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Background Intratumoral heterogeneity is a hallmark of diffuse gliomas. DNA methylation profiling is an emerging approach in the clinical classification of brain tumors. The goal of this study is to investigate the effects of intratumoral heterogeneity on classification confidence. Methods We used neuronavigation to acquire 133 image-guided and spatially separated stereotactic biopsy samples from 16 adult patients with a diffuse glioma (7 IDH-wildtype and 2 IDH-mutant glioblastoma, 6 diffuse astrocytoma, IDH-mutant and 1 oligodendroglioma, IDH-mutant and 1p19q codeleted), which we characterized using DNA methylation arrays. Samples were obtained from regions with and without abnormalities on contrast-enhanced T1-weighted and fluid-attenuated inversion recovery MRI. Methylation profiles were analyzed to devise a 3-dimensional reconstruction of (epi)genetic heterogeneity. Tumor purity was assessed from clonal methylation sites. Results Molecular aberrations indicated that tumor was found outside imaging abnormalities, underlining the infiltrative nature of this tumor and the limitations of current routine imaging modalities. We demonstrate that tumor purity is highly variable between samples and explains a substantial part of apparent epigenetic spatial heterogeneity. We observed that DNA methylation subtypes are often, but not always, conserved in space taking tumor purity and prediction accuracy into account. Conclusion Our results underscore the infiltrative nature of diffuse gliomas and suggest that DNA methylation subtypes are relatively concordant in this tumor type, although some heterogeneity exists.

The accumulation of fibrillar amyloid-β (Aβ) peptides alongside or within the cerebral vasculature is the hallmark of cerebral amyloid angiopathy (CAA). This condition commonly co-occurs with Alzheimer's disease (AD) and leads to cerebral microbleeds, intracranial hemorrhages, and stroke. CAA also occurs sporadically in an age-dependent fashion and can be accelerated by the presence of familial Aβ mutant peptides. Recent studies using Fourier transform infrared (FTIR) spectroscopy of vascular Aβ fibrils derived from rodents containing the double E22Q/D23N mutations indicated the presence of a novel antiparallel β-sheet structure. To address whether this structure is associated solely with the familial mutations or is a common feature of CAA, we propagated Aβ fibrils from human brain vascular tissue of patients diagnosed with nonfamilial CAA. Aβ fibrils were isolated from cerebral blood vessels using laser capture microdissection in which specific amyloid deposits were removed from thin slices of the brain tissue. Transmission electron microscopy revealed that these deposits were organized into a tight meshwork of fibrils, which FTIR measurements showed could serve as seeds to propagate the growth of Aβ40 fibrils for structural studies. Solid-state NMR measurements of the fibrils propagated from vascular amyloid showed they contained a mixture of parallel, in-register, and antiparallel β-sheet structures. The presence of fibrils with antiparallel structure derived from vascular amyloid is distinct from the typical parallel, in-register β-sheet structure that appears in fibrils derived from parenchymal amyloid in AD. These observations reveal that different microenvironments influence the structures of Aβ fibrils in the human brain.

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are closely related progressive disorders with no available causal therapy, neuropathologically characterized by intraneuronal aggregates of misfolded α-synuclein. To explore the role of DNA methylation changes in PD and DLB pathogenesis, we performed an epigenome-wide association study (EWAS) of 322 postmortem frontal cortex samples and replicated results in an independent set of 219 donors. We report novel differentially methylated replicating loci associated with Braak Lewy body stage near SFMBT2, PHYHIP, BRF1/PACS2 and DGKG. The DGKG locus also showed evidence of DNA methylation changes in the earliest, preclinical stage of disease. Differentially methylated probes were independent of known PD genetic risk alleles. Meta-analysis provided suggestive evidence for a differentially methylated locus within the chromosomal region affected by the PD-associated 22q11.2 deletion. Our findings elucidate novel disease pathways in PD and DLB and generate hypotheses for future molecular studies of Lewy body pathology.

Cognitive deficits in Alzheimer’s disease, specifically amnestic (memory dominant) deficits, are associated with cholinergic degeneration in the basal forebrain. The cholinergic nucleus within the basal forebrain, the nucleus basalis of Meynert, exhibits local atrophy and reduced cortical tract integrity on MRI, and reveals amyloid-β and phosphorylated-tau pathology at autopsy. To better understand the pathophysiology of nucleus basalis of Meynert atrophy and its neocortical projections in Alzheimer’s disease, we utilized a combined post-mortem in-situ MRI and histopathology approach. A total of 19 Alzheimer’s disease (10 amnestic and 9 non-amnestic) and 9 non-neurological control donors underwent 3T T1-weighted MRI for anatomical delineation and volume assessment of the nucleus basalis of Meynert, and diffusion-weighted imaging for microstructural assessment of the nucleus and its projections. At subsequent brain autopsy, tissue dissection and immunohistochemistry were performed for amyloid-β, phosphorylated-tau and choline acetyltransferase. Compared to controls, we observed an MRI-derived volume reduction and altered microstructural integrity of the nucleus basalis of Meynert in Alzheimer’s disease donors. Furthermore, decreased cholinergic cell density was associated with reduced integrity of the nucleus and its tracts to the temporal lobe, specifically to the temporal pole of the superior temporal gyrus, and the parahippocampal gyrus. The association between cholinergic cell density and alteration to cortical tracts was specific for amnestic, compared to non-amnestic Alzheimer’s disease donors. Our study illustrates that the nucleus basalis of Meynert is severely affected in amnestic Alzheimer’s disease, both in terms of pathology within the nucleus, but also in terms of damage to its cortical projections, specifically to the temporal lobe, which may contribute to the observed cognitive deterioration.

A repeat expansion in the C9orf72 gene is the most prevalent genetic cause of frontotemporal dementia (C9-FTD). Several studies have indicated the involvement of the unfolded protein response (UPR) in C9-FTD. In human neuropathology, UPR markers are strongly associated with granulovacuolar degeneration (GVD). In this study, we aim to assess the presence of UPR markers together with the presence of dipeptide pathology and GVD in post mortem brain tissue from C9-FTD cases and neurologically healthy controls. Using immunohistochemistry we assessed the presence of phosphorylated PERK, IRE1α and eIF2α in the frontal cortex, hippocampus and cerebellum of C9-FTD (n = 18) and control (n = 9) cases. The presence of UPR activation markers was compared with the occurrence of pTDP-43, p62 and dipeptide repeat (DPR) proteins (poly(GA), -(GR) & -(GP)) as well as casein kinase 1 delta (CK1δ), a marker for GVD. Increased presence of UPR markers was observed in the hippocampus and cerebellum in C9-FTD compared to control cases. In the hippocampus, overall levels of pPERK and peIF2α were higher in C9-FTD, including in granule cells of the dentate gyrus (DG). UPR markers were also observed in granule cells of the cerebellum in C9-FTD. In addition, increased levels of CK1δ were observed in granule cells in the DG of the hippocampus and granular layer of the cerebellum in C9-FTD. Double-labelling experiments indicate a strong association between UPR markers and the presence of dipeptide pathology as well as GVD. We conclude that UPR markers are increased in C9-FTD and that their presence is associated with dipeptide pathology and GVD. Increased presence of UPR markers and CK1δ in granule cells in the cerebellum and hippocampus could be a unique feature of C9-FTD.

