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Despite many reported associations, the direct cause of neurodegeneration responsible for cognitive loss in Alzheimer’s disease and some other common dementias is not known. The normal human plasma protein, serum amyloid P component, a constituent of all human fibrillar amyloid deposits and present on most neurofibrillary tangles, is cytotoxic for cerebral neurones in vitro and in experimental animals in vivo. The neocortical content of serum amyloid P component was immunoassayed in 157 subjects aged 65 or more with known dementia status at death, in the large scale, population-representative, brain donor cohort of the Cognitive Function and Ageing Study, which avoids the biases inherent in studies of predefined clinico-pathological groups. The serum amyloid P component values were significantly higher in individuals with dementia, independent of serum albumin content measured as a control for plasma in the cortex samples. The odds ratio for dementia at death in the high serum amyloid P component tertile was 5.24 (95% confidence interval 1.79–15.29) and was independent of Braak tangle stages and Thal amyloid-β phases of neuropathological severity. The strong and specific association of higher brain content of serum amyloid P component with dementia, independent of neuropathology, is consistent with a pathogenetic role in dementia.

Type 2 diabetes mellitus (T2D), characterised by peripheral insulin resistance, is a risk factor for dementia. In addition to its contribution to small and large vessel disease, T2D may directly damage cells of the brain neurovascular unit. In this study, we investigated the transcriptomic changes in cortical neurones, and associated astrocytes and endothelial cells of the neurovascular unit, in the ageing brain. Neurone, astrocyte, and endothelial cell-enriched mRNA, obtained by immuno-laser capture microdissection of temporal cortex (Brodmann area 21/22) from 6 cases with self-reported T2D in the Cognitive Function and Ageing Study neuropathology cohort, and an equal number of age and sex-matched controls, was assessed by microarray analysis. Integrated Molecular Pathway Level Analysis was performed using the Kyoto Encyclopaedia of Genes and Genomes database on significantly differentially expressed genes, defined as P

Diabetes mellitus is a risk factor for dementia, and nonenzymatic glycosylation of macromolecules results in formation of advanced glycation end-products (AGEs). We determined the variation in AGE formation in brains from the Cognitive Function and Ageing Study population-representative neuropathology cohort. AGEs were measured on temporal neocortex by enzyme-linked immunosorbent assay (ELISA) and cell-type specific expression on neurons, astrocytes and endothelium was detected by immunohistochemistry and assessed semiquantitatively. Fifteen percent of the cohort had self-reported diabetes, which was not significantly associated with dementia status at death or neuropathology measures. AGEs were expressed on neurons, astrocytes and endothelium and overall expression showed a positively skewed distribution in the population. AGE measures were not significantly associated with dementia. AGE measured by ELISA increased with Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) neurofibrillary tangle score (p = 0.03) and Thal Aβ phase (p = 0.04), while AGE expression on neurons (and astrocytes), detected immunohistochemically, increased with increasing Braak tangle stage (p

AIMS: Cellular senescence plays a role in organismal ageing and has been linked to persistent DNA damage in age-related diseases. Brain senescence has been described in astrocytes and microglia but is less well understood in neurones. Evidence suggests that neurones activate a senescence-like mechanism that could contribute to neurodegeneration. We aimed to determine whether a persistent DNA damage response (DDR) and senescence activation are features of Motor Neurone Disease (amyotrophic lateral sclerosis, ALS/MND). METHODS: We examined expression of senescence (p16 and p21) and DNA damage markers (8-OHdG and γH2AX) in motor cortex (MCx), frontal association cortex (FACx) and occipital cortex (OCx) in post-mortem tissue donated by patients with ALS/MND and controls. RESULTS: Nuclear expression of p16 and p21 was detected in glial cells; double immunofluorescence for p16/p21 and GFAP suggested that some of these cells were GFAP+ astrocytes. p21 nuclear expression was also found in neurones. Higher levels of p16+ (glia, p= 0.028) and p21+ (glia, p=0.003; neurones, p=0.008) cells were found in the FACx of ALS/MND donors but not in the MCx or OCx. Expression of p16 and p21 did not correlate with 8-OHdG or γH2AX. CONCLUSIONS: Expression of p16 and p21 in glia, mainly in astrocytes, suggests senescence induction in these cells; however, neuronal p21 expression might reflect a more general mechanism of age-related cell cycle dysregulation. The significantly higher proportion of cells expressing either p16 or p21 in the FACx of ALS/MND donors could indicate senescence activation and cell cycle dysregulation in early stages of the disease.

Aβ-amyloid deposition is a key feature of Alzheimer’s disease, but Consortium to Establish a Registry for Alzheimer's Disease (CERAD) assessment, based on neuritic plaque density, shows a limited relationships to dementia. Thal phase is based on a neuroanatomical hierarchy of Aβ-deposition, and in combination with Braak neurofibrillary tangle staging also allows derivation of primary age-related tauopathy (PART). We sought to determine whether Thal Aβ phase predicts dementia better than CERAD in a population-representative cohort (n = 186) derived from the Cognitive Function and Ageing Study (CFAS). Cerebral amyloid angiopathy (CAA) was quantitied as the number of neuroanatomical areas involved and cases meeting criteria for PART were defined to determine if they are a distinct pathological group within the ageing population. Agreement with the Thal scheme was excellent. In univariate analysis Thal phase performed less well as a predictor of dementia than CERAD, Braak or CAA. Logistic regression, decision tree and linear discriminant analysis were performed for multivariable analysis, with similar results. Thal phase did not provide a better explanation of dementia than CERAD, and there was no additional benefit to including more than one assessment of Aβ in the model. Number of areas involved by CAA was highly correlated with assessment based on a severity score (p < 0.001). The presence of capillary involvement (CAA type I) was associated with higher Thal phase and Braak stage (p < 0.001). CAA was not associated with microinfarcts (p = 0.1). Cases satisfying pathological criteria for PART were present at a frequency of 10.2% but were not older and did not have a higher likelihood of dementia than a comparison group of individuals with similar Braak stage but with more Aβ. They also did not have higher hippocampal-tau stage, although PART was weakly associated with increased presence of thorn-shaped astrocytes (p = 0.048), suggesting common age-related mechanisms. Thal phase is highly applicable in a population-representative setting and allows definition of pathological subgroups, such as PART. Thal phase, plaque density, and extent and type of CAA measure different aspects of Aβ pathology, but addition of more than one Aβ measure does not improve dementia prediction, probably because these variables are highly correlated. Machine learning predictions reveal the importance of combining neuropathological measurements for the assessment of dementia.

The blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC) that are tightly linked by tight junction (TJ) proteins, restricts the movement of molecules between the periphery and the central nervous system. Elevated systemic levels of neutrophils have been detected in patients with altered BBB function, but the role of neutrophils in BMEC dysfunction is unknown. Neutrophils are key players of the immune response and, when activated, produce neutrophil-derived microvesicles (NMV). NMV have been shown to impact the integrity of endothelial cells throughout the body and we hypothesize that NMV released from circulating neutrophils interact with BMEC and induce endothelial cell dysfunction. Therefore, the current study investigated the interaction of NMV with human BMEC and determined whether they altered gene expression and function in vitro. Using flow cytometry and confocal imaging, NMV were shown to be internalized by the human cerebral microvascular endothelial cell line hCMEC/D3 via a variety of energy-dependent mechanisms, including endocytosis and macropinocytosis. The internalization of NMV significantly altered the transcriptomic profile of hCMEC/D3, specifically inducing the dysregulation of genes associated with TJ, ubiquitin-mediated proteolysis and vesicular transport. Functional studies confirmed NMV significantly increased permeability and decreased the transendothelial electrical resistance (TEER) of a confluent monolayer of hCMEC/D3. These findings indicate that NMV interact with and affect gene expression of BMEC as well as impacting their integrity. We conclude that NMV may play an important role in modulating the permeability of BBB during an infection.

Hanna Jokinen tutkimusryhmän jäsenenä (VICCCS Grp) Introduction: Progress in understanding and management of vascular cognitive impairment (VCI) has been hampered by lack of consensus on diagnosis, reflecting the use of multiple different assessment protocols. A large multinational group of clinicians and researchers participated in a two-phase Vascular Impairment of Cognition Classification Consensus Study (VICCCS) to agree on principles (VICCCS-1) and protocols (VICCCS-2) for diagnosis of VCI. We present VICCCS-2. Methods: We used VICCCS-1 principles and published diagnostic guidelines as points of reference for an online Delphi survey aimed at achieving consensus on clinical diagnosis of VCI. Results: Six survey rounds comprising 65-79 participants agreed guidelines for diagnosis of VICCCS-revised mild and major forms of VCI and endorsed the National Institute of Neurological Disorders-Canadian Stroke Network neuropsychological assessment protocols and recommendations for imaging. Discussion: The VICCCS-2 suggests standardized use of the National Institute of Neurological Disorders-Canadian Stroke Network recommendations on neuropsychological and imaging assessment for diagnosis of VCI so as to promote research collaboration. (C) 2017 the Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

Mutation in the triggering receptor expressed on myeloid cells (TREM) 2 gene has been identified as a risk factor for several neurodegenerative diseases including Alzheimer’s disease (AD). Experimental studies using animal models of AD have highlighted a number of functions associated with TREM2 and its expression by microglial cells. It has therefore been assumed that this is also the case in humans. However, there is very limited information concerning the cellular expression of TREM2 in the human brain. As part of investigations of microglia using post-mortem resources provided by the Medical Research Council Cognitive Function and Ageing Studies (MRC-CFAS), we immunostained the cerebral cortex of 299 participants for TREM2 using the Sigma antibody HPA010917 and compared with the macrophage/microglial markers Iba1 and CD68. As expected, Iba1 and CD68 labelled microglia and perivascular macrophages. However, in most cases (284/299), the TREM2 antibody labelled monocytes within vascular lumens, but not microglia or perivascular macrophages. In contrast, in 5 out of 6 cases with acute infarcts, TREM2 immunoreaction identified cells within the brain parenchyma interpreted as recruited monocytes. Six cases with old infarcts contained phagocytic foamy macrophages which were CD68-positive but TREM2 negative. Our observations, using the HPA010917 anti-TREM2 antibody, suggest that TREM2 is not expressed by microglia but instead seems to be a marker of recruited monocytes in the human brain. This finding has implications with regards to the role of TREM2 as a risk factor, emphasizing the importance of systemic immune responses in the development and progression of Alzheimer’s disease.

AIMS: Amyotrophic lateral sclerosis / motor neurone disease (ALS/MND) is characterised by the presence of inclusions containing TDP-43 within motor neurones. In rare cases, ALS/MND may be associated with inclusions containing other proteins, such as fused in sarcoma (FUS), whilst motor system pathology may rarely be a feature of other neurodegenerative disorders. We here have investigated the association of FUS and tau pathology. METHODS: We report a case with an ALS/MND-plus clinical syndrome which pathologically demonstrated both FUS pathology and an atypical tauopathy. RESULTS: Clinical motor involvement was predominantly upper motor neurone, and was accompanied by extrapyramidal features and sensory involvement, but with only minimal cognitive impairment. The presentation was sporadic and gene mutation screening was negative. Post-mortem study demonstrated inclusions positive for FUS, including basophilic inclusion bodies. This was associated with 4R-tauopathy, largely as non-fibrillary diffuse phospho-tau in neurones, with granulovacuolar degeneration in a more restricted distribution. Double-staining revealed that neurones contained both types of protein pathology. CONCLUSION: FUS-positive basophilic inclusion body disease is a rare cause of ALS/MND, but in this case was associated with an unusual atypical tauopathy. The coexistence of two such rare neuropathologies raises the question of a pathogenic interaction.

