SAP; Aβ1-40 and Aβ1-42. Results We confirm that amyloid plaques in brain sections from AD patients stain for both NPTX1 and SAP.Thus, both pentraxins are present in the CNS and interact with plaques. We show that SAP can form complexes with NPTX family members when they are co-expressed in HEK293 cells, suggesting that SAP could interact with neuronal pentraxins. Application of nanomolar concentrations of each human pentraxin over 7 days reduced paired-pulse ratios at CA3-CA1 synapses. This indicates an increase in glutamate release probability, similar to that observed in APPswe+PSEN1M146V mice (Cummings et al. 2015 Brain 138:1992-2004). In contrast, acute application of SAP or NPTX1 to naive slices increased paired-pulse ratio, suggesting compensatory effects in response to prolonged exposure. The effects of co-application of SAP and NPTX1 were not additive, implicating a common pathway of action. Conclusion


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Both neuronal and peripheral pentraxins affect release of glutamate. SAP may alter synaptic transmission if it enters the brain, particularly under conditions of blood-brain barrier breakdown as may occur in AD or in acute inflammatory conditions.

A01.d. Disease Mechanisms, Pathophysiology: Synaptic plastcity & synapse pathology ADPD7-0792 NOVEL FACTOR REGULATING AGE RELATED COGNITIVE DECLINE AND AMYLOID LOAD IN ALZHEIMER’S DISEASE 1 F. Deak 1 University of Oklahoma Health Sciences Center, Geriatric Medicine, Oklahoma City, USA Aims The primary cause of cognitive impairment in the elderly is Alzheimer’s disease (AD). Amyloid production is known to strongly correlate with synaptic activity and destruction of synapses is the most consistent finding in AD brain pathology. However, the regulation of specific pathological steps leading to excessive amyloid production and secretion are unknown; the absence of key mechanistic data on molecular pathways for these processes impedes the development of effective therapies. As the SNARE complex is a key regulator of exocytosis and synaptic transmission we designed a study to test its role in cognition and AD etiology. Method We used the synaptobrevin1 knock-out mouse strain and Tg2576 mice, a model of AD carrying human APP with the Swedish mutation for behavioral, spatial learning and brain circuitry analysis. Further analysis was conducted on cultured hippocampal neurons using fluorescence imaging assays of synaptic function. Results Using heterozygous syb1 KO mice we have found that the lower levels of synaptobrevin1, a SNARE protein significantly decreases beta-amyloid production but detected no changes in spatial learning and synaptic neurotransmission. In aged Tg2576 mice the detrimental effect of amyloid load on neuronal function was markedly decreased and improved with chronic reduction of syb1 levels. Conclusion


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We propose that synaptobrevin1 is a key coupling factor between synaptic release and amyloid production. Based on these new results, we propose the synaptobrevin1 driven amyloidogenic pathway as a possible therapeutic target and a novel approach to improve cognitive function in the patients suffering from Alzheimer’s disease.

A01.d. Disease Mechanisms, Pathophysiology: Synaptic plastcity & synapse pathology ADPD7-0674 THE ROLE OF MITOCHONDRIA ASSOCIATED ER MEMBRANES (MAM) IN PRESYNAPTIC VESICLE RELEASE IN ALZHEIMER´S DISEASE PATHOLOGY 1 1 1 1 1 1 1 G. Dentoni , N.S. Leal , C.M. Pinho , B. Schreiner , B. Wiehager , M. Lindskog , M. Ankarcrona 1 Karolinska Institutet, NVS, Huddinge, Sweden Aims Structural and functional connection between mitochondria and ER, known as mitochondria associated ER 2+ membranes (MAM), regulate a variety of important processes in the cell such as Ca handling and apoptosis. In Alzheimer´s disease (AD), the aforementioned functions appear to be disrupted, along with dysfunctional MAM 2+ dynamics. Even though it is known that Ca shuttling from ER to mitochondria is important for synaptic activity, limited evidence exists on ER-mitochondria interplay in synapses. In this study, we report that synaptic vesicles (SV) are associated with MAM. Additionally, we seek to assess how modulation of ER-mitochondria contacts along with A-beta-load, affects presynaptic release mechanisms. Method C57BL6/J mice brains were homogenised, subcellular fractionation prepared and desired protein levels analysed with western blot. 21-24 DIV mouse primary hippocampal neurons were used for proximity ligation assay (PLA) to assess proximity of MAM to SV components. Results Subcellular fractionation experiments reveal that SV proteins are present in the enriched MAM fraction. In addition, we report a substantial amount of proximity (T (p.S115S), affecting splicing of exon 4 is responsible for X-linked Parkinsonism with Spasticity (XPDS). XPDS brain features 4R tauopathy in the striatum. Herein, we report studies on a family in which two brothers presented with intellectual disability, epilepsy and parkinsonism. Both brothers and their mother carry an intronic variant c.168+6T>A predicted to affect ATP6AP2 splicing. Our study addressed 1) effect of the c.168+6T>A on ATP6AP2 RNA splicing and global gene expression; 2) effect of different mutations in ATP6AP2 on autophagy. Method We established LCLs from c.168+6T>A and c.345C>T carriers. RNA expression and splicing in whole blood and LCL was profiled by RNA-Seq and qRT-PCR. Differential gene expression was analyzed by Gene Set Enrichment Analysis (GSEA). Autophagic flux in patients’ LCLs was analyzed by flow cytometry. Results We found that the c.168+6T>A causes exon 2 skipping that results in a frame-shift and production of a nonfunctional transcript. The amount of full-size protein-coding transcript was significantly diminished. GSEA of whole blood RNA of patients with c.168+6T>A identified differentially expressed gene signatures as a result of the mutation. Analysis of LCLs with c.345C>T and c.168+6T>A did not reveal substantial inhibition of the autophagic flux. Conclusion


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A spectrum of neurodegenerative conditions manifesting with PD-like movement abnormalities and 4-repeat tauopathy is caused by mutations that alter transcript level/balance of splice isoforms of the essential ATP6AP2 gene.

B01.f. Disease Mechanisms, Pathophysiology: Lysosomes, ubiquitin, proteasome, ER stress ADPD7-0513 ENDOCYTOSED EXOSOMES REQUIRE FUSION WITH LYSOSOMES IN ORDER TO INDUCE TAU INCLUSIONS IN THE CYTOPLASM OF RECIPIENT CELLS 1 2 J.C. Polanco , J. Götz 1 Queensland Brain Institute, Clem-Jones Centre for Ageing Dementia Research CJCADR, Brisbane, Australia 2 Queensland Brain Institute, Clem Jones Centre for Ageing Dementia Research CJCADR, Brisbane, Australia Aims 1) To track endocytosis of tau-containing exosomes during the induction of tau aggregation in tau biosensor cells. 2) To analyse whether the endosomal membrane has a role in tau aggregation. 3) To determine whether endocytosed exosomes fuse with lysosomes during tau aggregation. Method Exosomes were isolated from the brain extracellular space (Perez-Gonzalez, JBC 2012) of P301L tau-expressing rTg4510 mice (Santacruz, SCIENCE 2005). Cell-culture experiments were performed using tau biosensor cells (Holmes, PNAS 2014). Membranes of exosomes and plasma membrane of cells were labelled with lipid intercalating fluorescent dyes. Endocytosis of exosomes was tracked using confocal microscopy and standard immunofluorescence. Results We recently reported that only cells undergoing intracellular tau aggregation can release exosomes containing tau seeds, with the ability to induce tau misfolding and aggregation in naïve tau biosensor cells (Polanco, JBC 2016). Here, the plasma membrane of tau biosensor cells was tracked during endocytosis of exosomes using different fluorescent tags. The membranes of endocytic vacuoles appeared intact during the formation of exosomeinduced tau inclusions. Fusion with lysosomes occurred during the formation of tau inclusions and did not halt the tau-aggregation process. Therefore, we tested in several ways whether lysosome function was required for the induction of tau aggregation. First, overexpression of RAB7, required for fusion of late endosomes with lysosomes, strongly increased tau aggregation. Second, RAB7 knockdown decreased tau aggregation, indicating that the fusion with lysosomes strengthens tau aggregation. Furthermore, alkalinisation of lysosomes also showed a negative effect on tau aggregation. Conclusion


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Lysosome function is required for tau aggregation induced by exosomes.

B01.g. Disease Mechanisms, Pathophysiology: Mitochondrial dysfunction, oxidative damage ADPD7-0931 TAU DELETION BLOCKS STRESS-TRIGGERED DENDRITIC AND SYNAPTIC ATROPHY IN PREFRONTAL CORTEX: A ROLE FOR SYNAPTIC MITOCHONDRIA 1 1 1 1 2 2 1 I. Sotiropoulos , S. Lopes , C. Dioli , J. Silva , O.F.X. Almeida , M. Filiou , N. Sousa 1 ICVS Institute - Life and Health Sciences School, University of Minho, Braga, Portugal 2 Max Planck Institute of Psychiatry, NeuroAdaptations Group, Munich, Germany Aims Tau protein in dendrites and synapses has been recently implicated in synaptic degeneration and neuronal malfunction. Chronic stress, a well-known inducer of neuronal/synaptic atrophy, triggers hyperphosphorylation of Tau protein and cognitive deficits. However, the cause-effect relationship between these events remains to be established. Method To test the involvement of Tau in stress-induced impairments of cognition, we investigated the impact of stress on cognitive behavior and neuronal structure as well as on the synaptic proteome in the prefrontal cortex (PFC) of Tau knock-out (Tau-KO) and wild-type (WT) mice. Results Whereas exposure to chronic stress resulted in atrophy of apical dendrites and spine loss in PFC neurons and significant impairments in working memory in WT mice, such changes were absent in Tau-KO animals. Quantitative proteomic analysis of PFC synaptosomal fractions, combined with transmission electron microscopy analysis, suggested a prominent role for mitochondria in the mediation of the effects of stress. Specifically, chronically stressed animals exhibit Tau-dependent alterations in the levels of proteins involved in mitochondrial transport and oxidative phosphorylation as well as in the synaptic localization of mitochondria in PFC. Conclusion


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These findings provide evidence for a causal role of Tau in mediating stress-elicited neuronal atrophy in PFC and related cognitive deficits and indicating that stress and Tau may exert its synaptotoxic effects through mitochondria.

B01.h. Disease Mechanisms, Pathophysiology: Lipids, lipoproteins and membrane trafficking ADPD7-0178 APOE4 MARKEDLY EXACERBATES TAU-MEDIATED NEURODEGENERATION IN A MOUSE MODEL OF TAUOPATHY 1 2 3 4 5 1 1 6 1 1 Y. Shi , K. Yamada , S. Liddelow , S. Smith , L. Zhao , K. Wang , J. Roh , G. Robinson , M.B. Finn , H. Jiang , 7 8 9 7 4 3 5 1 S. Maloney , P. Sullivan , M. Wood , D. Wozniak , O. Butovsky , B. Barres , S. Paul , D. Holtzman 1 Washington University in St. Louis, Neurology, St.Louis- MO, USA 2 The University of Tokyo, Department of Neuropathology, Tokyo, Japan 3 Stanford University, Department of Neurobiology, Stanford, USA 4 Harvard Medical School, Brigham and Women's Hospital, Boston, USA 5 Weill Cornell Medical College of Cornell University, Appel Alzheimer's Disease Research InstituteFeil Family Brain and Mind Research Institute, New York, USA 6 University of Missouri School of Medicine, Department of Ophthalmology, Columbia, USA 7 Washington University in St. Louis, Department of Psychiatry, St. Louis, USA 8 Duke University, Department of Medicine, Durham, USA 9 AstraZeneca, R&D, Wilmington, USA Aims APOE4 is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). While ApoE4 increases brain Aβ pathology relative to other APOE isoforms, whether APOE independently influences tauopathy or taumediated neurodegeneration, a critical feature in AD, is not clear. Tau accumulation, in contrast to Aβ, strongly correlates with brain atrophy and cognitive deficits in AD. Several lines of evidence suggest direct interactions between ApoE and tau. This study aims to investigate how human ApoE isoforms vs. the absence of ApoE affect tau pathology and neurodegeneration. Method We generated P301S tau transgenic mice on either a human ApoE knockin(KI) or ApoE knockout (KO) background. We performed biochemical, histological, behavioral, and gene expression analyses to comprehensively assess the effect of ApoE. Results P301S/E4 mice had significantly higher soluble tau levels at 3 months of age compared to P301S/E2 and P301S/E3 mice. The elevated tau levels persisted at 9 months of age, but transited into a more insoluble state. At 9 months age, P301S/E4 mice had significantly more severe brain atrophy and neuronal loss. Concomitantly, P301S/E4 mice displayed massive gliosis, strong activation of neurodegenerative microglial genes, and neuroinflammation-associated A1 type and pan-reactive astroglial genes. P301S/EKO mice had similar tau levels as P301S/E2 and P301S/E3 mice, but were protected from neuroinflammation-related changes, neuronal loss, and brain atrophy. Conclusion


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ApoE directly regulates tau-related pathogenenic changes in P301S mice independent of Aβ. ApoE4 causes significantly higher tau accumulation and strikingly more severe brain atrophy. This may result from ApoEdependent neuroinflammation induced by tau pathology.

B01.i. Disease Mechanisms, Pathophysiology: Glial cells ADPD7-0366 INTERNALIZATION OF TAU BY MICROGLIA IN TAUOPATHIES 1,2 1,2,3 1,2 1,2 4 1,2,3 1,2 M. Bolós , M. Llorens-Martín , J. Jurado-Arjona , J.R. Perea , A. Rábano , F. Hernández , J. Avila 1 Centro de Biología Molecular Severo Ochoa, Molecular Neurobiology Function of microtubular proteins in neurons, Madrid, Spain 2 Centro de Biología Molecular "Severo Ochoa" CSIC-UAM, Nicolás Cabrera 1, 28049-Madrid, Spain 3 Department for Molecular Biology, Faculty of Sciences, Universidad Autónoma de Madrid (Spain) 4 Neuropathology Department, CIEN Foundation, Madrid, Spain Aims The microtubule-associated protein (MAP) Tau plays a critical role in the pathogenesis of tauopathies. Excess Tau can be released into the extracellular medium in a physiological or pathological manner to be internalized by surrounding neurons-a process that contributes to the spread of this protein throughout the brain. Such spreading may correlate with the progression of the abovementioned diseases. In addition to neurons, Tau can be internalized into other cells. Objective: Study the internalization of Tau by microglia. Method We have used primary microglia cultures, mouse model of tauopathies and human tissue derived from AD patients. Results Here we demonstrate that microglia take up Tau in vitro and in vivo. In this regard, microglia from primary cultures internalized Tau42 in vitro. Furthermore, using stereotaxic injection of Tau in mice in vivo, we show that murine microglia internalize human Tau. In addition, we demonstrate, for the first time, that microglia colocalize with various forms of Tau in postmortem brain tissue of patients with Alzheimer's disease and non-demented control subjects. Finally, some preliminary data regarding the mechanism of Tau internalization by microglia are shown. Conclusion


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Our data reveal a potential role of microglia in the internalization of Tau that might be relevant for the design of strategies to enhance the clearance of extracellular Tau in neurodegenerative diseases characterized by the accumulation of this protein.

B01.i. Disease Mechanisms, Pathophysiology: Glial cells ADPD7-1036 RELATIONSHIP BETWEEN TPPP/P25 IMMUNOREACTIVITY AND TAU OR ALPHA-SYNUCLEIN PATHOLOGY IN NEURODEGENERATIVE PROTEINOPATHIES WITH OLIGODENDROGLIAL INVOLVEMENT 1,2 3 3 1,2 3 Z. Rohan , I. Milenkovic , M. Lutz , R. Matej , G. Kovacs 1 Thomayer Hospital, Department of Pathology and Molecular Medicine, Prague 4, Czech Republic 2 First Faculty of Medicine of Charles University in Prague, Institute of Pathology, Prague, Czech Republic 3 Medical University of Vienna, Institute of Neurology, Vienna, Austria Aims To assess oligodendroglial response using the oligodendroglia-specific marker, TPPP/p25, and its correlation with tau and alpha-synuclein oligodendroglial inclusion pathology in neurodegenerative proteinopathies. Method Formalin-fixed, paraffin-embedded brain samples from multiple system atrophy (MSA; n=10), Lewy body diseases (LBD; n=10), globular glial tauopathy (GGT; n=7) and progressive supranuclear palsy (PSP; n=10) cases were evaluated for oligodendroglial response, white matter damage and inclusion pathology using antibodies against TPPP/p25, alpha-synuclein, tau, ubiquitin, myelin basic protein (MBP) and the microglia-marker HLA-DR. Results MSA and GGT were distinguished from PSP and LBD by the combination of significant loss of nuclear TPPP/p25 immunoreactivity, its increased accumulation in oligodendroglial cytoplasm and co-localization with alphasynuclein- and tau-immunoreactive inclusions and degeneration of white matter. In LBD cases, consistent presence of alpha-synuclein-immunoreactive oligodendroglial inclusions in the pallidothalamic tract was observed. TPPP/p25 co-localized more with alpha-synuclein than tau in inclusion bodies. Conclusion


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MSA a GGT can be linked together based on common features as revealed by TPPP/p25 immunostaining patterns. Together these features thus define and allow to group GGT and MSA as predominant oligodendroglial proteinopathies. Our novel finding of consistent presence of alpha-synuclein oligodendroglial cytoplasmic inclusions in the pallidothalamic tract in LBD cases suggests new role of oligodendroglia and white matter degeneration in the pathogenesis of LBDs. Our study raises awareness of the importance of oligodendroglial response in various dominant or nondominant oligodendroglial neurodegenerative proteinopathies.

B01.k. Disease Mechanisms, Pathophysiology: Vasculature, angiogenesis ADPD7-1591 ALTERED TRANSCRIPTION OF HYPOXIA AND ANGIOGENESIS GENES IN AGED TAU OVEREXPRESSING MICE 1 1 1 1 1 1 R. Bennett , A. Robbins , M. Hu , S. DeVos , S. Hopp , B. Hyman 1 Massachusetts General Hospital/ Harvard Medical School, Neurology, Charlestown, USA Aims Previously, we have observed an increase in the density and tortuosity of blood vessels in aged Tg4510 mice which overexpress tau. We hypothesized that this increased density was due to aberrant angiogenesis and was contributing to a state of chronic hypoxia. Method To test this hypothesis, we performed qPCR arrays on 84 hypoxia and angiogenesis-related genes from Tg4510 (n=3) and wild-type (n=3) mice at 15 months of age. In separate cohorts, we then dissociated brains from aged mice and used magnetic bead separation to isolate endothelial cells (CD31-positive), microglia (CD11b-positive), astrocytes (ASCA-2 positive) and neurons. Purity of cell-type specific preparations was performed by further qPCR analysis. Changes in gene expression identified by arrays were verified by western blot. Results Of the 84 putatively hypoxia associated genes that were assessed, seven were significantly upregulated and one was significantly downregulated (p0.99 and 0.88, respectively). Altogether, GBA CNVs occurred in 2.5% of Afrikaner participants in our cohort. Conclusion


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GBA CNVs are present in the Afrikaner population. An increase in GBA copies could also increase the enzymatic activity, which might be beneficial. Harboring an additional functional GBA gene can potentially ameliorate the effect of deleterious mutations and should be considered during genetic counseling and diagnostic testing in PD and GD.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-0006 TCTEX1 PLAYS A KEY ROLE IN THE Α-SYNUCLEIN AUTOPHAGY LYSOSOMAL DEGRADATION PATHWAY IN SH-SY5Y CELLS 1 Z. Cai 1 Affiliated Lianyungang Hospital of Xuzhou Medical College, Lianyungang, China Aims Tctex1 is an important structure of dynein light chain in mammalian cells, helping move targets toward the centrosome for degradation. Clarifying the role of Tctex1 in α-synuclein autophagy lysosomal degradation may offer insights into the formation of Lewy bodies and neuronal death. Method We constructed dsRED-N1-TcTex-1 overexpression, pDsRED2-N1-TcTex-1(T94E) mutation and Tctex1 siRNAvector and transfected into SH-SY5Y cells. Cell viability was detected by WST. Effect of Tctex1 siRNA on cell apoptosis was detected by flow cytometry. Relative protein expression of Tctex1、α-tublin、LC3II、α-synuclein and LAMP2 were measured by Western Blot. mRNA levels of α-synuclein、LC3-II and Tctex1 were measured by real-time quantitative PCR. Confocal microscopy was used to observe expression of Tctex1、α-tublin、LC3II、αsynuclein、LAMP2 and their sublocalizations in cells. Results 1. WST assay results show that cell activity decreased after Tctex1 mutation (T94E) and siRNA-Tctex1; Tctex1 overexpression increased cell activity. 2. Flow cytometry results show apoptosis increased after siRNA-Tctex1 interference. 3. In Tctex1 mutation and siRNA-Tctex-1 transfected cell lines Tctex1 and dynein protein levels decreased; α-synuclein, LC3-II and LAMP2 protein increased. However, α-synuclein, LC3-II and LAMP2 proteins were reduced in Tctex1 overexpression cell lines, with the same trend was found in mRNA levels. 4. In Tctex1 mutation and siRNA-Tctex1 transfected cell lines Tctex1 fluorescence intensity weakened; α-synuclein, LC3-II and LAMP2 fluorescence was enhanced. Conclusion


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Tctex1 mutants and siRNA-Tctex-1 interference lead to Tctex1 downregulation and dysfunction. Tctex-1 overexpression promoted autophagy lysosome fusion and effectively degraded α-synuclein with increased cell activity. Thus, Tctex-1 plays an important role in α-synuclein autophagic degradation and maintaining cell homeostasis.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-0116 TCTEX1 PLAYS A KEY ROLE IN THE Α-SYNUCLEIN AUTOPHAGY LYSOSOMAL DEGRADATION PATHWAY IN SH-SY5Y CELLS 1 1 1 1 Z. Cai , X. Zhang , J. Li , Y. Zhang 1 Affiliated Lianyungang Hospital of Xuzhou Medical College, Neurology, Lianyungang, China Aims Tctex1 is an important structure of dynein light chain in mammalian cells, clarifying the role of Tctex1 in αsynuclein autophagy lysosomal degradation may offer insights into the formation of Lewy bodies and neuronal death. Method We constructed dsRED-N1-TcTex-1 overexpression, pDsRED2-N1-TcTex-1(T94E) mutation transfected into SHSY5Y cells. Cell viability was detected by WST. Relative protein expression of Tctex1、α-tublin、LC3II、αsynuclein and LAMP2 were measured by Western Blot. mRNA levels of α-synuclein、LC3-II and Tctex1 were measured by real-time quantitative PCR. Confocal microscopy was used to observe expression of Tctex1、αtublin、LC3II、α-synuclein、LAMP2 and their sublocalizations in cells. Results 1. WST assay results show that cell activity decreased after Tctex1 mutation (T94E), while Tctex1 overexpression increased cell activity. 2. In Tctex1 mutation transfected cell lines, Tctex1 and dynein protein levels decreased; αsynuclein, LC3-II and LAMP2 protein increased. However, α-synuclein, LC3-II and LAMP2 proteins were reduced in Tctex1 overexpression cell lines, with the same trend was found in mRNA levels. 3. In Tctex1 mutation transfected cell lines, Tctex1 fluorescence intensity weakened; α-synuclein, LC3-II and LAMP2 fluorescence was enhanced. Fluorescence intensity of α-synuclein, LC3-II and LAMP2 weakened in Tctex1 over-expressing cells. Conclusion


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Tctex1 mutant’s interference lead to Tctex1 downregulation and dysfunction. Although autophagy (LC3-II levels) increased, targets did not relocate to perinuclear by Tctex-1, they fused with lysosomes, resulting in α-synuclein degradation disorder and cell activity decrease. Tctex-1 overexpression promoted autophagy lysosome fusion and effectively degraded α-synuclein with increased cell activity. Thus, Tctex-1 plays an important role in αsynuclein autophagic degradation and maintaining cell homeostasis.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-0655 ATP13A2 DEFICIENCY INDUCES LOSS OF DOPAMINERGIC NEURONS IN TRANSGENIC MICE EXPRESSING MUTANT A53T HUMAN ALPHA-SYNUCLEIN 1 1 1 2 2 M. Chiam , B.J. Turner , M.K. Horne , J.P. Liu , H. Li 1 University of Melbourne, Florey Department of Neuroscience and Mental Health, Parkville Victoria, Australia 2 University of Melbourne, Murdoch Childrens Research Institute, Parkville Victoria, Australia Aims Parkinson’s disease (PD) is characterized by alpha-synuclein aggregation which may reflect autophagy and lysosomal dysfunctions. Recently, PD familial genes such as ATP13A2 has been suggested to converge on autophagy pathways and therefore alpha synuclein metabolism.This study aims to investigate the interrelationship between ATP13A2 deficiency and alpha-synuclein accumulation. Method Novel ATP13A2 KO mice were crossed with transgenic human alpha-synuclein A53T mice to generate four genotypes for analysis: A53T/ATP13A2 KO (cross), A53T, ATP13A2 KO and wild-types. Brain regions including cortex, cerebellum and olfactory bulb were analysed for a-synuclein accumulation, autophagic and endocytic marker levels using Western blotting. Brain sections were analysed for nigral neuron and terminal counts using immunohistochemistry and stereology.

Results Cross mice revealed a significant 25 percent loss of dopaminergic phenotype in the substantia nigra at 9 months, compared to A53T Tg, ATP13A2 KO and WT mice. There was a corresponding 15 percent increase in dopaminergic terminals in the striatum of cross mice at 9 months, compared with wild types. Interestingly, detergent-soluble alpha-synuclein levels were similar in olfactory bulb, cortex and cerebellum of 9-month-old cross and A53T Tg mice. Protein expression levels of autophagy marker (p62) and endocytic marker (Rab11) were downregulated in a similar pattern in cross and A53T Tg mice as compared to WT mice. Conclusion


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ATP13A2 deficiency triggers loss of dopaminergic phenotype in nigral neurons and compensatory sprouting of dopaminergic terminals in the striatum. Importantly, it occurs without accumulation of soluble alpha-synuclein, suggesting that these phenomena are independent of soluble alpha-synuclein metabolism.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-1635 CHARACTERIZATION OF THE ΑLPHA-SYNUCLEINOPATHY IN GLUCOCEREBROSIDASE KNOCK-OUT MICE 1 2 3 4 3 2 G. Deangeli , M. Wegrzynowicz , A. Migdalska , P. Nilsson , A.H.V. Schapira , M.G. Spillantini 1 Scuola Superiore Sant'Anna, Instiute of Life Sciences, Pisa, Italy 2 University of Cambridge, Department of Clinical Neurosciences, Cambridge, United Kingdom 3 UCL Institute of Neurology, Department of Clinical Neurosciences, London, United Kingdom 4 Linköping University, Department of Chemistry, Linköping, Sweden Aims Glucocerebrosidase (GBA) is a lysosomal enzyme, which degrades glucocerebrosides, neuronal membrane components. Its functional impairment is causative for Gaucher’s disease. Such impairment also leads to chronic lysosomal stress, and thereby to a reduced α-synuclein clearance, possibly bringing about an α-synucleinopathy. In this project, heterozygous 24-month-old GBA knock-out mice were used to characterize such αsynucleinopathy. Method Preliminarily, the project aimed to optimize the methods to highlight the α-synucleinopathy. An antibody against phosphorylated α-synuclein was used to characterize a well-established model of progressive α-synucleinopathy: the pre-formed fibril inoculated mice. Several experimental fluorophores that bind β-sheet-rich proteins were tested on the same model. One of them, namely HS84, stained Lewy body/neurite-like structures in a highly specific way, with far less background noise compared to antibodies, and to the gold-standard fluorophore pFTAA. Results Different staining techniques were applied to GBA KO mice. Firstly, the α-synuclein staining revealed a massive accumulation, which appeared eminently at presynaptic level, and not at perinuclear or somatic level. Such pattern is typical of early stages of α-synucleinopathy. Secondly, the HS84 was used as a marker of large aggregates. However, no KO-specific aggregate could be observed in any brain region. Finally, GFAP staining was performed, to look for astrocytosis. Nevertheless, the astrocyte distribution did not show any obvious difference between KOs and WTs. The astrocyte counting in the hippocampus did not result in a significant difference either. Conclusion


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All the evidences confirm that the α-synucleinopathy is present in GBA KO mice, yet it is still at an early stage.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-1012 DIFFERENT MOLECULAR PATHWAYS THAT INDUCE AUTOPHAGY PROTECT AGAINST ALPHASYNUCLEIN INDUCED TOXICITY 1 N. Fussi 1 German Center for Neurodegenerative Diseases DZNE, Translational Neurodegeneration, München, Germany Aims Investigation of the protective potential of different autophagy activators against alpha-synuclein induced toxicity in Lund human mesencephalic (LUHMES) cells. In autophagy, protein structures or whole organelles are engulfed by a double layer membrane, building the autophagosome, which is thereafter transported to and fused with a lysosome, to form the autolysosome, in which degradation takes place. Pharmacological compounds can promote autophagy through different cellular pathways (e.g. mTOR dependent and mTOR-independent). Method Overexpression of alpha-synuclein was achieved by transduction with adenoviral vectors in LUHMES cells, which are immortalized human dopaminergic progenitor cells that can be differentiated to human postmitotic neurons. Treatments with different autophagy activators were started 24 hours after virus transduction. LDH release in the culture medium was performed to measure cytotoxicity levels. Furthermore the presence of autophagosomes was monitored by LC3II western blots. Results Different compounds modulating autophagy via the mTOR dependent or the mTOR independent pathway led to an induction of autophagy, visualized by LC3II Western blots. Overexpression of alpha-synuclein with adenoviral vectors led to ~50% cell death in LUHMES cells. The autophagy activating compounds protected against alphasynuclein induced toxicity, regardless of the intracellular pathway. Conclusion


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Different autophagy modulators rescued LUHMES neurons from alpha-synuclein induced cell death. However, we found that the pathway of pharmacological intervention was not relevant and the cells could be protected by mTOR dependent and independent pathways.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-1171 NANODISCS FORMATION BY SYNUCLEIN VARIANTS 1 P. Kumari 1 ETH Zurich, CHAB, Zurich, Switzerland Aims The essential physiological function of synuclein is still unknown but sequence similarity with apo-lipoprotein suggests it can interact with membranes and form nanodiscs to perform various function. Lose of formation of nanodiscs by synuclein mutants can also be related to diseases. In this study we tried to find the propensity of nanodiscs formation by disease-linked PD (Parkinson's Disease) mutants. What are the factors which increase the amount of nanodisc formation. Method Recombinant synuclein Protein was expressed in E.Coli and purified by using Ion exchange and hydrophobic chromatography. Nanodiscs were purified by size exclusion chromatography. To characterise the morphology of nanodisc negative stain electron microscopy has been done. Results All three variants of synuclein (alpha,beta and gamma) showed sequence similarity with apo-lipoprotein Secondary structure prediction showed high helix formation for gamma synuclein followed by beta synuclein and alpha synuclein. Nanodiscs formation was more for more helical variants, which suggested the increase in helicity may increase the nanodiscs amount. Different variant of Synuclein showed different propensity to form nanodiscs. Variants with more helicity has formed more nanodisc formation. Morphology of nanodiscs also showed some difference in the size as well as in shape which suggests a different mechanism of nanodiscs formation by synuclein varients. Conclusion


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Increase in the helix propensity increases the nanodiscs formation, disease-linked PD mutations change the nanodisc amount in the similar way. Decrease and increase in the amount of nanodiscs can be related with lose and gain of function. Further studies can be important by revealing the effect of nanodiscs on the aggregation pathway of synuclein.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-1909 A NEURONAL MODEL OF PARK20 (SYNJ1 MUTATION) USING PATIENT DERIVED IPSCS 1 1 1 1 2 3 1 R. Masius , M. Minneboo , S. Gordillo Sampedro , L. Stockmann , M. Piccillo , P. Barone , V. Bonifati , 1 W. Mandemakers 1 Erasmus MC, Clinical Genetics, Rotterdam, The Netherlands 2 University of Naples “Federico II”, Department of Neurological Sciences, Naples, Italy 3 University of Salerno, Department of Medicine and Surgery, Salerno, Italy Aims The SYNJ1 homozygous mutation (p.Arg258Gln) leads to juvenile Parkinsonism (PARK20). SYNJ1 plays an important role in synaptic vesicle cycling, and macroautophagy. The p.Arg258Gln mutation impairs the Sac1 domain phosphatase activity of SYNJ1 protein (Krebs et al, 2013). To investigate the effects of this mutation on the molecular disease mechanisms we have developed in vitro models for of PARK20 and control differentiated neurons and small molecule induced dopaminergic (iDA) neurons, derived from SYNJ1 p.Arg258Gln patient and unrelated control induced Pluripotent Stem Cells (iPSCs). Method SYNJ1 Patient derived and control iPSCs were differentiated for 3 with small molecules or 8 weeks with growth factors resulting in iDA and mixed neurons. After differentiation, cells were probed for markers for synaptic vesicle recycling pathways (i.e. clathrin, endophilin) using immunocytochemistry and Western blotting techniques. Macroautophagy markers, were also examined with and without induction of autophagy (i.e. LC3B, WIPI2). Expression levels were quantified using FIJI software. Results Beta-3 Tubulin positive neurons are present after both 3 and 8 weeks of differentiation. Furthermore Tyrosine Hydroxylase expressing neurons could be detected after 3 weeks of small molecule treatment both in control and patient. Immunohistochemistry analysis showed increased amount of WIPI2 positive vesicles in patient derived neurons as compared to control neurons before and after starvation. Conclusion


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Our preliminary data indicate that the SYNJ1 p.Arg258Gln mutation affects the autophagy pathway, indicated by increased levels of WIPI2 expression in patient derived neurons. Further research is needed to clarify the role of SYNJ1 in autophagy and synaptic vesicle cycling.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-1384 GLUCOCEREBROSIDASE DEFICITS IN BRAINS OF PARKINSON’S DISEASE AND DEMENTIA WITH LEWY BODIES PATIENTS SCREENED FOR GBA VARIANTS 1 2 1 3 3 2 4 4 T. Moors , S. Paciotti , A. Ingrassia , M. Quadri , G. Breedveld , A. Tasegian , D. Chiasserini , P. Eusebi , 5 5 4 3 2 1 G. Duran-Pacheco , T. Kremer , L. Parnetti , V. Bonifati , T. Beccari , W. van de Berg 1 VU University Medical Center, Anatomy and Neurosciences, Amsterdam, The Netherlands 2 University of Perugia, Department of Pharmaceutical Sciences, Perugia, Italy 3 Erasmus Medical Center, Department of Clinical Genetics, Rotterdam, The Netherlands 4 University of Perugia, Department of Medicine- section Neurology, Perugia, Italy 5 Roche Innovation Center- F. Hoffmann-La Roche Ltd, Roche Pharmaceutical Research and Early Development, Basel, Switzerland Aims The present study aims to characterize changes in activities and mRNA expression of lysosomal enzymes, including β-glucocerebrosidase (GCase), in selectively vulnerable brain regions of patients with Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB) compared to controls, screened for GBA variants. Method Enzymatic activities of GCase, β-hexosaminidase and cathepsin D (CathD) were measured using fluorimetric assays in frontal cortex (FC), putamen and substantia nigra (SN) of patients with PD, DLB and age-matched controls (n=15/group). Additionally, mRNA expression of GBA and some of its interactors, including LIMP-2, was measured using qPCR. The GBA genomic region was Sanger sequenced for all donors. Results GCase activity was decreased (-20%) in the SN, and CathD activity (-15%) in the FC, of PD and DLB patients compared to controls. Expression of GBA, but not of its interactors, was decreased in the PD SN (-24%). In total, 6 GBA variants, among which Asp140His and p.Leu444Pro, were detected. Population stratification based on genetic results showed substantially lower GCase (~-40%) and CathD (~-30%) activities in GBA-variant carriers compared to controls in all measured regions and in the FC, respectively. Furthermore, GCase activity was significantly lower (-18%) in the SN of PD and DLB patients without GBA variants compared to controls. Conclusion


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Our results show decreased GCase activity and mRNA expression in the brain of PD and DLB patients with and without GBA variants. Alterations were less pronounced for other lysosomal enzymes or GBA interactors. Our results indicate a key role for GCase in GBA-variant-related and nonrelated PD and DLB.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-0793 DISSECTING THE DEGRADATION PATHWAY INVOLVED IN PLK2-MEDIATED Α-SYNUCLEIN SELECTIVE TURNOVER 1 A. Oueslati 1 Centre de recherche du CHU de Québec, Molecular Medecine, Québec, Canada Aims Converging lines of evidence support the central role alpha-synuclein (α-syn) accumulation and toxicity in the Parkinson’s disease (PD) pathogenesis. This abnormal α-syn accumulation is triggered by its gene duplications/triplications or by the impairment of its degradation. Therefore, lowering α-syn protein levels may represent a viable therapeutic strategy for the treatment of PD and related disorders. However, the question on how α-syn is eliminated in vivo has yielded controversial results. In a recent study, we described a new selective α-syn autophagic degradation pathway (Oueslati et al., 2013). This process is governed by PLK2 kinase activity, α-syn phosphorylation at Ser129 and α-syn/PLK2 protein-protein interaction. Interestingly, activation of this degradation pathway reduced α-syn protein levels and mitigates its toxicity in a rat model of PD (Oueslati et al., 2013). These results suggest that PLK2-mediated α-syn turnover may offer a unique opportunity to modulate, in a selective manner, α-syn turnover and reduce its toxicity in vivo. Nevertheless, the exact molecular mechanisms underlining this cellular process remain unexplored.