The neuropathology of Alzheimer’s disease (AD) is characterized by hyperphosphorylated tau neurofibrillary tangles (NFTs) and amyloid-beta (Aβ) plaques. Aβ plaques are hypothesized to follow a development sequence starting with diffuse plaques, which evolve into more compact plaques and finally mature into the classic cored plaque type. A better molecular understanding of Aβ pathology is crucial, as the role of Aβ plaques in AD pathogenesis is under debate. Here, we studied the deposition and fibrillation of Aβ in different plaque types with label-free infrared and Raman imaging. Fourier-transform infrared (FTIR) and Raman imaging was performed on native snap-frozen brain tissue sections from AD cases and non-demented control cases. Subsequently, the scanned tissue was stained against Aβ and annotated for the different plaque types by an AD neuropathology expert. In total, 160 plaques (68 diffuse, 32 compact, and 60 classic cored plaques) were imaged with FTIR and the results of selected plaques were verified with Raman imaging. In diffuse plaques, we detect evidence of short antiparallel β-sheets, suggesting the presence of Aβ oligomers. Aβ fibrillation significantly increases alongside the proposed plaque development sequence. In classic cored plaques, we spatially resolve cores containing predominantly large parallel β-sheets, indicating Aβ fibrils. Combining label-free vibrational imaging and immunohistochemistry on brain tissue samples of AD and non-demented cases provides novel insight into the spatial distribution of the Aβ conformations in different plaque types. This way, we reconstruct the development process of Aβ plaques in human brain tissue, provide insight into Aβ fibrillation in the brain, and support the plaque development hypothesis. Electronic supplementary material The online version of this article (10.1186/s40478-020-01091-5) contains supplementary material, which is available to authorized users.

Alzheimer’s disease (AD) is characterized by amyloid-beta (Aβ) deposits, which come in myriad morphologies with varying clinical relevance. Previously, we observed an atypical Aβ deposit, referred to as the coarse-grained plaque. In this study, we evaluate the plaque’s association with clinical disease and perform in-depth immunohistochemical and morphological characterization. The coarse-grained plaque, a relatively large (Ø ≈ 80 µm) deposit, characterized as having multiple cores and Aβ-devoid pores, was prominent in the neocortex. The plaque was semi-quantitatively scored in the middle frontal gyrus of Aβ-positive cases (n = 74), including non-demented cases (n = 15), early-onset (EO)AD (n = 38), and late-onset (LO)AD cases (n = 21). The coarse-grained plaque was only observed in cases with clinical dementia and more frequently present in EOAD compared to LOAD. This plaque was associated with a homozygous APOE ε4 status and cerebral amyloid angiopathy (CAA). In-depth characterization was done by studying the coarse-grained plaque’s neuritic component (pTau, APP, PrPC), Aβ isoform composition (Aβ40, Aβ42, AβN3pE, pSer8Aβ), its neuroinflammatory component (C4b, CD68, MHC-II, GFAP), and its vascular attribution (laminin, collagen IV, norrin). The plaque was compared to the classic cored plaque, cotton wool plaque, and CAA. Similar to CAA but different from classic cored plaques, the coarse-grained plaque was predominantly composed of Aβ40. Furthermore, the coarse-grained plaque was distinctly associated with both intense neuroinflammation and vascular (capillary) pathology. Confocal laser scanning microscopy (CLSM) and 3D analysis revealed for most coarse-grained plaques a particular Aβ40 shell structure and a direct relation with vessels. Based on its morphological and biochemical characteristics, we conclude that the coarse-grained plaque is a divergent Aβ plaque-type associated with EOAD. Differences in Aβ processing and aggregation, neuroinflammatory response, and vascular clearance may presumably underlie the difference between coarse-grained plaques and other Aβ deposits. Disentangling specific Aβ deposits between AD subgroups may be important in the search for disease-mechanistic-based therapies.

Knowledge about the molecular mechanisms driving Alzheimer's disease (AD) is still limited. To learn more about AD biology, we performed whole transcriptome sequencing on the hippocampus of 20 AD cases and 10 age- and sex-matched cognitively healthy controls. We observed 2716 differentially expressed genes, of which 48% replicated in a second data set of 84 AD cases and 33 controls. We used an integrative network-based approach for combining transcriptomic and protein-protein interaction data to find differentially expressed gene modules that may reflect key processes in AD biology. A total of 735 differentially expressed genes were clustered into 33 modules, of which 82% replicated in a second data set, highlighting the robustness of this approach. These 27 modules were enriched for signal transduction, transport, response to stimulus, and several organic and cellular metabolic pathways. Ten modules interacted with previously described AD genes. Our study indicates that analyzing RNA-expression data based on annotated gene modules is more robust than on individual genes. We provide a comprehensive overview of the biological processes involved in AD, and the detected differentially expressed gene modules may provide a molecular basis for future research into mechanisms underlying AD.

Aims: The behavioural variant of frontotemporal dementia with a C9orf72 expansion (C9-bvFTD) is characterised by early changes in social-emotional cognition that are linked to the loss of von Economo neurons (VENs). Together with a subset of neighbouring pyramidal neurons, VENs express the GABA receptor subunit theta (GABRQ). It is not known if the selective vulnerability of VENs in C9-bvFTD also includes this GABRQ-expressing population. Methods: We quantified VENs and GABRQ immunopositive neurons in the anterior cingulate cortex (ACC) in C9-bvFTD (n = 16), controls (n = 12) and Alzheimer's disease (AD) (n = 7). Second, we assessed VENs and GABRQ-expressing populations in relation to the clinicopathological profiles. Results: We found the number of VENs and GABRQ-expressing neurons and their ratio over the total layer 5 neuronal population was lower in C9-bvFTD compared to control and AD. C9-bvFTD donors with underlying TDP43 type A pathology in the ACC showed the highest loss of GABRQ-expressing neurons. C9-bvFTD donors that did not present with motor neuron disease (MND) symptoms in the first half of their disease course showed a prominent loss of GABRQ-expressing neurons compared to controls. C9-bvFTD donors with no symptoms of psychosis showed a higher loss compared to controls. Across all donors, the number of VENs correlated strongly with the number of GABRQ-expressing neurons. Conclusion: We show that VENs, together with GABRQ-expressing neurons, are selectively vulnerable in C9-bvFTD but are both spared in AD. This suggests they are related and that this GABRQ-expressing population of VENs and pyramidal neurons, is a key modulator of social-emotional functioning.

Intratumoral heterogeneity is a hallmark of diffuse gliomas. We used neuronavigation to acquire 133 image-guided and spatially-separated stereotactic biopsy samples from 16 adult patients with a diffuse glioma, which we characterized using DNA methylation arrays. Samples were obtained from regions with and without imaging abnormalities. Methylation profiles were analyzed to devise a three-dimensional reconstruction of genetic and epigenetic heterogeneity. Molecular aberrations indicated that tumor was found outside imaging abnormalities, underlining the infiltrative nature of this tumor and the limitations of current routine imaging modalities. We demonstrate that tumor purity is highly variable between samples and largely explains apparent epigenetic spatial heterogeneity. Indeed, we observed that DNA methylation subtypes are highly conserved in space after adjusting for tumor purity. Genome-wide heterogeneity analysis showed equal or increased heterogeneity among normal tissue when compared to tumor. These findings were validated in a separate cohort of 61 multi-sector tumor and 64 normal samples. Our findings underscore the infiltrative nature of diffuse gliomas and suggest that heterogeneity in DNA methylation is innate to somatic cells and not a characteristic feature of this tumor type.

