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Abstract Background Progressive supranuclear palsy (PSP) is a rare neurodegenerative disease characterized by the accumulation of aggregated tau proteins in astrocytes, neurons, and oligodendrocytes. Previous genome-wide association studies for PSP were based on genotype array, therefore, were inadequate for the analysis of rare variants as well as larger mutations, such as small insertions/deletions (indels) and structural variants (SVs). Method In this study, we performed whole genome sequencing (WGS) and conducted association analysis for single nucleotide variants (SNVs), indels, and SVs, in a cohort of 1,718 cases and 2,944 controls of European ancestry. Of the 1,718 PSP individuals, 1,441 were autopsy-confirmed and 277 were clinically diagnosed. Results Our analysis of common SNVs and indels confirmed known genetic loci at MAPT, MOBP, STX6, SLCO1A2, DUSP10, and SP1, and further uncovered novel signals in APOE, FCHO1/MAP1S, KIF13A, TRIM24, TNXB, and ELOVL1. Notably, in contrast to Alzheimer’s disease (AD), we observed the APOE ε2 allele to be the risk allele in PSP. Analysis of rare SNVs and indels identified significant association in ZNF592 and further gene network analysis identified a module of neuronal genes dysregulated in PSP. Moreover, seven common SVs associated with PSP were observed in the H1/H2 haplotype region (17q21.31) and other loci, including IGH, PCMT1, CYP2A13, and SMCP. In the H1/H2 haplotype region, there is a burden of rare deletions and duplications (P = 6.73 × 10–3) in PSP. Conclusions Through WGS, we significantly enhanced our understanding of the genetic basis of PSP, providing new targets for exploring disease mechanisms and therapeutic interventions.

Abstract Lewy body dementia (LBD), a class of disorders comprising Parkinson’s disease dementia (PDD) and dementia with Lewy bodies (DLB), features substantial clinical and pathological overlap with Alzheimer’s disease (AD). The identification of biomarkers unique to LBD pathophysiology could meaningfully advance its diagnosis, monitoring, and treatment. Using quantitative mass spectrometry (MS), we measured over 9,000 proteins across 138 dorsolateral prefrontal cortex (DLPFC) tissues from a University of Pennsylvania autopsy collection comprising control, Parkinson’s disease (PD), PDD, and DLB diagnoses. We then analyzed co-expression network protein alterations in those with LBD, validated these disease signatures in two independent LBD datasets, and compared these findings to those observed in network analyses of AD cases. The LBD network revealed numerous groups or “modules” of co-expressed proteins significantly altered in PDD and DLB, representing synaptic, metabolic, and inflammatory pathophysiology. A comparison of validated LBD signatures to those of AD identified distinct differences between the two diseases. Notably, synuclein-associated presynaptic modules were elevated in LBD but decreased in AD relative to controls. We also found that glial-associated matrisome signatures consistently elevated in AD were more variably altered in LBD, ultimately stratifying those LBD cases with low versus high burdens of concurrent beta-amyloid deposition. In conclusion, unbiased network proteomic analysis revealed diverse pathophysiological changes in the LBD frontal cortex distinct from alterations in AD. These results highlight the LBD brain network proteome as a promising source of biomarkers that could enhance clinical recognition and management.

Abstract Our current understanding of the spread and neurodegenerative effects of tau neurofibrillary tangles (NFTs) within the medial temporal lobe (MTL) during the early stages of Alzheimer’s Disease (AD) is limited by the presence of confounding non-AD pathologies and the two-dimensional (2-D) nature of conventional histology studies. Here, we combine ex vivo MRI and serial histological imaging from 25 human MTL specimens to present a detailed, 3-D characterization of quantitative NFT burden measures in the space of a high-resolution, ex vivo atlas with cytoarchitecturally-defined subregion labels, that can be used to inform future in vivo neuroimaging studies. Average maps show a clear anterior to poster gradient in NFT distribution and a precise, spatial pattern with highest levels of NFTs found not just within the transentorhinal region but also the cornu ammonis (CA1) subfield. Additionally, we identify granular MTL regions where measures of neurodegeneration are likely to be linked to NFTs specifically, and thus potentially more sensitive as early AD biomarkers.

