ROYAL ACADEMY OF MEDICINE IN IRELAND IRISH JOURNAL OF MEDICAL SCIENCE

3rd Annual Meeting Frontiers in Neurology Friday 15th November 2013, Dublin, Ireland

Irish Journal of Medical Science Volume 183 Supplement 2 DOI 10.1007/s11845-014-1081-8

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Disclosure Statement Prof. Kumlesh K. Dev is supported by research grants from Novartis Ireland and Novartis Basel Switzerland.

Ó Royal Academy of Medicine in Ireland 2014

Kumlesh K. Dev Molecular Neuropharmacology, Drug Development, Department of Physiology School of Medicine, Trinity College Dublin, Ireland Tel.: ?353-1-8964180 e-mail: [email protected]

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Sponsorship

The conference was supported by Health Research Board, Block MS Ireland and Novartis Ireland, and in kind by Science Foundation Ireland and Trinity College Dublin.

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Meeting Programme Main Lecture Hall 11.00

Registration and Poster Setup

12.00

Welcome, Prof. Kumlesh K. Dev

12.10

Plenary Lecture: Prof. Sean Pittock (Mayo Clinic, USA): Autoimmune Mimics of MS and their Biomarkers

13.00

Lunch and Poster Viewing

Parallel Session 1 (14.30–16.20): Cellular Mechanisms Chairs: Prof. Kumlesh K. Dev and Prof. Marina A. Lynch 14.30

HRB Special Lecture: Prof. Trevor Owens (Univ. of Southern Denmark): Microglial regulation of CNS inflammation

15.10

Roisin McManus (Trinity College Dublin): Microglia in Amyloid Pathology

15.20

Niamh McGuinness (Trinity College Dublin): NK cells in animal models of MS

15.30

Dr. Charles Me´tais (Trinity College Dublin): Multiple sclerosis: the importance of the myelin sheath

15.40

HRB Special Lecture: Prof. Arthur Butt (University of Portsmouth, UK): Regulation of Astrocyte Pathophysiology

16.40

Plenary Lecture: Prof. George Ebers (University of Oxford, UK): Epigenetics of MS

17.40

Poster Prizes, Dr. Aisling Ryan

18.00

Closing Remarks, Prof. Orla Hardiman

Studio Lecture Room Parallel Session 2 (14.30–16.20): Clinical Studies Chairs: Dr. Aisling Ryan and Prof. Orla Hardiman 14.30

HRB Special Lecture: Dr. Elisa Canu (San Raffaele University, Italy): Brain Imaging Studies

15.10

Dr. Peter Bede (Trinity College Dublin): Subcortical brain imaging and gender

15.20

Dr. Anne Marie Miller (Trinity College Dublin): BIOMARKAPD: use of biomarkers in AD and PD

15.30

Adam Pritchard (Trinity College Dublin): Mechanisms of Demyelination in a brain slice cultures

15.40

Special Lecture: Dr. Frank Dahlke (Novartis Pharma AG): Evolution of S1P receptor drugs for treatment of MS

Local Organising Committee Prof Orla Hardiman, Dr. Aisling Ryan, Prof. Kumlesh K. Dev.

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Ir J Med Sci (2014) 183 (Suppl 2):S71–S85 DOI 10.1007/s11845-014-1081-8

1. Plenary Lecture

S1. Autoimmune mimics of MS and their biomarkers Sean J. Pittock Mayo Clinic, USA Multiple sclerosis (MS) remains poorly understood in terms of its etiology and pathogenesis. Assumption that MS is an autoimmune disease is based on inflammatory pathology and evidence of intrathecal immunoglobulin production. However, evidence for organspecific autoimmunity is in most cases unconvincing. The variable clinical course and inter-patient heterogeneity of active demyelinating lesions suggest that MS may not be a single disease entity. Water channels (aquaporin [AQP]-4) are a newly recognized target for CNS inflammatory autoimmune demyelinating diseases. The detection of AQP4-IgG, in serum or CSF, unifies a spectrum of idiopathic inflammatory demyelinating disorders previously classified amongst variants of multiple sclerosis (MS). This discovery represents a seismic shift in CNS demyelinating diseases research from historic emphasis on the oligodendrocyte and myelin to the astrocyte. Individual patient-specific NMO therapies are likely to result from advanced serological interpretive insights, coupled with increased understanding of the pathogenic impact of binding of AQP4-IgG to its target on the astrocytic end foot (complement activation, AQP4 downregulation and coupled glutamate transporter downregulation). Other less common MS-mimic autoimmune disorders include optic neuropathy and myelopathy (T cell-mediated and usually paraneoplastic) associated with collapsin responsemediator protein [CRMP5]-IgG and other neural autoantibodies recognized in the context of progressive myelopathy that are sometimes misdiagnosed as primary progressive MS. IgGs targeting the inwardly rectifying potassium channel (KIR4.1) were recently reported detectable in nearly 50 % of MS patient sera. Using recombinant and native sources of KIR4.1, our group did not identify KIR4.1 IgGs in sera from any of 50 MS patients tested [ANA 2013;S506].

Parallel session 1 2. Cellular Mechanisms

S2.1. Microglial regulation of CNS inflammation Trevor Owens Neurobiology, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark Microglia play a major role as contributors to or regulators of the inflammatory milieu in neuroinflammatory diseases such as multiple sclerosis (MS). Microglia are phagocytic, and can present antigen to CD4? T cells, which places them at the center of many schema for central nervous system inflammation. In neuromyelitis optica, even though astrocytes are the primary target of autoantibody attack, microglia are nevertheless also involved. Microglia recognize pathogen and danger signals via innate receptors and they are a source of pro-inflammatory cytokines such as IL1b and

Th1- and Th17-inducing IL12 and IL23, and chemokines. They are also a source of counter-inflammatory or neuroprotective mediators such as Type I interferons and Insulin-like Growth Factor-1. There is evidence for functional microglial heterogeneity in this regard. Although lineage tracking has identified distinct yolk sac origin, dissection of the role of microglia in neuroinflammation is complicated by phenotypic overlap with blood-derived cells such as macrophages. Recent studies point to selective expression of chemokine receptors in addition to previously-used differential CD45 levels as a means to distinguish these cell types. Cross-talk with astrocytes is bidirectional, with evidence for dependence of astrocytes on microglia for TLR response, as well as vice versa for microglial response in experimental demyelination. These concepts will be discussed with reference to our own recent data.

S2.2. Microglia in amyloid pathology Ro´isı´n McManus*, , Sarah Higgins*, Kingston HG Mills*, Marina A Lynch  *School of Biochemistry and Immunology;  Institute of Neuroscience, Trinity College Dublin, Dublin 2, Ireland Evidence from this laboratory has indicated that microglial activation is characteristic of the brain of aged mice, and also transgenic mice which overexpress amyloid precursor protein (APP) and presenilin 1 (PS1; APP/PS1 mice), a commonly-used animal model of Alzheimers disease (AD). This is accompanied by infiltration of peripheral immune cells, including T cells and an increase in blood brain barrier permeability, which together may contribute to the age-related microglial activation. Data from a number of studies suggest that exposure to infectious agents can trigger inflammatory changes in the brain and may accelerate the development of AD. As microglia play a key role in maintaining homeostasis in the central nervous system, the objective of this study was to assess the effect of infection on microglial activation and amyloid deposition in the brains of APP/PS1 compared with wildtype mice. Young (4 month-old) and middle-aged (10 monthold) wildtype and APP/PS1 mice were infected with the respiratory pathogen Bordetella pertussis. The bacteria persist in the lungs of infected mice for around 5 weeks. Eight weeks post infection, brain tissue was harvested for analysis of amyloid beta accumulation by ELISA and microglial activation by FACS. The data showed that there was a significant increase in the numbers of CD80? microglia in the brain of 6 and 12 month-old APP/PS1 mice in comparison to wildtype controls. Interestingly, infection resulted in significant enhancement in CD68? microglia cells in the brains of APP/PS1 mice, particularly in 12 month-old mice. Infection also resulted in a significant age-related increase in the number of CD3? T cells in the brains of APP/PS1 mice. The concentrations of both soluble and insoluble amyloid beta increased with age in APP/PS1 mice and interestingly, insoluble amyloid beta was significantly greater in the brain of infected compared with non-infected APP/PS1 mice at 12 months of age. The data indicate that a respiratory infection enhances T cell migration into the brain and this is accompanied by increased microglial activation in APP/PS1 mice. Amyloid beta accumulation was increased with age, but importantly, exposure to a respiratory pathogen for 5 weeks significantly enhanced amyloid beta concentrations in cortical tissue of older APP/PS1 mice.