The combined fluorescent and Aβ-binding properties of the dietary spice curcumin could yield diagnostic purpose in the search for a non-invasive Aβ-biomarker for Alzheimer’s disease (AD). However, evidence on the binding properties of curcumin, its conjugates and clinically used bio-available formulations to AD neuropathological hallmarks is scarce. We therefore assessed the binding properties of different curcumin forms to different neuropathological deposits in post-mortem brain tissue of cases with AD, other neurodegenerative diseases, and controls. Post mortem brain tissue was histochemically assessed for the binding of curcumin, its isoforms, conjugates and bio-available forms and compared to routinely used staining methods. For this study we included brains of early onset AD, late onset AD, primary age-related tauopathy (PART), cerebral amyloid angiopathy (CAA), frontotemporal lobar degeneration (FTLD) with tau or TAR DNA-binding protein 43 (TDP-43) inclusions, dementia with Lewy bodies (DLB), Parkinson’s disease (PD) and control cases without brain pathology. We found that curcumin binds to fibrillar amyloid beta (Aβ) in plaques and CAA. It does not specifically bind to inclusions of protein aggregates in FTLD-tau cases, TDP-43, or Lewy bodies. Curcumin isoforms, conjugates and bio-available forms show affinity for the same Aβ structures. Curcumin staining overlaps with immunohistochemical detection of Aβ in fibrillar plaques and CAA, and to a lesser extent cored plaques. A weak staining of neurofibrillary tangles was observed, while other structures immunopositive for phosphorylated tau remained negative. In conclusion, curcumin, its isoforms, conjugates and bio-available forms selectively bind fibrillar Aβ in plaques and CAA in post mortem AD brain tissue. Curcumin, being a food additive with fluorescent properties, is therefore an interesting candidate for in-vivo diagnostics in AD, for example in retinal fluorescent imaging.

While diffuse gliomas are notorious for their histopathological, genetic and transcriptional spatial heterogeneity, little is known about their epigenetic spatial heterogeneity. The result of spatial (epi)genetic analysis is strongly influenced by the proportion of cancer cells in a sample, so called tumor purity. However, a gold standard for assessment of tumor purity is lacking. We set out to analyze tumor purity using different measurement modalities and explore tumor purity-corrected DNA methylation spatial heterogeneity in glioma. DNA methylation(-derived), quantitative histological and radiological measurements of tumor purity, as well as DNA methylation profiles, were analyzed in 133 image-guided multi-sector stereotactic biopsy samples of 16 patients with newly diagnosed glioma. These biopsies were acquired in regions with and without abnormalities on MRI. Data was validated in two independent populations of respectively 102 multi-region samples in 24 glioma patients and 64 single-region samples from patients without glioma. DNA methylation profiles from The Cancer Genome Atlas and the patients without glioma was used to predict DNA methylation and transcriptional subtype. Consensus measurement of tumor purity estimates (CPE) ranged from 0 to 91% and was most correlated with the DNA methylation measurement of tumor purity. Neuropathological qualitative assessment of tumor presence generally corresponded well with CPE, but occasionally samples reported as ‘histologically normal’ demonstrated tumor purities up to 53%. After filtering specimens with tumor purity less than 50%, DNA methylation subtype showed little spatial heterogeneity, this in contrast to transcriptional subtype. Samples from core and peripheral regions showed similar DNA methylation profiles. Non-purity related intratumoral heterogeneity for promotor methylation of epigenetically regulated genes was minimal, but higher in IDH-wildtype than in IDH-mutant gliomas. In conclusion, after considering DNA methylation-based measurement of tumor purity DNA methylation in diffuse gliomas shows little spatial heterogeneity; incorporating such tumor purity information can further increase the reliability of methylation-profiling-based tumor classification.

ObjectiveDespite the strong genetic component of frontotemporal dementia (FTD), a substantial proportion of patients remain genetically unresolved. We performed an in-depth study of a family with an autosomal dominant form of FTD to investigate the underlying genetic cause.MethodsFollowing clinical and pathologic characterization of the family, genetic studies included haplotype sharing analysis and exome sequencing. Subsequently, we performed immunohistochemistry, immunoblotting, and a microtubule repolymerization assay to investigate the potential impact of the candidate variant in tubulin alpha 4a (TUBA4A).ResultsThe clinical presentation in this family is heterogeneous, including behavioral changes, parkinsonian features, and uncharacterized dementia. Neuropathologic examination of 2 patients revealed TAR DNA binding protein 43 (TDP-43) pathology with abundant dystrophic neurites and neuronal intranuclear inclusions, consistent with frontotemporal lobar degeneration–TDP type A. We identified a likely pathogenic variant in TUBA4A segregating with disease. TUBA4A encodes for α-tubulin, which is a major component of the microtubule network. Variants in TUBA4A have been suggested as a rare genetic cause of amyotrophic lateral sclerosis (ALS) and have sporadically been reported in patients with FTD without supporting genetic segregation. A decreased trend of TUBA4A protein abundance was observed in patients compared with controls, and a microtubule repolymerization assay demonstrated disrupted α-tubulin function. As opposed to variants found in ALS, TUBA4A variants associated with FTD appear more localized to the N-terminus, indicating different pathogenic mechanisms.ConclusionsOur findings support the role of TUBA4A variants as rare genetic cause of familial FTD.

Background: The majority of patients with Alzheimer’s disease (AD) exhibit amyloid- (A) deposits at the brain vasculature, a process referred to as cerebral amyloid angiopathy (CAA). In over 51% of AD cases, A also accumulates in cortical capillaries, which is termed capillary CAA (capCAA). It has been postulated that the presence of capCAA in AD is a specific subtype of AD, although underlying mechanisms are not yet fully understood. Sphingolipids (SLs) are implicated in neurodegenerative disorders, including AD. However, to date it remains unknown whether alterations in the SL pathway are involved in capCAA pathogenesis and if these differ from AD. 16 17 18 19 20 21 Objective: To determine whether AD cases with capCAA have an altered SL profile compared to AD cases without capCAA. 22 Methods: Immunohistochemistry was performed to assess the expression and localization of ceramide, acid sphingomyelinase (ASM), and sphingosine-1-phosphate receptors (S1P1, S1P3). In addition, we determined the concentrations of S1P as well as different chain-lengths of ceramides using HPLC-MS/MS. 23 24 25 Results: Immunohistochemical analysis revealed an altered expression of ceramide, ASM, and S1P receptors by reactive astrocytes and microglial cells specifically associated with capCAA. Moreover, a shift in the balance of ceramides with different chain-lengths and S1P content is observed in capCAA. 26 27 28 Conclusion: Here we provide evidence of a deregulated SL balance in capCAA. The increased levels of ASM and ceramide in activated glia cells suggest that the SL pathway is involved in the neuroinflammatory response in capCAA pathogenesis. Future research is needed to elucidate the role of S1P in capCAA. 29 30 31

INTRODUCTION: We performed a comprehensive quantitative proteomics study on human hippocampus tissue involving all Braak stages to assess changes in protein abundance over the various stages of Alzheimer's disease (AD).METHODS: Hippocampal subareas CA1 and subiculum of 40 cases were isolated using laser capture microdissection and analyzed using mass spectrometry. Immunoblotting and immunohistochemistry were used for validation.RESULTS: Over the Braak stages, an altered expression was found for 372 proteins including changes in levels of extracellular matrix components, and in calcium-dependent signaling proteins. Early changes were observed in levels of proteins related to cytoskeletal dynamics and synaptic components including an increase in RIMS1 and GRIK4. Several synaptic proteins, such as BSN, LIN7A, DLG2, -3, and -4, exhibit an early-up, late-down expression pattern.DISCUSSION: This study provides new insight into AD-dependent changes in protein levels in the hippocampus during AD pathology, identifying potential novel therapeutic targets and biomarkers.

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein alpha-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified alpha-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all alpha-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within alpha-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.

Mutations in presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP) are major genetic causes of early-onset Alzheimer's disease (EOAD). Clinical heterogeneity is frequently observed in patients with PSEN1 and PSEN2 mutations. Using whole exome sequencing, we screened a Dutch cohort of 68 patients with EOAD for rare variants in Mendelian Alzheimer's disease, frontotemporal dementia, and prion disease genes. We identified 3 PSEN1 and 2 PSEN2 variants. Three variants, 1 in PSEN1 (p.H21Profs*2) and both PSEN2 (p.A415S and p.M174I), were novel and absent in control exomes. These novel variants can be classified as probable pathogenic, except for PSEN1 (p.H21Profs*2) in which the pathogenicity is uncertain. The initial clinical symptoms between mutation carriers varied from behavioral problems to memory impairment. Our findings extend the mutation spectrum of EOAD and underline the clinical heterogeneity among PSEN1 and PSEN2 mutation carriers. Screening for Alzheimer's disease–causing genes is indicated in presenile dementia with an overlapping clinical diagnosis.