Abstract Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following fixation, staining, and sectioning, which limit resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) allows higher resolution imaging of unfixed cellular samples while preserving architecture, but it requires samples to be vitreous and thin enough for transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue via plunge-freezing and use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid at variable depth inside the tissue. Lamellae generated in Alzheimer’s disease brain tissue reveal intact subcellular structures including components of autophagy and potential pathologic tau fibrils. Furthermore, we reveal intact compact myelin and functional cytoplasmic expansions. These images indicate that plasma FIB milling with cryo-ET may be used to elucidate nanoscale structures within the human brain.

Abstract Objective Alzheimer's disease neuropathologic change and alpha‐synucleinopathy commonly co‐exist and contribute to the clinical heterogeneity of dementia. Here, we examined tau epitopes marking various stages of tangle maturation to test the hypotheses that tau maturation is more strongly associated with beta‐amyloid compared to alpha‐synuclein, and within the context of mixed pathology, mature tau is linked to Alzheimer's disease clinical phenotype and negatively associated with Lewy body dementia. Methods We used digital histology to measure percent area‐occupied by pathology in cortical regions among individuals with pure Alzheimer's disease neuropathologic change, pure alpha‐synucleinopathy, and a co‐pathology group with both Alzheimer's and alpha‐synuclein pathologic diagnoses. Multiple tau monoclonal antibodies were used to detect early (AT8, MC1) and mature (TauC3) epitopes of tangle progression. We used linear/logistic regression to compare groups and test the association between pathologies and clinical features. Results There were lower levels of tau pathology (β = 1.86–2.96, p

Abstract Astrocytes are one of the brain’s major cell types and are responsible for maintaining neuronal homeostasis via regulating the extracellular environment, providing metabolic support, and modulating synaptic activity. In neurodegenerative diseases, such as Alzheimer’s disease, astrocytes can take on a hypertrophic appearance. These reactive astrocytes are canonically associated with increases in cytoskeletal proteins, such as glial fibrillary acidic protein and vimentin. However, the molecular alterations that characterize astrocytes in human disease tissues have not been extensively studied with single cell resolution. Using single nucleus RNA sequencing data from normal, pathologic aging, and Alzheimer’s disease brains, we identified the transcriptomic changes associated with reactive astrocytes. Deep learning-based clustering algorithms denoised expression data for 17,012 genes and clustered 15,529 astrocyte nuclei, identifying protoplasmic, gray matter and fibrous, white matter astrocyte clusters. RNA trajectory analyses revealed a spectrum of reactivity within protoplasmic astrocytes characterized by a modest increase of reactive genes and a marked decrease in homeostatic genes. Amyloid but not tau pathology correlated with astrocyte reactivity. To identify reactivity-associated genes, linear regressions of gene expression versus reactivity were used to identify the top 52 upregulated and 144 downregulated genes. Gene Ontology analysis revealed that upregulated genes were associated with cellular growth, responses to metal ions, inflammation, and proteostasis. Downregulated genes were involved in cellular interactions, neuronal development, ERBB signaling, and synapse regulation. Transcription factors were significantly enriched among the downregulated genes. Using co-immunofluorescence staining of Alzheimer’s disease brain tissues, we confirmed pathologic downregulation of ERBB4 and transcription factor NFIA in reactive astrocytes. Our findings reveal that protoplasmic, gray matter astrocytes in Alzheimer’s disease exist within a spectrum of reactivity that is marked by a strong loss of normal function.

Abstract Single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cellular heterogeneity in health and disease. However, the lack of physical relationships among dissociated cells has limited its applications. To address this issue, we present CeLEry (Cell Location recovEry), a supervised deep learning algorithm that leverages gene expression and spatial location relationships learned from spatial transcriptomics to recover the spatial origins of cells in scRNA-seq. CeLEry has an optional data augmentation procedure via a variational autoencoder, which improves the method’s robustness and allows it to overcome noise in scRNA-seq data. We show that CeLEry can infer the spatial origins of cells in scRNA-seq at multiple levels, including 2D location and spatial domain of a cell, while also providing uncertainty estimates for the recovered locations. Our comprehensive benchmarking evaluations on multiple datasets generated from brain and cancer tissues using Visium, MERSCOPE, MERFISH, and Xenium demonstrate that CeLEry can reliably recover the spatial location information for cells using scRNA-seq data.