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S2.3. The differential role of natural killer cells in the induction and effector stages of experimental autoimmune encephalomyelitis Niamh C. Mc Guinness1,2, Lara S. Dungan1, Sarah Edwards1, Sarah C. Higgins1, Marina A. Lynch2, Kingston H.G. Mills1 1

School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute; 2Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin 2, Ireland Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model of multiple sclerosis and its induction is associated with the infiltration of encephalitogenic T cells into the CNS. Autoantigenspecific IL-17-producing Th17 cells and IFN–producing Th1 cells are central to the pathogenesis of EAE. However, cells of the innate immune system, including natural killer (NK) cells are also involved. In order to better understand the differential role of NK cells in the induction and acute stages of EAE, we depleted NK cells with antiasialo GM1 from mice with actively-induced disease at the time of initial immunisation, at the onset of symptoms, or throughout the disease. We also used flow cytometry to establish the kinetics of NK cell infiltration into the CNS. In contrast to Th1 cells which are present in highest numbers in the CNS at the peak of disease, NK cells maximally infiltrate the CNS before the manifestation of clinical signs. This observed increase in NK cell numbers in the CNS was associated with a corresponding peak in microglial TNF-alpha production, suggesting that NK cells may play a role in microglial activation early in disease. Depletion of NK cells at the onset of symptoms increased disease severity. In contrast, depletion at the induction of EAE delayed the onset of clinical signs and reduced disease severity in the early stages of disease; this correlated with a reduction in the numbers of Th1, Th17 and NKT cell in the CNS, and a decrease in microglial number. We observed a similar pattern of delayed onset but more severe disease following induction of EAE in IFN–/- mice (without NK cell depletion). Although the early clinical signs were delayed and less severe when NK cells were depleted at the induction of EAE, once symptoms developed, the disease was more severe. These findings suggest that IFN- production by NK cells is involved in the induction of pathogenic immune responses that mediate EAE but are protective at the acute stage of disease. The authors acknowledge grant support from the HEA under the Program for Research in Third Level Institutes (PRTLI) co-funded by the Irish Government and the European Union

Ir J Med Sci (2014) 183 (Suppl 2):S71–S85 suggested to have direct effects on S1PRs expressed in neuronal and glial cells in the CNS. To study the roles of S1PRs in demyelinating conditions, we prepared organotypic rat brain cerebellar slices and treated them with fingolimod in LPC-induced demyelinating conditions. We also treated these brain slice cultures with a novel S1PR inhibitor (MNP301) we have recently developed, to investigate the roles of S1PRs in the development of myelin. Immunofluorescence staining for myelin basic protein (MBP) and Western blot for myelin oligodendrocyte glycoprotein (MOG) analysis were performed to measure myelin state in our slice cultures. MBP immunostaining reveals that fingolimod rescues LPC-induced demyelination. Moreover, MOG immunoblot analysis shows that inhibition of S1PR1 by the pathway specific MNP301 reduces the levels of expression of MOG. Taking together, this data suggests that S1PRs rescue myelin state in demyelinating conditions and also that S1PR1s play an important role in the development and/or maintenance of myelin. This work has been funded by Health Research Board. CM is a Postdoctoral Fellow funded by the Health Research Board.

S2.5. Regulation of astrocyte pathophysiology Arthur M. Butt Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, UK The primary function of astrocytes is brain homeostasis and, to a certain extent, all neuropathologies can be considered disruption of normal homeostatic mechanisms. The pathology of astrocytes is broad-ranging, from dysregulation of K? and glutamate, to a fullblown glial scar that forms after injury. The glial scar is a major impediment to axon regeneration in the CNS, but is also inhibitory for myelination. We have been examining the physiological properties of astrocytes and how these may be involved in pathology, primarily using the optic nerve as a model CNS white matter tissue. We have identified a multiphase signaling mechanism in which astrocytes release ATP to propagate intercellular calcium waves through the glial network. I will describe how this signaling mechanism synchronises the astrocyte physiological function of potassium regulation to neuronal activity, but at high levels activation of P2X7 receptors and P2Y1 receptors are involved in reactive astrogliosis and glial cell death. Supported by the MRC and BBSRC.

Parallel session 2 S2.4. The involvement of shingosine-1-phosphate receptors (S1PRs) in myelinating and demyelinating conditions in cerebellar slice cultures Charles Me´tais, Kumlesh K. Dev Molecular Neuropharmacology, School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland The etiology of multiple sclerosis (MS) remains unclear, however, it is well accepted that auto-reactive T cells transmigrate into the central nervous system (CNS) causing axonal damage and myelin loss. In 2010, fingolimod, a pan-S1P receptor (S1PR) agonist, was the first oral drug to be approved for the treatment of relapsing-remitting MS. By targeting S1PRs, fingolimod restricts T cell egress from lymph nodes, thus limiting their inflammatory processes in the CNS. Importantly, fingolimod is blood brain barrier permeable and is

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2. Clinical Studies

S3.1. The MRI features of the frontotemporal lobar degeneration spectrum of disorders: from the behavioural variant of frontotemporal dementia to amyotrophic lateral sclerosis E. Canu, M. Filippi Neuroimaging Research Unit, Institute of Experimental Neurology, Division of Neuroscience, Scientific Institute and University Ospedale San Raffaele, Milan, Italy The clinical use of magnetic resonance imaging (MRI) has dramatically changed our ability to diagnose accurately several neurological

Ir J Med Sci (2014) 183 (Suppl 2):S71–S85 disorders, including dementia and motor neuron diseases. Over the past 10 years, there have been significant advances in the identification of the in vivo neuroimaging patterns of these syndromes, which improved our understanding of their pathophysiology. From this prospective, the talk will be focused on frontotemporal lobar degeneration (FTLD), which includes a large spectrum of syndromes with different clinical manifestations, such as the behavioral variant of FTD, the language variants (or primary progressive aphasia) and amyotrophic lateral sclerosis. Specifically, we will focus on their common and divergent patterns of brain damage that can be detected with imaging. Recently, the attention of imaging studies in the FTLD field has turned on the white matter integrity in motor and extra-motor brain regions using diffusion tensor MRI, as well as on the functional network connectivity using functional MRI. Currently, however, none of these techniques taken in isolation has yet been proven to be ‘‘the’’ diagnostic tool for these syndromes, whereas a ‘‘multiparametric’’ approach, carrying information on the various aspects of the complex pathological picture of these conditions, is likely to be the most useful strategy for an accurate diagnosis and treatment selection.

S3.2. Subcortical brain imaging and gender Peter Bede Academic Unit of Neurology, Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland While clinical neuroradiology mostly relies on qualitative MRI interpretation, novel quantitative imaging techniques offer unrivalled metabolic, structural and functional insights, capturing and characterising subtle changes in white and grey matter integrity. Amyotrophic lateral sclerosis (ALS) is primarily associated with motor cortex and pyramidal tract degeneration. However, on careful clinical assessment, extrapyramidal, sensory, and cognitive deficits can also be ascertained, suggestive of nigrostriatal, corticobasal and frontostriatal dysfunction. Using detailed genetic, neuropsychological and neurological profiling prior to imaging, we have described phenotype and genotype specific imaging signatures in ALS. Furthermore, our results indicate a sensitive correlation between clinical disability and imaging markers. We have recently demonstrated widespread basal ganglia involvement in ALS in vivo, which is consistent with large post mortem studies and explain many of the non-motor symptoms of ALS. Quantitative neuroimaging methods are readily applicable to a range of other neurological conditions, such as dementia syndromes, movement disorders, neuroinflammatory conditions etc. The relevance of these techniques is that they can potentially be utilised as monitoring markers, end-points of clinical trials or in diagnostic applications.

S3.3. BIOMARKAPD: harmonizing the clinical application of biomarkers in Alzheimer’s and Parkinson’s disease A-M Miller*1,2, Prof. B. Lawlor1,2,3 1 BIOMARKAPD, Mercer’’s Institute for Research on Aging, St. James’’s Hospital; 2Medical Gerontology, School of Medicine, Trinity College Dublin; 3Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin 2, Ireland

Alzheimer’s and Parkinson’s diseases (AD, PD), the most common neurodegenerative disorders, affect over 8 million individuals.