Alzheimer’s disease (AD) is characterized by a long pre-clinical phase (20–30 years), during which significant brain pathology manifests itself. Disease mechanisms associated with pathological hallmarks remain elusive. Most processes associated with AD pathogenesis, such as inflammation, synaptic dysfunction, and hyper-phosphorylation of tau are dependent on protein kinase activity. The objective of this study was to determine the involvement of protein kinases in AD pathogenesis. Protein kinase activity was determined in postmortem hippocampal brain tissue of 60 patients at various stages of AD and 40 non-demented controls (Braak stages 0-VI) using a peptide-based microarray platform. We observed an overall decrease of protein kinase activity that correlated with disease progression. The phosphorylation of 96.7% of the serine/threonine peptides and 37.5% of the tyrosine peptides on the microarray decreased significantly with increased Braak stage (p-value

In-vivo labeling of retinal amyloid-beta(Aβ) and tau has potential as non-invasive biomarker for Alzheimer’s disease (AD). However, literature on the presence of Aβ and phosphorylated tau (pTau) in AD retinas is inconclusive. We therefore assessed the presence of Aβ and pTau in post-mortem retinas in 6 AD and 6 control cases who donated brains and eyes to the Netherlands Brain Bank. Neuropathological diagnosis of AD was made according to NIA-AA criteria. Formalin fixed retinas were dissected in quadrants and cross-sections of medial and superior retinas were made. Immuno-histochemical stainings were performed for Aβ, amyloid precursor protein (APP) and pTau. To assess translation to an in-vivo set up using curcumin as labelling fluorophore, co-stainings with curcumin were performed. No typical Aβ-plaques and neurofibrillary tangles, like in the cerebral cortex, were observed in AD retinas. A diffuse immunoreactive signal for pTau was increased in the inner and outer plexiform layers of the retina in AD cases compared to control cases with absence of cerebral amyloid pathology. Immunostaining with anti-Aβ and anti-APP antibodies yielded signal in ganglion cells, amacrine cells, horizontal cells and Müller cells in both control and AD cases. We observed small extracellular deposits positive for anti-Aβ antibodies 12F4 and 6E10 and negative for 4G8 and curcumin. A subset of these deposits could be characterized as corpora amylacea. In conclusion we found that retinal manifestations of AD pathology appear to be different compared to cerebral AD pathology. Using a qualitative cross-sectional approach, we did not find Aβ/APP related differences in the retina between AD and control subjects. In contrast, tau related changes were found to be present in cases with cerebral AD pathology, suggesting retinal tau as a potential biomarker for AD.

SummaryParkinson’s disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites, which are neuronal inclusions that are immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of this Lewy pathology is crucial to understanding pathogenesis and progression of the disease. Using correlative light and electron microscopy/tomography on brain tissue from five Parkinson’s disease brain donors, we identified α-synuclein immunopositive Lewy pathology and could show that the majority of these features including Lewy bodies and Lewy neurites primarily consists of a crowded membranous medley of vesicular structures and dysmorphic organelles. Only a small fraction of observed Lewy bodies contained predominant proteinaceous filaments, as previously described. The crowding of organellar components was confirmed by STED- based super-resolution microscopy, and high lipid content within the α-synuclein immunopositive inclusions was corroborated by confocal imaging, CARS/FTIR imaging and lipidomics. Applying this correlative high-resolution imaging and biophysical approach, we discovered in the postmortem brain of Parkinson’s patients a subcellular protein-lipid compartmentalization not previously described in Lewy pathology.

The insular cortex is a heterogeneous and widely connected brain region. It plays a role in autonomic, cognitive, emotional and somatosensory functions. Its complex and unique cytoarchitecture includes a periallocortical agranular, pro-isocortical dysgranular, and isocortical granular sub-regions. In Parkinson’s disease (PD), the insula shows α-synuclein inclusions in advanced stages of the disease and its atrophy correlates with cognitive deficits. However, little is known regarding its regional neuropathological characteristics and vulnerability in Lewy body diseases. The aim of this study is to assess the distribution pattern of α-synuclein pathology in the insular sub-regions and the selective vulnerability of its different cell types in PD and dementia with Lewy bodies (DLB). Human post-mortem insular tissues from 10 donors with incidental Lewy body disease (iLBD), PD, DLB, and age-matched controls were immunostained for α-synuclein and glial fibrillary acid protein (GFAP). Results showed that a decreasing gradient of α-synuclein pathology was present from agranular to granular sub-regions in iLBD, PD and PD with dementia (PDD) donors. The agranular insula was heavily inflicted, revealing various α-synuclein immunoreactive morphological structures, predominantly Lewy neurites (LNs), and astroglial synucleinopathy. While dysgranular and granular sub-regions showed a decreasing gradient of inclusions and more Lewy bodies (LBs) in deeper layers. In DLB, this gradient was less pronounced and severe pathology was observed in the granular insula compared to PDD and regardless of disease stage. Protoplasmic astrocytes showed α-synuclein inclusions and severe degenerative changes increasing with disease severity. While few von Economo neurons (VENs) in the fronto-insular region revealed inclusions, particularly in PDD patients. Our study reports novel findings on the differential involvement of the insular sub-regions in PD and particular involvement of the agranular sub-region, VENs and astrocytes. Thus, the differential cellular architecture of the insular sub-regions portrays the topographic variation and vulnerability to α-synuclein pathology in Lewy body diseases.

An imbalance of tau isoforms containing either three or four microtubule-binding repeats causes frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) in families with intronic mutations in the MAPT gene. Here we report equivalent imbalances at the mRNA and protein levels and increased total tau levels in the brains of subjects with Huntington's disease (HD) together with rod-like tau deposits along neuronal nuclei. These tau nuclear rods show an ordered filamentous ultrastructure and can be found filling the neuronal nuclear indentations previously reported in HD brains. Finally, alterations in serine/arginine-rich splicing factor-6 coincide with tau missplicing, and a role of tau in HD pathogenesis is evidenced by the attenuation of motor abnormalities of mutant HTT transgenic mice in tau knockout backgrounds. © 2014 Nature America, Inc., Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CiberNed–Instituto de Salud Carlos III) and by grants from Ministerio de Ciencia e Innovación (MICINN), Ministerio de Economía y Competitividad (MINECO), Comunidad Autónoma de Madrid, Fundación Ramón Areces

Alzheimer's disease (AD) is the most common form of dementia and marked by deposition of amyloid-β (Aβ) within the brain. Alterations of Aβ transporters at the neurovasculature may play a role in the disease process. We investigated the expression of ABC transporters P-glycoprotein (P-gp) and breast cancer related protein (BCRP) in non-neurologic controls, AD, and severe capillary cerebral amyloid angiopathy (capCAA) cases, which are characterized by deposition of Aβ within cerebral capillaries. Our data show that microvascular expression of P-gp and BCRP is strikingly decreased in capCAA-affected vessels but not in AD and control samples. Messenger RNA levels of P-gp, but not of BCRP, were downregulated in brain endothelial cells on exposure to oligomeric Aβ42, but not fibrillar Aβ42 or Aβ40. Coincubating Aβ42 together with clusterin, an amyloid-associated protein highly expressed in capCAA-affected vessels, strongly reduced levels of P-gp. In conclusion, accumulation of Aβ, in combination with clusterin, within and around cerebral capillaries, may further aggravate the disease process in AD by affecting P-gp expression. Loss of P-gp expression or activity may serve as a selective biomarker for ongoing capCAA.