Abstract Objective Within Lewy body spectrum disorders (LBSD) with α‐synuclein pathology (αSyn), concomitant Alzheimer's disease (AD) pathology is common and is predictive of clinical outcomes, including cognitive impairment and decline. Plasma phosphorylated tau 181 (p‐tau181) is sensitive to AD neuropathologic change (ADNC) in clinical AD, and plasma glial fibrillary acidic protein (GFAP) is associated with the presence of β‐amyloid plaques. While these plasma biomarkers are well tested in clinical and pathological AD, their diagnostic and prognostic performance for concomitant AD in LBSD is unknown. Methods In autopsy‐confirmed αSyn‐positive LBSD, we tested how plasma p‐tau181 and GFAP differed across αSyn with concomitant ADNC (αSyn+AD; n = 19) and αSyn without AD (αSyn; n = 30). Severity of burden was scored on a semiquantitative scale for several pathologies (e.g., β‐amyloid and tau), and scores were averaged across sampled brainstem, limbic, and neocortical regions. Results Linear models showed that plasma GFAP was significantly higher in αSyn+AD compared to αSyn (β = 0.31, 95% CI = 0.065–0.56, and P = 0.015), after covarying for age at plasma, plasma‐to‐death interval, and sex; plasma p‐tau181 was not (P = 0.37). Next, linear models tested associations of AD pathological features with both plasma analytes, covarying for plasma‐to‐death, age at plasma, and sex. GFAP was significantly associated with brain β‐amyloid (β = 15, 95% CI = 6.1–25, and P = 0.0018) and tau burden (β = 12, 95% CI = 2.5–22, and P = 0.015); plasma p‐tau181 was not associated with either (both P > 0.34). Interpretation Findings indicate that plasma GFAP may be sensitive to concomitant AD pathology in LBSD, especially accumulation of β‐amyloid plaques.

SpaDecon is a semi-supervised learning-based method for cell-type deconvolution of spatially resolved transcriptomics (SRT) data that is also computationally fast and memory efficient for large-scale SRT studies.

Frontotemporal dementia (FTD) is a spectrum of clinically and pathologically heterogenous neurodegenerative dementias. Clinical and anatomical variants of FTD have been described and associated with underlying frontotemporal lobar degeneration (FTLD) pathology, including tauopathies (FTLD-tau) or TDP-43 proteinopathies (FTLD-TDP). FTD patients with predominant degeneration of anterior temporal cortices often develop a language disorder of semantic knowledge loss and/or a social disorder often characterized by compulsive rituals and belief systems corresponding to predominant left or right hemisphere involvement, respectively. The neural substrates of these complex social disorders remain unclear. Here, we present a comparative imaging and postmortem study of two patients, one with FTLD-TDP (subtype C) and one with FTLD-tau (subtype Pick disease), who both developed new rigid belief systems. The FTLD-TDP patient developed a complex set of values centered on positivity and associated with specific physical and behavioral features of pigs, while the FTLD-tau patient developed compulsive, goal-directed behaviors related to general themes of positivity and spirituality. Neuroimaging showed left-predominant temporal atrophy in the FTLD-TDP patient and right-predominant frontotemporal atrophy in the FTLD-tau patient. Consistent with antemortem cortical atrophy, histopathologic examinations revealed severe loss of neurons and myelin predominantly in the anterior temporal lobes of both patients, but the FTLD-tau patient showed more bilateral, dorsolateral involvement featuring greater pathology and loss of projection neurons and deep white matter. These findings highlight that the regions within and connected to anterior temporal lobes may have differential vulnerability to distinct FTLD proteinopathies and serve important roles in human belief systems.

