van Eersel et al.
Accepted Article
Early-onset Axonal Pathology in a Novel P301S-Tau Transgenic Mouse Model of Frontotemporal Lobar Degeneration1
Janet van Eersel1,*, Claire H Stevens1,*, Magdalena Przybyla1, Amadeus Gladbach1, Kristie Stefanoska1, Chesed KaiXin Chan2, Wei-Yi Ong2, John R Hodges3,4, Greg T Sutherland5, Jillian J Kril5, Dorothee Abramowski6, Matthias Staufenbiel6,#, Glenda M Halliday3,4, Lars M Ittner1,3,7,†
1
Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of
New South Wales, Sydney, NSW 2052, Australia. 2
Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119260
3
Neuroscience Research Australia, Sydney, NSW 2031, Australia.
4
Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia.
5
Discipline of Pathology, Sydney Medical School, University of Sydney, NSW 2006, Australia
6
Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland
7
Sydney Medical School, University of Sydney, NSW 2006, Australia
*J.v.E. and C.H.S. contributed equally to this publication.
†Correspondence should be addressed to L.M.I. (e-mail: [email protected])
#
Present address: Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of
Tübingen and German Center for Neurodegenerative Diseases, D-72076 Tübingen, Germany
Keywords: Tau, Neurofilament, Frontotemporal Lobar Degeneration, Mouse model, Alzheimer’s disease
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/nan.12233 Page 1 of 23
This article is protected by copyright. All rights reserved.
Accepted Article
van Eersel et al.
ABSTRACT
Aim: Tau becomes hyperphosphorylated in Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLDtau), resulting in functional deficits of neurons, neurofibrillary tangle (NFT) formation and eventually dementia. Expression of mutant human tau in the brains of transgenic mice has produced different lines that recapitulate various aspects of FTLD-tau and AD. In this study, we characterised the novel P301S mutant tau transgenic mouse line, TAU58/2.
Methods: Both young and aged TAU58/2 mice underwent extensive motor testing, after which brain tissue was analysed with immunohistochemistry, silver staining, electron microscopy and Western blotting. Tissue from various FTLD subtypes and AD patients was also analysed for comparison. Results: TAU58/2 mice presented with early-onset motor deficits, which became more pronounced with age. Throughout the brains of these mice, tau was progressively hyperphosphorylated resulting in increased NFT formation with age. In addition, frequent axonal swellings that stained intensively for neurofilament (NF) were present in young TAU58/2 mice prior to NFT formation. Similar axonal pathology was also observed in human FTLD-tau and AD. Interestingly, activated microglia were found in close proximity to neurons harbouring transgenic tau, but were not associated with NF-positive axonal swellings. Conclusions: In TAU58/2 mice, early tau pathology induces functional deficits of neurons associated with NF
pathology. This appears to be specific to tau, since similar changes are observed in FTLD-tau, but not in FTLD with TDP-43 inclusions. Therefore, TAU58/2 mice recapitulate aspects of human FTLD-tau and AD pathology, and will become instrumental in studying disease mechanisms and therapeutics in the future.
ABBREVIATIONS
Aβ
Amyloid β
NFT
Neurofibrillary tangle
AD
Alzheimer’s disease
Non-tg
Non-transgenic
BSA
Bovine serum albumin
PiD
Pick’s disease
CBD
Corticobasal degeneration
PSP
Progressive supranuclear palsy
FTLD
Frontotemporal lobar degeneration
TBS-T
Tris buffered saline with 0.1% Tween20
FTLD-tau
FTLD with tau pathology
WMT-GGT
White matter tract globular glial tauopathy
FTLD-TDP
FTLD with TDP-43 pathology
IHC
Immunohistochemistry
Page 2 of 23
This article is protected by copyright. All rights reserved.
van Eersel et al.
Neurofilament
NF200
Neurofilament of 200kDa
Accepted Article
NF
INTRODUCTION Frontotemporal lobar degeneration (FTLD) describes a heterogeneous group of disorders that are characterised by frontal and/or temporal lobe atrophy [1]. FTLD most commonly affects people between 50-75 years [2], and together with Alzheimer’s disease (AD) is one of the most common causes of early-onset dementia [3]. Typically, patients present with behavioural and/or language dysfunction, including social disinhibition, compulsive behaviour, hyperactivity and naming impairments [4]. Furthermore, approximately 15% of patients develop features of motor neuron disease, such as muscle weakness and wasting [5].