The insulin/insulin-like growth factor 1 (IGF1) signalling pathways are implicated in longevity and in progression of Alzheimer's disease. Previously, we showed that insulin-like growth factor 1 receptor (IGF1R) and downstream signalling transcripts are reduced in astrocytes in human brain with progression of Alzheimer's neuropathology and developed a model of IGF1 signalling impairment in human astrocytes using an IGF1R-specific monoclonal antibody, MAB391. Here, we have established a novel human astrocyte-neuron co-culture system to determine whether loss of astrocytic IGF1R affects their support for neurons. Astrocyte-neuron co-cultures were developed using human primary astrocytes and differentiated Lund Human Mesencephalic Cells (LUHMES). Neurite outgrowth assays, performed to measure astrocytic support for neurons, showed astrocytes provided contact-mediated support for neurite outgrowth. Loss of IGF1R did not affect neurite outgrowth under control conditions but when challenged with hydrogen peroxide IGF1R-impaired astrocytes were less able to protect LUHMES. To determine how loss of IGF1R affects neuronal support MAB391-treated astrocytes were FACS sorted from GFP-LUHMES and their transcriptomic profile was investigated using microarrays. Changes in transcripts involved in astrocyte energy metabolism were identified, particularly NDUFA2 and NDUFB6, which are related to complex I assembly. Loss of complex I activity in MAB391-treated astrocytes validated these findings. In conclusion, reduced IGF1 signalling in astrocytes impairs their support for neurons under conditions of stress and this is associated with defects in the mitochondrial respiratory chain in astrocytes.

INTRODUCTION: Microinfarcts, small ischaemic foci common in ageing brain, are associated with dementia and gait dysfunction. We determined their relationship to dementia, mobility and cerebrovascular disease in an older population-representative brain donor cohort. These data on microinfarcts were evaluated in relation to pathological assessments of clinically significant cerebral small vessel disease (SVD). METHODS: Microinfarcts were assessed in the MRC Cognitive Function and Ageing Study (n=331). Nine brain areas were staged according to the number of areas affected. RESULTS: 36% of brains showed at least 1 microinfarct. Higher cortical microinfarct stage was associated with dementia at death (OR 1.41, 95%CI 1.02; 1.96, p=0.038), whilst cortical and subcortical microinfarct stages were associated with impaired mobility (OR 1.36, 95%CI 1.05 - 1.74; p 0.018) and falls (OR 1.96, 95%CI 1.11 - 3.43; p= 0.02). Adding data on microinfarcts to a definition of SVD, based on white matter lesions, lacunes and significant arteriosclerosis, was assessed by comparing area under ROC curve (AUC) with and without microinfarcts. SVD was significantly related to dementia status with or without inclusion of microinfarcts. Modelling potential pathological definitions of SVD to predict dementia or impaired mobility indicated optimal prediction using combined assessment of white matter lesions, lacunes and microinfarcts. CONCLUSION: Cortical (dementia) and subcortical microinfarcts (impaired mobility) are related to diverse clinical outcomes. Optimal pathological assessment of significant SVD in brain ageing is achieved based on white matter lesions, lacunes and microinfarcts and may not require subjective assessment of the extent and severity of arteriosclerosis. This article is protected by copyright. All rights reserved.

Occludin is a component of tight junctions, which are essential structural components of the blood brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N- and C-terminal occludin was used to pull down proteins from 1321N1 cell lysates and protein binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell-types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states. This article is protected by copyright. All rights reserved.

Amyotrophic lateral sclerosis (ALS) is underpinned by an oligogenic rare variant architecture. Identified genetic variants of ALS include RNA-binding proteins containing prion-like domains (PrLDs). We hypothesized that screening genes encoding additional similar proteins will yield novel genetic causes of ALS. The most common genetic variant of ALS patients is a G4C2-repeat expansion within C9ORF72. We have shown that G4C2-repeat RNA sequesters RNA-binding proteins. A logical consequence of this is that loss-of-function mutations in G4C2-binding partners might contribute to ALS pathogenesis independently of and/or synergistically with C9ORF72 expansions. Targeted sequencing of genomic DNA encoding either RNA-binding proteins or known ALS genes (n = 274 genes) was performed in ALS patients to identify rare deleterious genetic variants and explore genotype-phenotype relationships. Genomic DNA was extracted from 103 ALS patients including 42 familial ALS patients and 61 young-onset (average age of onset 41 years) sporadic ALS patients; patients were chosen to maximize the probability of identifying genetic causes of ALS. Thirteen patients carried a G4C2-repeat expansion of C9ORF72. We identified 42 patients with rare deleterious variants; 6 patients carried more than one variant. Twelve mutations were discovered in known ALS genes which served as a validation of our strategy. Rare deleterious variants in RNA-binding proteins were significantly enriched in ALS patients compared to control frequencies (p = 5.31E-18). Nineteen patients featured at least one variant in a RNA-binding protein containing a PrLD. The number of variants per patient correlated with rate of disease progression (t-test, p = 0.033). We identified eighteen patients with a single variant in a G4C2-repeat binding protein. Patients with a G4C2-binding protein variant in combination with a C9ORF72 expansion had a significantly faster disease course (t-test, p = 0.025). Our data are consistent with an oligogenic model of ALS. We provide evidence for a number of entirely novel genetic variants of ALS caused by mutations in RNA-binding proteins. Moreover we show that these mutations act synergistically with each other and with C9ORF72 expansions to modify the clinical phenotype of ALS. A key finding is that this synergy is present only between functionally interacting variants. This work has significant implications for ALS therapy development.

Astrocytes have essential roles in the central nervous system and are also implicated in the pathogenesis of neurodegenerative disease. Forming non-overlapping domains, astrocytes are highly complex cells. Immunohistochemistry to a variety of proteins can be used to study astrocytes in tissue, labelling different cellular components and subpopulations, including GFAP, ALDH1L1, CD44, NDRG2 and amino acid transporters, but none of these label the entire astrocyte population. Increasing heterogeneity is recognised in the astrocyte population, a complexity that is relevant both to their normal function and pathogenic roles. They are involved in neuronal support, as active components of the tripartite synapse and in cell interactions within the neurovascular unit, where they are essential for blood brain barrier maintenance and neurovascular coupling. Astrocytes change with age, and their responses may modulate the cellular effects of neurodegenerative pathologies, which alone do not explain all of the variance in statistical models of neurodegenerative dementias. Astrocytes respond to both the neurofibrillary tangles and plaques of Alzheimer’s disease, to hyperphosphorylated tau and Aβ, eliciting an effect which may be neuroprotective or deleterious. Astrocyte hypertrophy, in the form of gliosis, occurs, but also astrocyte injury and atrophy. Loss of normal astrocyte functions may contribute to reduced support for neurons and dysfunction of the neurovascular unit. Understanding how astrocytes contribute to dementia requires an understanding of the underlying heterogeneity of astrocyte populations, and the complexity of their responses to pathology. Enhancing the supportive and neuroprotective components of the astrocyte response has potential translational applications in therapeutic approaches to dementia.