Abstract Objective To determine if plasma tau phosphorylated at threonine 181 (p‐tau181) distinguishes pathology‐confirmed Alzheimer's disease (AD) from normal cognition (NC) adults, to test if p‐tau181 predicts cognitive and functional decline, and to validate findings in an external cohort. Methods Thirty‐one neuropathology‐confirmed AD cases, participants with clinical diagnoses of mild cognitive impairment (MCI, N = 91) or AD dementia (N = 64), and NC (N = 241) had plasma collected at study entry. The clinical diagnosis groups had annual cognitive (Mini‐Mental State Examination, MMSE) and functional (Clinical Dementia Rating Scale, CDR) measures. NC (N = 70), MCI (N = 75), and AD dementia (N = 50) cases from the Alzheimer's Disease Neuroimaging Initiative (ADNI) were used as a validation cohort. Plasma p‐tau181 was measured using the Quanterix SiMoA HD‐X platform. Results Plasma p‐tau181 differentiated pathology‐confirmed AD from NC with negative amyloid PET scans with an AUC of 0.93. A cut point of 3.44 pg/mL (maximum Youden Index) had a sensitivity of 0.77, specificity of 0.96. p‐Tau181 values above the cut point were associated with the faster rate of decline in MMSE in AD dementia and MCI and a shorter time to a clinically significant functional decline in all groups. In a subset of MCI cases from ADNI, p‐tau181 values above the cut point associated with faster rate of decline in MMSE, and a shorter time to a clinically significant functional decline and conversion to dementia. Interpretation Plasma p‐tau181 differentiates AD pathology cases from NC with high accuracy. Higher levels of plasma p‐tau181 are associated with faster cognitive and functional decline.

Abstract Traumatic brain injury (TBI) is associated with the development of a range of neurodegenerative pathologies, including chronic traumatic encephalopathy (CTE). Current consensus diagnostic criteria define the pathognomonic cortical lesion of CTE neuropathologic change (CTE-NC) as a patchy deposition of hyperphosphorylated tau in neurons, with or without glial tau in thorn-shaped astrocytes, typically towards the depths of sulci and clustered around small blood vessels. Nevertheless, although incorporated into consensus diagnostic criteria, the contribution of the individual cellular components to identification of CTE-NC has not been formally evaluated. To address this, from the Glasgow TBI Archive, cortical tissue blocks were selected from consecutive brain donations from contact sports athletes in which there was known to be either CTE-NC (n = 12) or Alzheimer’s disease neuropathologic change (n = 4). From these tissue blocks, adjacent tissue sections were stained for tau antibodies selected to reveal either solely neuronal pathology (3R tau; GT-38) or mixed neuronal and astroglial pathologies (4R tau; PHF-1). These stained sections were then randomised and independently assessed by a panel of expert neuropathologists, blind to patient clinical history and primary antibody applied to each section, who were asked to record whether CTE-NC was present. Results demonstrate that, in sections stained for either 4R tau or PHF-1, consensus recognition of CTE-NC was high. In contrast, recognition of CTE-NC in sections stained for 3R tau or GT-38 was poor; in the former no better than chance. Our observations demonstrate that the presence of both neuronal and astroglial tau pathologies facilitates detection of CTE-NC, with its detection less consistent when neuronal tau pathology alone is visible. The combination of both glial and neuronal pathologies, therefore, may be required for detection of CTE-NC.

Abstract Background Synapse degeneration is an early event in pathological frontotemporal lobar degeneration (FTLD). Consequently, a surrogate marker of synapse loss could be used to monitor early pathologic changes in patients with underlying FTLD. The aim of this study was to evaluate the relationship of antemortem cerebrospinal fluid (CSF) levels of 8 synaptic proteins with postmortem global tau and TDP-43 burden and cognitive performance and to assess their diagnostic capacity in a neuropathological FTLD cohort. Methods We included patients with a neuropathological confirmation of FTLD-Tau (n = 24, mean age-at-CSF 67 years ± 11), FTLD-TDP (n = 25, 66 years ± 9) or AD (n = 25, 73 years ± 6) as well as cognitively normal controls (n = 35, 69 years ± 7) from the Penn FTD Center and ADRC. We used a semi-quantitative measure of tau and TDP-43 inclusions to quantify pathological burden across 16 brain regions. Statistical methods included Spearman rank correlations, one-way analysis of covariance, ordinal regression, step-wise multiple linear regression and receiver-operating characteristic curves. Result CSF calsyntenin-1 and neurexin-2a were correlated in all patient groups (r s = .55 to .88). In FTLD-TDP, we observed low antemortem CSF levels of calsyntenin-1 and neurexin-2a compared to AD (.72-fold, p = .001, .77-fold, p = .04, respectively) and controls (.80-fold, p = .02, .78-fold, p = .02, respectively), which were inversely associated with post-mortem global TDP-43 burden (regression r 2 = .56, p = .007 and r 2 = .57, p = .006, respectively). A multimarker panel including calsyntenin-1 was associated with TDP-43 burden (r 2 = .69, p = .003) and MMSE score (r 2 = .19, p = .03) in FTLD. A second multimarker synaptic panel, also including calsyntenin-1, was associated with MMSE score in FTLD-tau (r 2 = .49, p = .04) and improved diagnostic performance to discriminate FTLD-Tau and FTLD-TDP neuropathologic subtypes (AUC = .83). Conclusion These synaptic panels have potential in the differential diagnosis of FTLD neuropathologic subtypes and as surrogate markers of cognitive performance in future clinical trials targeting TDP-43 or tau.