S77 Treatments are not preventative, but only address the symptoms of these conditions. Biomarker analysis allows for the detection of biochemical changes which indicate when neurons are compromised and begin to die; this can be detected 10–20 years before symptoms become apparent. Early diagnosis is crucial for timely intervention, to delay the impairment of neuronal integrity, and tailor patient treatment and support. Established biomarkers exist for AD and candidates for PD are in development, yet the greatest problem facing clinicians and researches alike is the lack of standardisation regarding their analysis, use and interpretation. BIOMARKAPD is a multi-national research project comprising 51 sites in 21 countries, including Ireland. As part of this initiative the Irish Network for Biomarkers in Neurodegeneration (IN-BIND) was established, it is a research infrastructure aimed at creating a functional network for clinical and laboratory based scientists working in this area. The aims of both programmes are the standardisation of biomarker measurement across Ireland and Europe to include (1) sample collection, (2) measurement, (3) and interpretation. In addition, a national and central European biobank are being created with samples from characterised AD, PD, MCI patients and neurologically healthy controls. These samples will be used to better optimise biomarker assays and identify better biomarker candidates. The deliverables of the BIOMARKAPD project and IN-BIND initiative will have significant impact on clinical research and therapy development in general, and for AD and PD in particular. This is an EU Joint Programme—Neurodegenerative Disease Research (JPND) project. The project is supported through the Health Research Board, Ireland [BiomarkAPD JPND/2011/2], under the aegis of JPND http://www.jpnd.eu.

S3.4. Mechanisms of demyelination in a brain slice Adam J. Pritchard1, Anis K. Mir2, Kumlesh K. Dev1 1 Molecular Neuropharmacology, Department of Physiology, School of Medicine, Trinity College Institute of Neuroscience, Trinity College Dublin, Ireland; 2Autoimmunity, Transplantation and Inflammatory Disease, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland

Sphingosine-1-phosphate receptors (S1PRs) are a family of G-proteincoupled receptors, comprised of subtypes S1PR1–S1PR5 and activated by the endogenous ligand S1P. The phosphorylated version of Fingolimod (pFTY720), an oral therapy for multiple sclerosis (MS), induces S1PR1 internalisation in T cells, subsequent insensitivity to S1P gradients and sequestering of these cells within lymphoid organs, thus limiting immune response. S1PRs are also expressed in neuronal and glial cells where pFTY720 is suggested to directly protect against lysphosphatidylcholine-induced (LPC) deficits in myelination state in organotypic cerebellar slices. Of note, previous studies in this lab have found that such slice cultures contain immune cells, which could also be regulated by pFTY720 to maintain levels of myelin. Here, a mouse organotypic cerebellar slice and lymphocyte co-culture model was used to investigate the effects of pFTY720 on lymphocyte-induced demyelination. Spleen cells isolated from MOG-immunised mice (MOGlymphocytes) or from 2D2 transgenic mice (2D2-lymphocytes) both induced demyelination to a similar extent as LPC, when co-cultured with mouse organotypic cerebellar slices. As expected, in vivo treatment of MOG-immunised mice with FTY720 inhibited demyelination induced by MOG-lymphocytes. Importantly, in vitro treatment of MOG- and 2D2-lymphocytes with pFTY720 also attenuated demyelination caused by these cells. In addition, while in vitro treatment of 2D2-lymphocytes with pFTY720 did not alter cell phenotype, pFTY720 inhibited the release of the pro-inflammatory cytokines such as IFNc and IL6 from these cells. This work suggests that treatment of

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S78 lymphocytes by pFTY720 attenuates demyelination and reduces proinflammatory cytokine release, which likely contributes to enhanced myelination state induced by pFTY720 in organotypic cerebellar slices.

Ir J Med Sci (2014) 183 (Suppl 2):S71–S85 factors because it is here that prevention or reduction of MS burden can be most plausibly obtained.

5. Posters S3.5. The evolution of S1P receptor compounds for treatment of multiple sclerosis (MS)

P5.1. Multiple territory watershed infarct following spinal anaesthesia

Frank Dahlke Novartis, Basel, Switzerland

Tarig Abkur, Catherine Peters University Hospital Limerick, Limerick, Ireland

Modulation of the sphingosine 1-phosphate (S1P) receptors has emerged as an important novel therapeutic approach in MS. S1P receptor modulators target multiple sclerosis by binding to S1P1 expressed on lymphocytes and selectively retaining a subset of lymphocytes in lymphoid tissues. Selective retention prevents recirculation of potentially auto-aggressive lymphocytes and infiltration into the CNS. Preclinical data also suggest that S1P receptor modulators enter the CNS and might have direct effects on astrocytes, oligodendrocytes, and axons via modulation of S1P1 and S1P5. Fingolimod (FTY720, GilenyaÒ), is the first in class S1P receptor modulator and first approved oral treatment for RMS. In addition to anti-inflammatory effects as demonstrated by its robust effects on relapse rates and MRI outcomes, Fingolimod treatment reduced accumulation of disability and was associated with rapid and consistent reduction in brain volume loss. This suggests that S1P receptor modulation in both the immune system and CNS, may contribute to its efficacy in MS. Siponimod is a next-generation S1P receptor modulator that acts selectively on S1P1 and S1P5. Preclinical data show that it is also active in the CNS. Siponimod is largely eliminated within 7 days of discontinuation with a half-life of about 30 h. Strong anti-inflammatory effects on MRI parameters could be demonstrated in thorough phase 2 dose finding study, using a novel adaptive design. Its mode of action makes Siponimod a good candidate for the treatment of secondary progressive MS, where medical need is high.

Presenting Author: Tarig M. Mustafa: [email protected] Hypotension is one of the most common complications of spinal anaesthesia. The intra-operative drop in the blood pressure is clearly associated with increased morbidity and mortality. In order to increase the awareness of this complication, we report a case of devastating stroke following a spinal anaesthetic in a previously well lady with risk factors for vascular diseases. The patient developed multiple territory watershed infarct involving the territories of the Anterior Cerebral Artery and the Middle Cerebral Artery. She suffered from bilateral blindness from bilateral occipital lobes involvement. The patient eventually died from aspiration pneumonia 14 days after hospitalization. The case highlights the risk of hypotension complicating spinal anaesthesia.

P5.2. Astrocytes in the degenerating brain are primed to synthesize exaggerated levels of CXCL1 and CCL2 in response to IL-1/TNF-stimulation Edel Hennessy1,2, Colm Cunningham1,2 1

Trinity College Institute of Neuroscience; 2School of Biochemistry and Immunology, Trinity College Dublin, Republic of Ireland Presenting Author: Edel Hennessy [email protected]

4. Plenary Lecture S4. Epigenetics of MS George Ebers Department of Clinical Neurosciences, University of Oxford Nuffield, Oxford, UK The long standing debate about the relative importance of genetics and the environment in MS has given way to the growing realisation that susceptibility is determined by the interaction of environment with genetics. It seems impossible to explain the observed facts on the individual actions of genes or environment. Specific interactions have now been localised. There is now much evidence that interactions are taking place which encompass epigenetic changes notably in the MHC. Hints that this was the case had come from the genetic epidemiology which had shown a link between increasing sex ratio (increasing female predilection) and mediation of risk through maternal inheritance. There is now evidence from several directions that important maternal effects are not only epigenetic but appear to be transgenerational. The evidence for these conclusions will be reviewed with a view to identifying the importance of environmental

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Microgliosis and Astrogliosis are standard features of neurodegenerative disease. It has been shown in a number of animal models that microglia in the degenerating brain are primed to show exaggerated cytokine responses to subsequent stimulation with TLR agonists such as LPS and Poly I:C. It is not clear whether the degenerating brain shows similarly exaggerated responses to proinflammatory cytokines. In the current study we hypothesised that glial cells in the hippocampus of animals with chronic neurodegeneration (ME7 prion disease) would display abnormal responses to central cytokine challenges. Unilateral 1 l doses of TNF (300 ng/ l), IL-1 (10 ng/l) or saline were administered intrahippocampally via pulled glass microcapillary, in normal (NBH) and ME7 mice. These animals were terminally perfused at 2, 24 and 72 h post challenge. At 2 h post challenge ME7 microglia produced IL-1 following either IL-1 or TNF challenge while NBH microglia did not. Furthermore, there was very marked astrocytic synthesis of the chemokines CXCL-1 and CCL-2 in response to both cytokine challenges in ME7 animals. Conversely, very limited expression of these chemokines was apparent in NBH animals similarly challenged. Thus astrocytes are the primary chemokine synthesizing brain cell and in the prion-diseased brain are they primed to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines.