FTLD presents with a heterogeneous range of neuropathologies; approximately 45% of patients display tau proteincontaining lesions (FTLD-tau), while another 45% display TDP-43 protein-containing deposits (FTLD-TDP) [1]. The remaining cases display deposits made up of a variety of other proteins, including FUS, neurofilament, or as yet unidentified proteins. Unlike AD where tau deposition coincides with amyloid-β (Aβ) pathology, no overt Aβ pathology is found in FTLD-tau brains. Furthermore, the tau deposits in FTLD-tau differ from those in AD in their distribution and morphology. In AD, hyperphosphorylated tau aggregates present primarily as intraneuronal lesions known as neurofibrillary tangles (NFTs) as well as neuropil threads, whereas FTLD-tau displays a range of additional types of tau lesions, such as ovoid, intracytoplasmic neuronal inclusions known as Pick bodies, and glial inclusions such as coiled bodies, tufted astrocytes and astrocytic plaques. Why these different tau inclusions form is not known, however, it is commonly thought that deposits form when tau becomes hyperphosphorylated, causing it to detach from microtubules. Free tau then undergoes protein misfolding, aggregation, fibrillisation and eventually deposition [6].
Neurofilaments (NFs) are exclusively expressed in neurons where they are essential for maintaining cell shape and facilitating intracellular transport. NFs constitute the most abundant structures in large myelinated neurons. Abnormal accumulation of NF together with disease-specific protein aggregation has been identified in a number of neurodegenerative disorders, including FTLD-tau [7], amyotrophic lateral sclerosis [8], AD [9, 10], and Parkinson’s disease [11]. NF inclusions are also a characteristic feature of the FTLD subtype ‘neuronal intermediate filament inclusion disease’ together with FUS inclusions [12]. Accumulation of NF can be induced by a number of factors, including mutations, deregulated NF synthesis, defective axonal transport, and abnormal post-translational regulation [13]. It has been hypothesised that the abnormal accumulation of NFs in axons blocks the transportation of cargos along the axon, thereby causing neuronal death [14]. However, it remains to be determined whether this NF pathology plays a Page 3 of 23
This article is protected by copyright. All rights reserved.
van Eersel et al.
significant role in the pathogenesis of the various neurodegenerative disorders or is simply a secondary effect of the
Accepted Article
primary insult.
Approximately 40% of FTLD patients report a family history of dementia, indicating a strong genetic contribution [15]. The first genetic mutations to be linked to FTLD-tau were identified in the tau-encoding MAPT gene [16], and since then over 40 MAPT mutations linked to FTLD-tau have been described. Following the discovery of the MAPT
mutations, transgenic mice expressing human mutant tau have been generated and utilised for research into FTLD-tau and related tauopathies (reviewed by [6]). Depending on a range of factors, such as the promoter, the tau isoform and mutation, as well as the transgene integration site and copy number, these tau transgenic mice display a variety of phenotypic features. In this study, we present a detailed neuropathological and (motor) functional investigation of a novel P301S mutant tau transgenic mouse model that develops features similar to those observed in FTLD-tau patients, including a distinct NF pathology.
MATERIALS & METHODS Mice
All animal experiments were approved by the Animal Ethics Committees of the Universities of Sydney and New South Wales. All procedures complied with the statement on animal experimentation issued by the National Health and Medical Research Council of Australia. TAU58/2 mice express the human 0N4R tau isoform with the P301S mutation under the control of the mouse Thy1.2 promoter. For pronuclear injection (C57Bl/6xBALB/c background) the corresponding cDNA was inserted into vector pTSC21, linearized and vector sequences removed as described previously [17]. The TAU58/2 mice were backcrossed over 10 times and maintained on a C57Bl/6 background. Transgenic mice were identified by PCR as previously described [18]. Non-transgenic (non-tg) littermates were used as controls. Mice of both genders were analysed at indicated ages. Tau transgenic mice expressing K369I mutant human tau (1N4R, K3) [19], P301L mutant human tau (2N4R, pR5) [20] and non-mutant human tau (2N4R, Alz17) have previously been described [21].