Abstract TMEM106B, a type II lysosomal transmembrane protein, has recently been associated with brain aging, hypomyelinating leukodystrophy, frontotemporal lobar degeneration (FTLD) and several other brain disorders. TMEM106B is critical for proper lysosomal function and TMEM106B deficiency leads to myelination defects, FTLD related pathology, and motor coordination deficits in mice. However, the physiological and pathological functions of TMEM106B in the brain are still not well understood. In this study, we investigate the role of TMEM106B in the cerebellum, dysfunction of which has been associated with FTLD and other brain disorders. We found that TMEM106B is ubiquitously expressed in neurons in the cerebellum, with the highest levels in the Purkinje neurons. Aged TMEM106B-deficient mice show significant loss of Purkinje neurons specifically in the anterior lobe of the cerebellum. Increased microglia and astrocyte activation, as well as an accumulation of ubiquitinated proteins, p62 and TDP-43 were also detected in the cerebellum of aged TMEM106B deficient mice. In the young mice, myelination defects and a significant loss of synapses between Purkinje and deep cerebellar nuclei neurons were observed. Interestingly, TMEM106B deficiency causes distinct lysosomal phenotypes in different types of neurons and glia in the cerebellum and frontal cortex. In humans, TMEM106B rs1990622 risk allele (T/T) is associated with increased Purkinje neuron loss. Taken together, our studies support that TMEM106B regulates lysosomal function in a cell-type-specific manner and TMEM106B is critical for maintaining synaptic integrity and neural functions in the cerebellum.

Tau pathology drives neuronal dysfunction in 4- repeat tauopathies. Here, the authors combine tau-PET, resting-state fMRI and histopathology data, to show that brain connectivity is associated with tau deposition patterns in 4-repeat tauopathies.

Background: In previous studies of patients with frontotemporal lobar degeneration due to tau (FTLD-tau) and FTLD due to TDP (FTLD-TDP), cortical volumes derived from T1-weighted MRI have been used to identify a sequence of volume loss according to arbitrary volumetric criteria. Event-based modeling (EBM) is a probabilistic, generative machine learning model that determines the characteristic sequence of changes, or “events”, occurring during disease progression. EBM also estimates an individual patient’s disease “stage” by identifying which events have already occurred. In the present study, we use an EBM analysis to derive stages of regional anatomic atrophy in FTLD-tau and FTLD-TDP, and validated these stages against pathologic burden. Methods: Sporadic autopsy-confirmed patients with FTLD-tau (N = 42) and FTLD-TDP (N = 21), and 167 healthy controls with available T1-weighted images were identified. A subset of patients had quantitative digital histopathology of cortex performed at autopsy (FTLD-tau = 30, FTLD-TDP = 17). MRI images were processed, producing regional measures of cortical volumes. K-means clustering was used to find cortical regions with similar amounts of GM volume changes (n = 5 clusters). EBM was used to determine the characteristic sequence of cortical atrophy of identified clusters in autopsy-confirmed FTLD-tau and FTLD-TDP, and estimate each patient’s disease stage by cortical volume biomarkers. Linear regressions related pathologic burden to EBM-estimated disease stages. Results: EBM for cortical volume biomarkers generated statistically robust characteristic sequences of cortical atrophy in each group of patients. Cortical volume-based EBM-estimated disease stage was associated with pathologic burden in FTLD-tau (R2 = 0.16, p = 0.017) and FTLD-TDP (R2 = 0.51, p = 0.0008). Conclusions: We provide evidence that EBM can identify sequences of pathologically-confirmed cortical atrophy in sporadic FTLD-tau and FTLD-TDP.