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P5.3. Expression and functional analysis of DNA sensors in cells of the central nervous system Donal Cox1, Neil Kearney1, Andrew Bowie2, Marina Lynch3 and Aisling Dunne1 Molecular Immunology Group; 2Viral Immune Evasion Group, School of Biochemistry and Immunology; 3Neuroinflammation Group, Trinity College Institute of Neuroscience, Trinity College Dublin, Ireland 1

Presenting Author: Donal Cox [email protected] Sensing of viral DNA is mediated by the intracellular receptors IFI16/ p204, AIM2 and the newly characterised protein, cGAS. Activation of these receptors leads to the induction of anti-viral genes including type-1 interferons and chemokines such as RANTES. To date, most studies examining these receptors have focussed on cells of the peripheral immune system. We have carried out an extensive characterisation of these sensors in immune cells of the central nervous system. We have found that IFI16/p204, AIM2 and cGAS are induced in astrocytes and microglia in response to IFN-beta stimulation. Furthermore, these cells produce high levels of IFN-beta in response to both DNA and di-cyclic nucleotides with astrocytes being the more potent responders. Astrocytes were also more responsive to DNA stimulation in terms of chemokine production with higher levels of CCL5, CCL3 and CXCL2 being induced. AIM2, when activated, forms a multi-protein complex called the inflammasome leading to the production of mature IL-1beta. We show that microglia and astrocytes can respond to DNA by release of IL-1beta and IL-1alpha in an inflammasome dependent manner. These studies have implications not only for viral DNA sensing in the brain but also for focal epilepsy, traumatic brain injury and stroke where cell free DNA is known to be present and could therefore contribute to excessive brain inflammation. This research was carried out with the support of the Programme for Research in Third Level Institutions (PRTLI).

P5.4. Infection with a respiratory pathogen attenuates IL-17 mediated CNS autoimmunity through IL-10 induction Sarah C. Edwards, Sarah C. Higgins, Niamh McGuinness, Kingston H. G. Mills School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland Presenting Author: Sarah Edwards [email protected] Epidemiological studies have described an association between infection with certain bacteria or viruses and development of autoimmune diseases, such as multiple sclerosis (MS). Conversely, infection with helminth parasites has been associated with a reduced incidence and severity of autoimmunity. Whooping cough is a reemerging vaccine-preventable infectious disease caused by the bacteria Bordetella pertussis. Clearance of the bacteria is associated with the induction of Th1 and Th17 cells. However, the infection is persistent and results in the induction and recruitment of IL-10-secreting Treg cells to the lungs. In this study we have used a murine model of MS, experimental autoimmune encephalomyelitis (EAE), to examine the effect of infection on development of CNS inflammation. The data reveal that concurrent infection with B. pertussis significantly attenuates clinical disease and weight loss in mice with EAE. This reflected a significant reduction in the frequency of infiltrating IL17?, IFN-c? and IFN-c? IL-17? CD4 and cd T cells into the central

S79 nervous system (CNS) of B. pertussis infected compared with uninfected mice with EAE. Furthermore analysis of T cell populations in the spleens revealed that infected mice had higher numbers of IL-10producing Foxp3? cells as well as IL-17? IFN-c? CD4 T cells. The attenuation of EAE by B. pertussis was lost in IL-10-/- mice, suggesting that IL-10 induced by B. pertussis suppresses T cell responses that mediate CNS inflammation. Our findings demonstrate that a bacterial infection of the respiratory tract can attenuate the course of EAE in mice and this reflects the suppressive effect of pathogeninduced IL-10 produced by T cells or innate immune cells, which appear to be capable of preventing Th1 and Th17 cells from entering the CNS.

P5.5. Poly I:C in semaphorin 6A mutant and heterozygous adult mice, and the variable inflammatory response to poly I:C from different sources Niamh McGarry3, Kevin Mitchell1, Colm Cunningham2,3 1 Smurfit Institute of Genetics, Trinity College, Dublin; 2School of Biochemistry and Immunology; 3Trinity College Institute of Neuroscience, Trinity College, Dublin, Ireland

Presenting Author: Niamh McGarry: [email protected] Viral infection during pregnancy increases the risk of offspring developing disorders such as Schizophrenia, but does not do so invariably. Semaphorin 6A (Sema6A), is a gene involved in early brain development and mutants present a behavioural phenotype related to psychiatric disorders such as Schizophrenia. We proposed to examine gene x environment interactions in Sema6A deficient mice using an animal model of Maternal Immune Activation (MIA; systemic challenge with the synthetic double stranded RNA analogue poly I:C). First we ascertained that this strain of animals did not have a heightened inflammatory response to poly I:C. Since there were no differential effects of poly I:C on maternal inflammation in this strain we sought to investigate gene x environment interactions following MIA. To develop a model of MIA, poly I:C (20 mg/kg) was administered intraperitoneally on Gestation Day (GD) 12 in WT animals, as previously described in the literature, but both this dose, and lower doses (2 mg/kg) rendered the pregnancy inviable. Since multiple poly I:C preparations, that may vary in potency, have been widely used in inflammatory research we decided to compare two commonly used preparations. The results showed that Amersham poly I:C (12 mg/kg) induced significantly greater increases in plasma IL-6, TNF and IFN and significantly more robust sickness behaviour compared to Sigma poly I:C. MIA experiments with Sigma poly I:C have now produced viable offspring and the phenotype of these mice is currently under investigation.

P5.6. Studying neurodegeneration in drosophila: a small model organism with the potential for big discoveries Niamh C. O’Sullivan1 and Cahir J. O’Kane2 1

UCD Conway Institute, University College Dublin, Ireland; Department of Genetics, University of Cambridge, UK

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Presenting Author: Niamh O’Sullivan [email protected] I have recently established a research laboratory in University College Dublin using the fruit fly, Drosophila melanogaster, to investigate the

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S80 molecular events underpinning neurodegenerative diseases. Drosophila are particularly suitable for studying progressive degenerative diseases because they have a relatively short lifespan (*60 days), most genes controlling basic developmental processes are highly conserved between Drosophila and humans and there are many genetic tools available in Drosophila which can be used to determine the effect of mutations on specific cell/tissue types. This poster will present an example of how I have used Drosophila to uncover the function of a novel motor neuron disease-associated gene. The endoplasmic reticulum (ER)-shaping protein reticulon was recently found to be a causative gene for the neurodegenerative disorder hereditary spastic paraplegia (HSP). I generated flies lacking the Drosophila ortholog of reticulon, Rtnl1, using ubiquitous expression of a Rtnl1 RNAi insertion to investigate how loss of Rtnl1 causes neurodegeneration. Loss of Rtnl1 causes elevated levels of ER stress within neurons, likely due to the disruption of the smooth ER and expansion of the rough ER. Significantly, we detected abnormalities specifically within distal portions of longer motor axons and in their presynaptic terminals, including disruptions to the smooth ER, mictrotubule cytoskeleton and mitochondria. In contrast, the proximal axon portions appeared unaffected. These results provide direct evidence for reticulon function in the organisation of the smooth ER in long motor axons, and support a model in which HSP-causing mutations in reticulon may contribute to progressive axonal degeneration.

P5.7. EBI2 directs astrocyte migration and protects from demyelination A. Rutkowska1,2, A.W. Sailer2, K.K. Dev1 1

Molecular Neuropharmacology, School of Medicine, Trinity College, Dublin, Ireland; 2Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland Presenting Author: Aleksandra Rutkowska [email protected] Epstein Barr virus (EBV)-induced gene 2 (EBI2) is a G proteincoupled receptor (GPCR) expressed mainly in peripheral blood mononuclear cells and lymphoid tissue. The role of EBI2 in the immune system is crucial for appropriate T cell-dependant antibody response, B cell migration and correct positioning in the B cell follicles. Oxysterol 7, 25-dihydroxycholesterol (725HC) is a naturally occurring agonist for EBI2. Oxysterols are oxygenated derivatives of cholesterol which play various important physiological roles such as metabolism of vitamins and lipids, cholesterol trafficking, activation of nuclear hormone receptors, regulation of the inflammatory signalling pathways, and many others. Dysregulated expression of EBI2 and of the 725HC synthetic pathway have been implicated in a range of both, immune and central nervous system (CNS) diseases such as atherosclerosis, MS, diabetes, lupus, rheumatoid arthritis, and many others. Therefore, it is important to understand the role of EBI2 in the CNS as well as in the immune system. While our understanding of the function of EBI2 in the immune system has recently advanced, knowledge about its role in the CNS remains limited. Therefore, the current study aims to identify and characterise the functional role of EBI2 by investigating the expression and signalling of EBI2 in the CNS. Here we report that brain cells, specifically astrocytes, express both EBI2 and enzymes necessary for synthesis and degradation of 725HC. We also find that oxysterols stimulate migration of astrocytes via EBI2 and that agonism of EBI2 prevents LPC-induced demyelination in cerebellar slices. Taken together the data suggest that EBI2 is involved in glial cell function and that modulating EBI2 activity might be beneficial under pathophysiological conditions such

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Ir J Med Sci (2014) 183 (Suppl 2):S71–S85 as inflammation of the nervous system or neurodegenerative disorders. Acknowledgments: AR is an IRC/Novartis PhD Scholar. This work is supported by an Irish Research Council (IRC) Enterprise-Partnership Scheme (EPS) Award jointly with Novartis.