Human Brain Tissue Post-mortem human brain tissue from donors diagnosed with the FTLD subtypes Corticobasal Degeneration (CBD) (n=5), Progressive Supranuclear Palsy (PSP) (n=5), Pick’s disease (PiD) (n=6), FTLD-TDP (n=6) and white matter tract globular glial tauopathy (WMT-GGT) (n=5), as well as Alzheimer’s disease (AD) (n=6) and age-matched controls (n=6) were obtained from the New South Wales Brain Bank. For all brain tissue, clinical and pathological information was obtained with written consent from the brain donors and their next of kin as approved by the Human Ethics Page 4 of 23
This article is protected by copyright. All rights reserved.
van Eersel et al.
Committees of the South Eastern and Illawarra Area Health Service, the University of New South Wales and the
Accepted Article
University of Sydney. All procedures complied with the statement on human experimentation issued by the National Health and Medical Research Council of Australia. The average age at death ( standard deviation) was as follows, CBD and PSP, 71 7 years; PiD, 70 6 years; WMT-GGT, 83 6 years; FTLD-TDP, 71 4 years; AD, 70 6 years and for age-matched controls, 70 6 years. The average disease durations ( standard deviation) was CBD, 5 4 years; PSP, 4 2 years; PiD, 9 2 years; WMT-GGT, 6 3 years; FTLD-TDP, 8 5 years and AD 7 3 years. None of the cases had a family history suggestive of an autosomal dominant disease.
Histology
Mice. At the specified age, mice were anesthetised and transcardially perfused with phosphate buffered saline (pH7.4) to remove blood. Brains were removed and the hemispheres separated. One hemisphere was immersion-fixed in 4% paraformaldehyde for immunohistochemical analysis, whereas the other hemisphere was sub-dissected and then snapfrozen in liquid nitrogen for biochemical analysis. Fixed brains were processed in an Excelsior tissue processor (Thermo), embedded in paraffin and sagittally sectioned at either 3μm for immunohistochemistry (IHC) or 8μm for Gallyas and Bielschowsky silver staining. IHC procedures have been described previously [22]. Antigen retrieval was carried out in a temperature- and pressure-controlled microwave system (Milestone) in Tris/EDTA pH 9.0 or Citrate buffer pH 5.8. Primary antibodies were against human tau (Tau13, Abcam or Dako tau, Dako), tau phosphorylated at Ser396/Ser404 (PHF-1, P. Davies), Ser422, Ser214, or Thr231 (pS422, pS214 and pT231, Abcam), 200kDa heavy neurofilament (NF200, Abcam), GFAP (Sigma), IBA1 (Dako) and TDP-43 (Biosciences). For fluorescent staining, Alexa fluorophore-coupled secondary antibodies to rabbit and mouse IgG were used for visualisation (1:250, Invitrogen). Alternatively, for brightfield staining, biotin-coupled secondary antibodies were used together with the ABC-HRP detection kit (Vector) and the reaction product visualised using 3’3’-Diaminobenzidine tetrahydrochloride
(Sigma). Counterstaining was achieved with either DAPI (Molecular probes) or cresyl violet. Bielschowsky and Gallyas silver staining was carried out as previously described [19, 23].
Humans. Brains were removed at autopsy, fixed in 15% formalin for 14 days, and then weighed. The cerebrum was embedded in 3% agarose and sliced at 3mm intervals. Tissue was sampled from the hippocampal formation (at the level of the lateral geniculate nucleus region), motor cortex (primary motor gyrus immediately anterior to the central sulcus at the level of the mammillary bodies), frontal cortex (superior frontal gyrus at the level of the caudate nucleus) and temporal cortex (inferior temporal gyrus at the level of the lateral geniculate nucleus). Tissue blocks were embedded in
Page 5 of 23
This article is protected by copyright. All rights reserved.
van Eersel et al.
paraffin and 8m thick sections cut using a microtome for immunohistochemical analysis. Staining was carried out as
Accepted Article
outlined above.