Importance Delirium is associated with accelerated cognitive decline. The pathologic substrates of this association are not yet known, that is, whether they are the same as those associated with dementia, are independent, or are interrelated.\ud \ud Objective To examine whether the accelerated cognitive decline observed after delirium is independent of the pathologic processes of classic dementia.\ud \ud Design, Setting, and Participants Harmonized data from 987 individual brain donors from 3 observational cohort studies with population-based sampling (Vantaa 85+, Cambridge City Over-75s Cohort, Cognitive Function and Ageing Study) performed from January 1, 1985, through December 31, 2011, with a median follow-up of 5.2 years until death, were used in this study. Neuropathologic assessments were performed with investigators masked to clinical data. Data analysis was performed from January 1, 2012, through December 31, 2013. Clinical characteristics of brain donors were not different from the rest of the cohort. Outcome ascertainment was complete given that the participants were brain donors.\ud \ud Exposures Delirium (never vs ever) and pathologic burden of neurofibrillary tangles, amyloid plaques, vascular lesions, and Lewy bodies. Effects modeled using random-effects linear regression and interactions between delirium and pathologic burden were assessed.\ud \ud Outcomes Change in Mini-Mental State Examination (MMSE) scores during the 6 years before death.\ud \ud Results There were 987 participants (290 from Vantaa 85+, 241 from the Cambridge City Over-75s Cohort, and 456 from the Cognitive Function and Ageing Study) with neuropathologic data; mean (SD) age at death was 90 (6.4) years, including 682 women (69%). The mean MMSE score 6 years before death was 24.7 points. The 279 individuals with delirium (75% women) had worse initial scores (−2.8 points; 95% CI, −4.5 to −1.0; P

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). White matter lesions in MS are surrounded by areas of non-demyelinated normal appearing white matter (NAWM) with complex pathology, including blood brain barrier dysfunction, axonal damage and glial activation. Astrocytes, the most abundant cell type within the CNS, may respond and/or contribute to lesion pathogenesis. We aimed to characterise the transcriptomic profile of astrocytes in NAWM to determine whether specific glial changes exist in the NAWM which contribute to lesion development or prevent disease progression.\ud \ud Astrocytes were isolated from control and NAWM by laser capture microdissection (LCM), using glial fibrillary acidic protein (GFAP) as a marker, and the astrocyte transcriptome determined using microarray analysis. 452 genes were significantly differentially expressed (208 up-regulated and 244 down-regulated, FC ≥ 1.5 and p-value ≤ 0.05). Within the NAWM, astrocytes were associated with significant upregulation of genes involved in the control of iron homeostasis (including metallothionein-1 and -2, ferritin light chain and transferrin), oxidative stress responses, the immune response and neurotrophic support. These findings suggest a neuroprotective role of astrocytes in the NAWM in MS.

Astrocytes have essential roles in the central nervous system and are also implicated in the pathogenesis of neurodegenerative disease. Forming non-overlapping domains, astrocytes are highly complex cells. Immunohistochemistry to a variety of proteins can be used to study astrocytes in tissue, labelling different cellular components and subpopulations, including GFAP, ALDH1L1, CD44, NDRG2 and amino acid transporters, but none of these label the entire astrocyte population. Increasing heterogeneity is recognised in the astrocyte population, a complexity that is relevant both to their normal function and pathogenic roles. They are involved in neuronal support, as active components of the tripartite synapse and in cell interactions within the neurovascular unit, where they are essential for blood brain barrier maintenance and neurovascular coupling. Astrocytes change with age, and their responses may modulate the cellular effects of neurodegenerative pathologies, which alone do not explain all of the variance in statistical models of neurodegenerative dementias. Astrocytes respond to both the neurofibrillary tangles and plaques of Alzheimer's disease, to hyperphosphorylated tau and Aβ, eliciting an effect which may be neuroprotective or deleterious. Astrocyte hypertrophy, in the form of gliosis, occurs, but also astrocyte injury and atrophy. Loss of normal astrocyte functions may contribute to reduced support for neurons and dysfunction of the neurovascular unit. Understanding how astrocytes contribute to dementia requires an understanding of the underlying heterogeneity of astrocyte populations, and the complexity of their responses to pathology. Enhancing the supportive and neuroprotective components of the astrocyte response has potential translational applications in therapeutic approaches to dementia. This article is protected by copyright. All rights reserved.

Epidemiological and genetic studies have identified metabolic disorders and inflammation as risk factors for Alzheimer's disease (AD). Evidence in obesity and type-2 diabetes suggests a role for a metabolic inflammasome (“metaflammasome”) in mediating chronic inflammation in peripheral organs implicating IKKβ (inhibitor of nuclear factor kappa-B kinase subunit beta), IRS1 (insulin receptor substrate 1), JNK (c-jun N-terminal kinase), and PKR (double-stranded RNA protein kinase). We hypothesized that these proteins are expressed in the brain in response to metabolic risk factors in AD. Neocortex from 299 participants from the MRC Cognitive Function and Ageing Studies was analysed by immunohistochemistry for the expression of the phosphorylated (active) form of IKKβ [pSer176/180], IRS1 [pS312], JNK [pThr183/Tyr185] and PKR [pT451]. The data were analyzed to investigate whether the proteins were expressed together and in relation with metabolic disorders, dementia, Alzheimer's pathology and APOE genotype. We observed a change from a positive to a negative association between the proteins and hypertension according to the dementia status. Type-2 diabetes was negatively related with the proteins among participants without dementia; whereas participants with dementia and AD pathology showed a positive association with JNK. A significant association between IKKβ and JNK in participants with dementia and AD pathology was observed, but not in those without dementia. Otherwise, weak to moderate associations were observed among the protein loads. The presence of dementia was significantly associated with JNK and negatively associated with IKKβ and IRS1. Cognitive scores showed a significant positive relationship with IKKβ and a negative with IRS1, JNK and PKR. The proteins were significantly associated with pathology in Alzheimer's participants with the relationship being inverse or not significant in participants without dementia. Expression of the proteins was not related to APOE genotype. These findings highlight a role for these proteins in AD pathophysiology but not necessarily as a complex.