P5.8. Modulation of S1P receptors impairs LPS induced chemokine synthesis in astrocytes Sinead O’Sullivan, Luke M. Healy, Catherine O’Sullivan Graham K. Sheridan, Thomas Connor, Kumlesh K. Dev Molecular Neuropharmacology and Neuroimmunology Research Group, Department of Physiology, School of Medicine, Trinity College Institute of Neuroscience, Trinity College Dublin, Ireland Presenting Author: Sinead O’Sullivan [email protected] The Drug Fingolimod (FTY720) is the first oral therapy for patients with relapsing remitting multiple sclerosis (MS). The phosphorylated form of FTY720 (pFTY720) binds to and regulates the function of sphingosine-1-phosphate receptors (S1PR) by causing initial agonism then subsequent receptor internalisation and antagonism. These effects of pFTY720 limit S1P-mediated transmigration of lymphocytes to the central nervous system (CNS). S1PRs are also present on cells of the CNS and given that lipophilic FTY720 can cross the blood brain barrier, it likely has direct effects in the CNS. Here, the role of S1PRs in lipopolysaccharide (LPS) mediated changes in the levels of chemokines in astrocytes was investigated. pFTY720 was found to attenuate the LPS-induced increase of inflammatory cytokines in astrocytes, including RANTES/CCL5, LIX/CXCL5, IP10/CXCL10 and MCP1/CCL2. Moreover, the S1P1R selective agonist, SEW2871 also inhibited LPS-mediated increase of these chemokines. These effects were observed at the mRNA level indicating that S1P1Rs regulate synthesis of the chemokines investigated. Furthermore, inhibition of sphingosine kinase (SphK) attenuated LPS-induced increase in the mRNA levels of these chemokines, suggesting that activation of TLR4 by LPS may regulate these levels via a pathway that involves SphK, production of S1P and transactivation of S1PRs. These data highlight a role for S1P1Rs in regulating the levels of these chemokines in astrocytes, which could in turn alter T cell entry into the CNS, blood–brain-barrier function and/or neuronal and glia cell survival. These findings are important in further understanding the beneficial effects of pFTY720 in treatment of MS. This work was supported by research grants from the Health Research Board (HRB) Ireland. SOS is an HRB funded PhD Scholar.

P5.9. Amitriptyline acting on neuronal TRK receptors protects against cytokine-induced neuronal atrophy Eimear O’Neill, Billy Kwok, Andrew Harkin, Thomas J. Connor Neuroimmunology Research Group, Department of Physiology, School of Medicine and Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin 2, Ireland Presenting Author: Eimear O’Neill [email protected] Increased expression of pro-inflammatory cytokines is a common feature of various neurodegenerative disorders. There is a need to develop new treatments that target inflammatory degeneration in the brain. The tricyclic anti-depressant amitriptyline is used in treatment

Ir J Med Sci (2014) 183 (Suppl 2):S71–S85 of depressive, pain and anxiety disorders. Amitriptyline has been shown to act as an agonist for Trk receptors and to induce neurotrophic activity through their activation in PC12 cells. Here we report that amitriptyline (50, 500 nM) and its active metabolite nortriptyline (50, 500 nM) induce neurite outgrowth in rat primary cortical neurons. Neurons were treated for 24 h before complexity was measured using Sholl analysis. The neurotrophic action of amitriptyline is mimicked by the TrkA agonist nerve growth factor (1, 10 ng/ml) but not by anti-depressants clomipramine or fluoxetine. Amitriptylineinduced outgrowth is blocked by inhibition of Trk signalling using tyrosine kinase inhibitors K252a (200 nM) and Tyrphostin AG 879 (10 M) and rescues inhibition of outgrowth using neurotrophin antagonist Y1036 (40 M). Inhibition of the PI3K and MAPK pathways using wortmannin (100 nM), LY294002 (10 M) and PD98059 (10 M) also blocked amitriptyline-induced neuritic growth, implicating these pathways in the effect. Furthermore, pre-treatment of neurons with amitriptyline or nortriptyline prevented the atrophic effects of the pro-inflammatory cytokines TNF-alpha and IL-1beta (10 ng/ml). These findings suggest that amitriptyline and nortriptyline can exert neurotrophic and neuroprotective effects in cortical neurons via activation of Trk and PI3K/MAPK signalling. These compounds therefore have significant clinical potential to be utilised in the treatment of inflammatory neurodegenerative conditions. Grant support from the HEA under the Program for Research in Third Level Institutes (PRTLI), co-funded by the Irish Government and the European Union, is acknowledged.

P5.10. A novel role for tumor necrosis factor-A in synaptic plasticity after acute hypoxia in the rat dentate gyrus Audrey M. Wall*, Gatambwa Mukandala, John J. O’Connor UCD School of Biomolecular and Biomedical Science, UCD Conway Institute for Biomolecular and Biomedical Research, Belfield, Dublin 4, Ireland Presenting Author: John O’Connor [email protected] Hypoxia is one of the key components which can arise from neuropathological conditions such as stroke or Alzheimers disease. Hypoxic events can cause the release of pro-inflammatory cytokines such as TNF-a which can lead to either neurotoxicity or neuroprotection in the brain. We have investigated the effects of an acute hypoxic event on synaptic plasticity in the presence and absence of TNF-a and inhibitors in rat hippocampal brain slices. Slices were prepared from P21 young male Wistar rats and LTP was elicited by stimulation of the medial perforant pathway in the dentate gyrus region. The effect of acute hypoxic conditions (delivery of 95 % N2/ 5 % O2 for 30 min) on the fEPSP in the presence of picrotoxin (100 lM) and/or TNF-a (5 ng/ml), infliximab (10 lM), and thalidomide (20 lM), 3,6-dithiothalidomide (20 lM) and 1,6dithiorevlimid (20 lM; all TNF-a synthesis inhibitors) were examined. Application of TNF-a did not alter the effects of hypoxia on the fEPSP either during hypoxia or 30 min post hypoxia (46.5 ± 1.4 % and 87.7 ± 1.1 % respectively, n = 6). However in the presence of TNF-a, LTP post hypoxia was significantly increased compared to control hypoxia slices (125.9 ± 3.4 %, control LTP versus 186.9 ± 2.1 %, TNF-a LTP, P0.01, n = 4–6). Conversely perfusion of infliximab, 3,6-dithiothalidomide or 1,6-dithiorevlimid but not thalidomide, attenuated this LTP post hypoxia (LTP post hypoxia was 137.2 ± 7.6, 104.7 ± 5.5 % and 103.0 ± 6.9 %, respectively, versus control of 125.9 ± 3.4 % (infliximab) and 183.0 ± 15.6 % (thiothalidomide derivatives); P0.05 for both;

S81 n = 5–6). These findings implicate an important role for TNF-a in LTP post hypoxia.

P5.11. Investigations into the electroencephalography of visuospatial processing: an initial study into atypicalities in autism spectrum disorder Niall Cosgrave, Niamh McDevitt, Prof. Richard Reilly, Prof. Louise Gallagher Trinity Centre for Neuroscience, Trinity Centre for Bioengineering, Trinity College Dublin, Dublin 2, Ireland Presenting Author: Niall Cosgrave: [email protected] Autism spectrum disorder (ASD) is a neurodevelopmental condition which is associated with atypicalities in sensory processing, including visuospatial processing. Given widespread reports of enhanced behavioural performance in visuospatial processing tasks, and previous functional magnetic resonance imaging (fMRI) findings of differences between individuals with ASD and typically developing (TD) controls in a classic Shepard–Metzler task of three-dimensional mental rotation, the current study aimed to examine this task from an electrophysiological perspective using electroencephalography (EEG). Three males with ASD and eleven TD males were recruited to perform a parity judgment task requiring mental rotation. Eyes-closed resting state EEG data was also recorded, and a subset of TD controls also performed a two-dimensional mental rotation task. Analysis of event-related potentials found that there were differences between individuals with ASD and TD controls in the root mean square amplitude during a select time window associated with mental rotation. Similar differences were observed between two-dimensional and three-dimensional mental rotation in the TD control subset. It was also found that during eyesclosed resting state, individuals with ASD had reduced amplitude in the theta (4 8 Hz) and alpha (8 13 Hz) frequency bands. Reduced alpha power has previously been linked with increased cortical excitability, possibly offering an explanation for the sensory symptomatology seen in individuals with ASD. Together the results of this initial investigation indicate quantitative and qualitative differences associated with ASD pathology, and should provoke further research.