Immunohistochemical Analysis Sections, with the exception of the tissue stained with phosphorylated tau antibodies, were analysed using an Olympus BX51 microscope, and images taken using an Olympus DP70 camera and CellSens Standard software (Olympus). Quantification of staining was performed as outlined below on either 3 or 8μm thick sagittal brain sections at the level of the mid-hippocampus for each mouse. For all analyses, repeated measures on different days gave an intra-rater variability of
Accepted Article
Early-onset Axonal Pathology in a Novel P301S-Tau Transgenic Mouse Model of Frontotemporal Lobar Degeneration1
Janet van Eersel1,*, Claire H Stevens1,*, Magdalena Przybyla1, Amadeus Gladbach1, Kristie Stefanoska1, Chesed KaiXin Chan2, Wei-Yi Ong2, John R Hodges3,4, Greg T Sutherland5, Jillian J Kril5, Dorothee Abramowski6, Matthias Staufenbiel6,#, Glenda M Halliday3,4, Lars M Ittner1,3,7,†
1
Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of
New South Wales, Sydney, NSW 2052, Australia. 2
Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119260
3
Neuroscience Research Australia, Sydney, NSW 2031, Australia.
4
Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia.
5
Discipline of Pathology, Sydney Medical School, University of Sydney, NSW 2006, Australia
6
Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland
7
Sydney Medical School, University of Sydney, NSW 2006, Australia
*J.v.E. and C.H.S. contributed equally to this publication.
†Correspondence should be addressed to L.M.I. (e-mail: [email protected])
#
Present address: Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of
Tübingen and German Center for Neurodegenerative Diseases, D-72076 Tübingen, Germany
Keywords: Tau, Neurofilament, Frontotemporal Lobar Degeneration, Mouse model, Alzheimer’s disease
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/nan.12233 Page 1 of 23
This article is protected by copyright. All rights reserved.
Accepted Article
van Eersel et al.
ABSTRACT
Aim: Tau becomes hyperphosphorylated in Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLDtau), resulting in functional deficits of neurons, neurofibrillary tangle (NFT) formation and eventually dementia. Expression of mutant human tau in the brains of transgenic mice has produced different lines that recapitulate various aspects of FTLD-tau and AD. In this study, we characterised the novel P301S mutant tau transgenic mouse line, TAU58/2.
Methods: Both young and aged TAU58/2 mice underwent extensive motor testing, after which brain tissue was analysed with immunohistochemistry, silver staining, electron microscopy and Western blotting. Tissue from various FTLD subtypes and AD patients was also analysed for comparison. Results: TAU58/2 mice presented with early-onset motor deficits, which became more pronounced with age. Throughout the brains of these mice, tau was progressively hyperphosphorylated resulting in increased NFT formation with age. In addition, frequent axonal swellings that stained intensively for neurofilament (NF) were present in young TAU58/2 mice prior to NFT formation. Similar axonal pathology was also observed in human FTLD-tau and AD. Interestingly, activated microglia were found in close proximity to neurons harbouring transgenic tau, but were not associated with NF-positive axonal swellings. Conclusions: In TAU58/2 mice, early tau pathology induces functional deficits of neurons associated with NF
pathology. This appears to be specific to tau, since similar changes are observed in FTLD-tau, but not in FTLD with TDP-43 inclusions. Therefore, TAU58/2 mice recapitulate aspects of human FTLD-tau and AD pathology, and will become instrumental in studying disease mechanisms and therapeutics in the future.
ABBREVIATIONS
Aβ
Amyloid β
NFT
Neurofibrillary tangle
AD
Alzheimer’s disease
Non-tg
Non-transgenic
BSA
Bovine serum albumin
PiD
Pick’s disease
CBD
Corticobasal degeneration
PSP
Progressive supranuclear palsy
FTLD
Frontotemporal lobar degeneration
TBS-T
Tris buffered saline with 0.1% Tween20
FTLD-tau
FTLD with tau pathology
WMT-GGT
White matter tract globular glial tauopathy
FTLD-TDP
FTLD with TDP-43 pathology
IHC
Immunohistochemistry
Page 2 of 23
This article is protected by copyright. All rights reserved.
van Eersel et al.
Neurofilament
NF200
Neurofilament of 200kDa
Accepted Article
NF
INTRODUCTION Frontotemporal lobar degeneration (FTLD) describes a heterogeneous group of disorders that are characterised by frontal and/or temporal lobe atrophy [1]. FTLD most commonly affects people between 50-75 years [2], and together with Alzheimer’s disease (AD) is one of the most common causes of early-onset dementia [3]. Typically, patients present with behavioural and/or language dysfunction, including social disinhibition, compulsive behaviour, hyperactivity and naming impairments [4]. Furthermore, approximately 15% of patients develop features of motor neuron disease, such as muscle weakness and wasting [5].