Method In the present study, we sought to further investigate the molecular determinants of PLK2-mediated α-syn turnover in vivo, using a mammalian cell lines transiently overexpressing α-syn syn and PLK2 and by combining a battery of pharmacological and genetic approaches. Results Our results provide a detailed description of the new degradation route responsable for α-syn selective turnover. Conclusion


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In conclusion, our study provides new insight into the molecular mechanisms of α-syn elimination and offers new opportunities for the development of strategies aiming to reduce its accumulation and toxicity in vivo.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-0284 PROTEASOMAL PSMC1 ATPASE HETEROZYGOSITY CAUSES CELL CYCLE DEFECTS IN MOUSE EMBRYONIC FIBROBLASTS BUT NOT IN MOUSE BRAIN 1,2 N. Rezvani 1 University of Maryland School of Medicine, Neurology, Baltimore, USA 2 Nottingham University, Molecular Biology and Neuroscience, Nottingham, United Kingdom Aims The ubiquitin proteasome system (UPS) is a fundamental cellular pathway, degrading most unwanted intracellular soluble proteins. Dysfunction of the UPS has been associated with normal aging as well as various age-related pathological conditions, including chronic human neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, leading to a significant interest in the involvement of this degradative system in neurons. The 26S proteasome is essential for neuronal homeostasis and survival in the mouse brain following conditional genetic homozygous knockout of Psmc1. The aim of this study was to show the effect of Psmc1 heterozygosity on cell cycle in mouse embryonic fibroblasts (MEFs) and mouse brain. Method Cell culture, 26S proteasome purification, Protein immunoblotting, Histopathology, Cell cycle analysis Results Neuropathologically and biochemically, Psmc1 heterozygous (Psmc1(+/-)) knockout mice were indistinguishable from wild-type mice. However, we report a novel age-related accumulation of intraneuronal lysine 48-specific polyubiquitin-positive granular staining in both wild-type and heterozygous Psmc1 knockout mouse brain. In Psmc1(+/-) MEFs, we found a significant decrease in PSMC1 levels, altered 26S proteasome assembly and a notable G2/M cell cycle arrest that was not associated with an increase in the cell cycle regulatory protein p21. The disturbance in cell cycle progression may be responsible for the growth inhibitory effects in Psmc1(+/-) MEFs. Conclusion


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Heterozygosity for the proteasomal Psmc1 ATPase is insufficient to cause neuropathology in mouse brain, but causes cell cycle defects in mouse embryonic fibroblasts

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-1527 ANALYSIS OF THE LYSOSOMAL DYSFUNCTION IN THE PATHOGENESIS OF PARKINSON’S DISEASE 1 1 2 2 2 3 3 1 C. Ruz-Illescas , S. Bandrés-Ciga , F.J. Barrero , J. Gutiérrez , A. Mínguez , C. Gallo , M. Luna , F. Vives , 1 R. Durán 1 Institute of Neurosciences “Federico Olóriz”- Centre of Biomedical Research CIB, Department of PhysiologyUniversity of Granada, Armilla, Spain 2 Movement Disorders Unit- University Hospital “Health Campus”, Neurology Service, Granada, Spain 3 Centre of Biomedical Research, Department of Physiology- University of Granada, Granada, Spain Aims Lysosomes play a key role in protein clearance but also in catabolism of glucosphingolipids. Several studies have demonstrated that lysosomal dysfunction is a central event in neurodegenerative diseases such as Parkinson’s disease (PD). Here, we aimed to study the contribution of the lysosome to the pathogenesis of PD through the analysis of a group of lysosomal enzymes in peripheral blood from sporadic PD patients. Moreover, we explored their impact on protein and lipid catabolism and cognitive decline. Method 31 PD patients and 29 age-matched healthy controls were initially subjected under study.We first analysed the beta-glucocerebrosidase (b-GBA) activity in white blood cells according to van Dijk et al. (2013). We also assessed the b-GBA protein levels by western-blot. Statistical analysis was performed using the SPSS software. Continuous variables were presented as means (±standard deviations) and medians (ranges). Categorical variables were presented as count. We used Mann–Whitney U test for comparison between groups. Results Activity levels of b-GBA were significantly lower in PD patients compared to healthy controls (PD group: 10.44 ± 3.42 nmol/min/mg; healthy control group: 12.00 ± 4.22 nmol/min/mg; p value = 0.038). In addition, they were correlated with protein concentration in both groups. Further analyses of other lysosomal enzymes in an increased population are currently on course. Conclusion


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Although we are currently adding to this study new subjects and enzymes, our preliminary data support a defective lysosomal activity in PD. These results encourage us to develop new and efficient pharmacological approaches aimed to restore the impaired lysosomal enzyme activities.

C01.d. Disease Mechanisms, Pathophysiology: Autophagy, lysosomes, ubiquitin, proteasome ADPD7-0935 EFFECTS OF THE MODULATION OF GLUCOCEREBROSIDASE ACTIVITY IN EX VIVO AND IN VIVO MODELS OF PARKINSON’S DISEASE 1 1 1 1 1 F. Siani , C. Ghezzi , S. Cerri , J. Saiani , F. Blandini 1 C. Mondino National Neurological Institute, Laboratory of Functional Neurochemistry, Pavia, Italy Aims Heterozygous mutations in the GBA1 gene, encoding lysosomal enzyme glucocerebrosidase (GCase), increase the risk of developing Parkinson’s disease (PD). In this study we investigated the effects of GCase reduced activity, obtained using the specific inhibitor conduritol-ß-epoxide (CBE) to: 1) identify conditions differentiating GBA1-mutant patients; 2) identify synergistic interactions with PD-inducing toxin 1-methyl-4-phenyl-1,2,3,6tetraidropriridine (MPTP). Method Ex vivo experiments were conducted in fibroblasts from PD patients, with or without GBA1 mutations, and healthy controls. Cells were treated with increasing concentrations of CBE (10nM to 10µM) for 7 days and GCase activity and levels of GCase-related lysosomal proteins saposin C and LIMP2 were measured. For the in vivo experiments we used a PD-like murine model based on MPTP administration (30 mg/kg daily for 5 days) combined with CBE (1, 10, 100 mg/Kg/day, i.p.). Brain sections were processed for the immunohistochemical evaluation of nigrostriatal degeneration, α-Syn accumulation and microglia activation. Results CBE induced a dose-dependent decrease in GCase activity that differently affected the various groups. Fibroblasts from PD patients showed increased susceptibility to CBE, with those from GBA1-mutant patients showing dramatic GCase inhibition at a concentration (50nM) ineffective in the other groups. No further modifications were observed in saposin C and Limp2 levels. CBE also dramatically reduced brain GCase activity in MPTP-treated animals, showing synergistic interactions with the neurotoxin effects. Conclusion


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The fibroblasts study pointed out a selective susceptibility to CBE in patients with GBA1 mutation, while the in vivo study provided useful information on the role of GCase deficiency in the neurodegenerative process of PD.

C01.e. Disease Mechanisms, Pathophysiology: Lipids, lipoproteins and membrane trafficking ADPD7-0538 PARKINSON’S DISEASE-LINKED MUTATION IN DNAJC13 CAUSES SPECIFIC TRAFFICKING DEFECT IN ENDOSOMAL PATHWAY 1 1 2 1 1 3 4 1 T. Hasegawa , S. Yoshida , M. Suzuki , J. Kobayashi , A. Kikuchi , Y. Nagai , A. Takeda , M. Aoki 1 Tohoku University School of Medicine, Department of Neurology, Sendai, Japan 2 Tokyo Metropolitan Institute of Medical Science, Diabetic Neuropathy Project, Tokyo, Japan 3 Osaka University Graduate School of Medicine, Department of Neurotherapeutics, Osaka, Japan 4 Sendai Nishitaga Hospital, Department of Neurology, Sendai, Japan Aims Recently, missense mutations in DNAJC13/RME-8 gene have been identified in patients with PARK21 familial Parkinson’s disease (PD). DNAJC13 is the mammalian homolog of receptor-mediated endocytosis 8 (RME-8), a DnaJ domain-containing protein originally identified in a screen for endocytic defects in Caenorhabditis elegans. DNAJC13 localizes to the membrane of the endosomal system, where it plays a role in vesicle trafficking. The aim of this study is to investigate the effect of PD-linked mutant DNAJC13 on the vesicle transport and neurodegeneration process. Method COS7 cells were transfected with GFP-tagged DNAJC13 as well as mStrawberry-tagged Rab GTPase constructs. To determine how DNAJC13 mutant affect on the different endosomal pathway, cells over-expressing wt or mutant DNAJC13 were incubated in the culture media containing reference molecules including Alexa555® labeled transferrin and pHrodo EGF. Time-lapse images of internalized reference molecules were acquired using LSM. Fly culture and crosses were performed under standard conditions at 25 °C. The RME-8 RNAi fly lines were provided by the Vienna Drosophila RNAi Center. Results While wt-DNAJC13 was co-localized with rab5A-positive endosome, N855S mutant was co-localized with not only rab5A but Rab7 or Rab11A-positive endosome. Transport assay showed impaired cargo-trafficking from early endosome to late or recycling endosome in cells over-expressing N855S DNAJC13. Furthermore, mutant DNAJC13 expression resulted in abnormal endosomal retention of α-synuclein, a major constituent of Lewy body. RNAi-mediated knockdown of Drosophila RME8 promotes compound eye degeneration observed in transgenic flies overexpressing human wild-type α-synuclein. Conclusion


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PD-linked DNAJC13 mutation impairs specific endosomal trafficking and would thereby contribute to the pathogenesis of the disease.

C01.e. Disease Mechanisms, Pathophysiology: Lipids, lipoproteins and membrane trafficking ADPD7-1279 Α-SYNUCLEIN MEMBRANE INTERACTION AS A PARKINSON’S DISEASE TARGET 1 2 T. Amen , D. Kaganovich 1 Hebrew University of Jerusalem, Cell and Developmental Biology, Jerusalem, Israel 2 Hebrew University, Cell and Developmental Biology, Jerusalem, Israel Aims α-Synuclein is a membrane binding protein that is enriched in the brain and is linked to the membrane trafficking at the synapse. Mutations or overproduction of α-synuclein can trigger its uncontrolled aggregation, which is implicated in the pathology of Parkinson’s disease (PD). Interestingly, α-Synuclein membrane association appears to be critical for its function and the PD mutations described to date are situated in the N-terminal lipidbinding region of the protein. Recently, it has been shown in vitro that glycosphingolipids and cholesterol drive αsynuclein membrane insertion. Method We use the yeast model of α-synuclein aggregation to understand α-synuclein membrane association. Fullgenome screening and proteomics results indicate of non-protein mechanism regulating the association. Results We show that α-synuclein pronounced membrane association in the yeast S.cerevisiae is modulated solely by the lipid microenvironment. Blocking α-synuclein-lipid(sterol) interaction results in α-synuclein cytoplasmic aggregation. Modification of sterol levels in mammalian cells results in α-Synuclein redistribution to the membrane. Conclusion


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We demonstrate that α-Synuclein membrane association is regulated by lipid composition on the membrane in the living cell and is linked to α-Synuclein aggregation. Dynamic membrane association may underlie the physiological function of α-Synuclein in the synapse. We propose that cytoplasmic partners of α-synuclein ensure its solubility until the cell encounters changes in lipid composition, for example due to synaptic vesicle fusion with the plasma membrane. The change in the membrane-interaction kinetics due to the mutation in the lipid-binding region results in disrupted membrane trafficking. The overproduction of α-synuclein disrupts the ratio with its cytoplasmic binding partners leading to aggregation.

C01.e. Disease Mechanisms, Pathophysiology: Lipids, lipoproteins and membrane trafficking ADPD7-1201 DIFFERENTIAL MOBILITY SPECTROMETRY AS AN ORTHOGONAL SELECTIVITY TO LC-MS FOR QUANTIFICATION OF PLASMA CEREBROSIDES IN DEMENTIA WITH LEWY BODIES PATIENTS 1 1 H. Xu , S.A.L. Bennett 1 University of Ottawa, Biochemistry - Microbiology - Immunology, Ottawa, Canada Aims Cerebrosides, such as glucosylceramide and galactosylceramide, are the natural substrates of betaglucocerebrosidase and beta-galactocerebrosidase. Mutations in GBA1, encoding the lysosomal betaglucocerebrosidase, are the most common genetic risk factor for Parkinson’s disease. Differential quantification of isobaric glucosylceramide and galactosylceramide is particularly difficult due to their virtually identical structures. Therefore, methods allowing reliable quantification of monohexosylceramides are needed to better understand the disease mechanism with potential to identify biomarkers. Method Differential mobility separation (DMS) was performed using a SelexION DMS device coupled to a triple quadrupole-linear ion trap mass spectrometer QTRAP 5500 with a Turbo V ion source (AB SCIEX). DMS separation of isobaric monohexosylceramides was optimized using standards. A LC-DMS-ESI-MS method was then developed and validated for its capacity to separate and quantify isobaric monohexosylceramides. Results Isobaric monohexosylceramide species were sufficiently separated in the DMS cell with isopropanol as a gas phase chemical modifier under the optimized conditions. This method exhibits great quantitative performance, showing repeatability and precision with linear detection range of 2.8 – 355 nM. Lipidomic analysis using this method has revealed the presence of C16, C22, C23, C24:1, C24 glucosylceramide and galactosylceramide as well as C18, C20, C25 and C26 glucosylceramide in healthy human plasma with good quantification reproducibility. Conclusion


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SelexION DMS technology features orthogonal selectivity to LC-MS techniques and thus allows for the unambiguous assignment of cerebroside lipid species in biological samples. Our results show great promise for future applications of the DMS technology in the field of glycosphingolipid analysis for biomarker discovery and drug screening.

C01.f. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1839 CYTOKINE PROFILING IN THE PREFRONTAL CORTEX OF PARKINSON’S DISEASE AND MULTIPLE SYSTEM ATROPHY PATIENTS 1 2 1 1 1 3 1 1 S. Aznar , B. Elfving , M.D. Andersen , M.A. Langbøl , J. Folke , K. Winge , B. Pakkenberg , T. Brudek , 1 R. Rydbirk 1 Bispebjerg University Hospital - Copenhagen, Research Laboratory for Stereology and Neuroscience, Copenhagen, Denmark 2 Institute for Clinical Medicine- Aarhus University, Translational Neuropsychiatry Unit, Aarhus, Denmark 3 Bispebjerg University Hospital - Copenhagen, Department of Neurology, Copenhagen, Denmark Aims Parkinson’s Disease (PD) and Multiple System Atrophy (MSA) are characterized by alpha-synuclein accumulation in brain cells. This may induce neuroinflammation, which may participate in the pathogenesis behind MSA and PD. In PD patients, cytokines, the main inflammatory signalling molecules, have been identified in blood and cerebrospinal fluid, and neurotrophins, known to interact with cytokines and necessary for survival of brain cells, found to be altered in basal ganglia. Not much is known about cytokine or neurotrophin alterations in MSA brains. The aim of this study was to perform a thorough cytokine and neurotrophin profiling in cortical regions of PD and MSA brains. Method We measured protein levels of 18 cytokines and 5 neurotrophins in brain samples from the dorsomedial prefrontal cortex of MSA (n=19), PD (n=31) patients and a control group (n=17), using ELISA, Western blotting, and the Luminex xMAP® technology. Results In both PD and MSA, we found altered expression of the cytokines interleukin (IL)-2, IL-13, and granulocyte colony-stimulating factor, but no differences in neurotrophin levels. In MSA we identified increased mRNA levels of Glycogen Synthase Kinase 3-beta that is involved in neuroinflammatory pathways. Lastly, increased mRNA levels of the neurodegenerative marker S100 calcium binding protein beta, but not C-reactive protein, in PD and MSA, suggest inflammation to be local rather than systemic. There was further increased antigen-presentation indicative of reactive microglia in both diseases. Conclusion


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Neuroinflammatory states in both PD and MSA brains are present in areas distant to the disease epicentre and may precede neurotrophic changes.

C01.f. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1340 EXOSOMAL ALPHA-SYNUCLEIN MODULATES INNATE IMMUNITY AND POSSIBLY CONTRIBUTES TO INFLAMMATION IN PARKINSON'S DISEASE 1 2 3 4 4 4 3 1 V. Grozdanov , C. Meier , M. Hoffmeister , L. Bousset , L. Pieri , R. Melki , C. Behrends , J. Kassubek , 1 1 1 A.C. Ludolph , J.H. Weishaupt , K.M. Danzer 1 University Hospital Ulm, Department of Neurology, Ulm, Germany 2 University of Ulm, Department of Organic Chemistry, Ulm, Germany 3 University Hospital Frankfurt, Institute for Biochemistry II, Frankfurt am Main, Germany 4 Centre National de la Recherche Scientifique, Institut des Neurosciences Paris-Saclay, Gif-sur-Yvette, France Aims To investigate the inflammatory response of myeloid immune cells to extracellular alpha-synuclein oligomers and exosome-associated alpha-synuclein and assess its effects on alpha-synuclein aggregation. Method Peripheral blood monocytes from healthy controls and PD patients, human macrophages and murine microglia were stimulated with recombinant alpha-synuclein monomers, oligomers and fibrils. The immune response to exosome-associated alpha-synuclein was induced with exosomes from cell lines transiently overexpressing alpha-synuclein or with plasma exosomes from healthy controls and PD patients. Immune activation and cell polarization were measured with ELISA and qPCR, alpha-synuclein aggregation was measured using a protein complementation assay. Alpha-synuclein and exosome preparations were characterized by AFM, analytical ultracentrifugation, WB, nanoparticle analysis and mass spectrometry. Results We found that in addition to large extracellular aggregates, alpha-synuclein oligomers are a potent modulator of immune responses. Monocytes, macrophages and microglia are polarized to the pro-inflammatory M1 phenotype and secrete pro-inflammatory cytokines in response to stimulation with different species of alpha-synuclein oligomers. The activation of immune cells by alpha-synuclein oligomers is potentiated when oligomers are associated with exosomes. Furthermore, we show that vice versa the pro-inflammatory molecules can increase alpha-synuclein aggregation in vitro. Conclusion


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Exosome-associated alpha-synuclein is an important trigger for the activation of myeloid immune cells. Our findings strengthen the idea that extracellular alpha-synuclein-induced inflammation may be an important disease mechanism and add new insights to the non-cell-autonomous mechanisms of alpha synuclein toxicity.

C01.f. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1092 OLIGODENDROGLIAL ALPHA-SYNUCLEIN PATHOLOGY INITIATES NEUROINFLAMMATION IN MULTIPLE SYSTEM ATROPHY 1 1 2 3 1 A. Hoffmann , E. Benjamin , M. Elizier , R. Markus J. , W. Juergen 1 University Hospital Erlangen, Molecular Neurology, Erlangen, Germany 2 University of California San Diego, Department of Pathology- Department of Neuroscience, La Jolla, USA 3 Regensburg University Hospital, Department of Neuropathology, Regensburg, Germany Aims Multiple system atrophy (MSA) is a rare atypical parkinsonian disorder. Besides accumulation of alpha-synuclein (aSyn) in oligodendroglial cytoplasmic inclusions (GCIs) and myelin loss, MSA pathology is characterized by a profound microgliosis. Since the underlying pathomechanisms are still poorly understood, we hypothesized that there is an interplay of aSyn, oligodendrocytes and microglia. This study aims to characterize neuroinflammation in aSyn-related oligodendrogliopathy to identify potential interventional targets. Method We analyzed the number of microglial cells, their activation and proliferation in white (WMR) and gray matter regions (GMR) of MSA patients and in one of its transgenic mouse models (MBP29-ha-syn mice). Transcriptome analysis was performed in lentivirally transduced aSyn-overexpressing primary oligodendrocytes. Results The number of microglial cells was profoundly increased in the corpus callosum of MBP29-ha-syn mice. A moderate microgliosis was detected in the striatum, whereas no difference was present in the motor cortex. Similarly, a higher aSyn load was observed in the corpus callosum of MBP29-ha-syn mice in comparison to the motor cortex and striatum. Intriguingly, matching these findings in the MSA model, we detected an increased number of microglia associated with an increased aSyn density in WMR of MSA patients. RNA sequencing of aSyn-overexpressing oligodendrocytes revealed an increased expression of proinflammatory molecules such as CSF1 and CCL2. Conclusion


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aSyn-expressing oligodendrocytes are accompanied with a severe microgliosis predominantly present in WMR of the adult forebrain. These findings imply that there is an oligodendroglial-microglial crosstalk in which aSyn or oligodendrocytes with GCIs result in a widespread pro-inflammatory environment in MSA.

C01.f. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-1756 ANTI-VCAM-1 ANTIBODY PROTECTS DOPAMINERGIC NEURONAL LOSS THROUGH INHIBITION OF TRANSENDOTHELIAL MIGRATION. 1 2 3 3 2 4 G.R. Jeong , C.S. Park , A.R. Yoon , J. Chung , S. Lee , B.D. Lee 1 Kyung Hee University, Department of Neuroscience, Seoul, Republic of Korea 2 Scripps Korea Antibody Institute, Laboratory of Molecular Cancer Therapeutics, Chuncheon, Republic of Korea 3 Seoul National University College of Medicine, Department of Biochemistry and Molecular Biology, Seoul, Republic of Korea 4 Kyung Hee University, Department of Neuroscience- Department of PhysiologyNeurodegeneration Control Research Center, Seoul, Republic of Korea Aims Neuroinflammation is common pathological feature found in various neurodegenerative diseases. Therefore, treatment of anti-inflammatory agent has been suggested as a potential therapeutic application for alleviating the pathology of neurodegenerative diseases. In general, the brain is immunologically privileged site because peripheral immune cells are blocked by blood-brain barrier (BBB). However, in pathological condition, peripheral immune cells can infiltrate through altered BBB into the brain. Initial step of infiltration is the interaction between immune cell and endothelial cell, mediated by vascular cell adhesion molecule-1 (VCAM-1) on activated endothelial cells. In inflammatory diseases, inhibition of leukocyte binding to VCAM-1 or by inhibition of VCAM-1 signal transduction inflammation is blocked by antioxidants. Thus, we investigated whether blockage of immune cell infiltration through inhibition of VCAM-1 function can attenuate the pathology of neurodegenerative disease. Method The neuroinflammation was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), used for Parkinson’s disease animal model. To block the interaction between immune cell and endothelial cell, we used rabbit/human chimeric anti-VCAM1 antibody (VCAM-1-D6 IgG), specifically recognizes aa 511-599 within the sixth Ig-like domain. Results Firstly, we investigated whether VCAM-1 regulates transendothelial migration of leukocytes. The knockdown of VCAM-1 by siRNA in human brain microendothelial cells reduced the transendothelial migration ability of U937 cells. Furthermore, VCAM-1-D6 IgG blocked U937 cell transendothelial migration across hTNFa-treated human brain microendothelial cells. Next, VCAM-1-D6 IgG treatment significantly attenuated the MPTP-induced loss of tyrosine hydroxylase (TH) positive neuronal number and striatum density compared to Control IgG. Conclusion


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These findings suggest that blocking VCAM-1 might be used as therapeutic application for neurodegenerative disease.

C01.f. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0170 SYSTEMIC INFLAMMATION IN DEMENTIA WITH LEWY BODIES AND ALZHEIMER'S DISEASE 1 1 1 1 1 1 1 E. King , J. O'Brien , P. Donaghy , N. Barnett , K. Olsen , C. Martin-Ruiz , A. Thomas 1 Institute of Neuroscience, Campus for Ageing and Vitality, Newcastle upon Tyne, United Kingdom Aims Dementia with Lewy Bodies (DLB) is the second most common type of degenerative dementia. Whilst distinct from Alzheimer’s disease (AD) and Parkinson’s disease (PD), it shares many characteristics with both. Previous studies have shown that neuroinflammation plays a key role in the pathogenesis of both AD and PD. Many studies have also found raised systemic inflammatory markers in these conditions which appear to be associated with the disease progression. To our knowledge, the inflammatory profile in DLB has never before been investigated. This study therefore aims to characterise systemic inflammatory mediators in DLB and the prodromal Mild Cognitive Impairment (MCI) phase of DLB. Method We obtained plasma samples from patients in our clinical research studies with DLB (n=37), AD (n=20), MCI (n=80) and healthy comparison subjects (n=20). MCI patients included a mixture of MCI-AD (n=21), probable MCI-DLB (n=36) and possible MCI-DLB (n=18). Samples were stored at -80°C, and the following inflammatory biomarkers were measured: hsCRP, INF-gamma, IL-10, IL12-p70, IL-13, IL-1beta, IL-2, IL-4, IL-6, IL-8 and TNFalpha. Results The findings from the dementia and MCI groups will be presented at the meeting. Conclusion


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This study is the first to characterise the systemic inflammatory profile in patients with DLB, and we are also able to do so at the MCI stage. This will increase our understanding of the pathological inflammatory processes involved in this condition. Furthermore, raised inflammatory markers identified in this study may be utilised as potential targets in the future treatment of DLB.

C01.f. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0272 SYSTEMIC INFLAMMATION IN LEWY BODY DISEASES: A SYSTEMATIC REVIEW 1 1 E. King , A. Thomas 1 Institute of Neuroscience, Campus for Ageing and Vitality, Newcastle upon Tyne, United Kingdom Aims Few studies have investigated the role of inflammation in Lewy Body Dementia (LBD) and variable results have been found. The evidence of systemic inflammation in Alzheimer’s disease and the potential clinical importance that this could have on disease management for LBD led us to systematically review the literature for evidence of systemic inflammatory changes in Dementia with Lewy Bodies (DLB) and Parkinson’s Disease Dementia (PDD). However, due to the low numbers of studies available we also included Parkinson’s disease (PD) into our search. Method Key terms were used to search the relevant databases. Titles and abstracts were screened and potentially relevant articles were reviewed in full. References of included studies and relevant reviews were also searched. Results The database search returned 2166 results, 45 of which were included in the systematic review. These studies showed a general increase in inflammatory markers in the peripheral blood, most notably interleukin-1beta, tumor necrosis factor-alpha and interleukin-6. Studies examining cerebrospinal fluid (CSF) found interleukin-1beta, interleukin-6 and transforming growth factor-beta1 to be particularly increased, and interferon-gamma decreased. Overall C-reactive protein levels were increased, particularly in PDD. General cytokine levels of LBD compared to PD varied depending on the cytokine. Conclusion


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These results provide evidence that LBD is associated with an increased inflammatory response, but the difference in inflammatory markers between the blood and CSF warrants further investigation. Furthermore, it suggests that there may be a stronger general inflammatory response in LBD than in PD, whilst complex changes occur in the individual cytokines.

C01.f. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0345 TSPO RADIOLIGAND 18F-DPA-714 BINDING QUANTIFICATION AND CHARACTERIZATION IN NONHUMAN PRIMATES, HEALTHY HUMAN CONTROLS AND ALZHEIMER AND PARKINSON’S PATIENTS 1,2 3,4 3,4,5 1,2 1,2 2 4 4 S. Lavisse , B. Michel , H. Lorraine , I. Kentaro , J. Caroline , A.B. Romina , P. Marie-Ane , K. Bertrand , 5,6 1,2,7,8 1,2 S. Marie , R. Philippe , H. Philippe 1 CEA-DSV-I2BM, MIRCen, Fontenay aux Roses, France 2 CNRS-UMR9199- Universités Paris-Sud and Paris-Saclay, MIRCen, Fontenay aux Roses, France 3 Institut d’Imagerie Biomedicale- Direction des sciences du vivant- CEA, UNIACT- NeuroSpin, Gif-sur-Yvette, France 4 Laboratoire Imagerie Moleculaire In Vivo IMIV- CEA- Inserm- Univ Paris Sud- CNRS- Université Paris Saclay, CEA-SHFJ, Orsay, France 5 Centre de Psychiatrie et Neurosciences of Centre Hospitalier Sainte-Anne, INSERM-UMR S894, Paris, France 6 Unit of Memory and Langage, Université Paris Descartes Sorbonne, Paris, France 7 Paris-Est University, University, Créteil, France 8 Neurology Department- Centre Expert Parkinson, CHU Henri Mondor- AP-HP, Créteil, France Aims While increasing evidence suggests that neuroinflammation plays a crucial role in neurodegeneative diseases, the up-regulation of the translocator protein (TSPO) under inflammatory condition makes this protein an attractive target for PET imaging. Here we sought to evaluate the promising 18F-DPA-714 radiotracer in non-human primates, in healthy volunteers and in Alzheimer and Parkinson patients. Method Binding specificity of 18F-DPA-714 was evaluated in six cynomolgus primates by competition study using PK11195. A longitudinal imaging study was performed after quinolinate injection in the left putamen. Total distribution volume (VT) was estimated using the Logan plot. An immunohistochemistry analysis (IHC) was conducted using inflammatory and TSPO markers at different timepoints. In the ongoing clinical studies, 30 controls, 64 Alzheimer and 20 Parkinson’s patients have been enrolled. TSPO genotyping allowed the classification of all subjects into high and mixed affinity binders (HAB, MAB).The same outcome parameter VT was extracted for each studied region. Results PET competition studies in primates displayed a homogenous reduction of the intracranial activity compared to baseline. After lesion, VT increased in the ipsilateral side with a two-fold maximum at D21. IHC revealed an early microglial and delayed astrocytic activation with TSPO and CD68-positive-microglia co-localization. In all human subjects, VT in HABs was found significantly higher than in MABs and all patients display a higher binding compared to genotyped-matched controls. In Alzheimer patients, binding positively correlates with MiniMental State Examination scores and grey matter volume. Conclusion


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These preliminary results suggest that 18F-DPA-714 may be a suitable biomarker to study neuroinflammation longitudinally in patients affected by neurodegenerative diseases.

C01.f. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0619 ARE ASTROCYTES INVOLVED IN ANTIGEN PRESENTATION IN PARKINSON’S DISEASE? 1 2,3,4 5 1 1 2,3,4 1 J. Rostami , S. Holmqvist , J. Sigvardson , M. Ingelsson , J. Bergström , L. Roybon , A. Erlandsson 1 Uppsala University, Public health and caring sciences- Molecular geriatrics, Uppsala, Sweden 2 Lund University, Stem Cell laboratory for CNS Disease Modeling- Wallenberg Neuroscience CenterDepartment of Experimental Medical Science, Lund, Sweden 3 Lund University, Lund Stem Cell Center, Lund, Sweden 4 Lund University, Strategic Research Area MultiPark, Lund, Sweden 5 Bioarctic AB, Bioarctic AB, stockholm, Sweden Aims T-cell infiltration has been observed in the brain of patients with Parkinson disease (PD), but the mechanisms of T-cell activation remain unclear. Astrocytes, the most numerous cell type in the brain, respond to neurodegenerative disorders through a process referred to as reactive astrogliosis, in which they convert to an inflammatory state. Several studies have suggested that astrocytes may be involved in T-cell activation, as they express major histocompatibility complex class II (MHCII) and co-stimulatory molecules. The aim with this investigation was to clarify the role of astrocytes in antigen presentation and T-cell activation in PD. Method Human astrocytes, derived from embryonic stem cells were treated with Cy3-labeled oligomeric α-synuclein (αSYN) for 24 h. Following exposure, the cells were thoroughly washed and incubated for additional 0, 3 or 6 days in αSYN free medium. Using time lapse microscopy, immunocytochemistry and western blot analysis we investigated the change in expression and intracellular localization of MHCII following αSYN exposure. Results The human astrocytes engulfed large amounts of αSYN oligomers that entered the lysosomal pathway, but were not degraded. Instead, the αSYN accumulated in the trans-Golgi region. Interestingly, while the MHCII molecules were scattered throughout the cells in untreated astrocytes, they were redistributed to the Golgi region, following αSYN oligomer exposure.

Conclusion


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Our results demonstrate that the MHCII distribution in human astrocytes is altered by the presence of intracellular αSYN deposits. In ongoing studies we investigate if these cells are capable of presenting αSYN to T-cells.

C01.f. Disease Mechanisms, Pathophysiology: Inflammation ADPD7-0897 DIFFERENTIAL RESPONSE OF MICROGLIA TO DISTINCT ALPHA-SYNUCLEIN ASSEMBLIES 1 1 1 1 2 1 2 A. Van der Perren , G. Gelders , F. Macchi , W. Peelaerts , L. Bousset , C. Van den Haute , R. Melki , 1 V. Baekelandt 1 KU Leuven, Neurosciences, Leuven, Belgium 2 CNRS, Laboratoire d’Enzymologie et Biochimie Structurales, Gif-sur-Yvette, France Aims Misfolded protein aggregates are a critical aspect of several neurodegenerative diseases. There is emerging evidence that these protein aggregates can adopt distinct conformations characterized by noticeable differences in phenotypic features. The discovery of the prion-like transmissible nature of these proteins suggests a pathogenic trigger which propagate throughout the nervous system underlying the progression of the disease. In addition, there is mounting evidence that neuroinflammatory processes are closely linked to neurodegeneration during ageing. In the present study, we investigated the immunological properties of different alpha-synuclein assemblies to define the role of the innate immune system in Parkinson’s disease. Method We examined the microglia cell response to different recombinant alpha-synuclein assemblies via exogenous addition to primary microglia cultures. We studied uptake and degradation of the different alpha-synuclein assemblies in our primary microglial cultures by confocal microscopy and western blot. The microglial status was analyzed using qPCR and ELISA. Next, we added the microglial supernatant to primary neurons to investigate the impact of microglial priming by specific alpha-synuclein assemblies on neuronal toxicity. Results We showed that distinct fibrillar alpha-synuclein assemblies promote a pro-inflammatory activation of microglia, however differences between fibrillar assemblies could be observed. Oligomeric species, thought to be the primary species responsible for the disease, were unable to trigger the same cascades. Conclusion


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These results support our hypothesis that distinct fibrillar alpha-synuclein assemblies drive the pro-inflammatory activity of microglia and represent the toxic species contributing to the disease.