P5.12. Migration of astrocytes; investigating the role of S1P receptors in the treatment of multiple sclerosis Andrew Lockhart, Adam Pritchard, Kumlesh K. Dev Molecular Neuropharmacology, School of Medicine, Trinity College Dublin, Dublin 2, Ireland Presenting Author: Andrew Lockhart [email protected] Multiple sclerosis (MS) is a chronic inflammatory demyelinating neurodegenerative disease of the central nervous system. Fingolimod (FTY720) is the first oral therapy for MS and it acts as a modulator of Sphingosine-1-Phosphate Receptors (S1PRs). Here, the effects of FTY720 on astrocyte migration were studied. A walled culture system was used to create a free space between two populations of astrocytes and imaging used to measure the migration of astrocytes into this space over time, using various pharmacological tools to elucidate the mechanisms involved. It was found that FTY720 promotes astrocyte migration predominantly through S1PR1 signalling. Downstream PI3K/AKT signalling is also required for migration to occur. The S1PR1 modulating peptide MNP301 also promoted migration. This

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S82 study provides important information about FTY720’s mechanism of action and could aid the development of next generation S1PR modulators, more targeted to beneficial aspects of S1PR signalling, to improve MS therapy in future.

P.5.13. Characterisation of a novel rat model of Parkinson’s disease, combining intrastriatal 6-hydroxydopamine and intranigral lipopolysaccharide Mark Kelly1,*, Justin D.Yssel1,*, Thomas Connor1, Yvonne Nolan3, Andrew Harkin2 *Authors contributed equally to this study; 1Neuroimmunology Research Group; 2Neuropsychopharmacology Research Group, Trinity College Dublin, Dublin 2, Ireland; 3Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland Presenting Author: Mark Kelly [email protected] Parkinson’s disease (PD) is the worlds second most common neurodegenerative disorder with a prevalence of 0.3 % in the general population. The disease is characterised by progressive degeneration of dopaminergic neurons of the nigrostriatal tract. The aetiology of sporadic PD is not known, but the neurotoxin hypothesis provides evidence that endogenous and/or exogenous toxins may play a key role. Much of our understanding of the pathologies underlying PD have been obtained from neurotoxin-based animal models such as the 6-hydroxydopamine (6-OHDA) model. In addition, neuroinflammation is now recognised as a key player in the progression of PD and interest is increasing in the inflammation-based lipopolysaccharide (LPS) model. The aim of this study was to create an animal model using both 6-OHDA and LPS in an effort to combine two of the major hypotheses of PD pathogenesis and progression; the neuroinflammation and neurotoxin hypotheses. It is the intention that this model may be used to provide further insight into the underlying pathologies of PD and aid in the investigation of potential therapies. This 6-OHDA/LPS model was analysed and evaluated under three different parameters; (1) behavioural analysis, (2) biochemical analysis of DA concentrations and (3) immunohistochemical analysis of neuronal density. It was determined that the combination of intrastriatal 6-OHDA and intranigral LPS induced profound motor deficits. Using high-performance liquid chromatography and immunohistochemical analysis these deficits were correlated to the presence of a neurodegenerative lesion of the nigrostriatal tract. Grant support from the HEA under the Program for Research in Third Level Institutes (PRTLI), co-funded by the Irish Government and the European Union, is acknowledged.

P5.14. The role of glial cells in metachromatic leukodystrophy Amy Adair M.Sc. Neuroscience Course, Cellular Neuroscience Module, Department of Physiology, School of Medicine, Trinity College Institute of Neuroscience, Trinity College, Dublin, Ireland Presenting Author: Amy Adair [email protected] For over a century, research within the area of neuroscience has primarily focussed on neuronal functions. Yet this cell type accounts for only 10 % of cells in the human brain. The balance is made up of various types of glia, namely astrocytes, microglia and oligodendrocytes. It has become evident that astrocytes are responsible for a vast

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Ir J Med Sci (2014) 183 (Suppl 2):S71–S85 range of intricate functions in the healthy CNS, including primary roles in synaptic transmission and information processing by neural circuits. In addition, microglia act as the initial and chief form of active immune defence in the CNS. They incessantly scavenge the CNS for plaques, damaged neurons and infectious agents which they identify as foreign bodies, engulf them, and function as antigen presenting cells to activate T-cells. Moreover, oligodendrocytes are one type of glial cell that undergo a multistage transformation from early oligodendrocyte progenitor cell (OLPs) to a mature myelinating oligodendrocyte. Once matured, oligodendrocytes create fatty sheaths that encase long neuronal branches and help to speed up electrical impulses. Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder explicable by mutations in the ASA gene Arsa, leading to an inability to break down sulfatide. Its accumulation eventually results in demyelination, the hallmark of MLD. We can now confidently say that glial cells play a role in diseases of the CNS, although they have been not well researched in this disease. While astrocytes may play a pathogenic role in MLD, it appears that the function of both microglia and oligodendrocytes could play protective roles in MLD. Here, the known and putative roles of oligodendrocyte, astrocyte and microglial function in this illness will be reviewed.

P5.15 The role of glial cells in Tay–Sachs disease Melanie Lang-Orsini M.Sc. Neuroscience Course, Cellular Neuroscience Module, Department of Physiology, School of Medicine, Trinity College Institute of Neuroscience, Trinity College, Dublin, Ireland Presenting Author: Melanie Lang-Orsini [email protected] Tay–Sachs disease is an autosomal recessive condition caused by a mutation in the HEXA gene, encoding the enzyme N-acetylhexosaminidase A. This lysosomal enzyme provides the only breakdown pathway for GM2 gangliosides and loss of function of this enzyme results in GM2 accumulation. The accumulated GM2 causes massive neuronal apoptosis and neurodengeration, for which currently there is no cure or treatment. It is widely accepted that GM2 accumulation is the primary insult in Tay–Sachs disease, however the exact mechanism by which GM2 causes neurodegeneration is still not clear. In addition to this, the role of glial cells in Tay-Sachs disease is not well understood. In fact, some studies suggest that astrocytes, oligodendrocytes and microglia are all affected by GM2 accumulation and they can precipitate neuronal apoptosis by promoting inflammation, demyelination and excitotoxicity, and by interfering with neuronal repair and neurogenesis. Further studies investigating the role of glial cells in Tay–Sachs disease could shed some light onto the mechanistic pathways involved in these processes and may even allow the identification of new therapeutic targets to slow disease progression. Here, the known and putative roles of oligodendrocyte, astrocyte and microglial function in this illness will be reviewed.

P5.16 The role of glial cells in diabetic retinopathy (DR) Edel Lynch M.Sc. Neuroscience Course, Cellular Neuroscience Module, Department of Physiology, School of Medicine, Trinity College Institute of Neuroscience, Trinity College, Dublin, Ireland Presenting Author: Edel Lynch [email protected] Diabetic retinopathy (DR) is a leading cause of acquired blindness and its prevalence is predicted to double by 2020 in the US. To

Ir J Med Sci (2014) 183 (Suppl 2):S71–S85 understand the role of glia in DR, we hypothesise that insulin growth factor (IGF-1) and insulin deficiencies drive glial impairment to induce DR pathogenesis. Both hormones are reduced by approximately half in diabetes and are pivotal in astrocyte and oligodendrocyte function. In diabetic-induced decreases of IGF-1 and insulin, retinal astrocytes lose their function in maintaining the integrity of the blood-retinal barrier by means of reduced gap junction protein expression (e.g. connexin-26 and -43). This results in decreased blood flow which is insufficient to match the metabolic needs of the retina and leads to retinal ischemia. In turn, microglia become activated upon sensing the disrupted environment and migrate into the subretinal space, inducing neuroinflammation and exacerbating neuronal damage. Retinal cell dysfunction, ischemia, and background hyperglycemia trigger astrocyte reactivity and oligodendrocyte malfunction in the distal portion of the optic nerve. Impaired oligodendrocyte function results in demyelination, and in turn, enhanced vulnerability of the optic nerve axons to astrocyte-induced damage, resulting in axon loss and ultimately vision loss. Moreover, it has recently been shown IGF-1 and insulin prevent retinal degeneration, atrophy, and rescue markers of astrocyte and oligodendrocyte function (GFAP, MBP and PLP) in diabetic rats. Even more so, insulin and IGF are protective despite persistent hyperglycemia in diabetic rats. Thus this deduces a possible mechanistic picture of glia interplay in the pathogenesis of DR and highlights IGF-1 and insulin as novel therapeutic targets. Here, the known and putative roles of oligodendrocyte, astrocyte and microglial function in this illness will be presented.