FTLD presents with a heterogeneous range of neuropathologies; approximately 45% of patients display tau proteincontaining lesions (FTLD-tau), while another 45% display TDP-43 protein-containing deposits (FTLD-TDP) [1]. The remaining cases display deposits made up of a variety of other proteins, including FUS, neurofilament, or as yet unidentified proteins. Unlike AD where tau deposition coincides with amyloid-β (Aβ) pathology, no overt Aβ pathology is found in FTLD-tau brains. Furthermore, the tau deposits in FTLD-tau differ from those in AD in their distribution and morphology. In AD, hyperphosphorylated tau aggregates present primarily as intraneuronal lesions known as neurofibrillary tangles (NFTs) as well as neuropil threads, whereas FTLD-tau displays a range of additional types of tau lesions, such as ovoid, intracytoplasmic neuronal inclusions known as Pick bodies, and glial inclusions such as coiled bodies, tufted astrocytes and astrocytic plaques. Why these different tau inclusions form is not known, however, it is commonly thought that deposits form when tau becomes hyperphosphorylated, causing it to detach from microtubules. Free tau then undergoes protein misfolding, aggregation, fibrillisation and eventually deposition [6].
Neurofilaments (NFs) are exclusively expressed in neurons where they are essential for maintaining cell shape and facilitating intracellular transport. NFs constitute the most abundant structures in large myelinated neurons. Abnormal accumulation of NF together with disease-specific protein aggregation has been identified in a number of neurodegenerative disorders, including FTLD-tau [7], amyotrophic lateral sclerosis [8], AD [9, 10], and Parkinson’s disease [11]. NF inclusions are also a characteristic feature of the FTLD subtype ‘neuronal intermediate filament inclusion disease’ together with FUS inclusions [12]. Accumulation of NF can be induced by a number of factors, including mutations, deregulated NF synthesis, defective axonal transport, and abnormal post-translational regulation [13]. It has been hypothesised that the abnormal accumulation of NFs in axons blocks the transportation of cargos along the axon, thereby causing neuronal death [14]. However, it remains to be determined whether this NF pathology plays a Page 3 of 23
This article is protected by copyright. All rights reserved.
van Eersel et al.
significant role in the pathogenesis of the various neurodegenerative disorders or is simply a secondary effect of the
Accepted Article
primary insult.
Approximately 40% of FTLD patients report a family history of dementia, indicating a strong genetic contribution [15]. The first genetic mutations to be linked to FTLD-tau were identified in the tau-encoding MAPT gene [16], and since then over 40 MAPT mutations linked to FTLD-tau have been described. Following the discovery of the MAPT
mutations, transgenic mice expressing human mutant tau have been generated and utilised for research into FTLD-tau and related tauopathies (reviewed by [6]). Depending on a range of factors, such as the promoter, the tau isoform and mutation, as well as the transgene integration site and copy number, these tau transgenic mice display a variety of phenotypic features. In this study, we present a detailed neuropathological and (motor) functional investigation of a novel P301S mutant tau transgenic mouse model that develops features similar to those observed in FTLD-tau patients, including a distinct NF pathology.
MATERIALS & METHODS Mice
All animal experiments were approved by the Animal Ethics Committees of the Universities of Sydney and New South Wales. All procedures complied with the statement on animal experimentation issued by the National Health and Medical Research Council of Australia. TAU58/2 mice express the human 0N4R tau isoform with the P301S mutation under the control of the mouse Thy1.2 promoter. For pronuclear injection (C57Bl/6xBALB/c background) the corresponding cDNA was inserted into vector pTSC21, linearized and vector sequences removed as described previously [17]. The TAU58/2 mice were backcrossed over 10 times and maintained on a C57Bl/6 background. Transgenic mice were identified by PCR as previously described [18]. Non-transgenic (non-tg) littermates were used as controls. Mice of both genders were analysed at indicated ages. Tau transgenic mice expressing K369I mutant human tau (1N4R, K3) [19], P301L mutant human tau (2N4R, pR5) [20] and non-mutant human tau (2N4R, Alz17) have previously been described [21].