C01.g. Disease Mechanisms, Pathophysiology: Cellular signalling, kinases, phosphatases, calcium ADPD7-0108 TRPC3 PARTICIPATES IN Α-SYNUCLEIN-INDUCED ALTERATIONS IN APOPTOTIC ACTIVITY AND CELL CYCLE AND STAGE MARKER EXPRESSION IN MOUSE OLFACTORY NEURONS 1 1 1 J. Liu , G. Gao , H. Yang 1 capital medical university, neurobiology, beijing, China Aims Olfactory impairment is one of the earliest non-motor manifestations of Parkinson’s disease, which is accompanied by abnormal α-synuclein (α-syn) deposition in olfactory neurons. However, the mechanistic basis of this association is not well understood.To address this issue, we examined olfactory function in transgenic mice overexpressing human (h) α-syn in the olfactory bulb (OB) and olfactory epithelium (OE). Method Olfactory function of α-syn-overexpressing mice was measured by buried and surface pellet test. The localization of transient receptor potential canonical (TRPC) channel 3 in OB and OE were determined by immunocytochemistry, and its expressive level and activity were analysed by western blots. TUNEL staining were used for evaluating apoptosis, and cell stage markers measured by qRT-PCR to evaluate cell cycle. Results A decline in olfactory function was observed along with TRPC3 activated in the OB and OE. This was accompanied by apoptotic activity increasing and altered expressions of cell cycle and cell stage markers, including the upregulation of cell cycle marker (cyclin D1) and immature neuron markers (achaete-scute homolog 1, neurogenin 1, and growth-associated protein 43) as well as the downregulation of a mature neuron marker (olfactory marker protein). An increase activated TRPC3 was also detected in primary olfactory neurons overexpressing hα-syn. In addition, TRPC3 activation induced an abnormal elevation of intracellular 2+ Ca concentration, apoptosis induction, and changes of cell cycle markers similar to those observed in vivo, all of which were attenuated by TRPC3 knockdown. Conclusion


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Our results suggest that TRPC3 is involved in functional changes in olfactory neurons overexpressing α-syn.

C01.g. Disease Mechanisms, Pathophysiology: Cellular signalling, kinases, phosphatases, calcium ADPD7-0925 POLO-LIKE KINASE 2 REGULATES Α-SYNUCLEIN LEVELS IN CELLS AND BRAIN BUT NOT VIA AUTOPHAGY 1 1 1 1 2 3 1 R.H. Kofoed , N. Ferreira , J. Zheng , C. Betzer , M. Salvi , K. Fog , P.H. Jensen 1 Aarhus University, DANDRITE - Danish Research Institute of Translational Neuroscience- Dept. Biomedicine, Aarhus, Denmark 2 University of Padova, Department of Biomedical Sciences, Padova, Italy 3 Lundbeck A/S, Neurodegenerative research, Copenhagen, Denmark Aims Polo-like kinase 2 has been demonstrated to phosphorylate alpha-synuclein on serine129 and it has been hypothesised that PLK-2 drives autophagic degradation of alpha-synuclein based on studies using transgenic overexpression of PLK-2. We wanted to determine how endogenous PLK-2 regulates alpha-synuclein levels in cells and brain and characterize underlying mechanisms. Method Overexpression of recombinant PLK-2 and inhibition of endogenous PLK-2 using highly specific small molecule PLK-2 inhibitor. Cell lines, primary neurons and wild type mice treated orally with PLK-2 inhibitor. Biochemistry, cell biology, immunocytochemistry and immunohistochemistry. Results We confirmed overexpression of PLK-2 decreased alpha-synuclein in cells but this was not due to phosphorylation of serine129. Using the specific PLK-2 inhibitor we demonstrated that inhibition of PLK-2 causes increase in alpha-synuclein levels in cell lines, primary neurons and intact mouse brain but this was not due to decreased autophagic degradation of alpha-synuclein. The underlying mechanisms, which currently is being characterized, will be presented. Conclusion


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PLK-2 regulates alpha-synuclein levels but not via phosphorylation of alpha-synuclein and not via autophagy

C01.g. Disease Mechanisms, Pathophysiology: Cellular signalling, kinases, phosphatases, calcium ADPD7-1063 DYSREGULATION OF CALCIUM HOMEOSTASIS IN BRAIN NEURONS OF ZEBRAFISH WITH PINK1 MUTATION OBSERVED IN VIVO 1 1 1 2 J. Kuznicki , M. Bazała , S. Soman , O. Bandmann 1 International Institute of Molecular and Cell Biology, Laboratory of Neurodegeneration, Warsaw, Poland 2 University of Sheffield, Department of Neuroscience, Sheffield, United Kingdom Aims The goal of the project is to check whether there is abnormal calcium efflux/influx in mitochondria of the zebrafish pink1-/- mutant neurons. Loss of function mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of early onset Parkinson’s disease (PD). The mutations lead to mitochondrial dysfunction and neuronal cell death. We reported earlier that both genetic and pharmacological inactivation of the mitochondrial calcium uniporter (MCU) or its modulator MICU1 prevent dopaminergic neuronal cell loss in pink1Y431* zebrafish (Danio rerio) via rescue of mitochondrial respiratory chain function. Method We analyzed brains of fish with genetically encoded calcium indicator GCaMP5 in vivo. Wild type siblings were used as a control. To induce calcium efflux from mitochondria to cytoplasm, where GCaMP5g probe was located, mitochondrial uncoupling agent (CCCP, 10 μM) was used. Area postrema (AP) neurons were analyzed owing the facts they are large, separated from each other and behind the blood-brain barrier. Fluorescence transients of GCaMP5g were imaged by Light sheet Z.1 microscope. 3D tracking of neurons in time was performed by Bitplane Imaris. Results The pink1-/- mutant showed higher level of spontaneous neuronal activity in hindbrain than wild type fish. The dynamics of calcium released by CCCP treatment and calcium fluxes in AP neurons showed higher ratio of maximum to basal fluorescence in pink1-/- fish. This indicates that calcium level in mitochondria of mutant fish was higher than in wild type. Conclusion


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These data suggest that modulation of MCU-mediated mitochondrial calcium homeostasis can be a possible neuroprotective strategy in familial PD treatment.

C01.g. Disease Mechanisms, Pathophysiology: Cellular signalling, kinases, phosphatases, calcium ADPD7-1497 SYSTEMIC DEFICIENCY OF GM1 GANGLIOSIDE AS MAJOR RISK FACTOR IN SPORADIC PARKINSON'S DISEASE LEADING TO FAILURE OF GDNF NEUROTROPHIC SIGNALING 1 1 1 1 2 3 R. Ledeen , G. Wu , Z.H. Lu , M. Torres , R. Alcalay , S. DeFrees 1 Rutgers New Jersey Medical School, Pharmacology- Physiology- Neurosciences, Newark, USA 2 Columbia University, Neurology, New York, USA 3 Seneb BioSciences- Inc., Research, Doylestown, USA Aims (a) To assess GM1 levels in several tissues from sporadic PD patients. (b) To determine efficacy of GDNF signaling in dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc). (c) To study GM1 replacement therapy in a mouse PD model, B4galnt1(+/-), based on deficient GM1. Method (a) PD tissues obtained from PD tissue banks were analyzed for GM1 and GD1a (metabolic precursor) employing HPTLC. DA neurons in the SNpc were quantified via immunohistochemistry (IHC). (b) GDNF neurotrophic signaling was assessed by IHC determination of pRET levels (c) Use of GM1 analogs, GM1 replacement therapy was assessed with GM1-deficient mice which manifest numerous motor and non-motor symptoms of PD. Results (a) All examined anatomical regions of PD showed significant GM1 deficiency. Those studied by HPTLC (occipital cortex, colon, heart) were 34-48% below age-matched controls, approximating the reduced levels in the heterozygous B4galnt1(+/-) parkinsonian mice. Preliminary findings for PBMC (lymphocytes) and fibroblasts were similar. IHC revealed severe GM1 deficiency in DA neurons of the SNpc. (b) pRET levels in DA neurons of the SNpc were significantly below age-matched controls, similar to the above mice. (c) Administration of GM1 analogs to the above mice restored GDNF signaling and ameliorated the other PD symptoms. Conclusion


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Systemic GM1 deficiency is suggested as a major risk factor in sporadic PD, consistent with the extensive PD phenotype in GM1-deficient B4gallnt1(+/-) mice. A major consequence is failed GDNF neurotrophic support, suggesting the need for clinical testing of potent GM1 analogs capable of restoring effective neurotrophic signaling.

C01.g. Disease Mechanisms, Pathophysiology: Cellular signalling, kinases, phosphatases, calcium ADPD7-0467 PRPC MEDIATES SYNAPTIC DAMAGE TRIGGERED BY Α-SYNUCLEIN OLIGOMERS 1 1 2 1 3 4 3 D. G. Ferreira , M. Temido-Ferreira , H. Vicente Miranda , S. H. Vaz , M. Schmitz , J. Seop Rhee , I. Zerr , 1 4 L.V. Lopes , T.F. Outeiro 1 Instituto de Medicina Molecular, n/a, Lisboa, Portugal 2 NOVA Medical School- Faculdade de Ciências Médicas- Universidade NOVA de Lisboa, CEDOCChronic Diseases Research Center-, Lisbon, Portugal 3 University Medical Center Göttingen- and German Center for Neurodegenerative Diseases DZNE-site Göttingen, Department of Neurology, Goettingen, Germany 4 Center for Nanoscale Microscopy and Molecular Physiology of the Brain- University Medical Center GöttingenGöttingen- Germany and Max Planck Institute for Experimental Medicine, Department of Neurodegeneration and Restorative Research, Goettingen, Germany Aims The deposition of α-synuclein (aSyn) in pathognomonic inclusions known as Lewy bodies (LBs) is the neuropathological hallmark of Parkinson´s disease (PD). LBs accumulate throughout the brain as disease progresses, but prefibrillar, soluble aSyn oligomers are considered early and key intermediates in PD-related synaptic dysfunction. Method Long term potentiation (LTP) impairment associated with aSyn exposure was restored in PrP null mice and was C C rescued upon PrP blockade. We show that extracellular aSyn oligomers form a complex with PrP at the postsynaptic density, inducing the phosphorylation of intracellular Fyn kinase via the metabotropic glutamate receptor (mGluR5). Results C

Here, we identified the cellular prion protein (PrP ) as a key mediator in aSyn-associated synaptic dysfunction. C aSyn engagement of PrP /Fyn signaling causes NMDA receptor (NMDAR) activation and, consequently, dysregulation of calcium homeostasis. Strikingly, we completely rescued memory deficits in a transgenic mouse model of PD upon treatment with a specific antagonist for adenosine A2A receptors, which prevent the toxic activation of NMDAR. Conclusion


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Our results provide fundamental information on the molecular mechanisms underlying aSyn-induced synaptic malfunction and cognitive deficits and offer new mechanism-based therapeutic options to counteract downstream aSyn signaling.

C01.g. Disease Mechanisms, Pathophysiology: Cellular signalling, kinases, phosphatases, calcium ADPD7-1060 INTERFERON INDUCIBLE PKR KINASE PHOSPHORYLATES SERINE129 IN ALPHA-SYNUCLEIN AND CAUSES ALPHA-SYNUCLEIN AGGREGATION-DEPENDENT CELL DEATH IN AN OLIGODENDROGLIAL CELL MODEL OF MULTIPLE SYSTEM ATROPHY 1 1 1 2 1 1 1 1 P.H. Jensen , L. Reimer , C. Betzer , L.B. Vesterager , J. Zheng , L.D. Nielsen , L.B. Lassen , N. Ferreira , 2 3 K. Fog , S.R. Paludan 1 Aarhus University, DANDRITE - Danish Research Institute of Translational Neuroscience- Dept. Biomedicine, Aarhus, Denmark 2 Lundbeck A/S, Neurodegeneration, Copenhagen, Denmark 3 Aarhus University, Dept. Biomedicine, Aarhus, Denmark Aims Processes related are neuroinflammation is considered contributors to neurodegenerative processes and the interferon inducible serine-threonine kinase PKR is tightly associated to aspects of inflammation e.g. antiviral responses. We hypothesized PKR is involved in alpha-synuclein dependent cytotoxic processes and wanted to investigate if: 1) PKR can phosphorylate alpha-synuclein on serine129. 2) PKR contributes to serine129 phosphorylation in brain and this is modulated by proinflammatory stimuli. 3) PKR contribute to alpha-synuclein dependent cytotoxicity in a model of MSA dependent cytotoxicity. Method In vitro phosphorylation, cell, primary neuron and brain slice cultures, small molecule- and siRNA-dependent PKR inhibition, viral infection. Results PKR directly phosphorylates alpha-synuclein on Serine129 as demonstrated by phospho-specific immunoblotting. Inhibition of PKR in cells by small molecule inhibitor and siRNA knockdown reduces Serine129 phosphorylation in alpha-synuclein expressing SH-SY5Y and OLN-AS7 cell lines, primary mouse hippocampal neurons and acute brain slices from wild type and human alpha-synuclein expressing mice. Neuroinflammation-related factors known to activate PKR (herpes simplex virus, LPS and interferon IFN-β-1b) increased levels of phosphorylated alphasynuclein serine129 in cells and brain slice cultures. This phosphorylation has functional significance because inhibition of PKR with small molecule inhibitor and mRNA silencing of PKR kinase decrease cellular degeneration in the OLN/P25a multiple system atrophy model of alpha-synuclein aggregate-dependent degeneration. Conclusion


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PKR can phosphorylate alpha-synuclein serine129 and contributes to this phosphorylation in cells and brain tissue. This phosphorylation is increased by proinflammatory signals stimulating PKR and inhibition of PKR protects a oligodendroglial cell model of multiple system atrophy.

C01.i. Disease Mechanisms, Pathophysiology: Mitochondrial dysfunction, oxidative damage ADPD7-1138 MITOCHONDRIAL BIOENERGETICS IN PARKINSON'S DISEASE (PD) PATIENT-DERIVED SKIN FIBROBLASTS AND INDUCED PLURIPOTENT STEM CELL (IPSC) MODELS 1 2 3 1 N. Abboud , C. Biscarrat , B. Schüle , N. Compagnone 1 Innovative Concepts in Drug Development, Personalized medicine, Gemenos, France 2 Innovative Concepts in Drug Development, Mitochondrial function, Gemenos, France 3 Parkinson's Institute and Clinical Center, Gene Discovery and Stem Cell Modeling, Sunnyvale- CA, USA Aims To standardize patient-derived cell models for the selection of disease-modifying drugs we sought to understand the differences between idiopathic and LRRK2+ PD-patients with regards to mitochondrial bioenergetics in patient-derived cells (fibroblasts and iPSCs). Method We compared bioenergetics in patient-derived cell models from a cohort of 28 patients (10 healthy subjects, 12 idiopathic PD patients and 6 LRRK2+ PD patients). Patient's fibroblasts representative for each group were reprogrammed to develop iPSCs. 3-4 individual clones for each line were studied. Coupled dynamical measurements, in the same cell, of oxygen consumption rate, ATP production, glycolysis rate and cell viability were acquired with the BBS kit, using compatible fluorescent and luminescent detection agents. ATP production was explained through data modeling. Results Modeling of ATP production in both fibroblasts and iPSCs derived from the three studied groups showed that PD induced a steep mitochondrial dysfunction in both models. Mechanisms of reduced efficiency in energy production included ETC uncoupling, inhibition of respiratory complexes, and glycolysis reduction. Mitochondrial impairment was highest in idiopathic somatic PD cells while glycolysis was lowest in LRRK2+ cells. However, in iPSCs compared to fibroblasts from the same patient, nuclear reprogramming largely reduced mitochondrial respiratory efficiency, suggesting an immature phenotype. Conclusion


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Because nuclear reprogramming induces a significant impairment of mitochondrial function (loss of 96% of respiratory efficiency and reduction of 86% of glycolysis activity), study of mitochondrial dysfunction in PD cell models is more suitable in somatic cells. Studies are currently underway to evaluate the suitability of neurons derived from iPSCs as a model.

C01.i. Disease Mechanisms, Pathophysiology: Mitochondrial dysfunction, oxidative damage ADPD7-0949 PARKIN OVEREXPRESSION ALLEVIATES ALPHA-SYNUCLEIN-INDUCED CYTOTOXICITY VIA MAINTAINING MITOCHONDRIAL HOMEOSTASIS IN PC12 DOPAMINERGIC CELLS 1 2 2 A. Adamczyk , A. Lenkiewicz , A. Wilkaniec 1 Mossakowski Medical Research Centre, Warsaw, Poland 2 Mossakowski Medical Research Centre, Department of Cellular Signalling, Warsaw, Poland Aims Parkin is a Parkinson's disease (PD)-linked E3 ubiquitin ligase involved in sequestering misfolded or damaged proteins, fulfilling a protective role in PD and other proteinopathies. However, the mechanism underlying the neuroprotective function of parkin in dopaminergic neurons, especially its role in α-synuclein (ASN) evoked toxicity, remains unknown. Here we investigated the molecular mechaniasm of cytoprotective action of parkin against ASN-induced stress signalling pathways in dopaminergic cells. Method As a research model we used dopaminergic pheochromocytoma cells (PC12) treated with exogenous ASN oligomers as well as PC12 cells with parkin overexpression or Park2 silencing. The experiments were performed using spectrophotometrical, spectrofluorometrical and immunochemical methods as well as real-time PCR analysis. Results 2+

Our results demonstrated that extracellular ASN oligomers lead to dysregulation of intracellular Ca( ) homeostasis and down-regulation of endoplasmic reticulum (ER) stress related chaperone - protein disulfide isomerase (PDI). Furthermore, ASN induced nitric oxide (NO) and other free radicals generation, parkin Snitrosylation and activation of stress response genes. Nitrosative stress induced by ASN oligomers, as well as parkin knock-down, triggered mitochondrial dysfunction, followed by significant decrease in mitochondrial membrane potential, overproduction of mitochondrial superoxide anion and depletion of cellular ATP level. Parkin overexpression prevented mitochondrial superoxide production, ATP depletion and partially protected against ASN-evoked cell death. Conclusion Taken together, our findings reveal a parkin-mediated cytoprotective mechanism against exogenous ASN oligomers toxicity, which is a novel potential therapeutic target for treating PD and other disorders associated with protein misfolding and accumulation.


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Supported by NSC grants: 2012/05/B/NZ3/02047 (to A.A.) and 2013/09/D/NZ3/01359 (to A.W.).

C01.i. Disease Mechanisms, Pathophysiology: Mitochondrial dysfunction, oxidative damage ADPD7-1584 THE PARKINSONIAN NEUROTOXIN MPP+ AND ROTENONE DECREASE NURR1 NUCLEAR LOCALIZATION AND RNA OF ITS TRANSCRIPTIONAL TARGETS GENES 1 1 1 1 C. Carrasco , M.T. Nuñez , C. Gonzalez-Billault , J. Campos 1 Universidad de Chile, Biology, Santiago, Chile Aims Parkinson´s disease is characterized by dopaminergic neurons lost in substantia nigra pars compacta. Work of our laboratory has shown that treatments with iron chelators protect dopaminergic neurons from MPTP-induced degeneration, both in vitro and in vivo, suggesting that reactive oxygen species (ROS) resulting from iron accumulation are critical in this degenerative process. Nurr1 has been described as transcription factor essential for the development and maintenance of midbrain dopaminergic neurons, and with capacity of respond to oxidative stimulus, decreasing its activity. Method We studied the interplay between iron and MPP+ or rotenone in determining Nurr1 activity and dopaminergic cell death, utilizing a mesencephalic cell culture, SHSY-5Y cells and mouse model. To these end, we tested the effect of iron, MPP+ or rotenone and iron chelators on i) dopaminergic neuron death and neuritic tree lengh, ii) the nucleus/cytoplasm distribution of Nurr1 and iii) mRNA expression of Nurr1 transcriptional targets genes. Results We found that co-treatment with chelators protected dopaminergic neurons from MPP+-induced death, regenerating the neuritic tree lengh of dopaminergic neurons in culture. Iron, MPP+ and rotenone treatments induced decreased levels of Nurr1 in nuclei and this is reversed with iron chelators and reductant NAC treatment. Moreover, rotenone and iron treatment decreased mRNA of Nurr1 targets genes (AADC, RET, SOD1) in vitro. Conclusion


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Our data suggest that iron is a very important player in MPTP-induce degeneration and this process is mediated by Nurr1, because a decreased in this transcription factor and its targets genes increase the vulnerability of dopaminergic neurons to oxidative stimulus and toxins.

C01.i. Disease Mechanisms, Pathophysiology: Mitochondrial dysfunction, oxidative damage ADPD7-0957 REDUCED EXPRESSION LEVELS OF TOM RECEPTOR SUBUNITS IN DEMENTIA WITH LEWY BODIES BRAIN 1 1 1 2 3,4 1 1 Y.L. Chai , H. Xing , J.R. Chong , P.T. Francis , D. Aarsland , C.P. Chen , M.K.P. Lai 1 National University of Singapore, Department of Pharmacology- Yong Loo Lin School of Medicine, Singapore, Singapore 2 King's College London, Wolfson Centre for Age-Related Diseases- Institute of PsychiatryPsychology & Neuroscience, London, United Kingdom 3 King's College London, Department of Old Age Psychiatry- Institute of Psychiatry- Psychology & Neuroscience, London, United Kingdom 4 Karolinska Institutet, Department Neurobiology- Care Sciences and Society- Center for Alzheimer ResearchDivision of Clinical Geriatrics, Stockholm, Sweden Aims The translocase of the outer mitochondrial membrane (TOM) is one of the major mitochondrial import machineries. While TOM subunits were found to be downregulated in various neurodegenerative diseases including Alzheimer’s disease (AD), it is unclear about its involvement in Lewy Body Dementia (LBD) – the second most common form of neurodegenerative dementia. We aimed to investigate the protein expression levels of TOM subunits in LBD, which is subcategorized into Parkinson’s Disease Dementia (PDD) and Dementia with Lewy Bodies (DLB), and their potential associations with AD pathology. Method Postmortem brain samples from Brodmann area 40 were obtained from 20 PDD, 20 DLB and 19 non-demented age-matched controls. Samples were processed for amyloid beta (Abeta) 42 and 40 protein quantification using ELISA. Mitochondrial enriched fraction was also extracted from subcellular fractionation and used for immunoblotting with antibodies against TOM40, TOM22, TOM20 and TOM70. Results The surface receptor subunits TOM20 (p=0.036) and TOM70 (p90% and sensitivity about 80%. However, there have been only small numbers of cases with autopsy validation. We carried out a validation study of FP-CIT imaging in vivo diagnosis of DLB using autopsy as the gold standard. Method Patients with dementia who were over 60 years of age underwent FP-CIT imaging in research studies in Newcastle and London. All who donated their brain tissue to the Newcastle Brain Tissue Resource were included. Clinical diagnoses were applied by consensus panels using international diagnostic criteria. Scans were rated as normal or abnormal by blinded raters. All patients had detailed autopsy assessments and neuropathological diagnoses were applied using standard international criteria. Results 33 DLB and 22 AD patients were included. FP-CIT had a diagnostic accuracy of 86% (sensitivity 80%, specificity 92%). The accuracy of clinical diagnosis was 79% (sensitivity 87%, specificity 72%). Among DLB patients 10% met pathological criteria for Lewy body disease but had normal FP-CIT imaging Conclusion


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This autopsy assessment of imaging in dementia found FP-CIT to be a valid and accurate biomarker for DLB. The 20% higher specificity compared with clinical diagnosis alone is potentially important in clinical and research practice. Whilst an abnormal FP-CIT scan strongly supports Lewy body disease, the presence of a normal scan does not exclude DLB.

C04.d. Imaging, Biomarkers, Diagnostics: SPECT ADPD7-0392 APPLICATION OF DATSCAN (123 I-FP-CIT SPECT) IN DEMENTIA WITH LEWY BODIES CAN PREDICT THE RESPONSIVENESS TO TREATMENTS 1 C.K. Wu 1 University of California-Irvine School of Medicine, Neurology, Irvine, USA Aims It is a very challenging task for neurologists to diagnose and treat dementia with Lewy bodies (DLB). One major issue is to predict whether the dopaminergic treatments would improve the motor symptoms or worsening the behavioral problems (ie. visual hallucinations). In this observational study, the application of DaTSCAN (123 I-FPCIT SPECT) were studied for the treatment outcomes of patients with DLB. Method From January 1st 2011 to August 31 2016, all reports of DaTSCAN done for patients with DLB were reviewed. The clinical data, treatment history and outcomes were collected for analysis for this study. Results Overall, regarding the responsiveness to treatments, the patients who had a positive DaTSCAN usually responded to the dopaminergic treatment for motor symptoms, despite presenting "simple" visual hallucinations. The dopaminergic treatment did not cause the worsening condition of visual hallucinations. And the cognitive impairment usually was in mild degree. They can modestly benefit from treatment of a cholinesterase inhibitor.By contrast, patients who had a negative DaTSCAN often presented with complex visual hallucinations. Their motor symptoms were very mild; cognitive symptoms were worse. They did not respond to dopaminergic treatment and easily developed severe dopamine treatment induced psychotic symptoms.They usually needed the antipsychotic to treat psychotic behaviors. Moreover, they usually did not respond to the cognitive treatment with a cholinesterase inhibitor. Conclusion


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For DLB, the diagnosis has been difficulty to make with confidence with a reliable diagnostic tool. DaTSCAN (123 I-FP-CIT SPECT) can be a useful tool for diagnosis and be considered as the guidance for treatment plan.

C04.e. Imaging, Biomarkers, Diagnostics: Multimodal imaging ADPD7-0560 BRAIN STRUCTURAL AND FUNCTIONAL SIGNATURES OF IMPULSE CONTROL DISORDER IN PARKINSON’S DISEASE 1 1 1 2 3 2 2 4 1 F. Imperiale , F. Agosta , E. Canu , V. Markovic , A. Inuggi , M. Ječmenica , A. Tomić , M. Copetti , S. Basaia , 2 1,5 V. Kostic , M. Filippi 1 San Raffaele Scientific Institute- Vita-Salute San Raffaele University, Neuroimaging Research UnitInstitute of Experimental Neurology- Division of Neuroscience-, Milan, Italy 2 University of Belgrade, Clinic of Neurology- Faculty of Medicine, Belgrade, Serbia 3 Istituto Italiano di Tecnologia, Unit of Robotics- Brain and Cognitive Sciences, Genoa, Italy 4 IRCCS-Ospedale Casa Sollievo della Sofferenza, Biostatistics Unit, San Giovanni Rotondo FG, Italy 5 San Raffaele Scientific Institute- Vita-Salute San Raffaele University, Department of Neurology, Milan, Italy Aims To assess brain structural and functional connectivity alterations in patients with Parkinson’s disease and impulse control disorder (PD-ICD) compared with controls and PD no-ICD cases. Method Eighty-five PD patients (35 PD-ICD) and 50 controls underwent 3D T1-weighted, diffusion tensor (DT), and resting state functional MRI (RS fMRI). We assessed cortical thickness with surface-based morphometry, subcortical volumes using FIRST, DT MRI metrics using region of interest and tractography approaches, and RS fMRI using a model free approach. Results Compared with controls, both PD groups showed structural alterations in the basal ganglia (more evident in PD no-ICD patients), sensorimotor and associative systems. Compared with PD no-ICD, PD-ICD cases showed left precentral and superior frontal cortical thinning, and motor and extramotor white matter damage. Compared with controls, all patients had an increased functional connectivity (FC) within the visual network. Additionally, PD noICD showed increased FC of bilateral precentral and postcentral gyri within the sensorimotor network compared to controls and PD-ICD. Severity and duration of PD-ICD modulated FC between sensorimotor, visual and cognitive networks. Conclusion Relative to PD no-ICD, PD-ICD patients were characterized by a more severe involvement of frontal, meso-limbic and motor circuits. FC data suggest ICD in PD as the result of a disconnection between sensorimotor, associative and cognitive networks with increasing motor impairment, psychiatric symptoms, and ICD duration. MRI is promising as a tool for detecting brain changes in PD patients at risk to develop ICD.


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Funding. Ministry of Education and Science, Republic of Serbia (project 175090).

C04.e. Imaging, Biomarkers, Diagnostics: Multimodal imaging ADPD7-0567 CHARACTERISTICS OF PUNDING IN PARKINSON’S DISEASE: A MULTIMODAL NEUROIMAGING APPROACH 1,2 1 1 2 3 2 1,4 V. Markovic , F. Agosta , E. Canu , M. Ječmenica , A. Inuggi , V. Kostic , M. Filippi 1 San Raffaele Scientific Institute- Vita-Salute San Raffaele University, Neuroimaging Research UnitInstitute of Experimental Neurology- Division of Neuroscience-, Milan, Italy 2 University of Belgrade, Clinic of Neurology- Faculty of Medicine, Belgrade, Serbia 3 Istituto Italiano di Tecnologia, Unit of Robotics- Brain and Cognitive Sciences-, Genoa, Italy 4 San Raffaele Scientific Institute- Vita-Salute San Raffaele University, Department of Neurology, Milan, Italy Aims Punding is a poorly investigated clinical presentation of impulse control disorders (ICDs) in Parkinson’s disease (PD). This study aimed to assess resting state (RS) functional connectivity of the habenula and amygdala, as modulators of the reward circuit, in PD-punding patients compared to PD patients without any ICDs (PD no-ICD) and controls. Method We included 52 PD patients (22 PD-punding) and 30 controls. All subjects underwent 3D T1-weighted and RS functional MRI (RS-fMRI). We assessed cortical thickness measures using surface-based morphometry and RSfMRI data using a seed-based approach. Results PD-punding patients showed cortical thinning of the left superior frontal and precentral gyrus and right middle temporal gyrus and isthmus cingulate compared to controls, and of the right inferior frontal gyrus compared to both controls and PD no-ICD patients. Compared to both controls and PD no-ICD patients, PD-punding patients showed higher connectivity of habenula and amygdala with thalamus and striatum bilaterally; and lower connectivity between bilateral habenula and left frontal and precentral cortices. Finally, PD–punding patients showed lower connectivity between right amygdala and right hippocampus compared to PD no-ICD patients. Conclusion PD-punding is associated with a disconnection between brain regions which modulate the reward circuit and the frontal cortex, and a hyperconnectivity between such areas and subcortical regions. In PD-punding patients, these alterations may reflect their typical repetitive behavior regardless the reward. This study offers opportunity for the detection of PD patients at risk to develop punding.


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Funding. Ministry of Education and Science, Republic of Serbia (project 175090).

C04.f. Imaging, Biomarkers, Diagnostics: CSF, blood, body fluid biomarkers ADPD7-1657 DETECTION OF SER 129 PHOSPHORYLATED ALPHA-SYNUCLEIN IN HUMAN CSF USING AN ULTRASENSITIVE IMMUNOASSAY 1 2 3 2 1 R. Barbour , T. Kremer , B. Mollenhauer , C. Czech , W. Zago 1 Prothena Biosciences Inc, South San Francisco, CA, USA 2 Roche Pharmaceutical Research & Early Development, Translational Medicine Neuroscience & Biomarkers, Roche Innovation Center Basel, Basel, Switzerland 3 Paracelsus-Elena-Klinik, Kassel and University Medical Center Göttingen, Germany OBJECTIVES: alpha-synuclein protein phosphorylated at Serine 129 (PS129-aSyn) is a known histopathological hallmark in Lewy Body Dementia and Parkinson’s disease (PD). Here, we developed a well characterized, highly sensitive and specific immunoassay to investigate levels of PS129-aSyn in human cerebrospinal fluid (CSF) using the Singulex Erenna platform.

Methods: An ultra-sensitive assay was developed on the Erenna platform using PTR11A5, a monoclonal antibody with high affinity and specificity to PS129-aSyn, and PRT23E8, a monoclonal antibody to the N-terminus of alpha-synuclein. The PS129-aSyn calibrator was prepared by in-vitro phosphorylation of alpha-synuclein using recombinant PLK2, purified by Q HP HiTrap column, and confirmation of phosphorylation by mass spectrometry. After qualification, the assay was used to determine levels of PS129-aSyn in human CSF samples from PD patients and neurological controls. Blood-contaminated CSF’s (hemoglobin>500ng/ml) were not included in the analysis.

Results: Our newly developed immunoassay enables high sensitivity detection of pSer129-aSyn with a lower limit of quantitation of 100 fg/ml, equivalent to 400 fg/ml in CSF pre-dilution. Quantifiable levels of PS129-aSyn were found in all CSF samples studied, which ranged from 0.6 pg/ml to 13.0 pg/ml.


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Conclusions: A novel, ultra-high sensitivity assay to specifically detect alpha-synuclein phosphorylated at serine 129 in CSF was developed on the Singulex Erenna platform. Our results show that PS129 aSyn, albeit present at low concentrations, can be accurately measured in human CSF.

C04.f. Imaging, Biomarkers, Diagnostics: CSF, blood, body fluid biomarkers ADPD7-1445 DIAGNOSTIC BIOMARKERS IN THE CSF: AΒ40 AND AΒ42/AΒ40 RATIO ARE THE BEST PREDICTORS TO DIFFERENTIATE MCI DLB TO MCI AD 1 2 3 4 5 6 7 O. Bousiges , C. PAQUET , S. BOMBOIS , E. MAGNIN , A. GABELLE , D. WALLON , J.L. LAPLANCHE , 2 8 9 10 6 3 J. DUMURGIER , S. SCHRAEN , C. MIGUET-ALFONSI , S. LEHMANN , D. HANNEQUIN , F. PASQUIER , 11 F. BLANC 1 Hôpital de Hautepierre CHU de Strasbourg, Laboratoire de Biochimie et Biologie Moléculaire, STRASBOURG, France 2 Hôpital Lariboisière, Department of Neurology, Paris, France 3 CHU de Lille, Department of neurology, Lille, France 4 CHU de Besançon, Department of neurology, Besançon, France 5 CHU de Montpellier, Department of neurology, Montpellier, France 6 CHU de Rouen, Departmentr of neurology, Rouen, France 7 Hôpital Lariboisière, Department of biochemistry, Paris, France 8 CHU de Lille, Department of biochemistry, Lille, France 9 CHU de Besançon, Department of biochemistry, Besançon, France 10 CHU de Montpellier, Department of biochemistry, Montpellier, France 11 Hôpital de Hautepierre CHU de Strasbourg, Department of neurology, STRASBOURG, France Aims The differential diagnosis is difficult between Dementia with Lewy bodies (DLB) and Alzheimer’s disease (AD), especially early in the disease. We propose to compare AD biomarkers (Phospho-Tau, total-Tau, Aβ42 and Aβ40) in cerebrospinal fluid (CSF) between DLB and AD, with a particular focus on the prodromal stage. Method A total of 1,220 CSF samples were collected retrospectively at different memories French centers (ePLM). They were obtained from prodromal DLB (N = 52), DLB dementia (N = 154), prodromal AD (N = 132), and AD dementia patients (N = 783), and elderly controls (N = 95). Patients were classified according to the criteria of McKeith and DSMV for DLB patients and the criteria of McKhann and Dubois for Alzheimer's patients. Results At the prodromal stage, DLB patient’s Aβ42 levels in the CSF appeared much less disrupted as the demented stage. Whatever the stage of the DLB patients, Aβ40 is below the levels of our controls and much below the levels of AD patients. So the Aβ42/Aβ40 ratio of DLB patients whatever the stages remained closed of that of controls. Conclusion


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In the CSF of DLB patients, we find lower levels of Aβ42, but it happens at a later stage than in AD where Aβ42 levels are decreased since the prodromal stage. To note the Aβ40 level is decreased since the prodromal stage of DLB patients. Thus, it appears that the Aβ40 and Aβ42/Aβ40 ratio are the best tools for the differential diagnosis between DLB and AD patients, even at early stages of these pathologies.