Abstract Alzheimer’s disease (AD) is associated with inner retina (nerve fiber and ganglion cell layers) thinning. In contrast, we have seen outer retina thinning driven by photoreceptor outer nuclear layer (ONL) thinning with antemortem optical coherence tomography (OCT) among patients considered to have a frontotemporal degeneration tauopathy (FTLD-Tau). Our objective was to determine if postmortem retinal tissue from FTLD-Tau patients demonstrates ONL loss observed antemortem on OCT. Two probable FTLD-Tau patients that were deeply phenotyped by clinical and genetic testing were imaged with OCT and followed to autopsy. Postmortem brain and retinal tissue were evaluated by a neuropathologist and ocular pathologist, respectively, masked to diagnosis. OCT findings were correlated with retinal histology. The two patients had autopsy-confirmed FTLD-Tau neuropathology and had antemortem OCT measurements showing ONL thinning (66.9 μm, patient #1; 74.9 μm, patient #2) below the 95% confidence interval of normal limits (75.1–120.7 μm) in our healthy control cohort. Postmortem, retinal tissue from both patients demonstrated loss of nuclei in the ONL, matching ONL loss visualized on antemortem OCT. Nuclei counts from each area of ONL loss (2 – 3 nuclei per column) seen in patient eyes were below the 95% confidence interval (4 – 8 nuclei per column for ONL) of 3 normal control retinas analyzed at the same location. Our evaluation of retinal tissue from FTLD-Tau patients confirms ONL loss seen antemortem by OCT. Continued investigation of ONL thinning as a biomarker that may distinguish FTLD-Tau from other dementias is warranted.

Abstract Tau neurofibrillary tangle (NFT) pathology in the medial temporal lobe (MTL) is closely linked to neurodegeneration, and is the early pathological change associated with Alzheimer’s disease (AD). To elucidate patterns of structural change in the MTL specifically associated with tau pathology, we compared high-resolution ex vivo MRI scans of human postmortem MTL specimens with histology-based pathological assessments of the MTL. MTL specimens were obtained from twenty-nine brain donors, including patients with AD, other dementias, and individuals with no known history of neurological disease. Ex vivo MRI scans were combined using a customized groupwise diffeomorphic registration approach to construct a 3D probabilistic atlas that captures the anatomical variability of the MTL. Using serial histology imaging in eleven specimens, we labelled the MTL subregions in the atlas based on cytoarchitecture. Leveraging the atlas and neuropathological ratings of tau and TAR DNA-binding protein 43 (TDP-43) pathology severity, morphometric analysis was performed to correlate regional MTL thickness with the severity of tau pathology, after correcting for age and TDP-43 pathology. We found significant correlations between tau pathology and thickness in the entorhinal cortex (ERC) and stratum radiatum lacunosum moleculare (SRLM). When focusing on cases with low levels of TDP-43 pathology, we found strong associations between tau pathology and thickness in the ERC, SRLM and the subiculum/cornu ammonis 1 (CA1) subfields of the hippocampus, consistent with early Braak stages.

Abstract Primary age-related tauopathy (PART) is a form of Alzheimer-type neurofibrillary degeneration occurring in the absence of amyloid-beta (Aβ) plaques. While PART shares some features with Alzheimer disease (AD), such as progressive accumulation of neurofibrillary tangle pathology in the medial temporal lobe and other brain regions, it does not progress extensively to neocortical regions. Given this restricted pathoanatomical pattern and variable symptomatology, there is a need to reexamine and improve upon how PART is neuropathologically assessed and staged. We performed a retrospective autopsy study in a collection (n = 174) of post-mortem PART brains and used logistic regression to determine the extent to which a set of clinical and neuropathological features predict cognitive impairment. We compared Braak staging, which focuses on hierarchical neuroanatomical progression of AD tau and Aβ pathology, with quantitative assessments of neurofibrillary burden using computer-derived positive pixel counts on digitized whole slide images of sections stained immunohistochemically with antibodies targeting abnormal hyperphosphorylated tau (p-tau) in the entorhinal region and hippocampus. We also assessed other factors affecting cognition, including aging-related tau astrogliopathy (ARTAG) and atrophy. We found no association between Braak stage and cognitive impairment when controlling for age (p = 0.76). In contrast, p-tau burden was significantly correlated with cognitive impairment even when adjusting for age (p = 0.03). The strongest correlate of cognitive impairment was cerebrovascular disease, a well-known risk factor (p