The olfactory bulb (OB) is affected early in both Parkinson's (PD) and Alzheimer's disease (AD), evidenced by the presence of disease-specific protein aggregates and an early loss of olfaction. Whereas previous studies showed amoeboid microglia in the classically affected brain regions of PD and AD patients, little was known about such changes in the OB. Using a morphometric approach, a significant increase in amoeboid microglia density within the anterior olfactory nucleus (AON) of AD and PD patients was observed. These amoeboid microglia cells were in close apposition to β-amyloid, hyperphosphorylated tau or α-synuclein deposits, but no uptake of pathological proteins by microglia could be visualized. Subsequent analysis showed (i) no correlation between microglia and α-synuclein (PD), (ii) a positive correlation with β-amyloid (AD), and (iii) a negative correlation with hyperphosphorylated tau (AD). Furthermore, despite the observed pathological alterations in neurite morphology, neuronal loss was not apparent in the AON of both patient groups. Thus, we hypothesize that, in contrast to the classically affected brain regions of AD and PD patients, within the AON rather than neuronal loss, the increased density in amoeboid microglial cells, possibly in combination with neurite pathology, may contribute to functional deficits.

G. G. Kovacs, A. J. M. Rozemuller, J. C. van Swieten, E. Gelpi, K. Majtenyi, S. Al-Sarraj, C. Troakes, I. Bodi, A. King, T. Hortobagyi, M. M. Esiri, O. Ansorge, G. Giaccone, I. Ferrer, T. Arzberger, N. Bogdanovic, T. Nilsson, I. Leisser, I. Alafuzoff, J. W. Ironside, H. Kretzschmar and H. Budka (2013) Neuropathology and Applied Neurobiology39, 166–178 Neuropathology of the hippocampus in FTLD-Tau with Pick bodies: a study of the BrainNet Europe Consortium Aims: Frontotemporal lobar degeneration with Pick bodies (Pick's disease) is characterized by the presence of tau immunoreactive spherical structures in the cytoplasm of neurones. In view of confusion about the molecular pathology of Pick's disease, we aimed to evaluate the spectrum of tau pathology and concomitant neurodegeneration-associated protein depositions in the characteristically affected hippocampus. Methods: We evaluated immunoreactivity (IR) for tau (AT8, 3R, 4R), α-synuclein, TDP43, p62, and ubiquitin in the hippocampus, entorhinal and temporal cortex in 66 archival cases diagnosed neuropathologically as Pick's disease. Results: Mean age at death was 68.2 years (range 49–96). Fifty-two (79%) brains showed 3R immunoreactive spherical inclusions in the granule cells of the dentate gyrus. These typical cases presented mainly with the behavioural variant of frontotemporal dementia, followed by progressive aphasia, mixed syndromes or early memory disturbance. α-Synuclein IR was seen only in occasional spherical tau-positive inclusions, TDP-43 IR was absent, and 4R IR was present only as neurofibrillary tangles in pyramidal neurones. Aβ IR was observed in 16 cases; however, the overall level of Alzheimer's disease-related alterations was mainly low or intermediate (n = 3). Furthermore, we identified six cases with unclassifiable tauopathy. Conclusions: (i) Pick's disease may occur also in elderly patients and is characterized by a relatively uniform pathology with 3R tau inclusions particularly in the granule cells of dentate gyrus; (ii) even minor deviation from these morphological criteria suggests a different disorder; and (iii) immunohistological revision of archival cases expands the spectrum of tauopathies that require further classification.

The unfolded protein response (UPR) is a stress response activated upon disturbed homeostasis in the endoplasmic reticulum (ER). Previously, we reported that the activation of the UPR closely correlates with the presence of phosphorylated tau (p-tau) in Alzheimer's disease (AD). As well as increased presence of intracellular p-tau, AD brains are characterized by extracellular deposits of beta amyloid (A beta). Recent in vitro studies have shown that A beta can induce ER stress and activation of the UPR. The aim of the present study is to investigate UPR activation in sporadic tauopathies like progressive supranuclear palsy (PSP) and Pick's disease (PiD), and familial cases with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) which carry mutations in the gene encoding for tau (MAPT). The presence of phosphorylated pancreatic ER kinase (pPERK) and phosphorylated inositol requiring enzyme 1a (pIRE1), which are indicative of an activated UPR, was assessed by immunohistochemistry in cases neuropathologically defined as frontotemporal lobar degeneration with tau pathology (FTLD-tau). Increased presence of UPR activation markers pPERK and pIRE1 was observed in neurons and glia in FTLD-tau cases, in contrast to FTLD subtypes negative for tau pathology or in non-neurological controls. pPERK and pIRE1 were also prominently present in relatively young carriers of MAPT mutation. A strong association between the presence of UPR activation markers and p-tau was observed in the hippocampus of FTLD-tau cases. Double immunohistochemical staining on FTLD-tau cases revealed that UPR activation is predominantly observed in neurons that show diffuse staining of p-tau. These data demonstrate that UPR activation is intimately connected with the accumulation and aggregation of p-tau, and occurs independently from A beta deposits. Our findings provide new pathological insight into the close association between p-tau and UPR activation in tauopathies. Copyright (c) 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Item does not contain fulltext There is increasing evidence that frontotemporal dementia and amyotrophic lateral sclerosis are part of a disease continuum. Recently, a hexanucleotide repeat expansion in C9orf72 was identified as a major cause of both sporadic and familial frontotemporal dementia and amyotrophic lateral sclerosis. The aim of this study was to investigate clinical and neuropathological characteristics of hexanucleotide repeat expansions in C9orf72 in a large cohort of Dutch patients with frontotemporal dementia. Repeat expansions were successfully determined in a cohort of 353 patients with sporadic or familial frontotemporal dementia with or without amyotrophic lateral sclerosis, and 522 neurologically normal controls. Immunohistochemistry was performed in a series of 10 brains from patients carrying expanded repeats using a panel of antibodies. In addition, the presence of RNA containing GGGGCC repeats in paraffin-embedded sections of post-mortem brain tissue was investigated using fluorescence in situ hybridization with a locked nucleic acid probe targeting the GGGGCC repeat. Hexanucleotide repeat expansions in C9orf72 were found in 37 patients with familial (28.7%) and five with sporadic frontotemporal dementia (2.2%). The mean age at onset was 56.9 +/- 8.3 years (range 39-76), and disease duration 7.6 +/- 4.6 years (range 1-22). The clinical phenotype of these patients varied between the behavioural variant of frontotemporal dementia (n = 34) and primary progressive aphasia (n = 8), with concomitant amyotrophic lateral sclerosis in seven patients. Predominant temporal atrophy on neuroimaging was present in 13 of 32 patients. Pathological examination of the 10 brains from patients carrying expanded repeats revealed frontotemporal lobar degeneration with neuronal transactive response DNA binding protein-positive inclusions of variable type, size and morphology in all brains. Fluorescence in situ hybridization analysis of brain material from patients with the repeat expansion, a microtubule-associated protein tau or a progranulin mutation, and controls did not show RNA-positive inclusions specific for brains with the GGGGCC repeat expansion. The hexanucleotide repeat expansion in C9orf72 is an important cause of frontotemporal dementia with and without amyotrophic lateral sclerosis, and is sometimes associated with primary progressive aphasia. Neuropathological hallmarks include neuronal and glial inclusions, and dystrophic neurites containing transactive response DNA binding protein. Future studies are needed to explain the wide variation in clinical presentation. 01 maart 2012

Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and Pick's disease (PiD) are commonly known as tauopathies. Neurodegeneration observed in these diseases is linked to neuronal fibrillary hyperphosphorylated tau protein inclusions. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular cross-links. Both transglutaminase 1 (TG1) and transglutaminase 2 (TG2) are abundantly expressed in the brain and are associated with fibrillary hyperphosphorylated tau protein inclusions in neurons of AD, so-called neurofibrillary tangles (NFTs). However, other data obtained by our group suggested that tau pathology in the brain may be primarily related to TG1 and not to TG2 activity. To obtain more information on this issue, we set out to investigate the association of TG1, TG2, and TG-catalysed cross-links with fibrillary hyperphosphorylated tau inclusions in tauopathies other than AD, using immunohistochemistry. We found strong TG1 and TG-catalysed cross-link staining in neuronal tau inclusions characteristic of PSP, FTDP-17 with mutations in the tau gene (FTDP-17T), and PiD brain, whereas, in contrast to AD, TG2 was only rarely observed in these inclusions. Furthermore, using a biochemical approach, we demonstrated that tau is a substrate for TG1-mediated cross-linking. Interestingly, we found co-localization of the TG1 activator, tazarotene-induced gene 3 (TIG3), in the neuronal tau inclusions of PSP, FTDP-17T, and PiD, but not in NFTs of AD cases, indicating that these tau-containing protein aggregates are not identical. We conclude that TG1-catalysed cross-linking, regulated by TIG3, might play an important role in the formation of neuronal tau inclusions in PSP, FTDP-17T, and PiD brain. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pre-eclampsia and late-onset Alzheimer's disease (LOAD) share no clinical features. In contrast to these clinical dissimilarities, striking parallels exist between the (epi)genetic features associated with pre-eclampsia and LOAD for the genes located on 10q22. The parallels in identity between the 10q22 genes involved and active in the organs (placenta, brain) primarily affected in the respective diseases led us to explore, if the pre-eclampsia susceptibility gene STOX1 is functionally involved in LOAD. We demonstrate that isoform A of STOX1 is abundantly expressed in the brain, correlates with severity of disease, and selectively transactivates LRRTM3 in neural cells with increased amyloid-beta protein precursor processing. Similar in vitro results were seen in trophoblast. Our data indicate that STOX1 controls a conserved pathway shared between placenta and brain with overexpression in LOAD.

Nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration : an update

Accumulation of misfolded proteins in the endoplasmic reticulum triggers a cellular stress response called the unfolded protein response (UPR) that protects the cell against the toxic buildup of misfolded proteins. Previously, we reported that UPR activation is increased in Alzheimer's disease (AD) patients. How the UPR relates to the pathological hallmarks of AD is still elusive. In the present study, the involvement of UPR activation in neurofibrillary degeneration in AD was investigated. Immunoreactivity for the phosphorylated UPR activation markers pancreatic ER kinase (pPERK), eukaryotic initiation factor 2 alpha, and inositol-requiring enzyme la was observed in hippocampal neurons associated with granulovacuolar degeneration. The percentage of pPERK-immunoreactive neurons was increased in AD cases compared with nondemented control cases and with the Braak stage for neurofibrillary changes. Although absent from neurofibrillary tangles, pPERK immunoreactivity was most abundant in neurons with diffuse localization of phosphorylated tau protein. Additional analyses showed that pPERK immunoreactivity was associated with ubiquitin and the ubiquitin binding protein p62. A strong co-occurrence of immunoreactivity for both pPERK and glycogen synthase kinase 3 beta in neurons was also observed. Together, these data indicate that UPR activation in AD neurons occurs at an early stage of neurofibrillary degeneration and suggest that the prolonged activation of the UPR is involved in both tau phosphorylation and neurodegeneration in AD pathogenesis. (Am J Pathol 2009, 174:1241-1251; DOI: 10.2353/ajpath.2009.080814)

In a comprehensive proteomics study aiming at the identification of proteins associated with amyloid-beta (Abeta)-mediated toxicity in cultured cortical neurons, we have identified Thimet oligopeptidase (THOP1). Functional modulation of THOP1 levels in primary cortical neurons demonstrated that its overexpression was neuroprotective against Abeta toxicity, while RNAi knockdown made neurons more vulnerable to amyloid peptide. In the TgCRND8 transgenic mouse model of amyloid plaque deposition, an age-dependent increase of THOP1 expression was found in brain tissue, where it co-localized with Abeta plaques. In accordance with these findings, THOP1 expression was significantly increased in human AD brain tissue as compared to non-demented controls. These results provide compelling evidence for a neuroprotective role of THOP1 against toxic effects of Abeta in the early stages of AD pathology, and suggest that the observed increase in THOP1 expression might be part of a compensatory defense mechanism of the brain against an increased Abeta load.

Pathological accumulation of abnormally phosphorylated tau protein in astrocytes is a frequent, but poorly characterized feature of the aging brain. Its etiology is uncertain, but its presence is sufficiently ubiquitous to merit further characterization and classification, which may stimulate clinicopathological studies and research into its pathobiology. This paper aims to harmonize evaluation and nomenclature of aging-related tau astrogliopathy (ARTAG), a term that refers to a morphological spectrum of astroglial pathology detected by tau immunohistochemistry, especially with phosphorylation-dependent and 4R isoform-specific antibodies. ARTAG occurs mainly, but not exclusively, in individuals over 60 years of age. Tau-immunoreactive astrocytes in ARTAG include thorn-shaped astrocytes at the glia limitans and in white matter, as well as solitary or clustered astrocytes with perinuclear cytoplasmic tau immunoreactivity that extends into the astroglial processes as fine fibrillar or granular immunopositivity, typically in gray matter. Various forms of ARTAG may coexist in the same brain and might reflect different pathogenic processes. Based on morphology and anatomical distribution, ARTAG can be distinguished from primary tauopathies, but may be concurrent with primary tauopathies or other disorders. We recommend four steps for evaluation of ARTAG: (1) identification of five types based on the location of either morphologies of tau astrogliopathy: subpial, subependymal, perivascular, white matter, gray matter; (2) documentation of the regional involvement: medial temporal lobe, lobar (frontal, parietal, occipital, lateral temporal), subcortical, brainstem; (3) documentation of the severity of tau astrogliopathy; and (4) description of subregional involvement. Some types of ARTAG may underlie neurological symptoms; however, the clinical significance of ARTAG is currently uncertain and awaits further studies. The goal of this proposal is to raise awareness of astroglial tau pathology in the aged brain, facilitating communication among neuropathologists and researchers, and informing interpretation of clinical biomarkers and imaging studies that focus on tau-related indicators.

We examined the regulation of glutamate transporter protein expression after stimulation with selective metabotropic glutamate receptor (mGluR) agonists in cultured human glial cells. mGluR3 and mGluR5 are expressed in human astrocytes and in human glioma cells in vivo as well as in vitro, as shown by either RT-PCR or western blot analysis. The selective group I agonist (S)-3,5-dihydroxyphenylglycine produced a significant down-regulation of both GLAST and GLT-1 protein expression in astrocytes cultured in the presence of growth factors. This condition mimics the morphology of reactive glial cells in vivo including an increased expression of mGluR5 protein (observed in pathological conditions). In contrast, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine, a selective agonist of group II metabotropic glutamate receptors, positively modulates the expression of GLAST and GLT-1 proteins. A similar opposite effect of (S)-3,5-dihydroxyphenylglycine and (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine was observed for the expression of EAAT3 protein in U373 glioblastoma cell line. Selective group I and II antagonists prevented these effects. Pharmacological inhibition of mitogen-activated protein kinase and phosphatidylinositol-3-K pathways reduces the induction of GLT-1 observed in response to the group II metabotropic glutamate receptor agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine. Thus, mGluR3 and mGluR5 can critically and differentially modulate the expression of glutamate transporters and may represent interesting pharmacological targets to regulate the extracellular levels of glutamate in pathological conditions.