C04.f. Imaging, Biomarkers, Diagnostics: CSF, blood, body fluid biomarkers ADPD7-0936 EXOSOMAL SIGNATURE IN PARKINSON’S DISEASE: NEW TOOLS AND MARKERS 1 1 1 1 1 S. Cerri , C. Ghezzi , F. Siani , M. Sampieri , F. Blandini 1 "C. Mondino" National Neurological Institute, Laboratory of Functional NeurochemistryCenter for Research in Neurodegenerative Diseases, Pavia, Italy Aims Identification of sensitive and specific biomarker(s) for differential diagnosis, prognosis and effective treatment of Parkinson’s disease (PD) is a critical unmet need in the field. Different pathogenesis-related proteins (i.e. αsynuclein, α-syn) have been proposed as potential peripheral markers for PD. Recently nebulette, a cytoskeletal protein overexpressed in the substantia nigra of PD patients, has been suggested as a key pathogenic candidate. Its potential was further corroborated by the demonstration that α-syn can affect cytoskeletal function driving early neurite pathology. Blood is an ideal source for biomarkers due to its propensity to contain biological signals from relevant sources such as disease-associated cells. Growing evidence suggests that exosomes can provide more reliable biomarkers for neurodegenerative diseases than biological fluids, since they carry unique, diseasespecific cargos containing proteins and nucleic acids. In this study we investigated whether exosomal α-syn species (wild type and oligomers) and nebulette could represent valuable peripheral biomarkers in PD. Method Alpha-syn and nebulette contents were evaluated in exosomes isolated from blood of PD patients and age- and sex-matched controls by a modified ELISA assay and western blot, respectively. Results Modified ELISA exhibited good efficiency to detect wild type α-syn in exosomes. Specifically, we found a significant increase of exosomal wild type α-syn and augmented content of nebulette in PD patients, as compared to controls, this latter being more pronounced in patients with severe PD. Conclusion


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In summary, the identification of an exosomal panel of PD-related proteins, associated with α-syn and nebulette function, could provide a new biomarker-based diagnostic/prognostic tool for PD.

C04.f. Imaging, Biomarkers, Diagnostics: CSF, blood, body fluid biomarkers ADPD7-0815 DIAGNOSTIC UTILITY OF CSF ALPHA-SYNUCLEIN SPECIES IN PARKINSON’S DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS 1 2 1 3 1 3 1 1 P. Eusebi , D. Giannandrea , L. Biscetti , I. Abraha , D. Chiasserini , M. Orso , P. Calabresi , P. Lucilla 1 University of Perugia, Section of Neurology- Department of Medicine- Center for Memory Disturbances, Perugia, Italy 2 USL Umbria 1 North area- Gubbio and Gualdo Tadino Hospital, Neurology UnitDepartment of Specialist Medicine, Gubbio, Italy 3 Regional Health Authority of Umbria, Health Planning Service- Department of Epidemiology, Perugia, Italy Aims To assess the value of cerebrospinal fluid (CSF) alpha-synuclein (α-syn) for distinguishing subjects with Parkinson’s disease (PD) from controls and other parkinsonisms. Method We searched the Cochrane Library, Web of Science, MEDLINE (via PubMed) and EMBASE from inception to May 2016 using appropriate search strategies. Two independent reviewers have screened titles, abstracts and full-text articles, and completed data abstraction. We included studies that involved patients with PD, dementia with Lewy bodies, multiple system atrophy, progressive supra-nuclear palsy, corticobasal syndrome and vascular PD, and in which at least one between total α-syn, oligomeric α-syn and phosphorylated α-syn was measured in CSF. Standardized mean differences using Hedges' g were calculated. Results 44 of 37 independent cohorts were included. CSF total α-syn was significantly reduced in PD as compared to controls (SMD=-0.45; 95%CI=-0.56, -0.33) with significant heterogeneity (I-square=54%, p2 SD above the mean in seconds) according to Leong et al, 2015. Results Forty-five iRBD patients were studied. Mean age was 65.8 years, and 33 (73%) were men. The mean (± SD) KDT score for all patients was 59.8 (± 14.1) seconds. Fifteen (33%) patients had abnormal KDT scores (mean 73.94 ± 15.24), which included 4 (33%) women and 11 (33%) men in the cohort. Among those with abnormal KDT scores, 7 (47%) had scores >10 seconds beyond the cut-offs. Conclusion


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One third of our iRBD patients had abnormal KDT scores, indicating largely covert impairments in attention, processing speed, or visual scanning functions, which implies more diffuse neurological dysfunction consistent with a synucleinopathy. Future studies involving a larger longitudinal iRBD cohort and correlation with other neurodegenerative biomarkers may provide insights on the utility of the KDT in predicting phenoconversion to overt synucleinopathies.

C04.h. Imaging, Biomarkers, Diagnostics: Cognitive, psychometric, behavioral and motor tests ADPD7-1752 LACK OF NEURAL HABITUATION AND COGNITIVE BEHAVIORAL CORRELATE IN EARLY STAGE OF PARKINSON’S DISEASE: A COMPARISON WITH MIGRAINE SUFFERERS 1 2 3 M. Buonfiglio , G. Avanzini , F. Di Sabato 1 Policlinico Umberto I- Sapienza University of Rome, Clinical Medicine VI, Rome, Italy 2 Carlo Besta Neurological Institute, Division of Neurophysiology and Experimental Epilepsy, Milan, Italy 3 Policlinico Umberto I Sapienza University, Clinical Medicine Department, Rome, Italy Aims Lack of habituation to repeated sensory stimulations has been found both in Parkinson’s Disease (PD) and in migraine interictally, normalizing during the attack. It can be considered an impairment of a fundamental function of sensory system aimed at protecting the brain against sensory overload. Recently neurophysiological findings demonstrated lack of habituation to be correlated with analytic cognitive behavior (Buonfiglio et al. Neurol Sci 2015). The latter has been linked to migraine. We aimed at evaluating possible association between PD and analytic information processing style (Sternberg). Moreover we explored the relationship between migraine and PD.

Method 30 idiopathic PD patients at early stage of disease were randomly enrolled and matched with migraineurs with and without aura and healthy volunteers. All subjects underwent cognitive behavioral tests and migraine diagnosis according to IHCD II criteria. Results We highlighted significant higher scores in analytic style of information processing in both patients groups compared to controls. Scores in analytic information processing of auditory stimulations were higher in PD and in migraine with aura (MWA) compared to other groups (Anova: p < 0,005). Lack of association with migraine was observed in PD patients, whereas 45% of them reported having had MWA attacks before the onset of disease. Conclusion


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We are highlighting an analytic cognitive behavior linked to lack of habituation common to migraine and PD and improvement of migraine after the onset of PD. We discuss possible implications of deficit of habituation and cognitive behavioral correlate in PD pathophysiology, putting forward the idea of migraine attack as a compensatory mechanism.

C04.h. Imaging, Biomarkers, Diagnostics: Cognitive, psychometric, behavioral and motor tests ADPD7-0518 DEVELOPMENT OF DEMENTIA AND BURDEN OF WHITE MATTER HYPERINTENSITY IN PARKINSON’S DISEASE 1 1 S.M. Cheon , J.W. Kim 1 Dong-A University College of Medicine, Neurology, Busan, Republic of Korea Aims Dementia is major determinant of daily activities in patients with Parkinson’s disease (PD). The burden of white matter hyperintensity (WMH) has been known to contribute to cognitive and functional impairment. The purpose of this study was to address the relationship between the burden of WMH and development of dementia in PD. Method PD Patients without dementia, confirmed by comprehensive neuropsychological tests at baseline assessment, were included in this study. Dementia was identified during regular follow-up visit. Evaluation of WMH was done from baseline brain MRI based on Scheltens’ scale. Baseline cognitive performance and burden of WMH were analyzed in dementia-converters and non-converters. Results Total 159 patients with PD were recruited (age 64.1±7.6 years, Hoehn and Yahr stage 1-4 (median 2.5)). Dementia was developed in 31 patients (19.5%) during mean follow-up of 4.6 years. Dementia-conversion was increased in patients with old age, short education period, and vascular risk factors. Multi-domain mild cognitive impairment, especially language and visuospatial domain, was related with the development of dementia. Supratentorial WMH burden was significantly increased in dementia-converters. Language and visuospatial dysfunction was correlated with localized burden of WMH. Conclusion


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In this retrospective study, we demonstrated higher burden of WMH in dementia-converters and relationship between localized burden of WMH and specific cognitive performance. These results suggest that control of vascular risks and burden WMH could have role in management of dementia in PD.

C04.h. Imaging, Biomarkers, Diagnostics: Cognitive, psychometric, behavioral and motor tests ADPD7-1396 STROOP PERFORMANCE MAY HELP DISTINGUISH DEMENTIA WITH LEWY BODIES FROM ALZHEIMER’S DISEASE 1 2 3 4 J. Fields , A. Boeve , B. Boeve , T. Ferman 1 Mayo Clinic, Psychiatry & Psychology, Rochester, USA 2 Mayo Clinic, Sleep Medicine, Rochester, USA 3 Mayo Clinic, Neurology, Rochester, USA 4 Mayo Clinic, Psychology, Rochester, USA Aims To determine whether Stroop performance can help differentiate dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). Method Patients with DLB (n=171) and AD (n=85) with varying degrees of dementia were administered the Stroop as part of a larger cognitive battery. The Global Deterioration Scale (GLDS) was used to rate dementia severity on a scale of 1 to 6, with higher scores indicating greater severity. Additional analyses included only patients with a diagnosis of mild cognitive impairment (MCI). Analysis of variance compared age-corrected scaled scores for the three Stroop trials (Word, Color, Color-Word (C-W) Interference) between DLB and AD. Results DLB patients were on average younger than AD patients (72.4 vs. 75.4 years, p=.002). DLB and AD did not differ with regard to education (14.9 vs. 14.6 years) or GLDS score (3.67 vs. 3.64). DLB performed worse than AD on Word (5.4 vs. 7.8), Color (5.2 vs. 7.0) and C-W (4.6 vs. 6.1) trials, all p=.000. In MCI cases with a final diagnosis of DLB (n=22) or AD (n=22), DLB again performed worse than AD on Word (7.1 vs. 10.1, p=.001), Color (7.5 vs. 9.1, p=.04) and C-W (6.3 vs. 9.8, p=.000) trials. Conclusion


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Stroop performance was consistently worse in DLB than in AD. Furthermore, in the MCI groups, Stroop performance was within the normal range in AD, whereas it was low average to impaired in DLB. The Stroop test may aid in early detection and help differentiate DLB and AD.

C04.h. Imaging, Biomarkers, Diagnostics: Cognitive, psychometric, behavioral and motor tests ADPD7-1135 DIAGNOSTIC VALUE OF COGNITIVE TESTS FOR DETECTING PRODROMAL DEMENTIA WITH LEWY BODIES IN REM SLEEP BEHAVIOR DISORDER 1,2 1,3 1,4 1,2 D. Génier Marchand- BSc , J. Montplaisir- MD- PhD , R.B. Postuma- MD- MSc , S. Rahayel- BSc , 1,2 J.F. Gagnon- PhD 1 Hôpital du Sacre-Coeur de Montreal- Université de Montréal, Center of Advanced Research in Sleep Medicine, Montreal, Canada 2 Université du Québec à Montréal, Psychology, Montreal, Canada 3 Université de Montréal, Psychiatry, Montreal, Canada 4 Montreal General Hospital, Neurology, Montreal, Canada Aims REM sleep behavior disorder (RBD) is a major risk factor for synucleinopathies. As a specific and sensitive prodromal syndrome, RBD allows the investigation of cognitive markers for early detection of dementia. The purpose of this study was to evaluate the diagnostic value of cognitive tests for detecting prodromal dementia with Lewy bodies (DLB). Method Seventy-six polysomnography-confirmed RBD patients underwent a complete neuropsychological and neurological assessment and were then followed yearly. Receiver operating characteristic curves were calculated to assess the sensitivity and specificity of the cognitive tests in order to assess dementia predictability. The optiomal cutoff value was defined as the maximum accuracy value, calculated by the Youden Index. Results After a mean 3.6-year follow-up, 15 developed a dementia-first and 19 developed a parkinsonism-first. Dementiafirst patients were pair-matched at baseline with 30 healthy subjects without cognitive impairment. When comparing dementia-first convertors and controls, the best predictors (AUC ≥ 0.85) of dementia were the Stroop Color Word Test (III-II, time), Trail Making Test (part B, time), Verbal fluency test (phonetic and semantic), and Rey Auditory Verbal-Learning Test (sum of trials 1 to 5). When comparing dementia-first RBD and parkinsonismfirst RBD convertors, the best predictors (AUC ≥ 0.85) were the Stroop Color Word Test (III-II, time; IV-III, errors), Trail Making Test (part B, time), and Rey-Osterrieth Complex Figure (copy). Conclusion


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This study shows that cognitive tests assessing attention and executive functions strongly predict conversion to dementia in RBD patients. Moreover, these tests may be useful in future interventions to prevent cognitive deterioration in this population.

C04.h. Imaging, Biomarkers, Diagnostics: Cognitive, psychometric, behavioral and motor tests ADPD7-1809 SEX DIFFERENCES IN BIOMARKER PREDICTORS OF COGNITIVE PROGRESSION IN DE NOVO PARKINSON DISEASE PATIENTS 1 2 B. Hanna-Pladdy , H. Spenser 1 University of Maryland School of Medicine, Neurology- Psychiatry & Diagnostic Radiology, Baltimore, USA 2 University of Maryland School of Medicine, Neurology, Baltimore, USA Aims To evaluate sex differences in the relationship between baseline CSF biomarkers and risk factors in predicting cognitive progression at 4 year follow-up among newly diagnosed Parkinson’s disease (PD) patients. Method A sample of 137 de novo PD patients (males =100; females =37) in the Parkinson’s Progression Markers Initiative (PPMI) were assessed untreated at baseline, and at 4 year follow-up. CSF proteins (Aβ1-42, t-tau, ptau181, αsynuclein), APOE, REM sleep, and UPDRS were collected at baseline. Neuropsychological profiles determined attentional, memory, language and visuoperceptual progression across 4 years. Results Step-wise multiple regressions conducted separately for males and females evaluated how baseline variables of motor severity, REM sleep, CSF and cognitive biomarkers predicted 4 year cognitive outcomes. Cognition in males (16.3% variance) was predicted by REM (pC), HTR2A (rs6311 or -1438G>A) and HTR2C (rs6318 or Cys23Ser). Method When comparing the frequencies of alleles and genotypes χ2 criterion was applied in groups of patients and controls. SNP association with the disease were analyzed using the software package PLINK 1.07. To detect associations between clinical and neuropsychological characteristics and genotypes (alleles) was performed univariate (ANOVA) analysis. Results A significant association was observed between the genotype HTR2А*A/A and development of akinetic-rigid form PD in Bashkortostan Republic (OR=6,682; p=0,0003). The comparative analysis of the investigated polymorphic loci of genes 5-HTT, ТPН, HTR1B and HTR2C hasn’t revealed significant differences between PD samples and controls. The study found no effect of polymorphic variants of the genes of the serotonergic system in the development of neuropsychological disorders in patients with PD. Conclusion


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Genotype HTR2А*A/A, determining decrease of the enzyme expression, may be considered as genetic markers of the increased risk contributing to development of akinetic-rigid form PD in Bashkortostan Republic.

C05.c. Genetics, Epidemiology: GWAS, genetic associations, susceptibility & protective genes ADPD7-1471 SNCA RISK HAPLOTYPES IN Α-SYNUCLEINOPATHIES 1 2 1 3 3 4 4 4 C. Blauwendraat , A. Sailer , J.T. Geiger , T. Revesz , J. Holton , J.R. Gibbs , D.G. Hernandez , M.R. Cookson , 4 4 3 1 A.B. Singleton , F. or the North American Brain Expression Consortium , H. Houlden , S.W. Scholz 1 National Institute of Neurological Disorders and Stroke NINDS, Neurodegenerative Diseases Research Unit, Bethesda, USA 2 Universitätsklinik der Johann-Wolfgang-Goethe Universität Frankfurt, Klinik für Neurologie, Frankfurt, Germany 3 Institute of Neurology, Departments of Molecular Neuroscience and The Reta Lila Weston Institute, London, United Kingdom 4 National institute on aging NIA, Laboratory of Neurogenetics, Bethesda, USA Aims There is increasing recognition that the neurodegenerative disorders dementia with Lewy bodies (DLB), Parkinson disease (PD) and multiple system atrophy (MSA) overlap at pathological, clinical and molecular levels. These disorders, collectively named α-synucleinopathies, are characterized by the deposition of abnormally phosphorylated, fibrillar α-synuclein within the nervous system. We performed haplotype analysis of the SNCA region, encoding α-synuclein, to investigate whether distinct haplotypes are associated with increased risk for disease, and to study whether these are shared between different types of α-synucleinopathies. Method Genotype data was created of 176 pathology-proven DLB cases, 83 pathology-proven PD cases, 80 pathologyproven MSA cases, and 775 neurologically normal controls. Additionally, we used the Parkinson’s Progression Markers Initiative (PPMI) and the International Parkinson's Disease Genomics Consortium (IPDGC) NeuroX genotypes. To assess the potential molecular consequences of SNCA risk haplotypes, we investigated whether these haplotypes had an influence on the expression of SNCA by using genotype control data in combination with microarray, RNA-seq and CAGEseq expression data. Results Several haploblocks were identified spanning the SNCA region, each containing several different haplotypes. Next, we performed genotype and haplotype association analyses, which demonstrated a significant overrepresentation of both SNCA genotypes and haplotypes in DLB and PD cases compared to controls, but no significant associations were found between MSA and controls. Conclusion


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Our preliminary analysis indicates that certain haplotypes have a subtle influence on SNCA expression. In conclusion, these findings suggest that the identified risk haplotypes increase risk for disease via modest expression changes of SNCA.

C05.c. Genetics, Epidemiology: GWAS, genetic associations, susceptibility & protective genes ADPD7-0822 THE GENETIC BASIS OF DEMENTIA WITH LEWY BODIES 1 J. Bras 1 UCL Institute of Neurology, Molecular Neuroscience, London, United Kingdom Aims Dementia with Lewy bodies (DLB) is one of the most underserved common diseases. From clinical to pathological aspects, DLB is often a difficult disease to diagnose. The genetics of DLB has been tremendously understudied and only recently have we started to tease out the genetic aspects of DLB. This has been possible largely due to recent advances in technology and collaborative approaches that have allowed us to query the genome of large numbers of individuals. The vast majority of publications dealing with the genetics of DLB have been of very small size (in the region of 100 cases), which is not comparable with recent work performed in other neurodegenerative diseases. Method Here, I will discuss recent findings in the genetics of DLB, including large genome-wide association studies and sequencing efforts designed to identify novel causative and risk factors for this disease and that are currently underway. Results So far these studies have highlighted several genes known to be involved in neurodegenerative diseases, most notably: SNCA and APOE. The involvement of both genes is not entirely surprising and is in line with what we know occurs at the pathological level in this disease. DLB-specific causes and risk factors have so far not been identified. Conclusion


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We now know genetics plays a significant role in the etiology of DLB, despite the fact that the findings so far have only pointed to genes also involved in other neurodegenerative diseases.

C05.c. Genetics, Epidemiology: GWAS, genetic associations, susceptibility & protective genes ADPD7-1692 HUMAN PRDX6 SNP T177I MIGHT BE BENEFICIAL FOR PATIENTS SUFFERING FROM NEURODEGENERATIVE DISEASES: AN IN SILICO STUDY 1 R.K. Chowhan 1 University of Delhi, Dr. B. R. Center For Biomedical Research, New Delhi, India Aims Recent reports have shown that enhanced expression of an antioxidant enzyme Prdx6 instead of restraining further increases oxidative stress and aggravate neurodegenerative diseases(ND). Gain of phospholipase activity(PLA2) due to phosphorylation of Prdx6’s T177 residue is considered the reason. Interestingly, resequencing data registered by 1000genomes revealed existence of nsSNP T177I. Considering importance of T177 residue, we wanted to understand the effect of T177I mutation on Prdx6’s structure& investigate whether this polymorphism is deleterious to enzyme's physiological antioxidative peroxidase function thereby worsening ND. Method We generated MD simulated models for Wt and mutant Prdx6, and their trajectories were analysed to comprehend the difference in tertiary structure, shape, stability&flexibility. Systematic secondary structure comparison of Wt&T177I Prdx6 was done to study the effect of conformation change on active site catalytic behaviour. Results T177I&Wt Prdx6 has some structural differences which lie mostly in the regions that affect neither the alignment of a typical thioredoxin fold nor the active site localisation. Comparison of active site’s structure, flexibility, and ligand binding properties indicated T177I to have no damaging effect on Prdx6’s peroxidase activity. Conclusion


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T177I Prdx6 has lost its ability to gain phosphorylation mediated aiPLA2 activity but its peroxidase activity is intact. Since, enhanced aiPLA2 activity of Prdx6 is alleged to aggravate ND, it appears that T177I might prove to be a beneficial mutation for individuals suffering from ND as their antioxidant enzyme Prdx6 would be catalytically active but would no longer be a potential risk at higher concentrations due to phosphorylation mediated gain of aiPLA2 activity.

C05.c. Genetics, Epidemiology: GWAS, genetic associations, susceptibility & protective genes ADPD7-0390 REGIONAL DIFFERENCE OF PUTAMINAL TYROSINE HYDROXYLASE REDUCTION IN NEUROPATHOLOGICALLY PROVEN LEWY BODY DISEASE 1 2 1 1 1 1 K. Kasanuki , M.G. Heckman , M. Murray , S. Koga , O. Ross , D. Dickson 1 Mayo Clinic, Department of Neuroscience, Jacksonville, USA 2 Mayo Clinic, Division of Biomedical Statistics and Informatics, Jacksonville, USA Aims We performed imaging analysis of putaminal dopaminergic innervation and investigated its clinicopathologic correlates. Method We measured tyrosine hydroxylase immunoreactivity (TH) in dorsolateral and ventromedial putamen in 501 cases of Lewy body disease using image analysis. We scored neuronal loss in the midbrain and counted Lewy bodies (LB) in the neocortex with α-synuclein immunohistochemistry. Cases were assigned to clinical subgroups according to predominant syndrome – dementia, Parkinsonism and dementia (PDD) or parkinsonism (PD). Correlation tests and multivariate analyses were used to examine correlates of regional putaminal TH, neuronal loss, and LB counts with respect to 29 PD GWAS risk variants and to APOE4. Results TH was reduced in dorsolateral (79%) more than ventromedial putamen (57%). There was an inverse correlation -7 between dorsolateral TH and neuronal loss in substantia nigra (r=-0.49, p=2×10 ) and a weaker inverse -6 correlation with ventral tegmental area neuronal loss (-0.22, p=1.2×10 ). Dorsolateral TH was better preserved in dementia than in PD and PDD. Ventromedial TH showed no difference across clinical subgroups. Dorsolateral TH correlated with LB counts in neocortex, while ventromedial TH correlated with LB counts in limbic cortices. Genetic analyses failed to show an association between any of the 29 PD GWAS risk variants or APOE4 with putaminal TH. Conclusion


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The results demonstrate regional differences in putaminal dopaminergic degeneration, and confirm vulnerability of nigrostriatal pathway in Lewy body disease. The failure to find association with PD GWAS risk factors suggests that they are not associated with quantitative phenotypes, but more likely related to risk of disease per se.

C05.c. Genetics, Epidemiology: GWAS, genetic associations, susceptibility & protective genes ADPD7-1328 INTEGRATED WHOLE GENOME EXPRESSION PROFILING OF SUBSTANTIA NIGRA IN PARKINSON'S DISEASE TO COMPREHENSIVELY INVESTIGATE POTENTIAL GENETIC BIOMARKERS AND POSSIBLE DRUG TARGETS 1 M. Murthy 1 University of Mysore, DOS in Genetics and Genomics, Mysore, India Aims Whole genome expression profiling of substantia nigra was carried out in 171 samples from 9 datasets to identify differential expression pattern across different studies and their contribution to disease pathway. Method Total RNA was extracted from dissected brain tissues and expression profiling was performed using Affymetrix Human Gene Chip Primeview array. In addition, a meta-analysis was carried out using 9 publicly available datasets. The raw data sets was analyzed and normalization was carried out using the RMA (Robust Multi-array average) normalization algorithm. Principal component Analysis (PCA) was performed for quality control assessment. Only genes with P value 1 were considered significant. Multiple testing was corrected using the Benjamini–Hochberg FDR method to obtain the adjusted P-value. Subsequently, cluster analysis,gene ontology, functional annotation and enrichment was performed. Results Expression profiles of 92 cases and 79 controls identified 424 DEGS consistently in two or more studies with 36 genes with >5 fold change. Our study proposes diagnostic markers like GNAS and DDC and several therapeutic targets like PGC, RBM3, SLC18A2 and TH. The study also suggests regulatory partners which can be probable pharmacological modulators that might prove useful in treatment. Conclusion


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The study exclusively on expression profiles in substantia nigra uses top significant DEGs to identify the effect of DEGs on their interacting partners in the disease pathway.

C05.c. Genetics, Epidemiology: GWAS, genetic associations, susceptibility & protective genes ADPD7-1476 THE FAROESE PARKINSON’S DISEASES RESEARCH PROGRAM – MULTIFACTORIAL ANALYSES OF A COMPLEX SYNDROME 1 1 2 2 2 2 3 4 2 M.S. Petersen , S. Bech , L. Farrell , T. Candido , I. Guella , D. Evans , B. Ritz , J. Aasly , M. Farrer 1 The Faroese Hospital System, Department of Occupational Medicine and Publich Health, Tórshavn, Faeroe Islands 2 University of British Columbia, Centre for Applied Neurogenetics- Department of Medical Genetics, vancouver, Canada 3 UCLA School of Medicine and UCLA Fielding School of Public Health- University of California Los Angeles, Department of Neurology - Department of Epidemiology and Department of Environmental Health, Los Angeles, USA 4 Norwegian University of Science and Technology and St. Olav's Hospital, Department of NeuroscienceDepartment of Neurology, Trondheim, Norway Aims The program aim is to assess the contribution of genes and environment influencing susceptibility and progression underlying Parkinson’s disease (PD) utilizing a combined population and pedigree approach. The etiology of PD is unknown but genetic and environmental determinants seem to be involved in the etiology, probably in interaction. To decipher their relative contributions requires the homogeneity afforded by population isolates. The Faroe Islands have excellent historical genealogy, elevated risk of PD and high exposure to environmental risk factors. Comparing and contrasting findings within and between family-based cohorts provides a unique opportunity. Method As of January 2015, the Faroese cohort consists of 270 PD patients with clinical data, blood samples, detailed genealogic, lifestyle and environmental data available for ~80%. Also 253 controls and 156 unaffected relatives have provided a blood sample and lifestyle/environmental data. Ongoing clinical research exams detail autonomic, cognitive, motor, psychiatric, sensory and sleep components of PD, and explore potential prodromal features in asymptomatic relatives. Follow-up has been performed for 53 living patients and 46 unaffected relatives. Pedigrees have been constructed identifying two major clusters of increased incidence. Results Our genetic approach includes segregation and power analyses, under a variety of models, employing PD, related traits and liability classes. Targeted sequencing and genotyping has assessed pathogenic mutations in genes for parkinsonism. Genome-wide high-density genotyping (n=230) and exome sequencing (n=50) has been performed. In parallel, environmental research has explored exposure to persistent pollutants. Conclusion


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The Faroese Research Program offers a unique opportunity to study multiple aspects of PD risk and progression.

C05.c. Genetics, Epidemiology: GWAS, genetic associations, susceptibility & protective genes ADPD7-1393 GENETICALLY DIVERSE MOUSE MODELS - A NEW SYSTEM TO IDENTIFY THE GENE NETWORKS THAT UNDERLIE SUSCEPTIBILITY, VARIABILITY AND MECHANISMS OF NEURODEGENERATION. 1,2 3 4 4 5 6 1,2 S. Stephenson , D. Finkelstein , S. Rizzo , T. Green , C. Lutz , N. Rosenthal , P. Lockhart 1 Murdoch Childrens Research Institute, Bruce LeFroy Centre for Genetic Health Research, Melbourne, Australia 2 University of Melbourne, Department of Paediatrics, Melbourne, Australia 3 The Florey Institute of Neuroscience and Mental Health, Neurodegeneration, Melbourne, Australia 4 The Jackson Laboratory, Center for Biometric Analysis, Bar Harbor, USA 5 The Jackson Laboratory, Genetic Resource Science, Bar Harbor, USA 6 The Jackson Laboratory, Rosenthal Laboratory, Bar Harbor, USA Aims Neurodegenerative disease is thought to result from the culmination of life history and interactions between genetic background and the environment. Progress toward treatment and prevention requires new approaches that enable genetic analysis of complex systems. The Collaborative Cross (CC) is a panel of 8-way multiparental recombinant inbred mice strains designed for the analysis of diseases caused by the combined effect of multiple genes. To investigate the feasibility and suitability to neurodegenerative research, the acute MPTP-induced Parkinsonism model was employed in a panel of CC mice. Method Thirty two CC F1 hybrid strains (≥10 mice/strain) and a reference population of C57BL/6J mice were intoxicated with 60 mg/kg MPTP by intraperitoneal injection of 15 mg/kg or 0.5 ml saline given as four injections two hours apart. Mice were subjected to locomotor activity phenotyping (day -1, 7 & 20) and brains were collected (day 21) for stereological analysis of the dopaminergic population of the substantia nigra. Results The MPTP dose was tolerated by the CC F1 hybrid strains with minimal animal death during the acute toxicity phase (24h post MPTP). Strain-dependent variation in locomotor activity was identified. Stereological analysis of neurodegeneration within the substantia nigra is on-going and preliminary results will be presented. Conclusion


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Traditional model systems do not allow the study of the gene networks that underlie complex diseases. The CC panel of recombinant mouse strains now provides the opportunity to dissect the complex etiology of neurodegeneration in the context of an experimental model system that more accurately reflects the genetic structure of human populations.

C05.c. Genetics, Epidemiology: GWAS, genetic associations, susceptibility & protective genes ADPD7-0767 GENOME-WIDE ASSOCIATION OF THE PARK10 LOCUS IN IDIOPATHIC AUTOPSY-PROVEN LEWY BODY PARKINSON DISEASE AND AUTOPSY-PROVEN CONTROLS. 1 1 1 1 2 1 1 1 J. Vance , K. Nuytemans , L. Gomez , W. Scott , D. Gveric , G. Beecham , E. Martin , A. Consortium 1 University of Miami Miller School of Medicine, HIHG, Miami, USA 2 Imperial College London, Centre for Brain Sciences, London, United Kingdom Aims We have previously shown that the PARK10 locus is strongly associated with Parkinson disease (PD) using both autopsy-confirmed (AC) Lewy body (LB) PD and AC-controls, but not associated using clinically-diagnosed datasets. This finding supports the multiple reports that up to 20% of clinically diagnosed PD have additional neuropathological findings other than LB PD on autopsy. To confirm the PARK10 association, we genotyped a second independent AC-PD and AC-control dataset. Method 137 AC-PD cases and 55 AC-control DNA was obtained from the UK brain bank. These were genotyped for rs10788972, the SNP demonstrating the strongest previous association. This dataset was analyzed individually for association and jointly with the previous dataset collected by the Autopsy-confirmed PD Genetics Consortium (APDGC) (Beecham et al. 2015). Results Association results from the UK brain bank samples demonstrated positive association for rs10788972, with p = 0.02. Joint analysis (621 cases, 1200 controls) demonstrated genome-wide association (p = 4.43E-09). Conclusion


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This finding has several important implications for both PD and studies in neurodegeneration. First, this confirms PARK10 as a major locus for idiopathic, Lewy-body PD, based on genome-wide association and previous linkage studies. Second, the reason for the absence of association in clinically-defined cases and controls is unclear. While the most likely mechanism is neuropathological heterogeneity, it would seem that heterogeneity in PD cases AND controls are needed to contribute to this finding. This raises the question whether using clinicallyidentified controls is adding more genetic heterogeneity to current genetic analyses in neurodegenerative studies than previously thought.

C05.c. Genetics, Epidemiology: GWAS, genetic associations, susceptibility & protective genes ADPD7-1030 RARE GENETIC VARIANTS IN DEMENTIA WITH LEWY BODIES IDENTIFIED BY WHOLE EXOME SEQUENCING 1 1 L. Vergouw , F.J. de Jong 1 Erasmus MC, Neurology, Rotterdam, The Netherlands Aims Recent studies show that genetic factors may play an important role in Dementia with Lewy Bodies (DLB). Different genes have been shown to be associated with DLB, however evidence is still weak. Here, we used exome sequencing on patients with DLB to identify rare variants that were previously implicated in DLB (GBA,LRRK2,MAPT,APOE,APP,PSEN1,PSEN2,SCARB2,SNCA).Method DNA was extracted from blood samples of 10 unrelated cases conforming to both clinical and postmortem diagnostic criteria for DLB and sequenced using Nimblegen v2 Seqcap EZ Exome Kit. Variants were called using GATK and annotated by Annovar. We used the Exome Aggregation Consortium and Genome of the Netherlands as reference databases to filter for rare variants (MAF 1.2 in both directions, and FDR < 0.05 were considered significantly differently expressed between both groups. Biological functions enriched with these up- and down-regulated genes were identified using DAVID® bioinformatics resources. Results Results. Functional classification revealed cellular pathways differently affected in CADASIL vs sporadic SVD. The vascular compartment was more affected in CADASIL, with enhanced cytokine-related fibrosis, reduced oxidative phosphorylation switching to glycolysis, and induction of programmed cell death (apoptosis). Conclusion


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Conclusions. Elucidating the differences in molecular events participating in the pathologenesis of CADASIL and sporadic SVD will certainly identify novel and specific therapeutic approaches.