Abstract Efforts to characterize the late effects of traumatic brain injury (TBI) have been in progress for some time. In recent years much of this activity has been directed towards reporting of chronic traumatic encephalopathy (CTE) in former contact sports athletes and others exposed to repetitive head impacts. However, the association between TBI and dementia risk has long been acknowledged outside of contact sports. Further, growing experience suggests a complex of neurodegenerative pathologies in those surviving TBI, which extends beyond CTE. Nevertheless, despite extensive research, we have scant knowledge of the mechanisms underlying TBI-related neurodegeneration (TReND) and its link to dementia. In part, this is due to the limited number of human brain samples linked to robust demographic and clinical information available for research. Here we detail a National Institutes for Neurological Disease and Stroke Center Without Walls project, the COllaborative Neuropathology NEtwork Characterizing ouTcomes of TBI (CONNECT-TBI), designed to address current limitations in tissue and research access and to advance understanding of the neuropathologies of TReND. As an international, multidisciplinary collaboration CONNECT-TBI brings together multiple experts across 13 institutions. In so doing, CONNECT-TBI unites the existing, comprehensive clinical and neuropathological datasets of multiple established research brain archives in TBI, with survivals ranging minutes to many decades and spanning diverse injury exposures. These existing tissue specimens will be supplemented by prospective brain banking and contribute to a centralized route of access to human tissue for research for investigators. Importantly, each new case will be subject to consensus neuropathology review by the CONNECT-TBI Expert Pathology Group. Herein we set out the CONNECT-TBI program structure and aims and, by way of an illustrative case, the approach to consensus evaluation of new case donations.

Abstract Frontotemporal lobar degeneration proteinopathies with tau inclusions (FTLD-Tau) or TDP-43 inclusions (FTLD-TDP) are associated with clinically similar phenotypes. However, these disparate proteinopathies likely differ in cellular severity and regional distribution of inclusions in white matter (WM) and adjacent grey matter (GM), which have been understudied. We performed a neuropathological study of subcortical WM and adjacent GM in a large autopsy cohort (n = 92; FTLD-Tau = 37, FTLD-TDP = 55) using a validated digital image approach. The antemortem clinical phenotype was behavioral-variant frontotemporal dementia (bvFTD) in 23 patients with FTLD-Tau and 42 with FTLD-TDP, and primary progressive aphasia (PPA) in 14 patients with FTLD-Tau and 13 with FTLD-TDP. We used linear mixed-effects models to: (1) compare WM pathology burden between proteinopathies; (2) investigate the relationship between WM pathology burden and WM degeneration using luxol fast blue (LFB) myelin staining; (3) study regional patterns of pathology burden in clinico-pathological groups. WM pathology burden was greater in FTLD-Tau compared to FTLD-TDP across regions (beta = 4.21, SE = 0.34, p

Abstract Introduction Lewy body diseases are pathologically characterized by α‐synuclein pathology. Alzheimer's disease (AD) co‐pathology can influence phenotypes. In vivo AD biomarkers can suggest the presence of this co‐pathology in unusual cases, but pathological validation remains essential. Methods This patient originally presented with corticobasal syndrome and later developed visual hallucinations and parkinsonism consistent with a synucleinopathy. The patient underwent CSF sampling, 18F‐flortaucipir PET scanning, and brain donation with bilateral regions available for digital histological analysis. Results CSF Aβ42 and t‐tau were in the AD range. 18F‐flortaucipir scanning showed right‐lateralized retention in all lobes (t = 4.3‐10.0, P