P5.17 The role of glial cells in drug addiction Andrew Stokes M.Sc. Neuroscience Course, Cellular Neuroscience Module, Department of Physiology, School of Medicine, Trinity College Institute of Neuroscience, Trinity College, Dublin, Ireland Presenting Author: Andrew Stokes [email protected] Drug addiction is an increasingly common problem and to date no cure exists. While recent research has focused primarily on the effects of drugs on neurons, the role of glial cells in drug addiction is less well understood. Once thought to be merely supporting cells to neurons the roles of glial cells, namely astrocytes, microglia and oligodendrocytes have since been better elucidated. The many roles of glial cells include myelination of neurons by oligodendrocytes, phagocytosis performed by microglia, and neurotropic support by astrocytes. With such prominent functions, changes in the activity of glial cells have profound effects within the CNS. These glial cells display an activated state, known as reactive gliosis, in response to drugs of abuse which contributes to the damage caused by drugs and may even be involved in the acquisition of addiction itself. As microglia and astrocytes are heavily involved in the immune regulation of the CNS, activation of these cells by drugs of addiction leads to a state of inflammation within the brain. With this knowledge regarding the inflammatory response of CNS to illicit drugs it provides scope for the development of novel therapeutics that target glial cell function. Indeed, new compounds have been shown to modulate the activity of glial cells in response to illicit drugs such as cocaine and methamphetamine. Altering the response of these cells has also been demonstrated to have effects on behaviour and self-administration in rodent models of addiction. This poster looks at the effects of different drugs on the members of the glial tissue and the compounds that have shown promise in reducing such effects.

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P5.18 Glial involvement in pathophysiologies of narcolepsy with cataplexy Brendan Grafe M.Sc. Neuroscience Course, Cellular Neuroscience Module, Department of Physiology, Trinity College Institute of Neuroscience (TCIN), Dublin 2, Ireland Presenting Author: Brendan Grafe [email protected] Narcolepsy with cataplexy is a debilitating condition associated with symptoms of excessive daytime sleepiness, presented with cataplexy, episodic loss of muscle tone in response to emotional stimuli. Hypocretin (Hcrt) neurons of the Lateral Hypothalamus (LH) project widely throughout the brain and act as conductors of sleep-wake cycles and body energy balance. Loss of 90 % of these cells and hypocretin deficiency is found in patients diagnosed with narcolepsy with cataplexy. Though symptomatic treatments exist there are no systemic therapies, let alone known mechanisms mediating Hcrt cell loss. Even though approximately 90 % of narcoleptic patients exhibit Human Leukocyte Antigen DQB1*0602 the etiology of this pathology has yet to be linked to autoimmune attack. However, our expanding understanding of glia might explain pathophysiologies of this disease. For instance, a missense mutation in myelin oligodendrocyte glycoprotein (MOG) has been described in one family presenting 12 relatives afflicted with narcolepsy with cataplexy. Ectopic cytoplasmic clustering of this mutant in transfected oligodendrocyte cultures suggests dysfunction in myelination though additional studies are necessary to reveal the link between this event and a specific loss of LH neurons. As hereditable links to narcolepsy with cataplexy only account for 5 % of the disease population it is possible that issues arising in microglial and astroglial functioning, in concert, mediate Hcrt cell loss and dysfunction. Here, the known and putative roles of oligodendrocyte, astrocyte and microglial function in this illness will be reviewed.

P5.19 The effect of cannabinoid treatment on amyloidinduced inflammation via the hemichannel, PANX1 S. G. Fagan, V. A. Campbell Department of Physiology, Trinity College Dublin, Dublin, Ireland Presenting Author: Steven Fagan [email protected] Over the past two decades, neuroinflammation has emerged as an integral process in the pathogenesis of Alzheimers disease. Amyloid (A)-induced inflammasome activation in neurons, as well as the activation and migration of microglia results in the rise of proinflammatory cytokines to neurotoxic levels. Gating of the membrane channel panX1 has recently been linked to the cleavage and release of IL-1 and activation of immune cells. Furthermore, blocking the panX1 channel has been shown to reduce A induced neurodegeneration. The endocannabinoid system is a known modulator of intracellular Ca2?, a key regulator of panX1 and application of the endocannabinoid precursor arachidonic acid has been shown to close panX1 channels. Cortical neurons isolated from 1 day old male Wistar rats were cultured and treated with either URB597 (1 lM), O-2545 (1 lM) or cannabidiol (1 lM) in the presence or absence of amyloid beta (2 lM) for 48 h. Total panX1 levels were quantified by western blot and panX1 localization was evaluated through immunocytochemistry. Migration of BV2 microglial cells was assessed using a transwell assay. The

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S84 BV2 cells were exposed to conditioned media from neurons treated with either the panX1 mimetic peptide 10panx (200 lM), the nonspecific hemichannel blocker octanol (200 lM) in the presence or absence of URB597 (5 lM) and A (10 lM) for 72 h. IL-1 secreted from the neurons and BV2 cells was also quantified by ELISA. Results have shown that treatment of neurons with A or cannabinoids has no effect on total panX1 levels (n = 6–8; One way ANOVA; p0.05). However, panX1 localisation was found to be more somatic in amyloid-beta treated neurons when compared to control cells. Migration of BV2 microglial cells increases with A treatment, an effect that is attenuated when co-treated with hemichannels blocker or URB597 (n = 2; One way ANOVA; p0.05). No significant difference was found in IL-1 release from neurons or BV2 microglial cells between treatment groups (n = 2, One way ANOVA, p0.05).

P5.20. Psychosine-induced astrocyte cell death is attenuated by S1P receptor activation: implication for Krabbe’s disease C O’Sullivan, K.K. Dev Molecular Neuropharmacology, Department of Physiology, School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland Presenting Author: CatherineO’Sullivan [email protected] Krabbe’s disease is a rare autosomal recessive neurodegenerative disorder affecting 1:100,000 births. This illness is rapidly progressing, appearing within the first 3–6 months of life and is usually fatal by the age of 2 years. Krabbe’s disease is caused by a deficiency in the lysosomal enzyme galactocerebrosidase (GALC), which results in the accumulation of a toxic metabolite in the brain termed psychosine. The build-up of psychosine is believed to be the main pathogenic agent in Krabbe’s disease resulting in widespread oligiodendrocyte cell death and demyelination. To date, most studies have focused on the toxic effects of psychosine on oligiodendrocytes, however little is known about the effects of psychosine on astrocytes. Astrocytes, the most abundant cell type in the brain, have many important functions such as protection and support of neurons and oligodendrocytes. Astrocytes also play key roles in regulating metabolic and ion homeostasis in the CNS. Importantly, astrocytes express S1P receptors (S1PR) which are targets for the first oral therapy developed for multiple sclerosis, FTY720 (Fingolimod). S1PRs have various roles in the CNS, including astrocyte migration, oligodendrocyte survival, and neurogenesis. More recently, S1PRs have been found to play a role in inhibiting the release of proinflammatory cytokines from glial cells. Here we investigate the effect psychosine on human and rat astrocyte survival and demonstrate the protective effects of pFTY720 against cell-toxicity induced by psychosine. In brief, we find psychosine induces astrocyte cell death in a dose- and timedependent manner. Importantly, pre-treatment with FTY720 attenuates psychosine induced cell death and significantly prolongs astrocyte cell survival. Taken together, these results suggest that S1PRs could be potential drug targets for Krabbe’s disease. This work was supported in part by research grants from Trinity College Dublin, The Health Research Board Ireland, Science Foundation Ireland and The Higher Education Authority Ireland (Programme for Research in Third Level Institutions [PRTLI]). COS is a PRTLI funded PhD Scholar.