E01. Disease Mechanisms, Pathophysiology ADPD7-0298 INSIGHTS INTO THE MECHANISM OF PROTEIN FUNCTIONAL LOSS UPON COVALENT MODIFICATION BY HOMOCYSTEINE THIOLACTONE: OLIGOMERIZATION OR AGGREGATION 1 1 1 G.S. Sharma , T. Kumar , L.R. Singh 1 University of Delhi, Dr. B. R. Ambedkar Center for Biomedical Research, Delhi, India Aims Homocysteine thiolactone (HTL), a toxic metabolite accumulated in cells and serum under hyperhomocysteinemic conditions is known to covalently modify protein lysine residues resulting in functional loss. The present study aims at investigating the mechanism of enzyme functional loss due to protein post-translational modification by HTL. Method The effects of HTL on structural and functional activity parameters of Ribonuclease-A (RNase-A), Lysozyme (Lyz) and Carbonic anhydrase (CA) were studied using several spectroscopic techniques. Oligomerization and aggregation were monitored using Dynamic Light Scattering (DLS) measurements and Transmission Electron Microscopy (TEM). Results While RNase-A and Lyz did not exhibit any functional loss due to overnight incubation with HTL, CA was rendered non-functional at the same incubation time. However, gradual decrease in enzyme activities was observed upon prolonged incubation of RNase-A and Lyz, with complete inhibition in ten days. Conformational analyses revealed that CA undergoes unfolding, ultimately leading to aggregation which could be the reason for functional loss. RNase-A and Lyz experienced no structural alterations, but formed large oligomeric spherical ensembles with varying sizes (50-200 nm in diameter). We speculate that the substrate becomes inaccessible to the catalytic site of the modified enzyme due to generation of higher oligomeric species in case of RNase-A and Lyz, thereby resulting in functional loss. Conclusion


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The study revealed mechanisms for functional loss due to N-homocysteinylation, one occurring via generation of large oligomers thereby limiting substrate accessibility to the active site, or via structural alteration thereby disrupting the protein native conformation and stabilizing into amyloid-like structures.

E01. Disease Mechanisms, Pathophysiology ADPD7-1137 POTENTIAL THERAPEUTIC BENEFITS OF PERLECAN DOMAIN V IN PRECLINICAL VASCULAR DEMENTIA AND CEREBRAL ANGIOPATHY 1 1 1 1 1 A. Trout , Z. Zhang , J. Roberts , A. Hartz , G. Bix 1 University of Kentucky, Sanders-Brown Center on Aging, Lexington, USA Aims Cerebrovascular remodeling and angiogenesis may represent early compensatory changes to the reduced blood flow seen in VCID by increasing the proteolytic turnover of the surrounding extracellular matrix. We have demonstrated that one such extracellular matrix protein, perlecan (a heparan sulfate proteoglycan), possesses a C terminal domain V (DV) protein that upon cleavage from perlecan greatly enhances brain angiogenesis. Our objective is to determine if DV increases angiogenesis and promotes Amyloid βeta (Abeta)1-42 protein clearance in VCID. Method We characterized VCID induced changes in a distinct mouse model (diabetic APP/PS1 knock in (db/AD)) that has gradual cognitive decline by 9 months with microangiopathy, Abeta1-42 deposition, aneurysms, and microhemorrhages. We also utilized an in vitro model of the blood-brain barrier (BBB) to assess the transport of human Abeta1-42 in the presence of DV. Results In db/AD animals (3-6 months), we observed a decrease in BBB proteins (i.e.claudin-5), indicating that altered function correlated with an increase in brain DV expression during the asymptomatic angiogenic stage, which precedes cognitive changes (9-12 months). In vitro, DV doubled the transport of Abeta1-42 into the lumen of cerebral microvessels over 24 hours with increased activity and total protein expression of P-glycoprotein (P-gp), one of Abeta’s known transport proteins. Conclusion


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Collectively, these data indicate that early cerebrovascular changes induce angiogenic-remodeling that correlates with increased expression of DV. DV, in turn, may further enhance angiogenesis and increase brain Abeta clearance into the vascular compartment through P-gp, suggesting that DV could represent a novel therapeutic for VCID.

E01. Disease Mechanisms, Pathophysiology ADPD7-0070 COGNITIVE FINCTION, PHYSICAL FUNCITON, AND BODY COMPOSITIONS IN ELDERY WITH DIABETES 1 2 2 1 H. Umegaki , T. Makino , T. Hayashi , M. Kuzuya 1 Nagoya university, Geriatrics, Nagoya, Japan 2 Nagoya university, Institute for inovation of future society, Nagoya, Japan Aims Diabetes and/ or insulin resistance is a risk factor for dementia. Sarcopenia is also a risk for dementia. Recent studies have also shown that diabetes and/or insulin resistance is associated with sarcopenia. However, the profiles of cognitive function, physical function, and body composition in diabetic subjects have not well characterized. In the current study we compared the profiles of cognitive function, physical function, and body composition in the community dwelling elderly with diabetes to those in the subjects without diabetes. Method A total of 444 community dwelling elderly with subjective memory complaints were involved (61 diabetics and 383 non-diabetics, mean age 72.4±4.7). The subjects with the MMSE score of below 24 or diagnosis of dementia were excluded. Well-trained clinical psychologists examine the cognitive function of the participants via a neuropsychological battery of tests. Muscle strength, gait speed, aerobic capacity, and balance ability was measured. With a bioelectrical impedance data acquisition system (Inbody 430; Biospace Co., Ltd., Seoul, Korea) body compositions were analyzed. Blood samples were obtained after the participants have fasted for at least 12 h. Results The diabetics had lower executive function examined by Trail Making Tests and slower gait speed compared to non-diabetics. The multiple regression analysis showed that gait speed was significantly associated with executive function. Insulin resistance was not associated with cognition in this population. Conclusion


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Community dwelling elderly with diabetes had lower executive function and slower gait speed. The elucidation of the mechanism of these association would be warranted.

E01. Disease Mechanisms, Pathophysiology ADPD7-1052 CHRONIC CEREBRAL HYPOPERFUSION ENHANCES TAU HYPERPHOSPHORYLATION AND REDUCES AUTOPHAGY IN ALZHEIMER’S DISEASE MICE 1 L. zeng 1 National Neuroscience Institute, Research, Singapore, Singapore Aims Cerebral hypoperfusion and impaired autophagy are two etiological factors that have been identified as being associated with the development of Alzheimer's disease (AD). Nevertheless, the exact relationships among these pathological processes remain unknown. To elucidate the impact of cerebral hypoperfusion in AD, we created a unilateral common carotid artery occlusion (UCCAO) model by occluding the left common carotid artery in both young and old 3xTg-AD mice. Method We created a unilateral common carotid artery occlusion (UCCAO) model by occluding the left common carotid artery in both young (3 mths old) and old (16 mths old) 3xTg-AD and WT mice. Results Two months after occlusion, we found that ligation increases phospho-Tau (p-Tau) at Serine 199/202 in the hippocampus of 3-month-old AD mice, compared to sham-operated AD mice; whereas, there is no change in the wild type (WT) mice after ligation. Moreover, cerebral hypoperfusion led to significant increase of p-Tau in both the hippocampus and cortex of 16-month-old AD mice and WT mice. Notably, we did not detect any change in Aβ42 level in either young or old AD and WT mice after ligation. Interestingly, we observed a downregulation of LC3-II in the cortex of aged AD mice and WT mice after ligation. Conclusion


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Our results suggest that elevated p-Tau and reduced autophagy are major cellular changes that are associated with hypoperfusion in AD. Therefore, targeting p-Tau and autophagy pathways may ameliorate hypoperfusioninduced brain damage in AD.

E02. Therapeutic Targets, Mechanisms for Treatment ADPD7-1486 NEUROVASCULAR INTEGRATIVE EFFECTS OF ENVIRONMENTAL ENRICHMENT ON THE COGNITIVE IMPAIRMENT AND WHITE MATTER INJURY INDUCED BY CHRONIC CEREBRAL HYPOPERFUSION 1 1 1 2 Y.J. Kim , M.K. Song , Y. Kim , J. Lee 1 Kyung Hee University, College of Nursing Science, SEOUL, Republic of Korea 2 Kyung Hee University, College of Medicine, SEOUL, Republic of Korea Aims The aim of this study is to investigate whether environmental enrichment (EE) ameliorates cognitive impairment from blood-brain barrier (BBB) disruption and white matter injury induced by bilateral common carotid arteries occlusion (BCCAO). Method Wistar rats were randomly arranged in each of 7 rats as sham group, BCCAO group and BCCAO+EE group at 4 weeks of age. BCCAO was performed at 12 weeks. EE that includes running wheel, toys, etc. in larger cage than standard cage was applied for 8 weeks before BCCAO and for 6 weeks after BCCAO. Cognitive function was confirmed by 8-arm maze test and Morris water maze test. BBB destruction and white matter injury were confirmed by immunohistochemistry, western blot. Results BCCAO+EE group significantly increased the number of correct in 8-arm maze test and significantly reduced escape latency time in Morris water maze test. BCCAO+EE group significantly increased the length of RECA1 in motor cortex and hippocampal CA1 and ZO-1 in microvessel extracted from total cortex. BCCAO+EE group significantly increased optical density of myelin basic protein (MBP) staining in the corpus callosum and motor cortex Conclusion We demonstrated that EE alleviated the cognitive impairment in 8-arm maze test and Morris water maze test, and significantly inhibited the BCCAO-induced reduction of ZO-1 and MBP. Our results suggest that EE may ameliorate cognitive impairment by BBB disruption and white matter injury in the brain induced by BCCAO.


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Acknowledgement : This work was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2014R1A2A2A01007909)

E02. Therapeutic Targets, Mechanisms for Treatment ADPD7-0117 RESVERATROL PROTECTS AGAINST ICV COLLAGENASE-INDUCED NEUROBEHAVIORAL AND BIOCHEMICAL DEFICITS 1 A. Kuhad 1 Panjab University Chandigarh, UNIVERSITY INSTITUTE OF PHARMACEUTICAL SCIENCES, Chandigarh, India Aims Indeed intracerebral hemorrhage (ICH) account for only 15% of all strokes but it is one of the most devastating subtype of stroke associated with behavioral, cognitive and neurological deficits. The primary cause of neurological deficits in ICH is the hematoma growth, generation of free radicals, inflammatory cytokines and exhausting endogenous anti-oxidant machinery. It has been found that neuroinflammation following ICH leads to exaggeration of hallmarks of ICH. With this background the study was aimed to evaluate the protective effect of resveratrol in intracerebroventricular (ICV) collagenase (COL) induced neurological deficits in rats. Method ICV-ICH was induced by single unilateral intrastriatal injection of collagenase (1 IU in 2µL, ICV) over 10 minutes. From 2nd day onwards, resveratrol at three different doses were administered (5, 10 and 20 mg/kg; p.o.) till the end of the study. Results Chronic treatment with RSV (20 mg/kg) for 21 days restored various behavioral changes, including neurological scoring tests (cylinder test, spontaneous motility, righting reflex, horizontal bar test, forelimb flexion), actophotometer, rotarod, Randall Sellito and von Frey. RSV also restores increase in immobility time forced swim test used to evaluate post stroke depression and impaired memory deficit in Morris water maze. RSV administration also attenuated increased nitro-oxidative stress and TNF-α as well as MMP-9 levels. RSV being a potent antioxidant also restores changes in endogenous anti-oxidant levels. Conclusion


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In conclusion, our research demonstrates that RSV has a protective effect against ICH by virtue of it’s a) antiinflammatory property and b) antioxidant and nitrosative stress restoring property, c) MMP inhibitory action.

E02. Therapeutic Targets, Mechanisms for Treatment ADPD7-0280 NON-MEDICATION IN THE TREATMENT OF ASSOCIATED PAIN SYNDROMES IN THE STRUCTURE OF THE NON-MOTOR MANIFESTATIONS OF INITIAL STAGES OF THE VASCULAR PARKINSONISM 1 1 O. Tondiy , O. Zavalna 1 Kharkiv Medical Academy of Postgraduate Education, Neurology and Child Neurology, Kharkiv, Ukraine Aims To prove the feasibility of the inclusion in the comprehensive treatment of pain in patients with early stage of vascular parkinsonism, physical factors - sinusoidal modulated currents, balneotherapy and low-frequent variable magnetic field. Method We observed 74 patients 52 - 65 years with early-stage vascular parkinsonism - 1 - 2 points for Hoehn & Yahr, st with pains that remind those at spondylarthrosis, low back pain, frozen shoulder periarthrosis. 1 group (26 patients) - control, received only basic treatment - medication drugs - amantadine, MAO inhibitors, dopamine nd receptor agonists. 2 group (48 patients) received basic medical treatment, as well as the impact of physical factors in a combination of sinusoidal modulated currents, balneotherapy (sea salt baths), and low-frequent variable magnetic field magnetic field. Results In patients treated with the proposed inclusion complex therapeutic physical factors, reducing pain, as well as a subjective improvement and, ultimately, improved quality of life has come faster results are more pronounced in a proportion in comparison with the control group. Conclusion


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The combination of a combination of neuroprotective pathogenetic therapy and treatment of physical factors is effective and helps to keep the motor activity of patients with early stages of vascular parkinsonism.

E02. Therapeutic Targets, Mechanisms for Treatment ADPD7-0958 EFFECT OF TRADITIONAL CHINESE HERBAL MEDICINE ON MICROVASCULAR DISEASE AND RELATED PROTEIN OF IN BRAIN TISSUE OF SAMP8 MOUSE 1 1 2 2 J. Liu , D. Zhang , J. Feng , H. Li 1 China academy of Chines medical sciences, Cardiovascular department of xiyuan hospital, Beijing, China 2 China academy of Chines medical sciences, Geriatric department of xiyuan hospital, Beijing, China Aims To observe effect of traditional Chinese medicines ( Panax ginseng, Ligusticum wallichii and rhizoma coptidis) on behavioral and brain microvascular changes and expression of hypoxia induced factor-1α（HIF-1α) and vascular endothelial growth factor (VEGF) and telomerase in Senescence Accelerated Mouse-Prone 8 (SAMP8). Method SAMP8 mice were randomly divided into three groups, including model group, traditional Chinese medicine ( TCM ) high dose group ( 13g crude drug / kg body weight), TCM low dose group (2.6g crude drug / kg body weight), Senescence Accelerated Mouse Resistance-1 ( SAMR1) mice were the normal group, with 10 rats in each group, intragastric administration, continuous administration for 12 weeks. Behavior ability of learning and memory and spatial cognition were observed by Morris water maze and avoid dark test and step-down test, microvascular structure and numbers of neurons were measured, HIF-1α, VEGF and telomerase expression were measured. Results Compared with model group, platform quadrant swimming distance and total distance were significantly increased in the TCM group (P39) in huntingtin gene, encoding mutant Huntingtin protein (mHtt). HD is characterized by progressive atrophy of striatum as a result of degeneration in medium spiny neurons (MSN) which starts with the loss of postsynaptic dendritic spines. While precise link between accumulation of mHtt and 2+ selective vulnerability of MSN in HD remain elusive, a lot of evidence implicates that mHtt disturb Ca 2+ 2+ homeostasis in MSN and affect Ca regulation in spines. Abnormal Ca signaling in postsynapse initiate synaptic dysfunction in MSN which result in failures of synaptic maintenance machinery and the loss of synaptic connections between cortical and striatal neurons. Cortico-striatal synaptic dysfunction is a key component of early HD pathophysiology leading to cognitive decline and motor disorders. At the present study we focused on the neuroprotective properties of sigma 1 receptor (S1R) since one of its primary functions appear to involve 2+ modulation of cellular Ca homeostasis and recent study identified S1R in postsynapse. Method Study was performed on the cortico-striatal co-culture established from mouse model of HD (FVBTg(YAC128)53Hay/J; Jackson Laboratory). Results Administration of selective S1R agonists 2-(4-morpholinoethyl)-1-phenylcyclohexane-1-carboxylate (PRE-084) and R(+)-3-(3-Hydroxyphenyl)-N-propylpiperidine (3PPP) in cortico-striatal co-culture prevents spine elimination in MSN and restores synaptic connections between cortical and striatal neurons. Neuroprotective effect of PRE084 and 3PPP was completely blocked after RNAi-mediated knockdown of S1R. Conclusion


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Based on these findings we assume that S1R agonists may have therapeutic value for the treatment of HD.

G02. Therapeutic Targets, Mechanisms for Treatment ADPD7-1878 CLINICAL FINDINGS IN BRAIN WHITE MATTER PATHOLOGY IN PATIENTS OF MIDLANDS AND HIGHLANDS IN KYRGYZSTAN. 1 1 1 I. Lutsenko , D. Najmudinova , L. Ruslanova 1 Kyrgyz State Medical Academy, Neurology, Bishkek, Kyrgyzstan Aims To estimate the prevalence of white matter pathologies (WMP), such as frontotemporal dementia (FTD), Binswanger disease and CADASIL in patients of midlands and highlands of Kyrgyzstan and to study its predictors. Method We designed a case-control study to estimate the frequencies of WMP, where in basic group we included patients with WMP on MRI, T2 scans according to Fasekas scale and clinical criteria, while in control group we included patients without WMP and cognitive impairments (CI). We assessed CI according to MoCA test, the clockdrawing test, functional activity according to Barthel Index (ADL) and mood changes according to Zung depression test. Results A total of 114 patients (63 men and 51 women) with WMP were included, 52 patients with WMP and 52 in control group. Both FTD and Binswanger disease patients exhibited impaired performance on MoCA compared to controls.The prevalences of early-onset FTD (72%) was much higher on highlands and in total (p=0.01) compared to midlands group (OR for highlands 3.8, 95% CI, 0.8-5.6), with only one genetically confirmed case of CADASIL. In the mild dementia group, drawing clock test had a sensitivity of %, whereas the MoCA detected 100%.There were significant direct correlation between the MoCA test and ADL (p=0.003). More severe depression was in the group of midlands compared to highlands (p=0.001). Conclusion


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Frontotemporal dementia is a more common condition on highlands and midlands of Kyrgyzstan. Highlands is an independent statistically significant factor in combination of long untreated hypertension for WMP and cognitive impairments.

G02. Therapeutic Targets, Mechanisms for Treatment ADPD7-1885 DEVELOPING INHIBITORS OF THE ENZYME “TRMT2A” FOR THE TREATMENT OF POLYQ DISEASES 1,2 3 2,3 4 2,3 M.A. Margreiter , A. Voigt , J.B. Schulz , N.J. Shah , G. Rossetti 1 Institute of Advanced Simulations IAS-5, Forschungszentrum Jülich, Jülich, Germany 2 Institute of Neuroscience and Medicine INM-9, Forschungszentrum Jülich, Jülich, Germany 3 RWTH University Aachen, Department of Neurology, Aachen, Germany 4 Institute of Neuroscience and Medicine INM-4, Forschungszentrum Jülich, Jülich, Germany Aims Recently, experimental evidence produced by Dr. Voigt, in Prof. Schulz’s group, showed that inhibiting tRNA methyltransferase 2 homolog A (TRMT2A) function might cause an error-prone translation, leading to increased number of non Q insertions in the otherwise uninterrupted polyQ stretch. These interruptions, in turn, decrease the probability of polyQ stretches to form toxic aggregates. Upon silencing the TRMT2A gene, decreased polyQ aggregation was observed in yeast, flies and HEK293T cells. Here we report on our first steps towards small molecule inhibitors targeting TRMT2A. Method In order to develop a potent and non-toxic inhibitor, we built a ligand-based pharmacophore model across known tRNA methyltransferase inhibitors against the catalytic domain of TRMT2A. This was combined with structural models of the domain to allow educated guesses about spatial requirements of the binding pocket and proteinligand interaction hotspots. On the other hand, we are analyzing the structural and dynamic properties of the RNA recognition motif of TRMT2A (recently crystalized by Dr. Dierk Niessing’s lab) by molecular dynamics simulations. The aim here is to identify druggable binding pockets, specific for TRMT2A, to perform structure-based virtual screening. Results The resulting molecules of this first ligand-based in silico screening are currently being tested in HEK293 cells by Dr. Voigt’s lab. Conclusion


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In cell experiments will be complemented with ex vivo and in vivo magnetic resonance imaging on healthy and affected mouse brains in Prof. Shah’s lab to allow a more concise assessment of the effects of the hits discovered in the virtual screening.

G02. Therapeutic Targets, Mechanisms for Treatment ADPD7-1519 NEUROPROTECTIVE EFFECTS OF MELANIN-CONCENTRATING HORMONE IN PARKINSON’S DISEASE : A NOVEL FINDING FROM ACUPUNCTURE RESEARCH 1 2 3 4 5 6 6 7 8 H.J. Park , J.Y. Park , S.N. Kim , J. Kim , S. Jeon , M. Oh , Y.J. Kim , J. Yoo , S.U. Park 1 Kyung Hee University, Acupuncture and Meridian Science Research Institute, Seoul, Republic of Korea 2 Daejeon University, College of Korean Medicine, Seoul, Republic of Korea 3 Dongguk University, College of Korean Medicine, Goyangsi, Republic of Korea 4 Dongguk University, Dept. of Biomedical Engineering, Goyang, Republic of Korea 5 Dongguk University, UniversityResearch Institute of Bio-Medi Integration, Goyang-si, Republic of Korea 6 Kyung Hee University, Department of Oriental Pharmaceutical ScienceCollege of Pharmacy and Kyung Hee East-West Pharmaceutical Research Institute, Seoul, Republic of Korea 7 Daejeon University, Department of Biomedical Engineering, Seoul, Republic of Korea 8 Kyung Hee University, Stroke and Neurological Disorders Center, Seoul, Republic of Korea Aims Acupuncture has shown the therapeutic effect on various neurodegenerative disorders including Parkinson’s disease. While investigating the neuroprotective mechanism of acupuncture, we firstly found the novel function of melanin concentrating hormone (MCH) as a potent neuroprotective candidate. Here, we explored whether hypothalamic MCH mediates the neuroprotective action of acupuncture. In addition, we aimed at evaluating the neuroprotective effects of MCH and elucidating underlying mechanism in in vitro and in vivo Parkinson’s disease models. Method First, we tested whether hypothalamic MCH mediates the neuroprotective effects of acupuncture by challenging MCH-R1 antagonist (i.p.) in mice PD model. We also investigated whether MCH has a beneficial role in dopaminergic neuronal protection in in vitro primary midbrain and human neuronal cultures and in in vivo MPTP induced-, pitx3-/- and A53T mutant mice PD models. Transcriptomics followed by quantitative PCR and western blot analyses were performed to reveal the neuroprotective mechanism of MCH. Results We first found that hypothalamic MCH was directly activated by acupuncture treatment and that administration of an MCH R1 antagonist reverses the neuroprotective effects of acupuncture. A novel finding is that MCH showed a beneficial role in dopaminergic neuron protection via downstream pathways related to neuronal survival. Conclusion


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This is the first study to suggest the novel neuroprotective action of MCH as well as the involvement of hypothalamic MCH in the acupuncture effects in PD, which holds great promise for the application of MCH in the therapy of neurodegenerative diseases.

G02. Therapeutic Targets, Mechanisms for Treatment ADPD7-0803 PAK4 REGULATES DOPAMINE SYNTHESIS THROUGH TYROSINE HYDROXYLASE 1 1 2 1 S.Y. Won , S.W. Choi , K.H. Lee , E.G. Kim 1 Chungbuk National University, Biochemistry, Cheongju-Si, Republic of Korea 2 Chosun University, Biomedical Science, Gwangju, Republic of Korea Aims Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra (SN), which results in a biochemical decrease in striatal dopamine (DA) levels. DA producing neurons seem to be the key players and their deficiency contributes to the typical clinical features of the disease which include tremor, bradykinesia, rigidity and postural instability. Thus, it is important to maintain DA normal levels for normal brain function. Method 1. Stereotaxic injection 2. Measurement of dopamine levels 3. Immunohistochemistry 4. Immunoprecipitation 5. Behavioral testing 6. Tissue preparation for immunoblotting analysis Results Here we show that p21-activated kinase 4 (PAK4) increases DA synthesis and rescues 6-hydroxydopamine (6OHDA)/α-synuclein (α-syn)-induced PD behaviour in rat models. We observed that immunoprecipitation revealed a novel interaction between PAK4 and TH in human, rat brain homogenates and primary mesencephalic neuron cultures. Additionally, we found that PAK4 increases TH at Ser40 phosphorylation and activates TH promoter activity. Moreover, lenti-caPAK treatment in rat mesencephalic neuron cultures was increased levels of the neurotransmitter DA concentration compared with lenti-GFP treatment cultures. Conclusion


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Therefore, our results suggest that caPAK has therapeutic potential in the management of PD.

G03. Drug Development, Clinical Trials ADPD7-1804 TRUE SIGHT THE PURPOSE IS A MASS APPLICATION OF THE METHOD OF TREATMENT OF PEOPLE THE RESULTS ARE A COMPLETE TREATMENT THERE IS IRREFUTABLE EVIDENCE 1 G. Iliychovski 1 , Sofia, Bulgaria Aims I'm starting to directly explain the healing processes in my body that I've done over the years using the majority of my brain cells (12% above the normal) and by extrasensory perception. Method These “strings” or “strands” that I began to move after a few months increased their size up to about 60 cm. They were all over my body and usually were located symmetrically - in both hemispheres of the brain or in the left and right side of my body (Fig. 1). On the figure is shown the position of the particles prior to their movement in the direction towards the spine


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.

After intense meditation, channeling the energy from the inside out, I "came out" and managed to move the autonomic nervous system (both main stems) to their rightful position – the spinal column (cord). I moved the coating of both major stems of the autonomic nervous system, located on both sides of the spine (Fig. 2 condition before healing movements).


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The cyst, located in the center of the cerebrum, moved into the lower part of the pelvis and the severity in my head and eternal tiredness disappeared (Fig. 3). Upon relocation of the cyst to the bottom of the pelvis I felt a relief, as if I was freed from a heavily accusation for something I did not commit and cannot bear the guilt and

responsibility for it.

The health is movement! I wish everyone to find his way to it!

Results . Conclusion


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Revolutionary method of treatment, unmatched in history

G03. Drug Development, Clinical Trials ADPD7-1676 ASSESSMENT OF THE QUALITY OF LIFE OF PATIENTS WITH EXTRAPYRAMIDAL DISORDERS 1 2 1 S. Lukmonov , M. Ismailov , D. Tolibov 1 Tashkent Medical Academy, Neurology, Tashkent, Uzbekistan 2 Tashkent Pediatric Medical Institute, Neurology, Tashkent, Uzbekistan Aims Investigation of the effect of motor disorders to quality of life of patients with extrapyramidal disorders. Method Examined 28 patients with Parkinson's disease (PD) at the age from 51 to 74 years (mean age 62,5±7,1 years) and 30 patients with essential tremor (ET) at the age from 48 to 70 years (mean age 59±9,2 years). Quality of life have conducted using scales of merit rating of life in PD (Boer et al., 1996). Results The main symptoms of the disease in patients with PD were hypokinesia and rigidity, which occurred in all patients, as well as resting tremor (observed in 77,5% of patients) and postural instability (9,3%). The results of merit rating of life correlated with data of evidence of hypokinesia (p 0.85. Most subcortical items (immediate verbal memory, sustained attention, delayed verbal IV memory , alternating verbal fluency) differentiated PD-MCI group from PD-NC group, but only confrontation naming item scores—assesssing “cortical” dysfunction—independently differentiated PD-MCI group from PDD groups. A cut-off score of PD-CRS to discriminate PD-MCI from PD-ND is 80/81 yielding a sensitivity of 75.0% (95% CI = 54.8%, 88.6%) and a specificity of 86.4% (95CI% = 64.0%, 96.4%) . ROC curve analysis to discriminate PDD from PD-ND yielded a sensitivity of 87.5% (95% CI = 46.7%, 99.3%) and a specificity of 71.4% (95CI% = 55.2%, 83.8%) with a cut-off of 68/69. Conclusion


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Preliminary analysis of the Chinese version of the PD-CRS confirmed that the scale has high reliability and validity, and can effectively descriminate the severity of the disease.

G04. Imaging, Biomarkers, Diagnostics ADPD7-1898 DEVELOPMENT OF A FULLY AUTOMATED DIGITAL IMMUNOASSAY FOR NEUROFILAMENT LIGHT CHAIN FOR BLOOD AND CSF 1 1 1 1 1 D. Wilson , D. Shan , J. Wickman , M. Holdridge , L. Chang 1 Quanterix Corp, Product Dev, Lexington, USA Aims Neurofilament light chain (NF-L) has been shown to be a potential biomarker for AD progression. Here we develop a digital immunoassay to reliably measure NF-L in serum, plasma and CSF in healthy and diseased individuals. Method Reagents were developed for use on the Simoa HD-1 Analyzer. The Analyzer performs a 2-step sandwich immunoassay, transfers labeled capture beads to an array of microwells, and interrogates the wells for presence of enzyme label. A single labeled NF-L molecule provides sufficient fluorescence in 30 seconds to be detected. The concentration of NF-L is then interpolated from a calibration curve. We evaluated the assay for sensitivity, recovery, linearity, precision, and normal range. Mild brain injury and healthy control samples were evaluated for plasma NF-L. Results Limit of detection (2.5 SD) was 0.040 pg/mL and limit of quantification was 0.171 pg/mL. Recovery of NFL spiked into human serum and plasma averaged 89.5%. Mean dilution linearity of spiked serums was 97.4%. Mean interassay and intra-assay CVs were 10.2% and 7.8% respectively. In normal healthy individuals (n=20), median [NFL] was 8.87 pg/mL (Range 2.68-15.4) and 7.57 pg/mL (Range 3.23-16.0) in serum and EDTA plasma respectively. Median [NF-L] in CSF was 2127 pg/mL (range 258-87,559). Mild brain injury plasma samples (n=30) had a significantly higher median [NF-L] of 25.18 pg/mL (3.69-892.9) than healthy controls. Conclusion


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The Simoa NF-L assay reliably measures NF-L in plasma, serum and CSF samples from healthy and diseased individuals. The assay should facilitate the research and diagnostic development of NF-L for neurodegeneration.

G05. Genetics, Epidemiology ADPD7-0013 PREVALENCE OF ALZHEIMER DISEASE BETWEEN CEREBRAL DISEASES IN PATIENTS WITH ATTENTION IN PHYSICAL TEHRAPY SERVICES 1 G.A. Baquero Sastre 1 Manuela Beltran University, Physical Therapy, Bogotá, Colombia Aims The Alzheimer Disease in their evolution have implications in cerebral functions with impacts that progressively affect elements and capacities of movement that reduce social participation, independence, functionality and quality of life, and for this reasons is necessary interventions of physical therapy. The objective of this study is to know the prevalence of Alzheimer Disease with their characteristics of presentation between the cerebral diseases of patients with attention in Physical Therapy Services Method This investigation is a cross-sectional study and the people of study were patients with attention in Physical Therapy Services in the sites with clinical practices of the Physical Therapy Program of Manuela Beltran University, with diagnostic of cerebral diseases with over 15 years old. For the conformation of people of study was made one census de patients that for cerebral diseases were served in the Physical Therapy between months of February and November of 2012. For the analysis of results was job with general prevalences and specific prevalences with standard error Results In the period of observation of the study, 709 persons has cerebral diseases, of this persons 29.47% has Alzheimer Disease (n= 209, Standard Error=0.02), and this pathology was the second disease in prevalence between the cerebral disease after stroke that has prevalence of 57.54% (n=408, Standard Error=0.01). 77.51% of persons with Alzheimer Disease was of female gender (n=162, Standard Error=0.01) Conclusion


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The Alzheimer disease have one prevalence high between cerebral disease and their presentation have prevalence more high in persons of female gender

G05. Genetics, Epidemiology ADPD7-0184 ADORA1 MUTATION AS LIKELY CAUSE OF A NEUROLOGICAL DISEASE WITH EARLY-ONSET PARKINSONISM 1 2 2 3 4 5 E. Jaberi , M. Rohani , G.A. Shahidi , S. Nafisi , B. Klotzle , J.B. Fan 1 University of Tehran, Biology Cellular and Molecular Biology, tehran, Iran 2 Hazrat Rasool Hospital, Dept. of Neurology, Tehran, Iran 3 Tehran University of Medical Sciences, Dept. of Neurology, Tehran, Iran 4 Illumina, Illumina, San Diego, USA 5 Ilumina, Illumina, San Diego, USA Aims We aimed to identify the genetic cause of neurological disease in an Iranian family whose manifestations include symptoms of parkinsonism and cognitive dysfunction. Method Clinical data on the patients was gathered by interviews with parents, neurological examinations, and laboratory tests. Genetic analysis was performed by genome-wide SNP homozygosity mapping and exome sequencing. Effect of putative disease causing mutation was assessed on co-transfected HEK293 cells with wild type and mutated genes by immunocytochemistry, Western blotting, immunoprecipitation, ELISA, and In Situ Proximity Ligation Assay (PLA). Results Homozygosity mapping and exome sequencing led to identification of p.Gly279Ser causing mutation in ADORA1 that encodes adenosine A1 receptor (A1R) as the probable cause of disease. The mutation segregated with disease status in the family, affects a highly conserved amino acid, and was absent in 700 controls. Conclusion


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The known biological activities of A1R in brain functions including its physical interaction with and inhibitory effect on dopamine receptor D1 (D1R) provide supportive evidence that disruptions of A1R may result in neurological dysfunction. Also, recent evidence on the related adenosine A2B receptor marks the domain in which the mutation is positioned important for function. Finally, ADORA1 is located within the Parkinson’s disease locus PARK16 that has been identified in several populations. ADORA1 may be the PD susceptibility gene within this locus. The molecular mechanism by which the p.Gly279Ser disrupts A 1R function remains unknown, but a quantitative effect on interaction with dopamine receptor was not evidenced.

G05. Genetics, Epidemiology ADPD7-0403 EPIDEMIOLOGY AND MANAGEMENT FOR HUNTINGTON’S DISEASE IN JAPAN 1 M. Miyashita 1 National Hospital Organization- Sagamihara National Hospital, Neurology, Sagamihara, Japan Aims Huntington’s disease: HD is very rare in Asian country compared with European and American country. This discrepancy is reported by different frequency of haplotypes. So, Japanese neurologist have rare chance to manage patients with HD. To clarify the prevalence of HD in Japan, and to investigate their medical condition. Method We collected patients’ data from Japan Intractable Disease Information Center and department of the specific disease control, Japanese Ministry of Health, Labor and Welfare. Then, analyzed these data with statistical methods, and we also analyzed thirty patients with HD in our hospital in their detailed clinical conditions. Results 1.Prevalence rate of HD in Japan is estimated 0.7/100,000 population (number of patients are estimated less than 1000). 2.Mean age at onset is forties and mean duration of illness is estimated 15~20years same as Caucasians. 3.Most HD cases are under home care or long term hospitalization. Only a few patients are at work. Disturbed factors to work are both of psychiatric and movement disorders, especially disturbance of discrete movement, not chorea. 4.Their social states are isolated from social network even their family and friends because of their psychiatric symptoms and their heredity. 5.Most patients are under the medical treatment. They are prescribed tetrabenazine, typical and atypical antipsychotics, SSRI and so on.Conclusion


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Prevalence of Huntington’s disease of Japan is tenth part compared with Caucasian. It is important to social support avoid being closed off both patients and family.