BACKGROUND:\ud Laser capture microdissection (LCM) is an established technique for the procurement of enriched cell populations that can undergo further downstream analysis, although it does have limitations. Expression microdissection (xMD) is a new technique that begins to address these pitfalls, such as operator dependence and contamination.\ud \ud NEW METHOD:\ud xMD utilises immunohistochemistry in conjunction with a chromogen to isolate specific cell types by extending the fundamental principles of LCM to create an operator-independent method for the procurement of specific CNS cell types.\ud \ud RESULTS:\ud We report how xMD enables the isolation of specific cell populations, namely neurones and astrocytes, from rat formalin fixed-paraffin embedded (FFPE) tissue. Subsequent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirms the enrichment of these specific populations. RIN values after xMD indicate samples are sufficient to carry out further analysis.\ud \ud COMPARISON WITH EXISTING METHOD:\ud xMD offers a rapid method of isolating specific CNS cell types without the need for identification by an operator, reducing the amount of unintentional contamination caused by operator error, whilst also significantly reducing the time required by the current basic LCM technique.\ud \ud CONCLUSIONS:\ud xMD is a superior method for the procurement of enriched cell populations from post-mortem tissue, which can be utilised to create transcriptome profiles, aiding our understanding of the contribution of these cells to a range of neurological diseases. xMD also addresses the issues associated with LCM, such as reliance on an operator to identify target cells, which can cause contamination, as well as addressing the time consuming nature of LCM.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neurone loss resulting in muscle weakness, spasticity and ultimately death. 5-10% are caused by inherited mutations, most commonly C9ORF72, SOD1, TARDBP and FUS. Rarer genetic causes of ALS include mutation of optineurin (mt OPTN). Furthermore, optineurin protein has been localized to the ubiquitylated aggregates in several neurodegenerative diseases, including ALS. This study: (i) investigated the frequency of mt OPTN in ALS patients in England; (ii) characterized the clinical and neuropathological features of ALS associated with a mt OPTN; and (iii) investigated optineurin neuropathology in C9ORF72-related ALS (C9ORF72-ALS). We identified a heterozygous p.E322K missense mutation in exon 10 of OPTN in one familial ALS patient who additionally had a C9ORF72 mutation. This patient had bulbar, limb and respiratory disease without cognitive problems. Neuropathology revealed motor neurone loss, trans-activation response DNA protein 43 (TDP-43)-positive neuronal and glial cytoplasmic inclusions together with TDP-43-negative neuronal cytoplasmic inclusions in extra motor regions that are characteristic of C9ORF72-ALS. We have demonstrated that both TDP-43-positive and negative inclusion types had positive staining for optineurin by immunohistochemistry. We went on to show that optineurin was present in TDP-43-negative cytoplasmic extra motor inclusions in C9ORF72-ALS cases that do not carry mt OPTN. We conclude that: (i) OPTN mutations are associated with ALS; (ii) optineurin protein is present in a subset of the extramotor inclusions of C9ORF72-ALS; (iii) It is not uncommon for multiple ALS-causing mutations to occur in the same patient; and (iv) studies of optineurin are likely to provide useful dataregarding the pathophysiology of ALS and neurodegeneration.

INTRODUCTION: Deposition of abnormally phosphorylated tau (phospho-tau) occurs in Alzheimer'sdisease but also with brain ageing. The Braak staging scheme focused on neurofibrillary tangles, butabundant p-tau is also present in neuropil threads, and a recent scheme has been proposed by theBrainNet Europe consortium for staging tau pathology based on neuropil threads. We determined therelationship of threads to tangles, and the value of staging for threads in an unselected population-representative ageing brain cohort. We also determined the prevalence of astroglial tau pathologies, and their relationship to neuronal tau. Phospho-tau pathology was determined by immunohistochemistry (AT8 antibody) in the MRC-CFAS neuropathology cohort. Neuropil threads were staged using the BrainNet Europe protocol for tau pathology, and compared with Braak tangle stages. Astroglial tau pathology was assessed in neo-cortical, mesial temporal and subcortical areas. RESULTS: Cases conformed well to the hierarchical neuropil threads staging of the BrainNet Europe protocol and correlated strongly with Braak staging (r=0.84, p

OBJECTIVE: Dementia with Lewy bodies is an α-synucleinopathy characterized by neocortical Lewy-related pathology (LRP). We carried out a genome-wide association study (GWAS) on neocortical LRP in a population-based sample of subjects aged 85 or over. \ud \ud METHODS: LRP was analyzed in 304 subjects in the Vantaa 85+ sample from Southern Finland. The GWAS included 41 cases with midbrain, hippocampal, and neocortical LRP and 177 controls without midbrain and hippocampal LRP. The Medical Research Council Cognitive Function and Ageing Study (CFAS) material was used for replication (51 cases and 131 controls). \ud \ud RESULTS: By analyzing 327,010 markers the top signal was obtained at the HLA-DPA1/DPB1 locus (P = 1.29 × 10(-7)); five other loci on chromosomes 15q14, 2p21, 2q31, 18p11, and 5q23 were associated with neocortical LRP at P

White matter lesions (WML) are common in brain aging and are associated with dementia. We aimed to investigate whether oxidative DNA damage and occur in WML and in apparently normal white matter in cases with lesions. Tissue from WML and control white matter from brains with lesions (controls lesional) and without lesions (controls non-lesional) were obtained, using post-mortem magnetic resonance imaging-guided sampling, from the Medical Research Council Cognitive Function and Ageing Study. Oxidative damage was assessed by immunohistochemistry to 8-hydroxy-2′-deoxoguanosine (8-OHdG) and Western blotting for malondialdehyde. DNA response was assessed by phosphorylated histone H2AX (γH2AX), p53, senescence markers and by quantitative Reverse transcription polymerase chain reaction (RT-PCR) panel for candidate DNA damage-associated genes. 8-OHdG was expressed in glia and endothelium, with increased expression in both WML and controls lesional compared with controls non-lesional (P