Human Brain Tissue Post-mortem human brain tissue from donors diagnosed with the FTLD subtypes Corticobasal Degeneration (CBD) (n=5), Progressive Supranuclear Palsy (PSP) (n=5), Pick’s disease (PiD) (n=6), FTLD-TDP (n=6) and white matter tract globular glial tauopathy (WMT-GGT) (n=5), as well as Alzheimer’s disease (AD) (n=6) and age-matched controls (n=6) were obtained from the New South Wales Brain Bank. For all brain tissue, clinical and pathological information was obtained with written consent from the brain donors and their next of kin as approved by the Human Ethics Page 4 of 23
This article is protected by copyright. All rights reserved.
van Eersel et al.
Committees of the South Eastern and Illawarra Area Health Service, the University of New South Wales and the
Accepted Article
University of Sydney. All procedures complied with the statement on human experimentation issued by the National Health and Medical Research Council of Australia. The average age at death ( standard deviation) was as follows, CBD and PSP, 71 7 years; PiD, 70 6 years; WMT-GGT, 83 6 years; FTLD-TDP, 71 4 years; AD, 70 6 years and for age-matched controls, 70 6 years. The average disease durations ( standard deviation) was CBD, 5 4 years; PSP, 4 2 years; PiD, 9 2 years; WMT-GGT, 6 3 years; FTLD-TDP, 8 5 years and AD 7 3 years. None of the cases had a family history suggestive of an autosomal dominant disease.
Histology
Mice. At the specified age, mice were anesthetised and transcardially perfused with phosphate buffered saline (pH7.4) to remove blood. Brains were removed and the hemispheres separated. One hemisphere was immersion-fixed in 4% paraformaldehyde for immunohistochemical analysis, whereas the other hemisphere was sub-dissected and then snapfrozen in liquid nitrogen for biochemical analysis. Fixed brains were processed in an Excelsior tissue processor (Thermo), embedded in paraffin and sagittally sectioned at either 3μm for immunohistochemistry (IHC) or 8μm for Gallyas and Bielschowsky silver staining. IHC procedures have been described previously [22]. Antigen retrieval was carried out in a temperature- and pressure-controlled microwave system (Milestone) in Tris/EDTA pH 9.0 or Citrate buffer pH 5.8. Primary antibodies were against human tau (Tau13, Abcam or Dako tau, Dako), tau phosphorylated at Ser396/Ser404 (PHF-1, P. Davies), Ser422, Ser214, or Thr231 (pS422, pS214 and pT231, Abcam), 200kDa heavy neurofilament (NF200, Abcam), GFAP (Sigma), IBA1 (Dako) and TDP-43 (Biosciences). For fluorescent staining, Alexa fluorophore-coupled secondary antibodies to rabbit and mouse IgG were used for visualisation (1:250, Invitrogen). Alternatively, for brightfield staining, biotin-coupled secondary antibodies were used together with the ABC-HRP detection kit (Vector) and the reaction product visualised using 3’3’-Diaminobenzidine tetrahydrochloride
(Sigma). Counterstaining was achieved with either DAPI (Molecular probes) or cresyl violet. Bielschowsky and Gallyas silver staining was carried out as previously described [19, 23].
Humans. Brains were removed at autopsy, fixed in 15% formalin for 14 days, and then weighed. The cerebrum was embedded in 3% agarose and sliced at 3mm intervals. Tissue was sampled from the hippocampal formation (at the level of the lateral geniculate nucleus region), motor cortex (primary motor gyrus immediately anterior to the central sulcus at the level of the mammillary bodies), frontal cortex (superior frontal gyrus at the level of the caudate nucleus) and temporal cortex (inferior temporal gyrus at the level of the lateral geniculate nucleus). Tissue blocks were embedded in
Page 5 of 23
This article is protected by copyright. All rights reserved.
van Eersel et al.
paraffin and 8m thick sections cut using a microtome for immunohistochemical analysis. Staining was carried out as
Accepted Article
outlined above.
Immunohistochemical Analysis Sections, with the exception of the tissue stained with phosphorylated tau antibodies, were analysed using an Olympus BX51 microscope, and images taken using an Olympus DP70 camera and CellSens Standard software (Olympus). Quantification of staining was performed as outlined below on either 3 or 8μm thick sagittal brain sections at the level of the mid-hippocampus for each mouse. For all analyses, repeated measures on different days gave an intra-rater variability of