Epidemiological studies indicate that anti-inflammatory drugs, especially the non-steroidal anti-inflammatory drugs (NSAIDs), decrease the risk of developing Alzheimer's disease (AD). Their beneficial effects may be due to interference in the chronic inflammatory reaction, that takes place in AD. The best-characterized action of NSAIDs is the inhibition of cyclooxygenase (COX). There is special interest for anti-inflammatory treatment of AD using selective COX-2 inhibitors. These inhibitors reduce the inflammatory reaction but lack the side effects observed with non-selective NSAIDs. So far, clinical trials designed to inhibit inflammation or COX-2 activity have failed in the treatment of AD patients. Several lines of evidence can explain the failures of the anti-inflammatory and anti-COX-2 trials on AD patients. In this review we will focus on the role, expression and regulation of COX-1 and COX-2 in AD brain. Understanding the role of COX in AD pathogenesis could contribute to the development of an anti-inflammatory therapy for the treatment or prevention of AD.

Subjects with incidental Lewy body disease (iLBD) may represent the premotor stage of Parkinson's disease (PD). To elucidate molecular mechanisms underlying neuronal dysfunction and alpha-synuclein pathology in the premotor phase of PD, we investigated the transcriptome of the substantia nigra (SN) of well-characterized iLBD, PD donors and agematched controls with Braak alpha-synuclein stage ranging from 0-6. In Braak alpha-synuclein stages 1 and 2, we observed deregulation of pathways linked to axonal degeneration, immune response and endocytosis, including axonal guidance signaling, mTOR signaling, EIF2 signaling and clathrin-mediated endocytosis in the SN. In Braak stages 3 and 4, we observed deregulation of pathways involved in protein translation and cell survival, including mTOR and EIF2 signaling. In Braak stages 5 and 6, we observed deregulation of dopaminergic signaling, axonal guidance signaling and thrombin signaling. Throughout the progression of PD pathology, we observed a deregulation of mTOR, EIF2 and regulation of eIF4 and p70S6K signaling in the SN. Our results indicate that molecular mechanisms related to axonal dysfunction, endocytosis and immune response are an early event in PD pathology, whereas mTOR and EIF2 signaling are impaired throughout disease progression. These pathways may hold the key to altering the disease progression in PD. Copyright

Objective: To determine the clinical features and presence in CSF of antineuronal antibodies in patients with pathologically proven autoimmune encephalitis derived from a cohort of patients with suspected Creutzfeldt-Jakob disease (CJD). Methods: The Dutch Surveillance Centre for Prion Diseases performed 384 autopsies on patients with suspected CJD over a 14-year period (1998-2011). Clinical information was collected from treating physicians. Antineuronal antibodies were tested in CSF obtained postmortem by immunohistochemistry on fresh frozen rat brain sections, by Luminex assay for the presence of wellcharacterized onconeural antibodies, and by cell-based assays for antibodies against NMDAR, GABABR1/2, GABAAR GLUR1/2, LGI1, Caspr2, and DPPX. Results: In 203 patients, a diagnosis of definite CJD was made, while in 181 a variety of other conditions were diagnosed, mainly neurodegenerative. In 22 of these 181, the neuropathologist diagnosed autoimmune encephalitis. One patient was excluded because of lack of clinical information. Inflammatory infiltrates were predominantly perivascular and consisted mainly of T cells. The predominant locations were basal ganglia and thalamus (90%) and temporal lobes and hippocampus (81%). In 6 patients (29%), antineuronal antibodies were detected in postmortem CSF, directed against Hu, NMDAR, GABABR1/2, Caspr2, and an unidentified synaptic antigen in 2. The most frequent symptoms were dementia (90%), gait disturbance (86%), cerebellar signs (67%), and neuropsychiatric symptoms (67%). Immunopathologic and clinical findings did not differ between autoantibody-negative patients and patients with antineuronal antibodies. Conclusions: It is important to consider immune-mediated disorders in the differential diagnosis of rapidly progressive neurologic deficits.

Sir, We thank Pottier et al. , 2014 for their interest in our recent study, reporting the identification of a novel mutation in the PRKAR1B gene in a family with cases of FUS-negative neuronal intermediate filament inclusion disease (NIFID) …

which function in innate immunity also suggests that innate immunity may have a significant role in AD. Given that gliosis occurs at least decades earlier than the first appearance of clinical symptoms in AD, it is conceivable that glial dysfunction and mis-activation of the innate immune system drives AD type neurodegeneration.However, very few studies have directly addressed how inflammation per se drives Ab or tau pathology.To characterize the role of innate immunefactors on amyloid b (Ab) plaque and tau pathology, we overexpressed several mouse cytokines, chemokine receptors and soluble innate immune receptors in Amyloid precursor protein (APP) expressingTgCRND8 mice or tau transgenic mice. Methods: Using adeno associated virus (AAV2/1) mediated gene delivery in neonatal mice brain, we effectively demonstrate that this technique allows us to achieve high levels of transgene expression while 1) bypassing the need to create bigenic mice to test hypotheses in a rapid cost-effective manner, 2) testing different mediators on a uniform genetic background and 3) modeling cell non autonomous signaling in vivo. Results: We show that in TgCRND8 mice brain, expression of 1) inflammatory cytokines reduce Ab plaques; 2) anti-inflammatory cytokines exacerbate Ab pathology; and 3) modulating the innate immune receptors using soluble Toll-like receptors (sTLR4 and sTLR5) decrease Ab pathology. On the other hand, over-expression of inflammatory cytokines increase soluble phosphorylated tau while differentially affecting insoluble tau levels in tau transgenic mice. Conclusions: Understanding how innate immune activation states regulate tau and Abpathology could reveal novel therapeutic approaches for AD, including re-purposing current forms of immune therapy already tested in the clinic. Our ongoing studies show that manipulating innate immunity can be a double edged sword, and that development of any innate immune therapy for AD will need to be finely tuned and extensively validated in order to be truly effective. We demonstrate that though engagement of the innate immune system early on during disease pathogenesis may be beneficial in restricting the development of Ab plaques, this may potentially exacerbate tau pathology and negatively affect proteostasis. Therefore, our study strongly argues for a cautious re-examination of unwarranted side-effects of immune based therapies and calls for exploring the therapeutic potential of decoy receptors in preclinical mouse models ofAD as a means of restricting AD pathology while simultaneously minimizing bystander toxicity.

BRI2 protein binds amyloid precursor protein to halt amyloid-β production and inhibits amyloid-β aggregation via its BRICHOS-domain suggesting a link between BRI2 and Alzheimer's disease (AD). Here, we investigate the possible involvement of BRI2 in human AD pathogenesis. BRI2 containing BRICHOS-domain was increased up to 3-fold in AD hippocampus (p = 0.003, n = 14/group). Immunohistochemistry showed BRI2 deposits associated with amyloid-β plaques in early pathologic stages (Braak-III; Thal-2/3). We observed a decrease in the protein levels of ADAM10 (p = 0.02) and furin (p = 0.066), as well as an increase in SPPL2b (p < 0.0001) in AD hippocampus. Because these enzymes are involved in BRI2 processing, their changes may lead to aberrant processing of BRI2 promoting its deposition and likely affecting BRI2 function. Loss of BRI2 function in AD was supported by the decreased presence of BRI2-amyloid precursor protein complexes in the hippocampus of AD patients compared with control subjects. In conclusion, our data obtained from human samples indicate that in early stages of AD there is an increased deposition of BRI2, which likely leads to impaired BRI2 function thereby influencing AD pathophysiology.