BACKGROUND: The insulin/IGF1 signalling (IIS) pathways are involved in longevity regulation and are dysregulated in neurons in Alzheimer's disease (AD). We previously showed downregulation in IIS gene expression in astrocytes with AD-neuropathology progression, but IIS in astrocytes remains poorly understood. We therefore examined the IIS pathway in human astrocytes and developed models to reduce IIS at the level of the insulin or the IGF1 receptor (IGF1R). \ud \ud RESULTS: We determined IIS was present and functional in human astrocytes by immunoblotting and showed astrocytes express the insulin receptor (IR)-B isoform of Ir. Immunocytochemistry and cell fractionation followed by western blotting revealed the phosphorylation status of insulin receptor substrate (IRS1) affects its subcellular localisation. To validate IRS1 expression patterns observed in culture, expression of key pathway components was assessed on post-mortem AD and control tissue using immunohistochemistry. Insulin signalling was impaired in cultured astrocytes by treatment with insulin + fructose and resulted in decreased IR and Akt phosphorylation (pAkt S473). A monoclonal antibody against IGF1R (MAB391) induced degradation of IGF1R receptor with an associated decrease in downstream pAkt S473. Neither treatment affected cell growth or viability as measured by MTT and Cyquant® assays or GFAP immunoreactivity. \ud \ud DISCUSSION: IIS is functional in astrocytes. IR-B is expressed in astrocytes which differs from the pattern in neurons, and may be important in differential susceptibility of astrocytes and neurons to insulin resistance. The variable presence of IRS1 in the nucleus, dependent on phosphorylation pattern, suggests the function of signalling molecules is not confined to cytoplasmic cascades. Down-regulation of IR and IGF1R, achieved by insulin + fructose and monoclonal antibody treatments, results in decreased downstream signalling, though the lack of effect on viability suggests that astrocytes can compensate for changes in single pathways. Changes in signalling in astrocytes, as well as in neurons, may be important in ageing and neurodegeneration.

AIMS: Oxidative damage and an associated DNA damage response (DDR) are evident in mild cognitive impairment and early Alzheimer's disease, suggesting that neuronal dysfunction resulting from oxidative DNA damage may account for some of the cognitive impairment not fully explained by Alzheimer-type pathology. METHODS: Frontal cortex (Braak stage 0-II) was obtained from the Medical Research Council's Cognitive Function and Ageing Study cohort. Neurones were isolated from 8 cases (4 high and 4 low DDR) by laser capture microdissection and changes in the transcriptome identified by microarray analysis. RESULTS: 2378 genes were significantly differentially expressed (1690 up-regulated, 688 down-regulated, p

Aims\ud Population-based studies have shown that approximately 20% of the ageing population (aged 65 years and over) with dementia have little or no classical Alzheimer-type neuropathology. Cumulative DNA damage and a reduced capacity of DNA repair may result in neuronal dysfunction and contribute to cognitive impairment independent of Alzheimer-type pathology in the ageing brain.\ud \ud Methods\ud We investigated expression of the DNA damage response (DDR)-associated molecules γH2AX and DNA-PKcs using immunohistochemistry and western blotting, and senescence-associated β-galactosidase in the frontal association neocortex of cases with low levels of Alzheimer-type pathology (Braak & Braak stage 0–II), and explored their relationship to cognitive impairment in a population-representative sample from the Medical Research Council's Cognitive Function and Ageing Study cohort.\ud \ud Results\ud Increases in both γH2AX+ (rs = −0.36, P = 0.025) and DNA-PKcs+ (rs = −0.39, P = 0.01) neuronal counts were associated with a lower Mini-Mental State Examination score. Increasing levels of senescence associated-β-gal+ pyramidal neurones were weakly associated with the total number of DNA-PKcs+ neurones (P = 0.08), but not with traditional senescence-associated signalling molecules, including p53 and p16.\ud \ud Conclusion\ud The association between the neuronal DDR and cognitive impairment, independent of AD pathology in the ageing brain, may be suggestive of a causal link via neuronal dysfunction.

Introduction\ud \ud Studies with strong selection biases propose that alpha-synucleinopathy (AS) spreads upwards and downwards in the neuraxis from the medulla, that amygdala-dominant AS is strongly associated with Alzheimer’s disease (AD), and that a more severe involvement of the cerebral cortex is correlated with increasing risk of dementia. This study examines the association of AS patterns and observed neuropsychological symptoms in brains of a population-representative donor cohort.\ud \ud Methods\ud \ud Brains donated in 2 out of 6 cognitive function and ageing study cohorts (Cambridgeshire and Nottingham) were examined. Over 80% were >80 years old at death. The respondents were evaluated prospectively in life for cognitive decline and dementia. Immunocytochemistry for tau and alpha-synuclein (using LB509 by Zymed Laboratories) was carried out in 208 brains to establish Braak stage and the pattern and severity of AS following the dementia with Lewy bodies (DLB) consensus recommendations. Dementia, specific neuropsychological measures as measured using the Cambridge cognitive examination, the presence of hallucinations and Parkinson’s disease were investigated.\ud \ud Results\ud \ud Four patterns of AS were observed: no AS pathology (n = 92), AS pathology following the DLB consensus guidelines (n = 33, of which five were ‘neocortical’), amygdala-predominant AS (n = 18), and other AS patterns (n = 33). Each group was subdivided according to high/low neurofibrillary tangles (NFT) Braak stage. Results showed no association between dementia and these patterns of AS, adjusting for the presence of NFT or not. The risk of visual hallucinations shows a weak association with AS in the substantia nigra (odds ratio (OR) = 3.2; 95% confidence interval (CI) 0.5 to 15.5; P = 0.09) and amygdala (OR = 3.0; 95% CI 0.7 to 12.3; P = 0.07). The analysis is similar for auditory hallucinations in subcortical regions.\ud \ud Conclusions\ud \ud Among the whole population of older people, AS does not increase the risks for dementia, irrespective of Braak stage of NFT pathology. There was no evidence that the pattern of AS pathology in cortical areas was relevant to the risk of hallucination. In general, the hypothesis that AS as measured using these methods per se is a key determinant of cognitive clinical phenotypes is not supported.