G05. Genetics, Epidemiology ADPD7-1751 SCREENING OF GBA AND LRRK2 G2019S MUTATIONS IN EGYPTIAN PARKINSON’S DISEASE PATIENTS 1 2 1 1 L. R'Bibo , S. Hamed , H. Houlden , N. Wood 1 University College London, Institute of Neurology - Department of Molecular Neuroscience, LONDON, United Kingdom 2 Assiut University Hospital, Department of Neurology and Psychiatry - Faculty of Medicine, Assiut, Egypt Aims Mutations in the glucocerebrosidase gene (GBA) are the most common genetic risk factor for Parkinson’s disease (PD). The G2019S mutation in leucine-repeat rich kinase (LRRK2) gene is found in 1% sporadic PD cases and 4% familial PD cases worldwide. We aimed to estimate the frequency of these mutations in a yet unstudied Egyptian population from the region of Assiut Method DNA was extracted from blood samples from 70 PD cases and 294 controls and exons 8-11 of GBA as well as exon 41 of LRRK2 were screened using Sanger sequencing. Mutations frequencies are compared using Fisher’s exact test. Results GBA mutations were found in 5.1% (3/59) cases and 0.9% (2/217) controls; 2.9% (2/69) of cases and 0.7% (2/289) of controls carry LRRK2 G2019S. These frequencies are quite different despite not being significant (p>0.05 for both), probably due to small sample size. Conclusion This is the first study investigating the role of GBA and LRRK2 G2019S together in an Egyptian population. The most commonly found GBA mutation was L444P in 3 PD patients and 1 control, and has previously been characterised as a severe GBA mutation.


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Further analysis of the samples using NGS for screening of the common genes involved in PD risk is underway. Additional samples are being collected for a more robust estimation of the burden of these genes in PD in this population.

G05. Genetics, Epidemiology ADPD7-1747 SERUM CHOLESTEROL LEVELS OVER TIME AND RISK OF PARKINSON'S DISEASE 1 2 2 3 4 4 1 V. Rozani , T. Gurevich , N. Giladi , B. El-Ad , J. Tsamir , B. Hemo , C. Peretz 1 Tel-Aviv University, Epidemiology and Preventive Medicine, Tel-Aviv, Israel 2 Tel Aviv Sourasky Medical Center, Neurology, Tel Aviv, Israel 3 Maccabi Health Services, Neurology, Tel Aviv, Israel 4 Maccabi Health Services, Survey, Tel Aviv, Israel Aims To evaluate the association of serum cholesterol levels over time with the risk of Parkinson's disease (PD); among statin-free individuals. Method A Population-based large-scale cohort study using medical data of Maccabi Healthcare Services included statinfree individuals with repeated cholesterol measures during a 13-year period. Mean annual levels for total, low and high density lipoprotein cholesterol (TC, LDL-C and HDL-C, respectively) were included in a chronological order for each individual. PD incidence was assessed using a validated drug tracer approach. Cox proportional hazard models with time dependent covariates were applied to estimate PD risk (HR) associated with cholesterol levels over time. Results The cohort included 261,638 individuals (aged 40-79 at the first blood test, 42.7% men) with 2,093,104 repeated measures. During a mean follow up of 7.9 (±3.6) years, 764 (3.3% among individuals aged 65+ years) incident PD cases were detected. Among men, middle and upper tertiles of TC mg/dl (180-209; ≥ 210) and LDL-C (110139; ≥140), as compared to the lowest tertiles, were significantly associated with a lower PD risk. Age pooled HRs (95%CIs) were 0.91 (0.83-1.04) and 0.86 (0.77-0.96) for TC and 0.90 (0.82-0.99) and 0.86 (0.76-0.97) for LDL-C, respectively. Among women, insignificant associations were found between TC or LDL-C levels and PD risk. For HDL-C, null results were found for PD risk among both sexes. Conclusion


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Higher serum TC and LDL-C levels among men over time indicated a decreased PD risk. The potential role of serum cholesterol affecting PD etiology or as a marker of incipient PD warrants further investigation.

G05. Genetics, Epidemiology ADPD7-0166 SURVIVAL IN FINNISH PATIENTS WITH ADULT-ONSET HUNTINGTON’S DISEASE 1 2 3 J. Sipilä , T. Kauko , K. Majamaa 1 North Karelia Central Hospital, Department of Neurology, Joensuu, Finland 2 University of Turku, Department of Biostatistics, Turku, Finland 3 University of Oulu, Institute of Clinical Medicine- Department of Neurology, Oulu, Finland Aims Reports on the factors that contribute to survival in patients with Huntington’s disease (HD) are conflicting. We investigated predictors of survival in a nationwide adult-onset HD cohort. Method Survival after diagnosis of 206 Finnish HD patients (age at diagnosis ≥ 20 years) was analyzed. Data were obtained from national registries, medical charts and genetic laboratories. Results


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Age at diagnosis was 53.2 ± 11.9 years with no difference between genders (men, N=97; p = 0.93). The median survival after diagnosis was 12.3 years among women and 9.8 years among men (p = 0.0045). Gender and age at diagnosis independently, but not CAG repeat length, predicted survival (table). Age of death was 61.3 ± 12.4 years with no difference between genders (men, N=54; p = 0.47). Causes of death differed between genders (p=0.046, figure).

Conclusion


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Male gender and higher age at HD diagnosis, but not CAG repeat length, predicted shorter survival in adult-onset HD.

G05. Genetics, Epidemiology ADPD7-0496 PARKINSON’S DISEASE GWAS GPNMB LOCUS IS AN EXPRESSION QTL IN HUMAN BRAIN TISSUE (BRAINEAC): REVEALING A COMPLEX INTERACTION WITH ANTI-SENSE AND NON-CODING RNA 1 2 2 3 2,4 M.N. Murthy , J. Hardy , C. UKBEC , P.A. Lewis , D. Trabzuni 1 University of Mysore, Genetics and Genomics Laboratory, Karnataka, India 2 UCL- Institute of Neurology, Molecular neuroscience, LONDON, United Kingdom 3 University of Reading, School of Pharmacy, Reading, United Kingdom 4 King Faisal Specialist Hospital and Research Centre, Department of Genetics, Riyadh, Saudi Arabia Aims Exploring and investigating in depth Parkinson’s disease (PD) genome wide association studies (GWAS) result that revealed a significant association with glycoprotein nmb (GPNMB) locus (GPNMB, KLHL7, KLHL7-AS1, NUPL2 and AC005082.12), including the top SNP rs199347. Thus, to understand the risk SNPs/locus putative functional role on the expression in specific human tissues; mainly in brain. Method Integrating data from GWAS and expression quantitative trait loci (eQTL) public datasets such as Braineac (In house), GTEx and PheGenI for the GPNMB locus. Results rs199347 eQTLs show increased expression of GPNMB, KLHL7 and NUPL2 with the homozygouse major allele (AA) in brain tissues, with most significant eQTLs in cortical regions, followed by putamen (PUTM) and hippocampus (HIPP). On the other hand, same eQTL shows decreased expression of KLHL7-AS1. Furthermore, rs199347 is an eQTL with long non-coding RNA (AC005082.12) transcripts in other human tissues but not in brain. This suggesting the complex functional role of the eQTLs in specific tissues, cell type captured at specific time. Conclusion


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In conclusion, although our results suggesting that increase the expression of GPNMB is the rational explanation of the association; other transcripts functional roles in the locus cannot be excluded. This highlights the importance of further investigations to understand the functional interactions between the coding genes, antisense and non-coding RNA species in more depth considering the tissue and cell type specificity in order to understand the underlying biological mechanism for PD and build a holistic view.

G06. Cell, Molecular and Systems Biology ADPD7-0485 DIRECT EVIDENCE FOR THE AXONAL TRANSPORT OF A HTT-RAB4 COMPLEX VIA HIP1 AND RIP11 1 1 S. Gunawardena , J. White 1 SUNY at Buffalo, Biological Sciences, Buffalo, USA Aims The Huntington’s disease (HD) protein, HTT associates with molecular motors. However the cargo complex that HTT moves within axons remains elusive. Previously, we found that HTT differentially regulated the motility of a sub-set of small GTPase Rabs. While Rabs are restricted to particular cellular compartments, the localization of one of these HTT-mediated Rabs, Rab4 is unknown. Here, we tested the hypothesis that a synaptic HTT-Rab4 moving complex exists in vivo and that scaffolding proteins present on this complex mediate associations between this complex and motors during axonal transport. Method In simultaneous dual-view imaging HTT and Rab4, and Rab4 and SYNT/SYNB co-migrated within larval axons. Results Co-localization analysis showed that Rab4, HTT and motors co-localize in Drosophila and human neurons. Intriguingly reduction of the huntingtin interacting protein 1 (HIP1) and a known Rab effector, Rip11, perturbed both HTT and Rab4 motility in vivo. Both Rab4 and HIP1 were present on membranes, while kinesin and HIP1 co-IPed on Rab4 membranes. RIP11 and Rab4 were also co-localized within larval axons, indicating that HTTRab4-HIP1-RIP11 and motors likely form a complex during axonal transport. Strikingly, Rab4 motility was disrupted during HD in both Drosophila and humans. Larvae expressing polyQ HTT or neurons derived from HD patients exhibited axonal blockages that contained HTT and Rab4. Conclusion


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Collectively, our observations suggest that a moving HTT-Rab4 complex exists and that HIP1 and Rip11 function as scaffolding proteins to link this complex to motors for proper axonal transport. Our findings have important implications for how defects in Rab4 motility contribute to HD.

G06. Cell, Molecular and Systems Biology ADPD7-0924 COMPARISON STUDY OF DIFFERENT CELL MODELS RELEVANT TO PARKINSON’S DISEASE 1 1 2 2 2 P. Kitchener , F. Simon , C. Legrand , M. Ferderin , E. Esneault 1 FLUOFARMA, Cell Biology, Pessac, France 2 PORSOLT, Pharmacology, Le Genest Saint Isle, France Aims Parkinson’s disease (PD) is caused due to the loss of dopaminergic neurons in the substantia nigra. In vitro models have been developed using several cell types in order to elucidate the molecular mechanisms involved in the disease and better evaluate new therapies. Cell death induced by 6-hydroxydopamine (6-OHDA), which produces an oxidative-stress, is a phenomenon possibly involved in the loss dopaminergic neurons in PD.

Method The aim of this study was to compare three PD cell models and their response to 6-OHDA intoxication. Two cell lines (PC12 and SH-SY5Y) and dopaminergic neurons derived from human induced pluripotent stem cells (hiPSC) were investigated. Their sensitivity to 6-OHDA was evaluated by measuring lactate deshydrogenase release, mitochondrial function, or dopaminergic cell count. Results Neuronal processes were observed in cell lines but were far less developed than in dopaminergic neurons. Significant TH immunoreactivity in SH-SY5Y cells was observed, however, it was extremely low compared to PC12 and dopaminergic neurons derived from hiPSC. For all three cell types, 6-OHDA was dose-dependently toxic, with increased cytolysis and decreased numbers of viable cells. Conclusion


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These results show that the three cell models are sensitive to 6-OHDA although neuritic development and TH expression vary between cells. Additional experiments are needed to determine the reliability and the predictivity of these models for the evaluation of potential new and different neuroprotective compounds for PD.

G06. Cell, Molecular and Systems Biology ADPD7-1010 EMBRYONIC DEVELOPMENT OF SELECTIVELY VULNERABLE NEURONS IN PARKINSON'S DISEASE 1 1 1 M. Oliveira , R. Balling , R. Fleming 1 Luxembourg Centre for Systems Biomedicine, Luxembourg Centre for Systems Biomedicine, Esch-sur-alzette, Luxembourg Aims A specific set of brainstem nuclei have been proposed to be susceptible to degeneration by histopathological analysis of subjects with suggested prodromal or established early Parkinson’s disease. We hypothesise that neuronal vulnerability reflects a shared cellular and molecular phenotypic characteristics that confer selective vulnerability to degeneration. Neuronal cellular and molecular phenotypic specification is mainly the cumulative result of a transcriptional regulatory program active during the development of the nervous system. Method By manual curation of the developmental biology literature, we comprehensively reconstructed an anatomically resolved cellular developmental lineage for the adult neurons in five brainstem regions that are selectively vulnerable to degeneration in Parkinson’s disease. We synthesised the literature on transcription factors that are required to be active, or required to be inactive, in the development of each of these five brainstem regions, and at least two differentially vulnerable neuronal subtypes within each region. Results Certain transcription factors, e.g., Ascl1 and Lmx1b, seem to be required for specification of many neuronal populations that are susceptible to degeneration in early Parkinson’s disease. Conclusion


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Systematic in vivo assessment of fate determining transcription factors should be completed for all neuronal populations susceptible to degeneration in early Parkinson’s disease.

G06. Cell, Molecular and Systems Biology ADPD7-1767 DETECTION OF CELLULAR TOTAL AND PHOSPHO LRRK2 WITH HTRF® ASSAYS IN BIOMIMESYS® 3D CULTURE 1 F. Servent 1 Cisbio Bioassays, Marketing, Codolet, France Aims Leucine Rich Repeat Kinase 2 has been proven to have an important role in Parkinson’s disease. Mutations in this protein frequently result in the development of the disease. Among these mutations, the G2019S mutation is widely spread and results in a hyperphosphorylation of the LRRK2 protein [1]. This hyperphosphorylation can be monitored by assaying the phosphorylation status of LRRK2 Ser935 with Cisbio’s quantitative assays. Method EXPERIMENTAL PROCEDURE WITH HTRF® AND BIOMIMESYS® Results Phospho-LRRK2 assay was performed using a traditional 2D cell culture method in comparison to the Celenys 3D cell culture cells. While 3D NIH-3T3 cells were dispensed into Biomymesis 96-well plates at a concentration of 100 K cells/well, 2D cells were used at the optimal density of 500 K cells/wells. Serial titrations of CZC-54252 & GSK-257812A were used as inhibitors and added to the cells, and then corresponding IC50s were calculated. Despite observing a higher S/N with 2D cells, inhibitor pharmacology was very comparable between the 2 types of cell culture and in accordance with the literature. Conclusion


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HTRF detection is perfectly compatible with 3D cell models. Here it enabled the characterization of inhibitors targeting LRRK2 phosphorylation and determination of IC50, thus showing that Cisbio’s cell-based kits can be used in a wide variety of models from xenografts to 2D/3D cell culture.…

G06. Cell, Molecular and Systems Biology ADPD7-0779 NEURON PROTEOTOXIC RESPONSE MEDIATES NEURONAL VULNERABILITY TO ACUTE PROTEOTOXIC STRESS 1,2 3 4 1 T. Urban , A. Daub , A. Oliveira , S. Finkbeiner 1 The J. David Gladstone Institutes, Neurological Disease, San Francisco, USA 2 The University of Texas Medical Branch, School of Medicine, Galveston, USA 3 University of Washington Medical Center, Radiology, Seattle, USA 4 University of Zurich, Center for Molecular Cardiology, Zurich, Switzerland Aims Abnormal deposition of aggregated protein in neurons is a shared pathologic feature of the major neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, Huntington’s disease and ALS. This fact suggests that there is a mismatch between the production and clearance of misfolded proteins in neurons. The aim of this study is to understand whether and how neurons recognize and respond to misfolded protein. Method We used a combination of both in vitro an in vivo techniques including primary rodent neuron culture, longitudinal imaging, qRT-PCR, immunocytochemistry, Western Blot, and RNAseq to study the neuronal response to misfolded protein. Results We found evidence that the heat shock response (HSR), one of the principal coping responses to misfolded protein, is deficient in neurons, both in vitro and in vivo, compared with non-neuronal cells. Detailed analysis of the neuronal transcriptome following proteotoxic stress not only confirmed the absence of a recognizable HSR, but suggested a novel transcriptional response to proteotoxic stress. Conclusion


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Our findings suggest that the canonical HSR in neurons is deficient and that neurons may mount a unique response to proteotoxic stress. Ongoing studies in our lab suggest that this neuron-specific transcriptional response may help to protect neurons against proteotoxicity and may form part of an uncharacterized neuronal proteotoxic stress response.

G07. Animal Models ADPD7-0593 ULTRASONIC VOCALIZATIONS RELEASE CATALEPSY IN RATS: A NEW ANIMAL MODEL OF PARADOXICAL KINESIA 1 1 1 1 L. Tonelli , M. Wöhr , R. Schwarting , L. Melo-Thomas 1 Philipps-University of Marburg, Faculty of Psychology, Marburg, Germany Aims Paradoxical kinesia refers to a sudden transient ability of akinetic Parkinsonian patients to perform motor tasks they are otherwise unable to perform. The mechanisms underlying this phenomenon, which can be elicited by personally significant visual or auditory stimuli, are unknown due to a paucity of valid animal models that faithfully reproduce paradoxical kinesia. Our aim was to develop a new method to evaluate paradoxical kinesia in cataleptic rats by presenting species-relevant signals, namely rat ultrasonic vocalizations. Method To test the effects of such ultrasounds in cataleptic animals, male rats received haloperidol (0.5mg/kg, IP); 60 min after injection the bar test was performed during which a given rat was exposed to different playback presentations of appetitive 50-kHz USV, aversive 22-kHz USV, or relevant acoustic controls. The time until a rat steps down from the bar with both forepaws was considered the catalepsy time. Results Cataleptic rats rapidly stepped down from the bar when exposed to playback of appetitive 50-kHz USV, but not in response to either aversive 22-kHz USV or acoustic controls. In addition, rats exposed to 50-kHz USV showed clear approach behavior towards the sound source. Conclusion


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Our animal model fulfills the criterion of face validity and provides a completely new approach to studying paradoxical kinesia which might be useful for uncovering the mechanisms behind this phenomenon and improving behavioral therapies for Parkinson’s disease.

G07. Animal Models ADPD7-1684 EFFECT OF LITHIUM SALT OF TAURINE AT EXPERIMENTAL PARKINSON'S DISEASE 1 1 1 G. Ghazaryan , H. Zanginyan , L. Hovsepyan 1 Institute of Molecular Biology NAS RA, Molecular membranology, yerevan, Armenia Aims

Purpose of this study was to investigate the influence of the lithium salt of taurine on the development of free radical processes in experimental Parkinson's(PD) disease. Investigation of the effect of the synthesized product was performed in cell culture (in vitro), and animals with experimentally induced PD (in vivo).Method Cytotoxicity testing of the lithium salt of taurine. Cell line KCL-22 (human chronic myeloid leukemia) were cultured in a nutrient medium RPMI-1640 containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100ed / ml penicillin, and 100mcg / ml streptomycin at 37 ° C. KCL-22 cells at a concentration of 0.5 * 106 cells. / Ml were cultured in 24-well plates for 24 hours, after which it was added to the test substance at different concentrations (1-1000 mg / ml). After 48 hours of incubation of 0.4% trypan blue dye and cell viability determined. Cytotoxicity of the drug was determined by the values of IC50 - the concentration of test compound causing 50% inhibition of cell viability. Results The results showed that the lithium salt of taurine is not cytotoxic towards human KCL-22 cells ( human chronik myeloid leukemia). Experiments on animals have shown that the introduction of the lithium salt of taurine animals with experimental PD at a dose of 50mg / kg results in a decrease in the content of lipid peroxides and hydroperoxides, and also reduces the oxidative modification of proteins. Conclusion


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The data are interpreted in connection with the anti-oxidant properties of the drug.

G07. Animal Models ADPD7-0205 PHENOTYPIC CHARACTERIZATION OF THE CAENORHABDITIS ELEGANS STRAIN N2 WITH ETHANOLIC EXTRACTS OF OF WITHERINGIA COCCOLOBOIDES 1 M. Gomez Jimenez 1 UNIVERSIDAD COLEGIO MAYOR DE CUNDINAMARCA, CUNDINAMARCA, Bogotà D.C, Colombia Aims The neurodegenerative diseases have a high prevalence, causing a major public health problem. Also, it has a great social and economic impact. The lack of treatments has allowed the use of alternative therapies in the pharmaceutical industry and the use of plant secondary metabolites (Witheringia coccoloboides native of Colombia) to prevent and control the disease. Using a biological model as Caenorhabditis elegans (Ce), which has been used in different studies of neuroscience, allowing to will evaluate in vivo the ethanol extract of the plant, as a promising treatment for these diseases. Aim: Analyze the phenotypic characteristics of the strain N2 (Ce) with the aim to be used as a model for screening the extract of plant. Method The nematode was cultured and grown in NGM medium with E. coli OP50. Strain was synchronized from eggs to L1 larvae. The extract was performed and standardized for phenotypic characterization of the strain N2 using the length, longevity, reproduction thermal stress trials. Results The longevity of the control strain N2 was 16 to 22 days; however using the extract with a concentration of 1 mg was 28 days. The offspring reproduction was 225, when used extract the reproduction was affected at the highest concentration where there was a decreasing the number of offspring. The nematode length was approximately 1100 +/- 50 µm using extract where there was on length increment of 1400µm. Conclusion


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it was showed that the extract does not affect nematode thermo tolerance and it is not toxic to the N2 Strain.

G07. Animal Models ADPD7-0598 THE ROLE OF ROSEMARY EXTRACT (CARNOSIC ACID) IN DEGRADATION OF HIPPOCAMPAL NEURONS INDUCES BY KAINIC ACID IN THE RAT (THE BEHAVIORAL AND HISTOCHEMICAL APPROACH) 1,2 3 4 5 H. Rasoolijazi , E. Naderali , F. Nikbakht , S. Noruzi ofogh 1 Iran university of medical sciences- school of medicine, Department of anatomy, Tehran, Iran 2 Iran University of Medical Sciences, Cellular & Mulecular Research Center, Tehran, Iran 3 Tabriz University of medical sciences- School of medicine, Department of Anatomy, Tabriz, Iran 4 Iran university of medical sciences- School of medicine, Department of Physiology, Tehran, Iran 5 Iran university of medical sciences- School of advanced technologies in medicine, Department of Neuroscience, Tehran, Iran Aims Systemic Kainic Acid (KA) administration has been used to induce experimental temporal lobe epilepsy (TLE) in rats. The aim of this study was to evaluate the neuroprotective effect of Rosemary Extract (RE, 40% Carnosic acid) against KA-induced neurotoxicity and impaired learning and memory. Method Animals received a single dose of KA (9.5 mg/kg) intraperitoneally (i.p.) and during 2 h, were scored according to a modified version of the scale devised by Racine. RE (100 mg/kg, orally) or vehicle, was administered daily for 23 days, starting a week before KA injection. KA-induced neuronal degeneration and the number of pyramidal cells in hippocampus were evaluated by using flouro-jade B immunofluorescence and Nissl staining. Also, the Morris Water Maze (MWM) and passive avoidance task have been used to assess spatial memory and passive avoidance learning, respectively. Results KA-induced neuronal degeneration was demonstrated by using flouro-jade B immunofluorescence. Our results revealed that, after treatment with RE, neuronal loss decreased significantly in hippocampus in the KA+RE group compared to in the KA group. The Morris water navigation and passive avoidance tasks results revealed that spatial memory and working memory impairment decreased in the KA+RE group compared to in the KA group. Conclusion


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These results showed that RE potentially might moderate the protective effect of neurons in KA-induced neurotoxicity and memory impairment due to its antioxidant activities.

G07. Animal Models ADPD7-1460 EVALUATION OF ETHANOL EXTRACT FROM GARCINIA MANGOSTANA (GM) IN THE MUTANT STRAIN CAENORHABDITIS ELEGANS (HA759). 1 R. Sanchez 1 Universidad Colegio Mayor de Cundinamarca, Bacteriologia, Bogotá, Colombia Aims The aim of this project was to analyze the neuroprotective effects of ethanol extract from Gm in the HA759 strain Neuronal damage is the lead cause of neurodegenerative diseases in the population. Method The mutant strain of C. elegans HA759 , was obtained from the Caenorhabditis Genetics Center at the University of Minnesota. Phenotypic characterization of the mutant strain was performed using a Leica DM IL inverted microscope. The nematode was maintained on NGM Nematode growth medium, the Escherichia coli strain OP:50 used as a food source (donated by Calixto A, Chile Mayor University), and kept at 20°C. The synchronization protocol was standardized according to the characteristics of the mutant strain adjusting the protocols. The extract was added to the agar NGM and S medium in concentrations of 2mg and 1mg. The longevity, sensory-chemo, and neuroprotective assays were performed Results of this study showed that the exposure of the nematodes to extract concentrations (1 mg and 2mg) increases their longevity over the control (approximately 7 days). Also, the nematodes were able to evade the aversive component at concentrations of 2mg, which could relate to the neuroprotective trial where there was evidence of nematodes with ASH neurons, suggesting fluorescent cytotoxicity inhibition Conclusion


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The polyphenols of Gm have a neuroprotective effect, due to their ability to capture free radicals. Finally, it has been suggested that the strain (HA759) can be used as a model for screening analysis of natural extracts in neurodegenerative diseases.

G07. Animal Models ADPD7-1729 QUERCETIN IN COMBINATION WITH PIPERINE INHIBITS MPTP INDUCED STRIATAL NEUROTOXICITY IN RAT MODEL OF PARKINSON’S DISEASE: A NEUROCHEMICAL AND NEUROINFLAMMATORY ANALYSIS 1 1 S. Singh , P. Kumar 1 I.S.F.College of Pharmacy, Pharmacology, moga, India Aims To investigate the neuroprotective effect of quercetin (QC) in combination with piperine against MPTP induced Parkinsonian rats. QC is a natural polyphenolic bioflavonoid with potent antioxidant, anti-inflammatory and free ion scavenging properties but has low oral bioavailability, whereas piperine as bioavailability enhancer was combines with QC for better bioavailability and neuroprotective effect. Method st

th

th

MPTP (100 μg/1 μL /rat) was injected intranigrally on 1 , 4 and 7 days using stereotaxic apparatus. QC (25 and 50 mg/kg/po) and combination of QC (25 mg/kg/p.o) with piperine (2.5 mg/kg/po) was administered daily for 21 th st nd days starting from the 7 day of 1 MPTP injection. On 22 day the animals were sacrificed and brains were subjected to biochemicals (lipid peroxidation, glutathione and nitrite), neuroinflammatory (IL-1β, IL-6 and TNF- α) and neurochemical analysis (DA, NE, 5- HT, GABA, Glutamate, DOPAC, HVA and 5-HIAA). Results st

th

th

Bilateral infusion of MPTP (100 μg/1 μL intranigrally/rat on 1 , 4 and 7 days) into substantia nigra pars compacta leads to impairment of motor skill confirmed from locomotor, rotarod, grip strength and narrow beam walk performance. Co-administration QC in combination with piperine had significantly improved the behavioral, biochemical, neuroinflammatory and neurochemical changes than the alone QC treated group. Conclusion


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The present finding implicated that the anti-Parkinsonism effect afforded by QC and its combination with piperine might be mediated through antioxidant, anti-inflammatory and free ion scavenging mechanisms.

H01. Disease Mechanisms, Pathophysiology ADPD7-1122 OLIGODENDROCYTE PRECURSOR CELLS PROLIFERATION DEFECT IN THE RARE DEMYELINATING DISEASE AGC1 DEFICIENCY 1 1 2 2 3 2 2 3 S. Petralla , L.E. Pena Altamira , L. Sbano , A. Danese , E. Profilo , M. Corricelli , C. Giorgi , F. Palmieri , 2 4 1 P. Pinton , F.M. Lasorsa , B. Monti 1 University of Bologna, Department of Pharmacy and Biotechnology, Bologna, Italy 2 University of Ferrara, Department of Morphology- Surgery and Experimental Medicine- Section of PathologyOncology and Experimental Biology- Laboratory for Technologies of Advanced Therapies LTTA, Ferrara, Italy 3 University of Bari, Department of Biosciences- Biotechnologies and Biopharmaceutics, Bari, Italy 4 University of Bari, Institute of Biomembranes and Bioenergetics IBBE- CNR, bari, Italy Aims AGC-1 deficiency is a rare genetic syndrome caused by mutations in the SLC25A12 gene, which encodes for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC-1). The disease is characterized by reduced AGC1 activity with a profound developmental delay, epilepsy, abnormal myelination and low levels of N-acetyl aspartate, myelin precursor in the CNS, suggesting an oligodendrocytes alteration. To study whether reduced AGC-1 activity due to it reduced expression could influence proliferation and differentiation of oligodendrocyte precursor cells (OPCs) in in vitro (Oli-Neu cells) and in vivo (mice) models. Method OPCs proliferation and differentiation studies by cell count, BrdU incorporation assay and TGF-β/PDGF-α pathway western blot and immunofluorescence analysis in cell models; in vivo analysis of the same pathways, as well as immunohistochemistry and CNPase activity both in adult and in 21-day old mice, since OPCs proliferation peaks in at this developmental stage. Results the in vitro model show a OPCs proliferation defect with an increased expression of differentiation inducing factors (TGF-βs and receptors) and a decreased of proliferation inducing factors (PDGF-α), confirmed in the in + vivo model, in which immunohistochemistry data show a reduced number of Olig2 cells. Conclusion


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alterations of the analyzed pathways may disrupt the physiological cross-talk between neurons and OPCs, crucial for OPCs proliferation and differentiation into oligodendrocytes and for myelin synthesis in the CNS. Future studies will focus on human iPS from AGC1-deficiency patients in the framework of the project from the Italian Ministry of Foreign Affairs (MAE) for Italy-USA cooperation (2016-2017).

H01. Disease Mechanisms, Pathophysiology ADPD7-0870 EMERGENCE OF BRANCHED O-MANNOSE GLYCAN IN A CUPRIZONE-INDUCED DEMYELINATION MOUSE MODEL 1,2 1 1 1 3 3 2 1 K. Sakuda , S. Kitazume , K. Kanekiyo , Y. Kizuka , K. Ogiwara , I. Matsuo , H. Ogawa , N. Taniguchi 1 RIKEN, Disease Glycomics Team, Wako-shi, Japan 2 Ochanomizu University, Graduate School of Humanities and Science, Bunkyo-ku, Japan 3 Gunma University, Graduate School of Science and Technology, Kiryu-shi, Japan Aims Branched O-mannose glycan is specifically expressed in the brain and found on the protein tyrosine phosphatase receptor-type Z (PTPRZ) in vivo. In our previous study, we found that in a cuprizone-induced demyelination mouse model, this glycan is upregulated in GFAP-positive astrocytes in the corpus callosum. Additionally, mice deficient in the O-mannose branching enzyme GnT-IX showed enhanced remyelination in the cuprizone-induced demyelinaion model. These results suggest that this glycan could be a therapeutic target for demyelinating diseases. Therefore, in the present study, we aimed to investigate in detail the expression of branched Omannose glycan during cuprizone-induced demyelination. Method GnT-IX–deficient and wild type mice were treated with cuprizone for 2-8 weeks. Brain sections were obtained and immunohistochemical analysis was performed using Cat-315 antibody as a probe for branched O-mannose glycan. To clarify the origin of the Cat-315–positive cells, cuprizone was administered to Olig2-CreER;ROSA-GFP and Aldh1l1-EGFP mice to specifically visualize oligodendrocyte precursor cells and astrocytes, respectively. Results Although activated astrocytes were observed after 2 weeks of cuprizone treatment, Cat-315–positive astrocytes emerged after 6 weeks of treatment in wild type mice. In GnT-IX–deficient mice, astrogliosis was attenuated after 6 weeks of cuprizone treatment. By analysis of cuprizone–treated Olig2-CreER;ROSA-GFP and Aldh1l1-EGFP mice, cell lineage analysis of Cat-315–positive cells is now conducted. Conclusion


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Cat-315–positive astrocytes originate from limited astrocyte subtypes in the late phase of demyelination. These data suggest that branched O-mannose glycan could be a diagnostic marker for the progressive phase of demyelinating disorders.

H02. Therapeutic Targets, Mechanisms for Treatment ADPD7-1610 LONG-TERM CLINICAL AND NEUROVISUAL RESULTS AFTER TRANSLUMINAL BALLLON ANGIOPLASTY (TPA) IN PATIENT WITH MULTIPLE SCLEROSIS (MS) 1 2 I. Rukhadze , A. Archvadze 1 Central University Clinic after Academic N.Kipshidze, Neurology, Tbilisi, Georgia 2 Central University Clinic after Academic N.Kipshidze, Cardiology, Tbilisi, Georgia Aims Our aim was to look at clinical and neurovisual results after endovascular treatment in patient with MS. Those patient didn’t underwent any other treatment. Method Under our study were 33 patient with different form of MS. From them 17 patient with MS relapsing-remitting (RR), 9 patient with secondary-progressive (SP), 6 patient –primary-progressive (pp) and 1 patient with progressive-relapsing(PR). In all patients we found different degrees stenos in proximal part of jugular vein. From them 28-left-side and 5-right sides. TPA was done in all cases. Every 6 month during two years after endovascular treatment we used: Clinico-Neurological assessment (Kurtzke Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Functional Composite (MSFC) (every 6 month); MRI (every 6 month). Results Our clinical study showed in 4 cases-positive dynamic (3-MS-RR, 1-MS-SP); in 24 cases – stable dynamic-15 – MS-RR,6-MS-SP,4-MS-PA). In 5 there were observed worse MRI finding without clinical picture. Conclusion


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Chronic cerebrospinal venous insufficiency is important in parthenogenesis in MS. Endovascular treatment acts a vital role in the stability of different form of MS.

H03. Drug Development, Clinical Trials ADPD7-1800 EFFICACY OF MOTOR IMAGERY IN MOTOR REHABILITATION OF UPPER EXTREMITY IN MULTIPLE SCLEROSIS 1 1 1 A. Ghassemi , N. Zangiabadi , M. Azin 1 Kerman University of Medical Sciences, Neuroscience Research Center, Kerman, Iran Aims Motor dysfunction is one of the prevalent and morbid sequels in multiple sclerosis (MS). While physical practice is an established therapy for MS, the imagination of a physical activity without any associated overt movement, motor imagery (MI), may also yield promising results. This study was designed to evaluate the ability to perform MI and efficacy of MI in rehabilitating motor tasks in MS patients. Method 24 patients with relapsing-remitting MS were subjected to an immediate pre-treatment and post-treatment singlegroup study. The abilities to perform MI and motor tasks were measured by Nine Hole Peg Test (9HPT), Hand Mental Rotation, and Box and Block Task (BBT) in physical forms. A total of 10 MI training sessions (60 minutes each, with 10 minutes break in the middle) were scheduled for each patient in two weeks. MI training was adjusted according to patient feedback. Results Six patients were not able to perform MI and were excluded from the study. The time required to complete the 9HPT was significantly decreased after MI training (p=0.003). The BBT results revealed a significantly higher number of blocks transferred in 1 minute (p=0.02). MI training increased the response accuracy rate of Hand Mental Rotation (p=20 mild; 19 to 10 moderate; /= 500 mg /day. One patient developed symptoms on increasing pramipexole >2.0 mg/day while 2 patients had it after addition of amantadine. All of them had erectile dysfunction leading to feeling of guilt as well as shame, leading to genesis of thoughts of infidelity by the spouse. All these patients improved after multipronged approach including counselling, reduction in the dose of levodopa or dopa agonist and addition of 50-100mg/day of quetiapine. Conclusion


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OS is a very disturbing, often underestimated, symptom predominantly in males with YOPD, with erectile dysfunction.