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P5.21. Iron loading in brain slices: development of a novel in vitro platform to assess the impact of an iron-loaded environment in multiple sclerosis (MS) Sinead Healy, Michelle Naughton, Jill McMahon, Una FitzGerald Galway Neuroscience Centre, NCBES, NUI Galway, Galway, Ireland Presenting Author: Sinead Healy [email protected] Background: Iron is known to accumulate in the brain of MS patients, but its role in disease remains unclear. Clarification of how brain iron is regulated could reveal a role for iron in MS progression. Aim: To develop an organotypic hippocampal slice model of iron loading in the brain. Methods: Organotypic hippocampal slices derived from postnatal P10/ P11 rats were cultured for 10 days before being exposed to iron. Endogenous iron content was also assessed in non-cultured postnatal hippocampus (P8-45; n = 3–5). Iron levels were quantified using a ferrozine colorimetric assay and viability of cells assessed using a LDH assay. Results: For the first time in organotypic brain slices, we demonstrate that treatment with 0.01 mM ferrous ammonium sulfate (12 h) produces a significant 1.7-fold increase in iron content when compared with vehicle (8.4 ± 0.2 versus 4.9 ± 0.7 nmol/mg) without affecting viability. Similarly, 0.01 mM ferrocene exposure under serum free conditions produced a significant 1.7-fold increase without affecting toxicity. Encouragingly, levels of iron detected in cultured hippocampal slices were similar to those in the equivalent aged non-cultured hippocampus. The amount of iron detected was highest at P08 (13.6 ± 1.3 nmol/mg), declined sharply at P10/P11 (5.5 ± 0.8 nmol/ mg) before a transient peak at P14 (12.5 ± 2.8 nmol/mg) and a final levelling off during the third postnatal week (6.0 ± 1.0 nmol/mg). There was no difference in iron content from the fourth postnatal week onwards (6.0 ± 0.9 nmol/mg at P21). There were also regional variations in iron content in P10/11 brain. Conclusions: This slice model of iron loading appears to be a promising platform for the study of iron regulation in MS.

P5.22. Increased endoplasmic reticulum stress signalling in rat white matter development M. Naughton, J. McMahon, U. Fitzgerald Multiple Sclerosis and Stroke Research Group, Galway Neuroscience Centre, NCBES, National University of Ireland, Galway, Ireland Presenting Author: Michelle Naughton [email protected] nuigalway.i.e. Myelinating oligodendrocytes dramatically expand their cell membrane to simultaneously ensheath multiple axons. Membranous proteins and lipids are primarily synthesised in the endoplasmic reticulum which, when placed under stress, may trigger the Unfolded Protein Response (UPR) in order to increase its capacity. In this study we assessed the activation status of the UPR in developing white matter tracts. FFPE cerebella from Sprague–Dawley rats at postnatal days 7, 10, 14, 17 and adult (min. n = 2) were sagittally sectioned at 7 m. Chromogenic immunohistochemistry was used to characterise myelin development and ER stress. Numbers of positive cells per mm2 for activated ER stress sensors (pIRE1, ATF6, pPERK) and their associated targets were calculated from randomised and blinded

Ir J Med Sci (2014) 183 (Suppl 2):S71–S85 images of developing tracts in lobules III and IV. Although expression of pPERK and associated molecules (peIF2alpha, CHOP) was negligible, nuclear ATF6 (active) increased at P10 (vs P17 and adult, p less than 0.01), and pIRE1 expression peaked at P14 (vs P7 and adult, p less than 0.01). Targets of the UPR including the potent transcription factor XBP1, ER chaperone GRP94 and folding enzyme PDI, were also significantly upregulated in this active period of myelination. To summarise, ATF6 and pIRE1-mediated signalling is upregulated in a time-course that corresponds with early stages of myelination. In agreement with Lin et al. [1], PERK signalling is not activated. To our knowledge this is the first demonstration of ER stress signalling during developmental myelination. This data may be relevant for understanding mechanisms underlying remyelination in disorders such as multiple sclerosis. Grant support from the Health Research Board and MS Ireland (MRCG/2011/21) is gratefully acknowledged. Dr. McMahon is supported by the NUIG Foundation Office. References: 1. Lin W et al (2008) Enhanced integrated stress response promotes myelinating oligodendrocyte survival in response to interferon-gamma. Am J Pathol 173:150817

P5.23. Investigating pro-inflammatory cytokine levels in the serum of patients with mental illness treated with psychotropic medications Kara E. O’Connell1,2, Jogin Thakore2, Kumlesh K. Dev1 1 Molecular Neuropharmacology, Department of Physiology, School of Medicine, Trinity College Institute of Neuroscience, Trinity College Dublin, Ireland; 2Neuroscience Centre, St. Vincent’s Hospital Fairview, Fairview, Dublin, Ireland

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P5.24. Investigating the potential use of par-1/-2 antagonists as therapeutics for the prevention of T. brucei blood–brain barrier traversal Tara McDonald1,2, Kumlesh K Dev1, Andrew Knox2 1

Molecular Neuropharmacology, Department of Physiology; 2School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland Presenting Author: Tara McDonald [email protected]

During human African trypanosomiasis (HAT), also known as sleeping sickness, subspecies of the protozoan parasite Trypanosoma brucei cross the blood–brain barrier (BBB) to invade the central nervous system (CNS). Recent studies suggest that the parasites cross the BBB through generating Ca2? activation signals in endothelial cells, causing barrier modifications and subsequently penetrate into the CNS. Findings suggest that T. brucei mediated BBB dysfunction is linked to interaction of the parasite and/or parasite cysteine proteases, such a brucipain, with the G-protein coupled receptor known as proteaseactivated receptors (PARs). Both PAR-1 and PAR-2 are expressed on endothelial cells and astrocytes and activation of these receptors may provide a method of entry of trypanosomes into the CNS. Here we demonstrate that PAR compounds may be used to examine parasite interactions with the cells of the BBB and that in vitro examination of these cells show parasite-induced activation of BMECs and astrocytes and subsequent observations of preliminary barrier-crossing.

P5.25. Investigating changes in neuroinflammation in middle-aged and aged mice

Presenting Author: Kara E. O’Connell [email protected] Background and objectives: Schizophrenia occurs in approximately 1 % of the population and presents as a well-defined heterogenous set of clinical symptoms that affects cognitive function, perceptual experiences and belief systems. The immune system is suggested to play a role in schizophrenia, as evidenced by altered serum and plasma cytokines levels in this illness. To date, however, the use of cytokines as markers for this illness is curtailed as they fail to separate patients from healthy controls with high enough fidelity. Methods: Here, the levels of six cytokines (IL-1b, IL-6, IL-8, IL-17, IL-23, TNF-a) were measured in serum samples obtained from patients with schizophrenia, treated with clozapine (n = 91) or depot medication (n = 36), and compared with healthy controls (n = 50). Individual cytokine levels were measured using dot-immunoblotting methods. Treatment time, patient age, gender, illness severity and metabolic parameters were correlated with these cytokine levels. In addition, the summing of all 6 cytokines generated a ‘cytokine signature’ and the benefit of this analysis, over single cytokine measurements, was examined. Results: The levels of pro-inflammatory cytokines were raised, predominately in females, in depot and clozapine treated groups compared to healthy controls. Compared to individual cytokines, the ‘cytokine signature’ analysis showed less scatter of data. This ‘cytokine signature’ method thus improved separation between patient and control groups. Conclusions: The use of analysing a ‘cytokine signature’ resulted in a greater than twofold increase in differentiation of patient from control groups compared to analysis of individual cytokines. This work supports the idea that ‘cytokine signatures’ may be better suited to investigate altered immune response in schizophrenia. Furthermore, the incorporating of additional cytokines to those analysed here, may lead to a ‘cytokine signature’ characteristic for schizophrenia.

Colin Dempsey, Claire McDonald Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin, Ireland Presenting Author: Colin Dempsey [email protected] Microglia are the resident immune cells of the CNS and are the main source of inflammatory mediators in the brain. Microglia can adopt different phenotypes depending on the stimulus and these are referred to as the M1 and M2 activation states which are associated with production of pro-inflammatory, and anti-inflammatory mediators respectively. The aim of this study was to assess whether age modulates the balance between microglial activation states. Hippocampal tissue from young (3–4 months), middle-aged (14 months) and aged (16–22 months) mice were examined for markers of the M1 and M2 activation states using RT-PCR. An enriched microglial population was generated from the remainder of the brain and flow cytometry was used to examine protein expression. Expression of M1 markers, specifically TNFalpha, IL-6 and IFNGR was increased in the hippocampus of middle-aged animals, and similar changes were observed in tissue from aged mice where these changes were accompanied by increases in expression of MHCII and CD11b. We also saw increases in M2 markers, specifically IL-4R (in both middle-aged and aged tissue), and MR and arginase-1 (in aged tissue only). These data allow us to conclude that there is a significant age-related increase in the M1 activation state. These increases were maximal in tissue from aged animals. The enhanced expression of M2 markers may be part of a compensatory mechanism by microglia in response to the enhanced pro-inflammatory environment.

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Abstracts of the 3rd Annual Meeting, Frontiers in Neurology, 15 November 2013, Dublin, Ireland.

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