Aims\ud \ud β-amyloid (Aβ) plaques are a key feature of Alzheimer’s disease pathology but correlate poorly with dementia. They are associated with astrocytes which may modulate the effect of Aβ-deposition on the neuropil. This study characterised the astrocyte response to Aβ plaque subtypes, and investigated their association with cognitive impairment.\ud \ud Methods\ud \ud Aβ plaque subtypes were identified in the cingulate gyrus using dual labelling immunohistochemistry to Aβ and GFAP+ astrocytes, and quantitated in two cortical areas: the area of densest plaque burden and the deep cortex near the white matter border (layer VI). Three subtypes were defined for both diffuse and compact plaques (also known as classical or core-plaques): Aβ plaque with (1) no associated astrocytes, (2) focal astrogliosis or (3) circumferential astrogliosis.\ud \ud Results\ud \ud In the area of densest burden, diffuse plaques with no astrogliosis (β = -0.05, p = 0.001) and with focal astrogliosis (β = -0.27, p = 0.009) significantly associated with lower MMSE scores when controlling for sex and age at death. In the deep cortex (layer VI), both diffuse and compact plaques without astrogliosis associated with lower MMSE scores (β = -0.15, p = 0.017 and β = -0.81, p = 0.03, respectively). Diffuse plaques with no astrogliosis in layer VI related to dementia status (OR = 1.05, p = 0.025). In the area of densest burden, diffuse plaques with no astrogliosis or with focal astrogliosis associated with increasing Braak stage (β = 0.01, p

Aims\ud \ud β-amyloid (Aβ) plaques are a key feature of Alzheimer’s disease pathology but correlate poorly with dementia. They are associated with astrocytes which may modulate the effect of Aβ-deposition on the neuropil. This study characterised the astrocyte response to Aβ plaque subtypes, and investigated their association with cognitive impairment.\ud \ud Methods\ud \ud Aβ plaque subtypes were identified in the cingulate gyrus using dual labelling immunohistochemistry to Aβ and GFAP+ astrocytes, and quantitated in two cortical areas: the area of densest plaque burden and the deep cortex near the white matter border (layer VI). Three subtypes were defined for both diffuse and compact plaques (also known as classical or core-plaques): Aβ plaque with (1) no associated astrocytes, (2) focal astrogliosis or (3) circumferential astrogliosis.\ud \ud Results\ud \ud In the area of densest burden, diffuse plaques with no astrogliosis (β = -0.05, p = 0.001) and with focal astrogliosis (β = -0.27, p = 0.009) significantly associated with lower MMSE scores when controlling for sex and age at death. In the deep cortex (layer VI), both diffuse and compact plaques without astrogliosis associated with lower MMSE scores (β = -0.15, p = 0.017 and β = -0.81, p = 0.03, respectively). Diffuse plaques with no astrogliosis in layer VI related to dementia status (OR = 1.05, p = 0.025). In the area of densest burden, diffuse plaques with no astrogliosis or with focal astrogliosis associated with increasing Braak stage (β = 0.01, p

The BrainNet Europe consortium assessed the reproducibility in the assignment of the type of frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein (TDP) 43 following current recommendations. The agreement rates were influenced by the immunohistochemical (IHC) method and by the classification strategy followed. p62-IHC staining yielded good uniform quality of stains, but the most reliable results were obtained implementing specific Abs directed against the hallmark protein TDP43. Both assessment of the type and the extent of lesions were influenced by the Abs and by the quality of stain. Assessment of the extent of the lesions yielded poor results repeatedly; thus, the extent of pathology should not be used in diagnostic consensus criteria. Whilst 31 neuropathologists typed 30 FTLD-TDP cases, inter-rater agreement ranged from 19 to 100 per cent, being highest when applying phosphorylated TDP43/IHC. The agreement was highest when designating Type C or Type A/B. In contrast, there was a poor agreement when attempting to separate Type A or Type B FTLD-TDP. In conclusion, we can expect that neuropathologist, independent of his/her familiarity with FTLD-TDP pathology, can identify a TDP43-positive FTLD case. The goal should be to state a Type (A, B, C, D) or a mixture of Types (A/B, A/C or B/C). Neuropathologists, other clinicians and researchers should be aware of the pitfalls whilst doing so. Agreement can be reached in an inter-laboratory setting regarding Type C cases with thick and long neurites, whereas the differentiation between Types A and B may be more troublesome. © 2014, Springer-Verlag Wien.

GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and\ud frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed\ud repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding\ud partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first\ud identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic\ud lateral sclerosis with a repeat expansion of C9orf72 (C9orf72 + ), but not from those patients without a repeat expansion\ud of C9orf72 (C9orf72) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated\ud with the clinical phenotype (t-test P5 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified\ud foci in fibroblasts from an asymptomatic C9orf72 + carrier. We next performed pulldown assays, with GGGGCC5, in conjunction\ud with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing\ud RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched.\ud Immunohistochemistry in central nervous system tissue from C9orf72 + patients with amyotrophic lateral sclerosis demonstrated\ud co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP\ud H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins\ud to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small\ud proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry\ud demonstrated that neurons with and without RNA foci were equally likely to show nuclear depletion of TDP-43 (2 P = 0.75) or\ud poly-GA dipeptide repeat protein inclusions (2 P = 0.46). Our findings suggest two non-exclusive pathogenic mechanisms: (i)\ud functional depletion of RNA-processing proteins resulting in disruption of messenger RNA splicing; and (ii) licensing of expanded\ud C9orf72 pre-messenger RNA for nuclear export by inappropriate association with messenger RNA export adaptor\ud protein(s) leading to cytoplasmic repeat associated non-ATG translation and formation of potentially toxic dipeptide repeat\ud protein.\ud d