J05. Genetics, Epidemiology ADPD7-0683 THE ALZHEIMER’S DISEASE SEQUENCING PROJECT (ADSP): DATA PRODUCTION, MANAGEMENT, AND AVAILABILITY 1 2 3 1 4 4 5 6 7 A. Kuzma , A. DeStefano , K. Faber , L. Cantwell , M. Feolo , A. Stine , N. Gupta , W. Salerno , R. Fulton , 1 8 1 1 2 9 6 1 O. Valladares , J. Bis , D. Childress , R. Cweibel , S. Choi , D. Reyes , E. Boerwinkle , G. Schellenberg , 10 3 1 S. Seshadri , T. Foroud , L.S. Wang 1 University of Pennsylvania, Department of Pathology and Laboratory Medicine, Philadelphia, USA 2 Boston University, School of Public Health, Boston, USA 3 Indiana University, Department of Medical and Molecular Genetics, Indianapolis, USA 4 National Institutes of Health, National Center for Biotechnology Information, Bethesda, USA 5 Broad Institute of Harvard and MIT, Medical Population Genetics Program, Cambridge, USA 6 Baylor College of Medicine, Human Genome Sequencing Center, Houston, USA 7 Washington University, The Genome Institute, St. Louis, USA 8 University of Washington, Department of Medicine, Seattle, USA 9 Columbia University, Taub Institute for Research on Alzheimer's Disease, New York, USA 10 Boston University, Department of Neurology, Boston, USA Aims The Alzheimer’s Disease Sequencing Project (ADSP) was established in 2012 as a Presidential Initiative to fight Alzheimer’s Disease (AD). The aims are to identify protective and risk variants and find a therapeutic target for disease prevention. Method The ADSP Data Flow Work Group (DFWG) and the NIA Genetics of Alzheimer’s Disease Data Storage Site (NIAGADS) support data production, sharing and management for the ADSP, and facilitation of data access to the community. Samples are contributed by the AD Genetics Consortium and Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium. Sample plating and shipping is coordinated by the National Cell Repository for AD. Whole-exome (WES) and whole-genome (WGS) sequencing data were generated by three NHGRI funded Sequencing Centers and QC’ed by the ADSP. The DFWG maintains the ADSP web site, providing study design, cohort information, news releases, and application instructions for ADSP access. The ADSP Data Portal is a collaboration between the Database of Genotypes and Phenotypes (dbGaP), Sequence Read Archive, and NIAGADS. The portal allows authenticated users to identify and download sequencing data archived at dbGaP. Results The ADSP has completed WES of 11,053 unrelated cases and controls and WGS of 892 members from 190 families. As of September 2016, an additional 3,000 genomes are being sequenced. Conclusion


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The DFWG provides support to all ADSP Work Groups for data related issues, coordinates with dbGaP for data transfers, and reviews, posts, and notifies members of new results generated by other work groups. Additional information is located at www.niagads.org/adsp or dbGaP.

J05. Genetics, Epidemiology ADPD7-0123 DIFFERENTIAL A-TO-I RNA EDITING OF THE SEROTONIN-2C RECEPTOR G-PROTEIN-COUPLED, HTR2C, IN PORCINE BRAIN TISSUES 1 2 3 4 K. Larsen , J. Momeni , L. Farajzadeh , C. Bendixen 1 Molecular Biology and Genetics, Molecular Genetics and Systems Biology, Århus C, Denmark 2 Department of Molecular Biology and Genetics, Department of Molecular Biology and Genetics, Tjele, Denmark 3 Deparment of Molecular Biology and Genetics, Deparment of Molecular Biology and Genetics, Tjele, Denmark 4 Department of Molecular iology and Genetics, Department of Molecular iology and Genetics, Tjele, Denmark Aims Parkinson’s disease is a chronic neurodegenerative disorder characterized by the cardinal motor symptoms: bradykinesia, tremor, rigidity and postural instability. Also non-motor symptoms such as chronic fatigue, depression, dementia and sleep disturbances play a significant role with negative consequences in the quality of life of patients with Parkinson’s disease. Several studies have indicated that serotonergic dysfunction in Parkinson’s disease is associated with the development of motor and non-motor symptoms (Politis and Niccolini 2015). The HTR2C gene encodes the 5-Hydroxytryptamine (serotonin) receptor 2C, G-protein-coupled protein which functions as a serotonin receptor. The HTR2C mRNA is subject to A-to-I RNA editing mediated by adenosine deaminases acting on RNA 1 and 2. Method In the current study we examined the molecular characteristics of the porcine HTR2C gene and determined the mRNA editing of the HTR2C transcript in different tissues. Results The A-to-I RNA editing of HTR2C was shown to be conserved in the porcine homologue with five nucleotides edited in exon 5. A differential editing was demonstrated with a high editing frequency in the frontal cortex, parietal cortex, occipital cortex, hypothalamus, brain stem and spinal cord and significantly lower in the cerebellum. Conclusion


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Similarities between pigs and humans in differential RNA editing support the use of the pig as a model organism for the study of neurological diseases.

J05. Genetics, Epidemiology ADPD7-0241 IS ANGIOTENSIN CONVERTING ENZYME GENE ASSOCIATED WITH THE BEHAVIORAL AND PSYCHOLOGICAL SYMPTOMS OF ALZHEIMER’S DISEASE? 1 2 2 3 4 M. Liu , L. Hui-Tzu , S. Yi-Chun , C. I-Ting , Y. Yuan-Han1 kaohsiung Medial University, Neurology, Kaohsiung, Taiwan R.O.C. 2 Kaohsiung Municipal Ta-Tung Hospital, Teaching and Research Center, Kaohsiung, Taiwan R.O.C. 3 Kaohsiung Municipal Ta-Tung Hospital, Department of Library, Kaohsiung, Taiwan R.O.C. 4 Kaohsiung Municipal Ta-Tung Hospital, Department of Neurology, Kaohsiung, Taiwan R.O.C. Aims The indel polymorphism of angiotensin converting enzyme (ACE) gene is correlated with the levels of β –amyloid (Aβ) in serum and cerebrospinal fluid (CSF). Deposition of Aβ in synaptic and cerebral vessels will lead to the white matter change (WMC) that often found at frontal lobe area of Alzheimer’s disease (AD) patient with behavioral and psychological symptoms of dementia (BPSD). Therefore, this study aimed to investigate whether the ACE gene acts as a risk factor of BPSD status in AD patients. Method 360 clinically diagnosed AD patients have recruited together with their demographic characteristics, age, gender, and education, polymorphisms of apolipoprotein E (APOE) and ACE gene polymorphisms. The comprehensive neuropsychological tests including Cognitive Ability Screening Instrument (CASI), mini-mental status examination (MMSE), clinical dementia rating (CDR) scale, and Neuropsychiatric Inventory (NPI) annually to evaluate the cognitive function and BPSD for each recruited patient. All data have recruited into statistical analyses. Results At the base measurement of the first year patient recruited, there shows no significant differences of ages, gender, education and APOE gene within the ACE gene indel group, also no significant contributory factors to NPI score(p>.05). In the two-year follow-up, ACE insert polymorphism shows significant positive correlation (B=.05, p=.019, OR=1.6, 95%CI=1.1~2.5) with progression (worsening) of NPI score. This result implicates that ACE gene involves in aggravating symptoms of BPSD. Conclusion


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This result implicates that the ACE insert polymorphism might involves in aggravating symptoms of BPSD through contributing to lower levels of angiotensin converting enzyme.

J05. Genetics, Epidemiology ADPD7-0892 GLUCOCEREBROSIDASE GENE MUTATION AND APOPROTEIN E GENOTYPE ON DEMENTIA AND PSYCHOSIS IN PARKINSON’S DISEASE 1 1 2 1 1 1 3 3 4 T. Oeda , A. Umemura , Y. Mori , S. Tomita , M. Kohsaka , K. Park , K. Inoue , H. Fujimura , H. Hasegawa , 1 1 H. Sugiyama , H. Sawada 1 Utano National Hospital, Neurology and Clinical research center, Kyoto, Japan 2 Utano National Hospital, Clinical research center, Kyoto, Japan 3 Toneyama National Hospital, Neurology, Osaka, Japan 4 Minami-Kyoto National Hospital, Neurology, Kyoto, Japan Aims Glucocerebrosidase gene (GBA) heterozygote mutations, one of the most prominent genetic risks in Parkinson’s disease (PD), are associated with the development of dementia and psychosis in PD. The relationship between these symptoms and apolipoprotein E (APOE) genotype in PD is controversial, though pathological studies have revealed the co-existence of Lewy body and Alzheimer pathologies to various degrees. We investigated the relationship of these genes and dementia or psychosis in PD. Method 213 PD patients (age 66.8 [10.8], mean [SD]) were examined. We performed the full sequence of GBA and determined APOE genotype using restriction fragment length polymorphism analysis. Odds ratios (ORs) of GBA mutations and APOE alleles for dementia and psychosis history were calculated using logistic regression analysis (adjusted for sex, age, PD duration, and Hoehn & Yahr stage). Results We identified GBA mutations in 19 subjects (8.8%). APOE4 and E2 alleles were found in 45 (21.1%) and 24 subjects (11.3%), respectively. Forty-three subjects (21.2%) were diagnosed with dementia and 76 (35.7%) had a history of psychosis. GBA mutations showed a significant impact on dementia (OR 8.7, 95% CI 2.3-33.1, p=0.002); by contrast, APOE4 allele did not. Psychosis history was significantly associated with GBA mutations and APOE4 allele (3.5, 1.1-11.7, p=0.038 and 2.4, 1.1-5.1, p=0.028, respectively). APOE2 allele did not show any significant impact on these symptoms. Conclusion


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Not only GBA mutations but also the APOE4 allele were significantly related to the development of psychosis in PD.

J05. Genetics, Epidemiology ADPD7-1795 PREVALENCE IN DEPRESSION POST STROKE 1 2 3 V. Qemalli , D. Dobi , A. Prifti 1 The Regional Health Authority, Tirana, Albania 2 Neurology, QSUT, Tirana, Albania 3 The Regional Health Authority, Laboratory, Tirana, Albania Aims To determine the prevalence of depression after stroke; To determine differences in prevalence of depression between the sexes and between age

Method 186 (83F&103M) patients admitted at University Clinic of Neurology in UHC “Mother Teresa”, Tirana, Albania; diagnosed for ischemic stroke were assessed initially, by examination and interview. This patients are followed from 4 to 12 months by the psicosocial staff of our center in their houses. back inventory of used to measure the degree of depression clinical interviews .For each patient was prepared a folder to compare changes in time as a result of intervention of psychosocial.Results

Results The prevalence of depressive illness 4 months after stroke in 186 patients was 28%,major depression and 12% minor depression. There were smoll differences between the sexes and smoll diferenc from the ages . With a non-hierarchic approach to diagnosis of those with depression, 43% of men and 31% of women had an associated anxiety disorder. 12% of male and 15% of female patients interviewed had evidence of depression at the time of stroke. 12 months after stroke 42% of the men were still depressed , as were 26% of the woman and 22% joungest patients were still depressed and 37% older .

Conclusion The prevalence of depression after stroke was comparable with that reported from other studies.


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They have small differences between sexes was revealed and small differences for ages.

J05. Genetics, Epidemiology ADPD7-1700 ATTENTION DEFICIT/HYPERACTIVITY DISORDER, DRUG ADDICTION AND ALZHEIMER 1 1 2 3 1 1 1 1 S. Salman , T. Nadeem , F. Hassan Shah , K. Rubya , S. Karim , M. Usman , R. Ashfaq , A. Iqbal , 1 4 H. Rafiq Khattak , S. Siddique 1 University of Lahore, Department of Pharmacy, Islamabad, Pakistan 2 University of Peshawar, Center of Biotechnology and Microbiology, Peshawar, Pakistan 3 MRC Center, Department of Psychiatry, London, United Kingdom 4 Abdul Wali Khan, Department of Pharmacy, Mardan, Pakistan Aims Attention deficit/hyperactivity disorder (ADHD) and Substance Use Disorder (SUD) co-occur with each other. ADHD effects on SUD or Alzheimer’s have been independent of any other psychiatric co-morbidity. However, the majority of the studies were conducted with a smaller sample size did not cover many sub-types of SUD. We attempted to evaluate the co-morbid condition in relatively larger sample size along with covering the SUD’s subtypes and Alzheimer’s which have not been previously reported. Method It was a cross-sectional, multinational, multi-stage study conducted in Pakistan and Afghanistan (76 hospitals, 153 drug detoxification clinics and centers) and about 4553 (1300 females, 3253 males) patients were enrolled. They were assessed for the prevalence of adult ADHD diagnosed in paint thinners (inhalants) and paan addicts, prescription drug abuse (PDA) and heroin addiction disorder (HD) (according to DSM-IV criteria for SUDs), Alzheimer through ICD-11 and DSM-V. Results The prevalence of adult ADHD in patients with Alzheimer varied from 1.1% (CI 95%: 2.5-5.8) to the highest among heroin addicts 14.3% (CI 95%:9.6–21.2). Conclusion


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In our study, adults with SUD had an elevated risk of ADHD as compared to Alzheimer. Patients with cocaine addiction are more at risk for adult ADHD than any other kind of SUD. This is the first largest study to demonstrate the comorbid condition of ADHD with Alzheimer, inhalants, paan, HD, and PDA.

J05. Genetics, Epidemiology ADPD7-1422 PREVALENCE OF MILD COGNITIVE IMPAIRMENT (MCI) – FIRST RESULTS FROM A POPULATION BASED CHILEAN STUDY 1 2 2 3 2 4 2 2 P. Toro Espinoza , C. Lopez , C. Bambs , J. Schröder , V. Alejandra , H. Andrea , A. JOhanna , C. Sandra , 2 F. Catterina 1 Pontificia Universidad Católica de Chile, Psychiatry, Santiago, Chile 2 Pontificia Universidad Católica de Chile, Public health, Santiago, Chile 3 Heidelberg University, Psychiatry, Heidelberg, Germany 4 Universidad Católica del Maule, Public Health, Talca, Chile Aims Little is known about the prevalence of MCI in different world regions and the effects of education in different social systems. We therefore thought to establish the MCI prevalence according to different diagnostic concepts in Chile. Method Up to now, 3135 residents of the Maule region-Chile (mean age= 53,7 years, SD= 9.3) were investigated by using the Mini Mental State Examination (MMSE), the Adenbrooke´s Cognitive Examination and Trail Making Test. Normative values were obtained from the population. MCI was diagnosed according to the "aging-associated cognitive decline (AACD)" criteria. Complainers were defined as subjects with cognitive complaints but without objective deficits in neuropsychological testing. Results 9,7% of the sample were diagnosed with MCI, 16,9% as non- complainers with cognitive deficits. 32,2% presented with cognitive complaints but showed no objective deficits in neuropsychological testing, and 50,24% were diagnosed as cognitively healthy, without any complaints. With respect to education effects, 7,68% of those with a high education were diagnosed with MCI while 10,9% in the lower education group had MCI. Conclusion


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Similar to other countries, MCI is a rather frequent condition in Chile and effects app. 10% of the young old. That education is associated with a lower prevalence of MCI conforms with the cognitive reserve hypothesis and underlines the importances of preventive measures.

J06. Cell, Molecular and Systems Biology ADPD7-1650 THE EFFECTS OF SEX AND APOE GENOTYPE ON NEURONAL AND AD-RELATED MARKERS 1 1 1 R. Bar , A. Boehm-Cagan , D.M. Michaelson 1 The department of neurobiology, the george S. wise faculty of life science, Tel-Aviv university, Israel


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Background: Age, apoE4 and sex are the three most pronounced risk factors for Alzheimer`s disease (AD). We previously established an animal model in which young male apoE4 and apoE3 mice were used in order to explore apoE4-driven brain pathologies. This revealed that apoE4 is associated with Aβ and phosphorylated tau accumulation, as well as neuronal and synaptic impairments. Objectives: The present study was directed at assessing the extent to which these effects are also apparent in homozygotes and heterozygotes apoE4 female mice. Methods: This was pursued by immunohistological and immunoblot stainings of young apoE3, apoE4 and apoE3/4 female mice directed at AD-related and neuronal parameters. Results: This revealed a significant effect of apoE4 genotype in female mice on the AD-related parameters tau and Aβ. Furthermore, pronounced synaptic effects were observed, showing a decrease in glutamatergic and gabaergic pre-synaptic markers. The magnitude of the effects observed in females was more pronounced than those observed in males allowing the detection of significant effects in apoE3/4 heterozygotes female mice. Accordingly, experiments with the heterozygotes mice revealed intermediate effects to those of homozygotes in Aβ, tau and GFAP. In contrast, in the synaptic markers: VGluT and VGAT, the levels in heterozygotes were similar to those of apoE4 homozygotes mice. Conclusion: The effect of apoE4 genotype, like in AD, is more pronounced in female mice than in male mice. Accordingly, apoE4 in females provides an excellent model for studying the effects of apoE4 both in homozygotes and heterozygotes mice.

J06. Cell, Molecular and Systems Biology ADPD7-1654 DIFFERENTIAL EFFECTS OF APOE4 AND ACTIVATION OF ABCA1 ON BRAIN AND PLASMA LIPOPROTEINS 1 1 1 2 3 3 4 5 Z. Menahem , A. Boehm-Cagan , R. Bar , D. Harats , A. Shaish , H. Levkovitz , J.K. Bielicki , J.O. Johansson , 1 D.M. Michaelson 1 The Department of Neurobiology, The George S. Wise Faculty of Life Sciences, The Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel 3 The Bert W. Strassburger Lipid Center, Sheba Medical Center, Tel-Hashomer 5265601, Israel 4 Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA 5 Artery Therapeutics, Inc. San Ramon, CA, USA


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Background: Apolipoprotein E4 (apoE4), the leading risk factor for Alzheimer's disease (AD), is hypolipidated compared to the most common and AD-benign allele, apoE3. We have recently shown that the ATP-binding cassette A1 (ABCA1) agonist peptide CS-6253 reverses the hypolipidation of apoE4 in the brain and the associated molecular and cognitive deficits. Objectives: We presently investigated the extent to which apoE genotype and CS-6253 treatment affect the plasma levels and lipid particle distribution of apoE, apoA-I and apoJ. Methods: This was pursued by CS-6253 treatment of apoE3 and apoE4-targeted replacement mice and FPLC and Western blot analysis of brain and plasma samples. Results: This revealed that plasma levels of apoE4 were lower and eluted faster following FPLC than plasma apoE3. Treatment with CS-6253 increased the levels of plasma apoE4 and rendered the elution profile of apoE4 similar to that of apoE3. Similarly, the levels of plasma apoA-I were lower in the apoE4 mice compared to apoE3 mice, and this effect was partially reversed by CS-6253. Conversely, the levels of apoA-I in the brain which were higher in the apoE4 mice, were unaffected by CS-6253. The plasma levels of apoJ were higher in apoE4 mice than apoE3 mice and this effect was abolished by the CS-6253. Similar but less pronounced effects were obtained in the brain. Conclusion: In conclusion, these results suggest that apoE4 affects the levels of apoA-I and apoJ and that the anti-apoE4 beneficial effects of CS-6253 may be related to both central and peripheral mechanisms.

J06. Cell, Molecular and Systems Biology ADPD7-1651 APOE4 EXACERBATES HIPPOCAMPAL PATHOLOGY FOLLOWING ACUTE BRAIN PENETRATION INJURY 1 1 1 1 H. Ninio Ben-Moshe , I. Luz , O. Liraz , D.M. Michaelson 1 Department of Neurobiology, The George S. Wise Faculty of Life Sciences, The Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel


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Background: Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with increased risk for hemorrhage and poor outcome following traumatic brain injury (TBI). Objectives: The aim of this study was to develop a model for studying the mechanism underlying the pathological effects of apoE4 following acute brain injury. Methods: This present study used apoE3 and apoE4 TR mice to investigate the effects of apoE genotype following hemorrhage. This was pursued utilizing a needle penetration paradigm, in which the needle is inserted through the cortex into the hippocampus to produce a lesion. This was followed by monitoring the resulting neuronal, vascular and inflammatory effects at different time points following the hemorrhage. Results: The results thus obtained revealed increased levels of activated microglia at the site of injury which was most detectable at 3 days following injury and decreased by 14 days. Importantly, these effects were more pronounced in the apoE4 mice than in the apoE3 mice. Parallel measurements of Aβ42 revealed increased accumulation following injury at the lesion area which was most pronounced at 14 days following injury. Like in the microglia this effect was more pronounced in the apoE4 mice then in the apoE3 mice. The extent to which the observed microglia and Aβ accumulation are associated with additional neuronal and vascular effects of apoE4 will be discussed. Conclusion: The present study shows that the needle penetration model is a good paradigm for studying the isoform specific effects of apoE4 following acute brain injury.

J06. Cell, Molecular and Systems Biology ADPD7-1652 EXPLORING THE SIGNALING PATHWAYS ASSOCIATED WITH THE VEGF-MEDIATED PATHOLOGICAL EFFECTS OF APOE4 1 2 2 1 S. Salomon-Zimri , T. Ben-Zur , D. Offen , D.M. Michaelson 1 The Department of Neurobiology, The George S. Wise Faculty of Life Sciences, The Sagol School of Neuroscience, Tel Aviv University, Israel 2 Sackler School of Medicine, Sagol School of Neuroscience, Tel Aviv University, Israel


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Background: ApoE4, the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with increased neurodegeneration and vascular impairments. Vascular endothelial growth factor (VEGF), originally described as a key angiogenic factor, has recently been shown to play a crucial role in the nervous system. Objectives: To examine the role of VEGF in mediating the apoE4-driven pathologies and the underlying mechanism. Methods: Gene therapy assays (e.g lentivirus and adeno-associated-virus) were used to examine the apoEVEGF related crosstalk utilizing young apoE3 and apoE4 targeted replacement mice. Results: We recently showed that hippocampal VEGF levels are lower in apoE4 mice compared to the corresponding apoE3 mice and that this effect is accompanied by a specific decrease in both VEGF receptor-2 and HIF1-α. Up-regulation of the VEGF system by VEGF-expressing virus reversed the apoE4-driven cognitive deficits and brain pathologies. The signaling cascade leading to the apoE4-driven VEGF pathology was next examined. This was perused by focusing on AKT, ERK and p38 MAPK as markers of key VEGF signaling pathways. This revealed marked activation of phosphorylated p38 in apoE4 mice compared to apoE3 mice and no difference in AKT or ERK phosphorylation. Importantly, the specific activation of p38 in apoE4 mice was reversed by the VEGF treatment. The extent to which additional component of the MAPK cascade are effected by apoE4 is presently examined. Conclusion: These findings suggest that apoE4-driven pathologies in-vivo are mediated by VEGF and that MAPK signaling are involved in this process. These results have important therapeutical implications for AD treatment tailored to apoE4 carriers.

J07. Animal Models ADPD7-0018 THE NEUROPROTECTIVE EFFECT OF CREATINE IS SUPERIOR TO CYCLOSPORINE IN PROPIONIC ACID- INDUCED NEUROTOXICITY. 1 2 3 E. Abdelzaher , A. El-Ansary , H. Nounou 1 Faculty of Medicine, Pathology, Alexandria, Egypt 2 King Saud University- KSA, Biochemistry Department, Riyadh, Kingdom of Saudi Arabia 3 Faculty of Medicine- Alexandria University- Egypt.., Biochemistry Department, Alexandria, Egypt

Objectives: to study the neuroprotective effect of creatine and cyclosporine in ameliorating the neurotoxic effects of orally administrated propionic acid to young rats. Methods: Young male rats (n= 32) were divided into four groups; oral buffered propionic acid - treated group, cyclosporine and creatine protected groups followed by propionic acid. A fourth control group. Several markers were assayed in brain homogenates. Results: propionic acid caused brain degeneration as measured by reduced glutathione and vitamin C depletion together with the increase of malondialdehyde as a marker of oxidative stress. Decreased lactate oxidase activity in brain homogenates of propionic acid-treated rats was attributed to the leakage of the enzyme from brain, while increase of creatine kinase activity suggested a compensatory mechanism to overcome ATP depletion. In addition, propionic acid induced DNA strand breaks. Creatine administration prior to propionic acid treatment was greatly protective showing elevated reduced glutathione and vitamin C levels, decreased lipid peroxidation and lactate oxidase release from brain to plasma, and DNA damage. On the other hand, cyclosporine treatment was less protective compared to creatine. Moreover histopathological examination showed neural loss which is considered as a neurotoxic marker in propionic acid treated group. Creatine and cyclosprine showed high efficacy in ameliorating the induced pathological changes.


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Conclusion: These findings prove that propionic induced brain degeneration in young rats and confirm the protective efficacy of creatine as a nutritional supplement that could be used to treat early diagnosed patients with autism, a disease related to propionic acid neurotoxicity.

J07. Animal Models ADPD7-1395 NEONATAL IMMUNE STIMULATION INDUCES LONG-LASTING DEPRESSION LIKE BEHAVIOR, LESS ANXIETY-LIKE RESPONSE AND HIPPOCAMPAL INJURY IN AGED RATS 1 2 2 2 I. Berkiks , A. mesfioui , A. ouichou , A. el hessni 1 , Kenitra, Morocco 2 ibn tofail university, biology, kenitra, Morocco Aims Postnatal Acute immune stimulation by lipopolysaccharide lead to alterations in affective and cognitive behavior, including the reduction in locomotion, food ingestion and environmental exploration. Generally, sickness behavior represents a conserved strategy for animals to overcome the disease. The aging process is associated with a variety of changes in immunity, which are referred to as immunosenescence, and include higher mortality by infectious diseases. The current study was to evaluate the impact of postnatal immune stimulation on the behavior and the microglia response at the middle age in both of sex Method The pups rat males and females were treated in with LPS (0.250 mg/kg) after 48h the half of each group were sacrificed for the analysis assay. The remained animals were kept until the middle age (10 months) and were tested in the cognitive and affective behavior. Hippocampal proinflammatory cytokine TNF-alpha concentration and the number of astrocytes and microglia were estimated in the dentate gyrus, CA1, and CA3. Results Our results showed that the administration of LPS resulted in alterations in anxiety and depressive behaviors in female but not in male rats. LPS resulted in increased number of astrocytes in the hippocampus in both sexes, although the response was higher in females. Furthermore, LPS increased the number of microglia in the hippocampus, but only in females. Conclusion


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These findings indicate that an immune challenge in infantile rats induces sex differences in affective behavior in late-adulthood, which may be the consequence of sex differences in the number of astrocytes and microglia in the hippocampus.

J07. Animal Models ADPD7-0160 ANTI-PARKINSONIAN EFFECTS OF FLUVOXAMINE MALEATE IN MATERNALLY SEPARATED RATS 1 2 1 E. Dalle , W. Daniels , M. Mabandla 1 University of KwaZulu-Natal, Human Physiology, Durban, South Africa 2 University of the Witwatersrand, School of Physiology, Johannesburg, South Africa Aims Exposure to early life stress has been shown to result in anxiety-like symptoms and exacerbates degeneration of dopaminergic neurons in a rat model of Parkinson’s disease (PD). First line treatment for anxiety disorders includes the use of Fluvoxamine maleate (FM). In this study, we investigated whether treating anxiety-like symptoms with FM has an effect in alleviating the neurotoxic effects of 6-OHDA in a parkinsonian rat model. Method Early maternal separation was used to create a rat model that depicts anxiety-like symptoms. Maternally separated adult Sprague-Dawley rats were treated with FM prior to and following lesion with 6-hydroxydopamine (6-OHDA). The elevated plus-maze (EPM) and the forelimb akinesia tests were used to evaluate anxiety-like symptoms and motor impairment respectively. Blood plasma was used to measure corticosterone concentration, and striatal tissue was collected for dopamine (DA) and serotonin (5-HT) analysis. Results Our results show that animals exposed to early life stress displayed increased anxiety-like symptoms and elevated basal plasma corticosterone concentration which were attenuated by treatment with FM. A 6-OHDA lesion effect was evidenced by impairment in the forelimb akinesia test as well as decreased DA and 5-HT concentrations in the lesioned striatum. These effects were attenuated on DA neurons by FM treatment in the pre-lesion treated as opposed to the post-lesion treated rats. Conclusion


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This study shows that early treatment of anxiety-like behavior decreases the vulnerability of DA neurons to neurotoxic insults later in life thus slowing down DA degeneration in PD.

J07. Animal Models ADPD7-0163 FLUVOXAMINE MALEATE EFFECTS ON DOPAMINE SIGNALING IN THE PREFRONTAL CORTEX OF STRESSED PARKINSONIAN RATS: IMPLICATIONS FOR LEARNING AND MEMORY 1 2 1 E. Dalle , W. Daniels , M. Mabandla 1 University of KwaZulu-Natal, Human Physiology, Durban, South Africa 2 University of the Witwatersrand, School of Physiology, Johannesburg, South Africa Aims Parkinson’s disease (PD) affects extra-striatal midbrain cells resulting in reduced extrinsic supply of dopamine (DA) to the prefrontal cortex (PFC). In the present study, we investigated the effects of reduced DA presence in the PFC on cognitive function and whether treatment with Fluvoxamine maleate (FM) attenuated these effects. Method Maternal separation was used to develop an animal model for early life stress that has chronic effects on brain and behavior. Sprague-Dawley rats were treated with the antidepressant FM prior to 6-hydroxydopamine (6OHDA) lesion to model motor deficits in rats. The Morris water maze (MWM) and the forelimb use asymmetry (cylinder) tests were used to assess learning and memory impairment and motor deficits respectively. Blood plasma was used to measure corticosterone concentration and prefrontal tissue was collected for lipid peroxidation, DA, and serotonin (5-HT) analysis. Results Our results show that animals exposed to early life stress displayed learning and memory impairment as well as elevated basal plasma corticosterone concentration which were attenuated by treatment with FM. A 6-OHDA lesion effect was evidenced by impairment in the cylinder test as well as decreased DA and 5-HT concentration in the PFC. These effects were attenuated by FM treatment resulting in higher DA concentration in the PFC of treated animals than in non-treated animals. Conclusion


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This study suggests that FM may ameliorate cognitive impairment in PD by preserving DA and 5-HT transmission in the PFC.

K01.a. Dementia and Cognitive Dyfunction: Caregiver support ADPD7-1139 A MULTI-CENTER RANDOMISED CONTROLLED PILOT STUDY OF AN E-LEARNING COURSE FOR FAMILY CAREGIVERS OF PATIENTS WITH YOUNG ONSET DEMENTIA 1 2 3 4 5 6 J. Diehl-Schmid , A. Metcalfe , B. Jones , U. Schwertel , A. Kurz , R. Rhapsody Study Group 1 Technical University of Munich, Department of Psychiatry, München, Germany 2 APHP Pitié Salpêtrière, Department of Neurology, Paris, France 3 University of Surrey, Health Economics Centre, Surrey, United Kingdom 4 Imc information multimedia communication AG, Imc information multimedia communication AG, Saarbrücken, Germany 5 Technical University of Munich, Department of Psychiatry, Munich, Germany 6 n.a., n.a., n.a., Germany Aims RHAPSODY (Research to Assess Policies and Strategies for Dementia in the Young) is an international research initiative conducted by eight partner institutions from six countries, supported by the EU Joint Program Neurodegenerative Disease Research (JPND). Based on an analysis of the policy and information environment in six countries, focus-group assessments of individual needs of people with young onset dementia (YOD) and their relatives, and the clinical experience of the participating sites, an e-learning course for family carers of patients with YOD was developed. This is composed of 7 modules with a focus on practical problem solving, and suggestions for available support. The e-learning program is currently being evaluated in a pilot study carried out at three sites (France, Germany, and UK). Method In a multi-center, randomized, controlled trial the e-learning course is being compared with standard support. 60 family carers of patients that had been diagnosed with young onset Alzheimer's dementia or frontotemporal dementia within the last 3 years have been included at the three study sites. Results This is - to the best of our knowledge - the first randomised study of an e-learning course for family carers of patients with YOD. Study closure is scheduled February 2017. Data will be presented about 1) user adherence and online user behavior; 2) impact of the intervention; 3) user satisfaction. Conclusion


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Positive results in this pilot study are a precondition for the implementation and dissemination of the e-learning program as well as larger-scale trials about the program's efficacy.

K01.a. Dementia and Cognitive Dyfunction: Caregiver support ADPD7-0113 FAMILY CAREGIVERS OF PEOPLE WITH DEMENTIA HAVE POORER PSYCHOLOGICAL AND PHYSICAL HEALTH OUTCOMES COMPARED TO NON-CAREGIVERS 1 2 3 4 5 G. Dowling , A. Verstaen , A. Snowberg , J. Merrilees , M. Judith 1 University of California, Physiological Nursing, San Francisco, USA 2 University of California- Berkeley, Psychology, Berkeley, USA 3 Unviersity of California- San Francisco, Physiological Nursing, San Francisco, USA 4 Unviersity of California- San Francisco, Neurology, San Francisco, USA 5 Northwestern University, Medical Social Sciences, Chicago, USA Aims The purpose of this abstract is to describe symptoms of stress, depression, anxiety and burden associated with caregiving of a family member with dementia. Method As part of a larger study, participants completed a series of questionnaires online. The questionnaires included: Perceived Stress Scale, Zarit Burden Inventory, Caregiver Strain Index, PROMIS Global Health Scale, Positive Aspects of Caregiving, Differential Emotions Scale. Dementia type and severity of the care recipient were also assessed. Results To date, 117 family caregivers (97 female, 20 male, mean age = 63 years) have completed the questionnaires. The mean duration of caregiving is 4.3 years with 65% of caregivers being the spouse of the person with dementia and 35% being the child or other family member. Females reported experiencing greater perceived stress (p=.005), burden (p=.002), strain (p=.000) and negative emotion (p=.01) than males. Both male and female caregivers scored, on average, >2 standard deviations (sd) above the mean perceived stress scale score for non-caregiver normative data with younger caregivers scoring even higher (30-44 years >4 sd, 45-54 years >3 sd). Caregivers also reported higher depression and anxiety symptoms, and lower physical and mental health compared to NIH published normative data. Conclusion


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Family caregiving is associated with significantly poorer perceived psychological and physical health than what is reported by non-caregivers. Women and younger caregivers seem particularly at risk for these negative outcomes. It is important to assess psychological and physical health in all caregivers and initiate appropriate treatment early to mitigate negative health outcomes.