Background Neuron Glial 2 (NG2) cells are glial cells known to serve as oligodendrocyte progenitors as well as modulators of the neuronal network. Altered NG2 cell morphology and up-regulation as well as increased shedding of the proteoglycan NG2 expressed on the cell surface have been described in rodent models of brain injury. Here we describe alterations in the human NG2 cell population in response to pathological changes characteristic of Alzheimer’s disease (AD). Results Immunohistological stainings of postmortem brain specimens from clinically diagnosed and postmortem verified AD patients and non-demented controls revealed reduced NG2 immunoreactivity as well as large numbers of NG2 positive astrocytes in individuals with high amyloid beta plaque load. Since fibrillar amyloid beta (Aβ)1-42 is the major component of AD-related senile plaques, we exposed human NG2 cells to oligomer- and fibril enriched preparations of Aβ1-42. We found that both oligomeric and fibrillar Aβ1-42 induced changes in NG2 cell morphology. Further, in vitro exposure to fibrillar Aβ1-42 decreased the NG2 concentrations in both cell lysates and supernatants. Interestingly, we also found significantly decreased levels of soluble NG2 in the cerebrospinal fluid (CSF) from clinically diagnosed AD patients compared to non-demented individuals. Additionally, the CSF NG2 levels were found to significantly correlate with the core AD biomarkers Aß1-42, T-tau and P-tau. Conclusion Our results demonstrate major alterations in the NG2 cell population in relation to AD pathology which highlights the NG2 cell population as a new attractive research target in the search for cellular mechanisms associated with AD pathogenesis.

Amyloid β-peptide (Aβ) is a key molecule in Alzheimer's disease (AD). Reliable immunohistochemical (IHC) methods to detect Aβ and Aβ-associated factors (AAF) in brain specimens are needed to determine their role in AD pathophysiology. Formic acid (FA) pre-treatment, which is generally used to enable efficient detection of Aβ with IHC, induces structural modifications within the Aβ, as well as in AAF. Consequently, interpretation of double IHC stainings becomes difficult. Therefore, serial stainings of two newly produced monoclonal antibodies (mAbs) VU-17 and IC16 and two other mAbs (6E10 and 3D6) were performed with four different pre-treatments (no pre-treatment, Tris/EDTA, citrate and FA) and additionally six IHC characteristics were scored: diffuse/compact/classic plaques, arteries with cerebral Aβ angiopathy, dyshoric angiopathy, capillaries with dyshoric angiopathy. Subsequently, these stainings were compared with IHC procedures, which are frequently used in a diagnostic setting, employing mAbs 4G8 and 6F/3D with FA pre-treatment. IHC Aβ patterns obtained with VU-17 and, IC16 and 3D6 without the use of FA pre-treatment were comparable to those obtained with 4G8 and 6F/3D upon FA pre-treatment. Omission of FA pre-treatment gives the advantage to allow double IHC stainings, detecting both Aβ and AAF that otherwise would have been structural modificated upon FA pre-treatment.

Cerebral deposits of amyloid-β peptides (Aβ) form the neuropathological hallmarks of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). In the brain, Aβ can aggregate as insoluble fibrils present in amyloid plaques and vascular amyloid, or as diffuse plaques consisting of mainly non-fibrillar Aβ. Previously, magnetic resonance imaging (MRI) has been shown to be capable of detecting individual amyloid plaques, not only via the associated iron, but also Aβ itself has been suggested to be responsible for a decrease in the image intensity. In this current study we aim to investigate the MRI properties of the different cerebral Aβ deposits including diffuse plaques and vascular amyloid. Postmortem 60-μm-thick brain sections of AD, CAA, and Down's syndrome patients, known to contain Aβ, were studied. High resolution T2*- and T2-weighted MRI scans and quantitative relaxation maps were acquired using a microcoil on a Bruker 9.4T MRI system. Specific MRI characteristics of each type of Aβ deposit were examined by co-registration of the MRI with Congo Red and Aβ-immunostainings of the same sections. Our results show that only fibrillar Aβ, present in both vascular and parenchymal amyloid, induced a significant change in T2* and T2 values. However, signal changes were not as consistent for all of the vessels affected by CAA, irrespective of possible dyshoric changes. In contrast, the non-fibrillar diffuse plaques did not create any detectable MRI signal changes. These findings are relevant for the interpretation and further development of (quantitative) MRI methods for the detection and follow-up of AD and CAA.

Aβ is the main component of amyloid deposits in Alzheimer disease (AD) and its aggregation into oligomers, protofibrils and fibrils is considered a seminal event in the pathogenesis of AD. Aβ with C-terminus at residue 42 is the most abundant species in parenchymal deposits, whereas Aβ with C-terminus at residue 40 predominates in the amyloid of the walls of large vessels. Aβ peptides with other C-termini have not yet been thoroughly investigated. We analysed Aβ38 in the brains of patients with Aβ deposition linked to sporadic and familial AD, hereditary cerebral haemorrhage with amyloidosis, or Down syndrome. Immunohistochemistry, confocal microscopy, immunoelectron microscopy, immunoprecipitation and the electrophoresis separation of low molecular weight aggregates revealed that Aβ38 accumulates consistently in the brains of patients carrying APP mutations in the Aβ coding region, but was not detected in the patients with APP mutations outside the Aβ domain, in the patients with presenilin mutations or in subjects with Down syndrome. In the patients with sporadic AD, Aβ38 was absent in the senile plaques, but it was detected only in the vessel walls of a small subset of patients with severe cerebral amyloid angiopathy. Our results suggest that APP mutations in the Aβ coding region favour Aβ38 accumulation in the brain and that the molecular mechanisms of Aβ deposition in these patients may be different from those active in patients with familial AD associated with other genetic defects and sporadic AD.

Background: In cases with a long (>1 year) clinical duration of prion disease, the prion protein can form amyloid deposits. These cases do not show accumulation of 4-kDa β-amyloid, which is observed in amyloid deposits in Alzheimer’s disease (AD). In AD, amyloid is associated with inflammation and neurofibrillary degeneration, and it is elusive whether prion amyloid is associated with these changes as well. Objectives: The presence of inflammation and neurofibrillary degeneration was evaluated in prion amyloidosis. Material and Methods: Cortical areas of variant Creutzfeldt-Jakob disease (CJD; n = 3), young sporadic CJD (n = 4), different Gerstmann-Sträussler-Scheinker’s disease patients (n = 5) and AD cases (n = 5) were examined using immunohistochemistry and specific stainings for amyloid. Results: In both AD and prion disease cases, which were negative for 4-kDa β-amyloid, parenchymal and vascular amyloid deposits were positive for amyloid-associated proteins such as complement protein and were associated with microglia clusters. Tau and ubiquitin were found near prion plaques in some of the Gerstmann-Sträussler-Scheinker’s disease and sporadic CJD cases and also near vascular prion amyloid deposits. In variant CJD cases, occasionally, microglia clustering was found in plaques but no ubiquitin or complement proteins and hardly tau protein. Conclusions: In both AD and prion disease amyloid formation, irrespective of the protein involved, there seems to be a neuroinflammatory response with secondary neurofibrillary degeneration.

Background: Neuropathological studies supported by experimental animal studies show that the constituents of the innate immunity are intimately involved in the early steps of the pathological cascade of Alzheimer’s disease (AD). Objectives: To show the evidence that constituents of the innate immunity contribute to the etiology of late-onset AD. Methods: Evaluation of the relationship between the constituents of the innate immunity and genetic risk factors for late-onset AD. Results: Complement activation and activated microglia are early neuropathogical events in AD brains. Genome-wide association studies have demonstrated gene loci that are linked to the complement system. The production capacity for inflammatory cytokines is under genetic control and the offspring with a parental history of late-onset AD have a higher production capacity for inflammatory cytokines. Conclusion: Epidemiological and genetic data suggest that the innate immunity is involved in the etiology of late-onset AD.