Cerebral white matters lesions (WML) are seen in 94% of the population aged 64 and over and are associated\ud with cognitive decline and depression. We used immunohistochemistry and stereological methods on post mortem\ud brain samples derived from the Medical Research Council Cognitive Function and Ageing Study (MRC-CFAS) cohort\ud to investigate the axonal density within deep subcortical lesions. There was no significant difference between the\ud lesional and control white matter, therefore, we conclude that there is axonal preservation within these lesions that\ud are characterized by demyelination.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the progressive degeneration of motor neurons. Substantial evidence implicates oxidative stress and mitochondrial dysfunction as early events in disease progression. Our aim was to ascertain whether mutation of the SOD1 protein increases metabolic functional susceptibility to oxidative stress. Here we used a motor neuron-like cell line (NSC34) stably transfected with various human mutant SOD1 transgenes (G93A, G37R, H48Q) to investigate the impact of oxidative stress on cell viability and metabolic function within intact cells. NSC34 cells expressing mutant SOD1 showed a dose dependent reduction in cell viability when exposed to oxidative stress induced by hydrogen peroxide, with variation between mutations. The G93A transfectants showed greater cell death and LDH release compared to cells transfected with the other SOD1 mutations, and H48Q showed an accelerated decline at later time points. Differences in mitochondrial bioenergetics, including mitochondrial respiration, coupling efficiency and proton leak, were identified between the mutations, consistent with the differences observed in viability. NSC34 cells expressing G93A SOD1 displayed reduced coupled respiration and mitochondrial membrane potential compared to controls. Furthermore, the G93A mutation had significantly increased metabolic susceptibility to oxidative stress, with hydrogen peroxide increasing ROS production, reducing both cellular oxygen consumption and glycolytic flux in the cell. This study highlights bioenergetic defects within a cellular model of ALS and suggests that oxidative stress is not only detrimental to oxygen consumption but also glycolytic flux, which could lead to an energy deficit in the cell.\ud

The finding of TDP-43 as a major component of ubiquitinated protein inclusions in amyotrophic lateral sclerosis (ALS) has led to the identification of 30 mutations in the transactive response-DNA binding protein (TARDBP) gene, encoding TDP-43. All but one are in exon 6, which encodes the glycine-rich domain. The aim of this study was to determine the frequency of TARDBP mutations in a large cohort of motor neurone disease patients from Northern England (42 non-superoxide dismutase 1 (SOD1) familial ALS (FALS), nine ALS-frontotemporal dementia, 474 sporadic ALS (SALS), 45 progressive muscular atrophy cases). We identified four mutations, two of which were novel, in two familial (FALS) and two sporadic (SALS) cases, giving a frequency of TARDBP mutations in non-SOD1 FALS of 5% and SALS of 0.4%. Analysis of clinical data identified that patients had typical ALS, with limb or bulbar onset, and showed considerable variation in age of onset and rapidity of disease course. However, all cases had an absence of clinically overt cognitive dysfunction.

The aim of this study was to characterize myelin loss as one of the features of white matter abnormalities across three common dementing disorders. We evaluated post-mortem brain tissue from frontal and temporal lobes from 20 vascular dementia (VaD), 19 Alzheimer’s disease (AD) and 31 dementia with Lewy bodies (DLB) cases and 12 comparable age controls. Images of sections stained with conventional luxol fast blue were analysed to estimate myelin attenuation by optical density. Serial adjacent sections were then immunostained for degraded myelin basic protein (dMBP) and the mean percentage area containing dMBP (%dMBP) was determined as an indicator of myelin degeneration. We further assessed the relationship between dMBP and glutathione S-transferase (a marker of mature oligodendrocytes) immunoreactivities. Pathological diagnosis significantly affected the frontal but not temporal lobe myelin attenuation: myelin density was most reduced in VaD compared to AD and DLB, which still significantly exhibited lower myelin density compared to ageing controls. Consistent with this, the degree of myelin loss was correlated with greater %dMBP, with the highest %dMBP in VaD compared to the other groups. The %dMBP was inversely correlated with the mean size of oligodendrocytes in VaD, whereas it was positively correlated with their density in AD. A two-tier regression model analysis confirmed that the type of disorder (VaD or AD) determines the relationship between %dMBP and the size or density of oligodendrocytes across the cases. Our findings, attested by the use of three markers, suggest that myelin loss may evolve in parallel with shrunken oligodendrocytes in VaD but their increased density in AD, highlighting partially different mechanisms are associated with myelin degeneration, which could originate from hypoxic–ischaemic damage to oligodendrocytes in VaD whereas secondary to axonal degeneration in AD.

When 22 members of the BrainNet Europe (BNE) consortium assessed 31 cases with α-synuclein (αS) immunoreactive (IR) pathology applying the consensus protocol described by McKeith and colleagues in 2005, the inter-observer agreement was 80%, being lowest in the limbic category (73%). When applying the staging protocol described by Braak and colleagues in 2003, agreement was only 65%, and in some cases as low as 36%. When modifications of these strategies, i.e., McKeith's protocol by Leverenz and colleagues from 2009, Braak's staging by Müller and colleagues from 2005 were applied then the agreement increased to 78 and 82%, respectively. In both of these modifications, a reduced number of anatomical regions/blocks are assessed and still in a substantial number of cases, the inter-observer agreement differed significantly. Over 80% agreement in both typing and staging of αS pathology could be achieved when applying a new protocol, jointly designed by the BNE consortium. The BNE-protocol assessing αS-IR lesions in nine blocks offered advantages over the previous modified protocols because the agreement between the 22 observers was over 80% in most cases. Furthermore, in the BNE-protocol, the αS pathology is assessed as being present or absent and thus the quality of staining and the assessment of the severity of αS-IR pathology do not alter the inter-observer agreement, contrary to other assessment strategies. To reach these high agreement rates an entity of amygdala-predominant category was incorporated. In conclusion, here we report a protocol for assessing αS pathology that can achieve a high inter-observer agreement for both the assignment to brainstem, limbic, neocortical and amygdala-predominant categories of synucleinopathy and the Braak stages. © The Author(s) 2009.