K01.a. Dementia and Cognitive Dyfunction: Caregiver support ADPD7-0654 NEW CARE STRUCTURE FOR DEMENTIA PATIENTS AN INTERSECTORAL CARE CONCEPT IN FRANKFURT 1 B. Fahl 1 Neuro-psych.Zentrum, Psychiatrie, Frankfurt, Germany Aims Introduction The Project ist a direction sign wow the social Task and the challenge of the demografic Change with a chronical and progressiv disease as shown with dementia could been acepted and realised i. Method Using the german Guidelines S 3 on diagnosis and tratment of dementia and developing a cross-sectoral integral therapyconcept for patients with medical and non-medical structures. using the General practitioners the psychiatrists and neurologists in ccomination of the care managerist in prozessevaluted konferences to find the best way in a Treatment in the Family a central Office with a elektronical data collection with the help of sozialworkers coordinates the Organisation of home Treatment,non medical activities . The target of this strategyis the improve the detection of dementia patients in die General practise,the Koordination of prevention ,the Kommunikation between the different Groups,to coordinatate the different offers of mrdical and non medical Treatment. the Support of the care giving relatives and avoiding of clinical Treatment .s. Method is written above Results after one year projekt 34 patients wer entiltes. 20 male an 18.female the aceptanz for the patients showed an 100 % aceptanz, with improving the Quality of life. the identical Effekt with the relatives with imprvment of 96% even in the aceptanz and understanding her dementia patients and a significant relief in the daily care giving. 93 % of the General practioniers were complet satisfied in the diagnosis medical and non-medical treatmen as the relief in the burden in the every day work in the doctors office Conclusion


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the Project will continue with the help of the North west hosipital foundation

K01.a. Dementia and Cognitive Dyfunction: Caregiver support ADPD7-1737 CARING FOR ME AND YOU: CO-PRODUCTION OF AN ONLINE COGNITIVE BEHAVIOUR THERAPY PACKAGE FOR CARERS OF PEOPLE WITH DEMENTIA 1 1 2 J. Fossey , S. Hales , W. Clare 1 Oxford Health NHS Foundation Trust, Psychological Services, Oxford, United Kingdom 2 Alzheimer's Society, Communications, London, United Kingdom Aims Carers of people with dementia often experience stress but they may have difficulty accessing support due to the lack of flexibility in delivery of services. Cognitive Behavioural Therapy (CBT) is an effective psychological intervention but there is no online programme in the UK addressing these needs. The aim was to develop a bespoke online CBT programme based on the experiences of carers of people with dementia and involving carers in the co-production of materials at every stage. The package would be used in a 3 arm randomised controlled trial to evaluate its effectiveness. Method Reviews of literature and interviews with therapists and online package developers identified the elements of successful online CBT programmes and of face to face treatments for carers of people with dementia. Carers who had previously attended CBT groups shared their stories and ideas about which elements of their treatment they thought was helpful. They and carers of people with dementia who had not previously received CBT, tested and provided feedback on the materials which were then optimised for use in the trial. Results A bespoke on-line CBT programme adapted to meet the needs of carers of people with dementia in terms of format, session length, language use , video and case examples has been created. It is being used in an RCT which is currently underway, the results of which will be available in 2017 Conclusion


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We illustrate the value of co-production in research and services and the importance of engaging with intended participants at the earliest possible stage

K01.a. Dementia and Cognitive Dyfunction: Caregiver support ADPD7-0115 LOOKING FOR POSITIVE VIEWPOINTS FOR ALZHEIMER’S CAREGIVERS: CASE EVALUATION OF POSITIVE ENERGY GROUP IN TAIWAN 1 W.C. Hsieh 1 Kaohsiung Municipal Ta-Tung Hospital, social work department, Kaohsiung City, Taiwan

Purposes of the study: 1. Explore the needs and burdens of Alzheimer’s caregivers in the treatment process of patients. 2. Design a program that meets the needs of caregivers. 3. Evaluate the effects of implementation of the program. Methods: The study is a qualitative study employing in-depth interviewing. After interviews were conducted from September to December in 2015, the interview results were reviewed and a positive energy group program was designed. Pre-test and post-test were carried out for the program so as to evaluate the effects of the program. Results: The study is a qualitative study employing in-depth interviewing. After investigation was made from September to December in 2015, according to the interview results of caregivers, we understand the caring needs and caring process of caregivers. According to the interview results, we know that caregivers’ act of continuous care mainly comes from positive energy in psychological, social and relationship aspects. And positive energy really has been affecting caregivers’ caring ability and willingness.Conclusion Conclusion:


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Therefore, a two-stage group activity program was designed to provide recollective handmade craft group and art therapy group. Research results of the study show that when the main caregivers of Alzheimer’s patients took part in group activities, positive energy in social and relationship aspects would be increased to them (e.g. caregiver group). The caregivers could obtain more knowledge of patient care, and could more effectively lighten the burden of the caregivers.

K01.a. Dementia and Cognitive Dyfunction: Caregiver support ADPD7-1631 FACTORS ASSOCIATED WITH MULTIDIMENSIONAL CARE BURDEN ANALYSES IN CAREGIVERS FOR DEMENTIA PATIENTS: A CREDOS SUBGROUP STUDY 1 1 2 T.S. Lim , S.M. Lee , Y.H. Lee 1 Ajou university school of medicine, Neurology, Suwon, Republic of Korea 2 Ajou university school of medicine, Department of Preventive Medicine and Public Health, Suwon, Republic of Korea Aims Care burden analysis in dementia patients is important in this rapidly aging society. However, its multidimensional evaluation is difficult and complex process. In this study, the authors evaluated caring time, burden interview, depressive symptoms, health-related quality of life in caregivers for dementia and tried to find out patient and caregiver factors associated with them. Method Among dementia patients enrolled in CREDOS study, 335 caregivers of patients from 12 hospitals in Korea were interviewed in 2012. We assessed patient factors with CGA-NPI, B-ADL, S-IADL, CDR-SOB. Also, care burden was evaluated with Burden interview, Beck Depression Inventory (BDI), Korean Version of Caregiver Activity Survey (CAS-K) and caregiver’s quality of life (QOL) with SF-36v2 health survey. Results There were 218 women and 117 men in the dementia patient group with mean age of 75.9 years. In the matched caregiver group, there were 226 women and 109 men with mean age of 57.1 years. Caregiver gender, caregiver age, CDR-SOB, NPI had significant importance regarding the burden interview score. Caregiver gender, caregiver age, CDR-SOB, and NPI were significantly with caregiver’s QOL, while caregiver gender, BADL and NPI with caregiver’s depression. Concerning caring time for patient support, patient gender, patient age and NPI were significant. Conclusion


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In terms of caregiver’s subjective measurement for care burden such as the burden interview, QOL and depression, caregiver’s demographic factors were more important than patient’s factors. In contrast, the objective caring time was more correlated with patient’s demographic factors.

K01.a. Dementia and Cognitive Dyfunction: Caregiver support ADPD7-0323 THE INFLUENCE OF THE HEALTH CONDITION OF THE CAREGIVER THE CARE ENVIRONMENT AND THE PROGNOSIS OF PATIENTS WITH ALZHEIMER’S DISEASE 1 M. Matsumura 1 Geriatrics, Neurology, Shibuya-ku, Japan Aims The caregiver has a strong influence on the quality of dementia care; therefore, healthcare professionals acknowledge the importance of providing mental support to caregivers. I examined that the influence of the caregiver’s health on the prognosis of the dementia patient.

Method Subjects were 23 patients with AD. Further, 12 of these patients lived at home (HL group) and 11 of them lived at a nursing home (NH group). I evaluated the patients’ functions using MMSE, MENFIS, DAD, Digit Span Test and Trail Making Test. I compared the two groups in terms of number of caregivers, sex, and whether they used the day service. In the NH group, I also examined the reason for admission into the nursing home. Results Six patients with AD were admitted to the nursing home because their caregiver died or fell sick; the other five entered the nursing home because of caregiver burden. Two of the participants died about six months after admission into the nursing home. The mean MENFIS score of the HL group was 2±1, which was significantly lower than that of the NH group 3±2. Conclusion


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This study revealed that more than half of the AD patients entered the nursing home because of the illness or death of their caregivers. This indicates that it is extremely important to manage the health of the caregivers in order to ensure that patients with AD live at home for long. Doctors, nurses, and other healthcare staff should also focus on the health of the caregiver during the course of treatment of dementia.

K01.a. Dementia and Cognitive Dyfunction: Caregiver support ADPD7-0555 LIVING WITH ALZHEIMER: HOW FAMILIES DEAL WITH IT? 1 1 2 G. Ribas Miquel , A. Del Valle , M. Ferré Munté 1 Universitat de Girona, Psicologia, Girona, Spain 2 INSTITUT D'ASSITÈNCIA SANITÀRIA IAS, CAP CASSÀ DE LA SELVA, CASSÀ DE LA SELVA, Spain Aims qualitative study Method 509 surveys filled by careers who take care of people with Alzheimer Results 1- Struggle: Everyday is a struggle against the clock with the diagnosis, with your fear and with the sick person who doesn’t admit what is happening and with their difficulty to accept their need of help. When you help them you don’t know if it is right or not. Will disease develop faster? Will them stop thinking? Will they be able to face any trouble? or they may not react. They start to adopt unusual routines for them and with some luck they will not want to get out of the house. 2-Fear: The fear sensation is constant, the head is full of questions without any answer that might be acceptable. Their character and personality change, not always in a better way, they are irritable. The biggest fear is that they might get lost. Even though just one for every four people affected feel like walking around with no direction, you are thinking: will this happen to him? Will be him the next one who will appear on the news? 3-Anxiety: You don’t sleep well, you are anxious, you have your nerves on edge. You are worried and concerned about everything and always keeping an eye on them. Your job can be affected for this situation. When the disease gets worse you have to consider if you may resign to take care of them or if you send them to a care center. Conclusion


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caregivers need information about grants, psichological suport and assessment trhoughout the disease phases

K01.a. Dementia and Cognitive Dyfunction: Caregiver support ADPD7-0733 SOLUTIONS WITH ALZHEIMER: HOW FAMILIES ARE SUFFERING IT? 1 1 2 2 2 G. Ribas Miquel , A. Del Valle , J.M. Gifre Hipolit , E. Boix Roqueta , M. Ferré Munté 1 UNIVERSITAT DE GIRONA, PSICOLOGIA, GIRONA, Spain 2 INSTITUT D'ASSISTÈNCIA SANITARIA IAS, CAP CASSÀ DE LA SELVA, CASSÀ DE LA SELVA, Spain Aims Qualitative study Method 509 interviews of Alzheimer patients' caregivers Results 1-INFORMATION: Once back at home with the diagnosis, families start looking for more information in the internet or discreetly asking to others. "It is not easy to find useful answers". 2-Families using GPS TRACKER: Many ways can be found to locate the patient. It can be through GPS tracker located in watches, cell phones, wristbands, etc. The most effective one are watches and cell phones. We looked for the best solution for our case agreeing in the family. "It is not easy, nor cheap to be able to wear the device, but the relief obtained out of this worth it". "Economical help is required" 3- Families no using GPS TRACKER: The families who did not wear any GPS tracker feel the following: -Stuck to someone: The family is all the day long looking after the patient. I left my life for focusing in his life. This means that I give up going other places for him, I have no space for me. I only live for him. -Closed indoors: We had to lock the door he wanted to go back to his childhood home, he always try to go back there. At home, the doors are locked and the keys out of the lock. The patient tries to open the door, but when he realises that he cannot open, he just sits next to the door and do not do anything else for a long time. Conclusion


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Caregivers need financial assistance in order to Access technologies GPS

K01.c. Dementia and Cognitive Dyfunction: Cognitive training ADPD7-0461 IMPACT OF COGNITIVE INTERVENTIONS ON COGNITIVE SYMPTOMS IN IDIOPATHIC PARKINSON'S DISEASE 1 1 1 M. Couture , A. Giguère-Rancourt , M. Simard 1 Laval University, School of psychology, Quebec, Canada Aims The present systematic review addressed the effectiveness of cognitive stimulation (CS), cognitive training (CT) and cognitive rehabilitation (CR) programs on cognitive functions in individuals presenting Parkinson’s disease with or without mild cognitive impairment. Method EBSCO, EMBASE, MEDLINE (OVID), PsycNET and Cochrane databases were searched using the key terms such as Parkinson’s Disease, Parkinson Disease and Mild Cognitive Impairment, Cognitive Impairment and Parkinson’s Disease Dementia, Parkinson Dementia and Cognitive Rehabilitation, Cognitive Training, Cognitive Stimulation, Executive Function Training or Executive Function Rehabilitation. Hedges’g were calculated. Results Samples sizes varied from 5 to 76 (most of them with less than 30). Among the 13 studies, 11 were randomized and two were quasi-experimental trials. Twelve intervention programs were CT, four were CS and the last one, a combination of CT and CR. Four were offered in small groups, and nine took place on an individual basis. Most programs targeted multiple aspects of cognition while others focused on a cognitive domain only. Results demonstrated a significant benefit of cognitive interventions in 26% of between-group comparisons, and in 29% of within-group comparisons on information processing speed, attention, working memory, executive functions, visuospatial and visuoconstructive abilities and episodic memory. The data also showed significant improvements on psychological, behavioral and functional symptoms. Conclusion


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Larger samples, robust experimental design, standardized and validated measures of efficacy and standardized cognitive interventions are recommended in future. Guidelines on how to design cognitive intervention programs should be established to improve cognitive interventions in Parkinson’s disease.

K01.c. Dementia and Cognitive Dyfunction: Cognitive training ADPD7-1417 EARLY-LIFE ADVERSITY INCREASES THE VULNERABILITY TO INDUCE THE COGNITIVE DECLINE LINKED TO MICROGLIA ALTERATION IN AGING 1 2 2 2 A. Elhessni , I. berkiks , A. ouichou , A. mesfioui 1 Faculty of Science, biology, Kenitra, Morocco 2 ibn tofail university, biology, kenitra, Morocco Aims Aging is one of many factors associated with an increased susceptibility to neurodegenerative disorders which can be related to early life inflammation. Early life immune stimulation, however, can be characterized by the increase in cytokines, oxidative stress as well as changing in microglia phenotypes from activated to priming during the age. Indeed the early neuroinflammation can profoundly affect brain function which can elicit behavioral impairments and cognitive deficits. The aim of our study is to explore the possibilities to accelerate the cognitive decline associated with aging after a neonatal immune stimulation.

Method Male Wistar rats were treated on a postnatal day 14 with PBS or LPS, and then tested for learning & memory at 3 or 10 months of age, using novel object, Y-maze, and a spatial water maze task. Results Neonatally-infected rats exhibited memory impairments in the water maze, but only at 10 months. And no significant differences in novel object and Y-maze. Neonatally-infected rats also exhibited greater aging-induced increases in a number of microglia-activating in DG, CA1, and CA3, as well as an increase in Nitrite Oxide and lipid peroxidation but not TNFα within the hippocampus, but not in prefrontal cortex compared to controls. Conclusion


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Taken together, these data suggest that early-life infection leads to less successful cognitive aging, which may be linked to changes in microglial reactivity.

K01.c. Dementia and Cognitive Dyfunction: Cognitive training ADPD7-1829 MOOD IMPROVEMENT BOOSTED COGNITIVE TRAINING GAINS THROUGH HIPPOCAMPUS-AMYGDALA CONNECTIVITY IN ELDERLY WITH SUBJECTIVE MEMORY COMPLAINTS 1 J. Li 1 Chinese Academy of Sciences, Beijing, China Aims This randomized controlled trial examined the effect of mood improvement on cognitive training gains and the underlying brain mechanisms using functional connectivity (FC) in elderly with subjective memory complaints (SMC). Method A sample of 124 older adults with SMC were randomly assigned into a group counseling plus cognitive training (GC+CT) group, a cognitive training (CT) group or a group counseling group (GC) (Figure 1). The GC+CT group (n=42) received 6-hour group counseling followed by 4-week cognitive training. The CT group (n=38) completed 6-hour reading followed by 4-week cognitive training. The GC group (n=44) received 6-hour group counseling followed by 4-week health lectures. A battery of neuropsychological tests was assessed at baseline, before (midtest) and after CT (post-test). The GC+MT and GC group were MRI-scanned at mid- and post-tests. Results The GC+MT group showed improved memory performance after training compared to the GC group, indicating the efficacy of cognitive training. GC reduced depression and anxiety, and memory improvement in the CG+CT group was doubled compared to the improvement in the CT group, indicating mood improvement enhanced cognitive training gains (Figure 2). The change in depression was correlated to amygdala-hippocampus FC at mid-test. Moreover, FC between hippocampus and amygdala at mid-test was correlated to improvement in working memory in the GC+CT group. Conclusion


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Mood improvement facilitated benefits from cognitive training applied to individuals with subjective memory complaints. The boosting effect of mood improvement on training gains was related to the hippocampusamygdala network.

K01.c. Dementia and Cognitive Dyfunction: Cognitive training ADPD7-0539 VALIDATION OF A NEWLY DEVELOPED AUTOMATIC MCI EVALUATION SYSTEM; A COGNITIVE ASSESSMENT TABLET (COGNITIVE ASSESSMENT TESTS USING A TABLET COMPUTER; CAT) 1 2 1 3 3 K. Shigematsu , H. Takeuch , N. Oka , T. Tumatani , R. Kakehashi 1 Minami Kyoto Hospiata- National Hospital Organization, Neurology, Kyoto, Japan 2 Minami Kyoto Hospital- National Hospital Organization, Neurology, Joyo, Japan 3 Uji-Tokusyukai Medical Center, Clinical Reseach Center, Uji, Japan Aims Assessment of the cognitive function by area is necessary for the early discovery of dementia. We developed a system (CAT) capable of automatically evaluating and recording the cognitive function in various areas and in a detailed fashion. Method We validated CAT and confirmed its reliability. The correlations of the total score, score of each item, and time required with MMSE were investigated. In addition, ROC curves of the CAT cognitive function score by each area and each answer time were drawn for dementia and mild cognitive impairment, the AUCs were calculated, and the sensitivity and specificity were determined. Results CAT evaluated cognitive functions in various ways and recorded the results in a detailed fashion, so that we could elucidate the features of the cognitive dysfunction. The correlation with MMSE was high for calling names of objects, time required for it, immediate recall, and distinguishing objects, whereas it was relatively low for recalling in the reverse order. The correlation coefficients of Spearman’s and of Pearson’s between CAT and MMSE were 0.851 (P0.05). Of the MeDi components, greater cereal and legume intake was significantly associated with decreased beta-amyloid deposition (p0.05). Conclusion


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These findings demonstrated the MeDi displayed no association with beta amyloid deposition in a cohort of healthy Australian women. However, of the MeDi’s individual components, a significant association was observed between greater cereal and legume intake and decreased beta-amyloid deposition.

K01.h. Dementia and Cognitive Dyfunction: Behavioral & psychiatric symptoms ADPD7-1238 DELUSIONS, AGITATION AND ABERRANT MOTOR BEHAVIOR AT BASELINE ARE ASSOCIATED WITH THE PROGRESSION OF ALZHEIMER’S DISEASE 1 2 3 4 5 1,5 I. Hallikainen , K. Hongisto , T. Välimäki , J. Martikainen , T. Hänninen , A.M. Koivisto 1 University of Eastern Finland, Institute of Clinical Medicine- Neurology, Kuopio, Finland 2 University of Eastern Finland, Institute of Public Health and Clinical Nutrition- Department of Geriatrics, Kuopio, Finland 3 University of Eastern Finland, Department of Nursing Science, Kuopio, Finland 4 University of Eastern Finland, Pharmacoeconomics & Outcomes Research Unit- School of Pharmacy, Kuopio, Finland 5 Kuopio University Hospital, NeuroCenter, Kuopio, Finland Aims Importance of neuropsychiatric symptoms (NPS) in the course of Alzheimer’s disease (AD) has emerged during recent years. NPS associate with caregiver stress, risk of nursing home placement, and hospitalization. NPS prevalence and spectrum of different NPS varies during the course of the disease. Our knowledge is still limited, whether the manifested NPS and which NPS are related to the rate of AD progression and prognosis. Our aim was to study which baseline NPS are associated with AD progression, during the 5-year follow-up. Method This study was a part of prospective, 5-year ALSOVA study. Study participants (n= 236) had very mild (CDR 0.5) or mild (CDR 1) AD at baseline. Their caregivers were also interviewed. The Neuropsychiatric Inventory (NPI) was used to assess NPS, and the Clinical Dementia Rating Sum of Boxes (CDR-sb) to evaluate AD progression. Data was analyzed with Generalized Estimated Equations. Results We present the prevalence of all 12 NPS, which are included in the NPI Inventory, during the 5-year follow-up. Delusions, agitation, and aberrant motor behavior at baseline associated significantly with AD progression during the five-year follow-up. Conclusion


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AD may progress faster in persons with AD who have delusions, agitation, and aberrant motor behaviour already at the time of diagnosis. Results highlight the importance of evaluating NPS at the time of diagnosis. We suggest to offer support especially to the persons with AD who have delusions, agitation and aberrant motor behaviour, and also to their caregivers.

K01.h. Dementia and Cognitive Dyfunction: Behavioral & psychiatric symptoms ADPD7-1739 PSYCHOLOGICAL SUPPORTS FOR THE DLB PATIENTS PRESENTING VISUAL HALLUCINATIONS 1 2 1 3 K. Ota , H. Fujishiro , K. Sato , E. Iseki 1 Juntendo Tokyo Koto Geriatric Medical Center, PET-CT Dementia Research Center, Tokyo, Japan 2 Graduate School of Medicine- Nagoya University, Department of Psychiatry, Aichi, Japan 3 Seniro Mental Clinic, Mental clinic, Tokyo, Japan Aims Onset of visual hallucinations (VH) in DLB may be associated not only with the brain organic factors but also with the psychosocial and environmental factors. In this study, we present DLB patients who exhibit VH and suggest psychological supports to reduce the burden associated with VH in patients with DLB. Method The first step is to assess whether the patients recognize that VH is illusion. If they can recognize or have a sense that VH may be illusion, we explain the characteristics of the disease and the mechanism of VH occurrence to the patients along with their care partners. If the patients have difficulties in recognizing that VH is not real, we give suggestions mainly to the care partners on how to reduce risk of VH occurrence and to deal with the patients when they are experiencing VH. Results The psychological supports can facilitate reduced frequency of VH, reduction of fear and confusion about VH, and less frightening contents of VH. Conclusion


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For the treatment and management for VH in DLB, non-pharmacological approach including psychological supports can be important and effective.

K01.h. Dementia and Cognitive Dyfunction: Behavioral & psychiatric symptoms ADPD7-0657 CYPROTERONE ACETATE TO TREAT MODERATE TO SEVERE INAPPROPRIATE SEXUAL BEHAVIOR IN DEMENTIA 1 1 Y.H. Park , S. Kim 1 Seoul National University Bundang Hospital, Department of Neurology, Seongnam, Republic of Korea Aims Although inappropriate sexual behavior (ISB) is not uncommon and causes much distress in patients with dementia and their caregivers, treatments for ISB have not been well studied. We investigated whether cyproterone acetate is effective in reducing moderate to severe ISB and whether it induces side effects. Method A consecutive series of 9 elderly male patients with dementia who visited Neurocognitive Behavior Center of Seoul National University Bundang Hospital between April 2014 and September 2016 and exhibited moderate to severe ISB which did not respond to nonpharmacological treatments was treated with cyproterone acetate. Their diagnoses were as follows: four with Alzheimer’s disease, three with mixed dementia, one with vascular dementia and one with dementia with Lewy bodies. Results Cyproterone acetate effectively reduced ISB in all the patients except one patient with mixed dementia. Four patients responded to cyproterone acetate 25mg twice a day, whereas three patients responded 50mg twice a day. One patient with Alzheimer’s disease responded to 75mg twice a day, but he discontinued cyproterone acetate due to depression and fatigue. There were no laboratory abnormalities associated with cyproterone acetate in all of the patients. Conclusion


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In this small pilot study, cyproterone acetate at a dose of 25 to 50mg twice a day could be considered as an effective treatment for moderate to severe ISB. Although the side effects were not serious, it needs to be studied in a larger sample size.

K01.h. Dementia and Cognitive Dyfunction: Behavioral & psychiatric symptoms ADPD7-0786 SUBJECTIVE COGNITIVE COMPLAINTS AND OBJECTIVE COGNITIVE DISORDERS IN PATIENTS WITH IDIOPATHIC PARKINSON’S DISEASE 1 1 2,3 2,3 1 M. Plourde , M. Doiron , D. Nicolas , L. Mélanie , S. Martine 1 Laval University, School of Psychology, Quebec, Canada 2 Laval University, Department of Medicine, Quebec, Canada 3 CHU de Québec Enfant-Jésus, Department of Neurological Sciences, Quebec, Canada Aims Subjective cognitive complaints guide clinicians in the assessment of objective cognitive deficits and can provide vital additional information in the clinical setting. It is therefore important to evaluate its clinical value, and the awareness of the patients to their cognitive difficulties, in order to support the diagnosis. The overall objective of this study is to investigate cognitive, psychological and behavioral as well as motor, sociodemographic and clinical variables in relationship to subjective cognitive complaints in patients with Parkinson's Disease (PD) Method Data from 50 patients with PD at three levels of cognitive functioning, i.e. cognitively intact (CI), mild cognitive impairment (MCI) and dementia, were analyzed. A model was created using the LASSO method to determine the variables most correlated with self-reported executive complaint measured with the Dysexecutive Questionnaire (DEX). The memory complaint was analyzed according to the diagnostic groups and objective memory deficits. Results Score on the Apathy Scale, rigidity as a dominant symptom and executive deficits were the variables the most correlated with the executive complaint. The Stroop test (inhibition condition) correlated significantly with the DEX complaint score. There was no difference between CI, MCI and dementia patients regarding the objective score in episodic memory and the memory complaint. Conclusion


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Patients with PD seem to be aware of their difficulties with respect to executive functions. The Stroop test shall be added to neuropsychological protocol when assessing PD patients. The association between symptoms of apathy, rigidity and executive complaints warrant further research.

K01.j. Dementia and Cognitive Dyfunction: Other ADPD7-1047 COUNTRY VARIATIONS IN THE DIAGNOSTIC EXPERIENCE OF PATIENTS WITH COGNITIVE IMPAIRMENT 1 2 3 2 1 1 C. Black , R. Wood , C. Ritchie , E. Jones , R. Khandker , B. Ambegaonkar 1 Merck & Co.- Inc., CORE, Rahway, USA 2 Adelphi Group, Adelphi Real World, Manchester, United Kingdom 3 University of Edinburgh, Centre for Clinical Brain Sciences, Edinburgh, United Kingdom Aims To describe country variations in the physicians patients with cognitive impairment (CI) consult during diagnosis. Method Data were taken from the 2015 Adelphi Real World Dementia Disease Specific Programme, a cross-sectional survey of physicians, and their patients with CI in 5 European countries, Canada and the US. Physicians completed record forms for each patient documenting their diagnosis experience, including physicians who evaluated and diagnosed the patient. Results are presented by country using descriptive statistics. Results Variations exist in the types of physicians evaluating CI patients across countries. Frequency of referrals and types of physicians referred to also vary between countries.

First Consulting Diagnosing Physician Physician Global (n=7620) Canada (n=600) France (n=1100) Germany (n=1100) Italy (n=1100) Spain (n=1095)

PCP/GP (71.8%) PCP/GP (77.4%) PCP/GP (69.4%) PCP/GP (75.7%) PCP/GP (60.9%) PCP/GP (75.0%)

UK (n=1125)

PCP/GP (83.3%)

USA PCP/GP (n=1500) (65.6%) Conclusion

Neurologist (47.5%)

Referred 71.0%

PCP/GP (40.6%) 71.8% Neurologist (61.1%) Neurologist (43.3%) Neurologist (61.7%) Neurologist (74.3%) PsychoGeriatrician (55.2%) Neurologist (46.9%)

71.5% 58.9% 73.6% 77.3% 86.2% 61.1%

Referred Physician Neurologist (58.8%) Neurologist (40.5%) Neurologist (71.1%) Neurologist (71.9%) Neurologist (66.3%) Neurologist (85.8%) PsychoGeriatrician (57%) Neurologist (69.5%)

Diagnosing Physician without Referral

Diagnosing Physician after Referral

PCP/GP (39.7%)

Neurologist (52.6%)

PCP/GP (62.2%)

PCP/GP (32.6%)

Neurologist (40.9%) Neurologist (69.1%) PCP/GP (53.6%)

Neurologist (53.8%)

Neurologist (49.3%) Neurologist (66.2%) Neurologist (61.7%) Neurologist (78%) PCP/GP (41.1%)

Psycho-Geriatrician (59.8%)

PCP/GP (48.1%)

Neurologist (58%)


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Variations exist in the types of physicians involved in the evaluation and diagnosis of CI patients, as well as the use of referrals.

K01.j. Dementia and Cognitive Dyfunction: Other ADPD7-1086 COGNITION AND ACTIVITY IMPAIRMENT OF NEWLY DIAGNOSED ALZHEIMER DISEASE PATIENTS IN THE MEDICARE MINIMUM DATA SET 1 2 3 1 3 3 3 1 C. Black , H. Fillit , L. Xie , R. Khandker , M.F. Kariburyo , O. Baser , H. Yuce , B. Ambegaonkar 1 Merck & Co.- Inc., CORE, Rahway, USA 2 The Mount Sinai Hospital, The Icahn School of Medicine, New York, USA 3 StatinMed Research, StatinMed Research, Ann Arbor, USA Aims Describe the cognitive performance and activity impairment of newly diagnosed Alzheimer Disease (AD) patients in the Medicare Minimum Data Set (MDS). Method Patients with ≥1 primary or ≥2 secondary AD diagnoses claims [(ICD-9-CM) code 331.0] were identified from Medicare fee-for-service claims from 01JAN2011–30JUN2014. Study sample included Medicare beneficiaries age 65-100 years with continuous medical and pharmacy benefits for ≥12 months pre-index (baseline period) and ≥6 months post-index date (first AD diagnosis date). The MDS is a federally mandated nursing home assessment that includes all residents in Medicare and Medicaidcertified nursing facilities in the United States. MDS assessments include the Cognitive Performance Scale (CPS), and Activities of Daily Living (ADL) Dependency Scale. Results


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Among 672 patients with baseline scores, average baseline CPS and ADL were 2.9 and 9.6 respectively. Among 1,469 patients with scores within 2 years post-index, the last observed CPS and ADL were on average 3.9 and 11.4 respectively. For patients with both baseline and follow-up score within 2 years (N=399), mean CPS and ADL scores increased by 0.6 and 1.79 respectively, with eating and locomotion on unit being the categories with most increased scores.

Conclusion


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Newly diagnosed AD patients who reside in a nursing home have severe cognitive and activity impairment. As patients progressed after index diagnosis, increase in both ADL and CPS scores were observed.

K01.j. Dementia and Cognitive Dyfunction: Other ADPD7-1119 MOST FREQUENTLY UTILIZED DIAGNOSTIC TEST AND SCANS ACROSS TWO NORTH AMERICAN AND FIVE EUROPEAN COUNTRIES 1 2 3 2 1 1 R. Khandker , R. Wood , C. Ritchie , E. Jones , B. Ambegaonkar , C. Black 1 Merck & Co.- Inc., CORE, Rahway, USA 2 Adelphi Group, Adelphi Real World, Manchester, United Kingdom 3 University of Edinburgh, Centre for Clinical Brain Sciences, Edinburgh, United Kingdom Aims To describe the most frequently utilized AD diagnostics across two North American and five European countries. Method Data were taken from the 2015 Adelphi Real World Dementia Disease Specific Programme, a cross-sectional survey of physicians, and their patients with CI in 5 European countries, Canada and the US. Physicians completed record forms for each patient, containing patient demographics, clinical characteristics, diagnosis and tests/scales and scans/imaging performed to aid patients’ diagnosis. Results are presented by country using descriptive statistics. Results The most frequently used tests to aid diagnosis were MMSE (86.5%), blood tests (64.6%) and thyroid examinations (61.7%). Results varied among Canada, Spain, the UK and US, which tested B12 more frequently.


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The most frequently used imaging technology to aid diagnosis were the MRI-Volumetric (43.3%), CT (33.0%), and no testing/scale (17.3%). Results varied in France, Germany, Italy and US which utilized MRI-Diffusion more frequently.

Conclusion


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More advanced diagnostic tools are still not widely used. More consistent and wider use of validated tools and scans may aid the diagnostic process, thus leading to early detection and opportunity for early intervention.

K01.j. Dementia and Cognitive Dyfunction: Other ADPD7-1136 COUNTRY VARIATIONS IN THE DURATION OF THE DIAGNOSTIC PROCESS 1 2 3 2 1 1 R. Khandker , R. Wood , C. Ritchie , E. Jones , B. Ambegaonkar , C. Black 1 Merck & Co.- Inc., CORE, Rahway, USA 2 Adelphi Group, Adelphi Real World, Manchester, United Kingdom 3 University of Edinburgh, Centre for Clinical Brain Sciences, Edinburgh, United Kingdom Aims To describe country variations in the timing of the diagnostic experience of patients with cognitive impairment (CI). Method Data were taken from the 2015 Adelphi Real World Dementia Disease Specific Programme, a cross-sectional survey of physicians, and their patients with CI in 5 European countries, Canada and the US. Physicians completed patient record forms containing patient demographics and patients’ diagnosis experience, including the duration since: onset of symptoms, first consultation, referral to second physician, and initial diagnosis. Descriptive statistics by country are presented below. Results The mean duration between onset of CI symptoms and first consultation was 6.1 months. Depending on the need for a referral, the mean duration between first consultation and diagnosis was 5.3 months. Three-quarters (71%) of patients were referred on. Results varied between countries, as shown below. Mean Duration (months) from: Symptom onset to First Consultation Global 6.1 (n=7620) Canada 7.8 (n=600) France 6.2 (n=1100) Germany 5.0 (n=1100) Italy 4.4 (n=1100) Spain 6.0 (n=1095) UK 8.0 (n=1125) USA 6.0 (n=1500) Conclusion

First %Referred Consultation to Referral

First Consultation to Referral to Diagnosis, Diagnosis including Referral

First First Consultation to Consultation to Diagnosis, Diagnosis (All) without Referral

71.0%

5.8

2.1

6.0

3.6

5.3

71.8%

8.0

1.0

5.2

2.9

4.6

71.5%

7.0

2.4

7.8

5.5

7.1

58.9%

6.0

1.1

4.5

1.2

3.1

73.6%

5.5

2.2

5.8

5.7

5.8

77.3%

6.1

3.4

8.1

4.5

7.3

86.2%

4.1

1.6

4.9

4.5

4.8

61.1%

5.8

2.0

5.2

3.0

4.4


Downloaded by: 80.82.77.83 - 4/6/2017 2:34:30 PM

The timings experienced within the diagnostic process vary between countries and differ depending on referrals.

K01.j. Dementia and Cognitive Dyfunction: Other ADPD7-1155 RATES OF INSTITUTIONALIZATION AMONG NEWLY DIAGNOSED AD PATIENTS 1 2 3 1 3 3 3 1 C. Black , H. Fillit , L. Xie , R. Khandker , M.F. Kariburyo , O. Baser , H. Yuce , B. Ambegaonkar 1 Merck & Co.- Inc., CORE, Rahway, USA 2 Mount Sinai Hospital, The Icahn School of Medicine, New York Ciry, USA 3 StatinMedResearch, StatinMedResearch, Ann Arbor, USA Aims To compare the rate of institutionalization and time-to-institutionalization between treated and untreated newly diagnosed AD patients. Method Patients with ≥1 primary or ≥2 secondary AD diagnoses claims [(ICD-9-CM) code 331.0] were identified from Medicare fee-for-service claims from 01JAN2011–30JUN2014. Study sample included Medicare beneficiaries age 65-100 years with continuous medical and pharmacy benefits for ≥12 months pre-index (baseline period) and ≥6 months post-index date (first AD diagnosis date). Patients were followed until the earliest of death, disenrollment or 31DEC2014 (follow-up period). Patients were assigned to Treated and Non-treated cohorts based on anti-dementia treatment received post-index date. Using the Minimum Data Set, patients who spent 120 consecutive days in a nursing home were considered institutionalized, and the time-to-event and incidence were calculated to compare the two cohorts. Results A total of 8,995 incident AD patients were identified, 4,037 received anti-dementia medication (44.8%) during the follow-up period. The untreated cohort had a higher Charlson Comorbidity Index (3.54 vs. 3.22, p

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