Frontotemporal lobar degeneration (FTLD) is a heterogeneous spectrum of age-associated neurodegenerative diseases that include two main pathologic categories of tau (FTLD-Tau) and TDP-43 (FTLD-TDP) proteinopathies. These distinct proteinopathies are often clinically indistinguishable during life, posing a major obstacle for diagnosis and emerging therapeutic trials tailored to disease-specific mechanisms. Moreover, MRI-derived measures have had limited success to date discriminating between FTLD-Tau or FTLD-TDP. T2*-weighted (T2*w) ex vivo MRI has previously been shown to be sensitive to non-heme iron in healthy intracortical lamination and myelin, and to pathological iron deposits in amyloid-beta plaques and activated microglia in Alzheimer’s disease neuropathologic change (ADNC). However, an integrated, ex vivo MRI and histopathology approach is understudied in FTLD. We apply joint, whole-hemisphere ex vivo MRI at 7 T and histopathology to the study autopsy-confirmed FTLD-Tau (n = 4) and FTLD-TDP (n = 3), relative to ADNC disease-control brains with antemortem clinical symptoms of frontotemporal dementia (n = 2), and an age-matched healthy control. We detect distinct laminar patterns of novel iron-laden glial pathology in both FTLD-Tau and FTLD-TDP brains. We find iron-positive ameboid and hypertrophic microglia and astrocytes largely in deeper GM and adjacent WM in FTLD-Tau. In contrast, FTLD-TDP presents prominent superficial cortical layer iron reactivity in astrocytic processes enveloping small blood vessels with limited involvement of adjacent WM, as well as more diffuse distribution of punctate iron-rich dystrophic microglial processes across all GM lamina. This integrated MRI/histopathology approach reveals ex vivo MRI features that are consistent with these pathological observations distinguishing FTLD-Tau and FTLD-TDP subtypes, including prominent irregular hypointense signal in deeper cortex in FTLD-Tau whereas FTLD-TDP showed upper cortical layer hypointense bands and diffuse cortical speckling. Moreover, differences in adjacent WM degeneration and iron-rich gliosis on histology between FTLD-Tau and FTLD-TDP were also readily apparent on MRI as hyperintense signal and irregular areas of hypointensity, respectively that were more prominent in FTLD-Tau compared to FTLD-TDP. These unique histopathological and radiographic features were distinct from healthy control and ADNC brains, suggesting that iron-sensitive T2*w MRI, adapted to in vivo application at sufficient resolution, may eventually offer an opportunity to improve antemortem diagnosis of FTLD proteinopathies using tissue-validated methods.

Summary: Neurodegenerative diseases are challenging for systems biology because of the lack of reliable animal models or patient samples at early disease stages. Induced pluripotent stem cells (iPSCs) could address these challenges. We investigated DNA, RNA, epigenetics, and proteins in iPSC-derived motor neurons from patients with ALS carrying hexanucleotide expansions in C9ORF72. Using integrative computational methods combining all omics datasets, we identified novel and known dysregulated pathways. We used a C9ORF72 Drosophila model to distinguish pathways contributing to disease phenotypes from compensatory ones and confirmed alterations in some pathways in postmortem spinal cord tissue of patients with ALS. A different differentiation protocol was used to derive a separate set of C9ORF72 and control motor neurons. Many individual -omics differed by protocol, but some core dysregulated pathways were consistent. This strategy of analyzing patient-specific neurons provides disease-related outcomes with small numbers of heterogeneous lines and reduces variation from single-omics to elucidate network-based signatures.

Abstract Pituicytoma is a rare, poorly characterized tumor of the sellar region that is thought to be derived from neurohypophyseal pituicytes. Resection of pituicytomas is often associated with significant morbidity including diabetes insipidus and panhypopituitarism. Most of the literature on this tumor exists as small case series or case reports. Here we describe a cohort of fourteen pituicytoma resections from eleven patients. The average follow-up on these cases is 3.7 years with some patients having over 10 years of follow-up data available in the electronic medical record. Pituicytomas were frequently misdiagnosed on pre-operative imaging, and surgical resection was associated with persistent endocrine abnormalities. Histologically, the tumors showed a range of morphologies from epithelioid to spindled. All tumors were positive for TTF-1 with variable immunostaining for other markers including GFAP, EMA, S100, SSTR2A, and synaptophysin. Within this cohort are two patients with atypical pituicytomas which showed increased cellularity, pleomorphism, mitoses and elevated Ki-67 proliferation indexes when compared to non-atypical pituicytomas. Next generation sequencing performed on three tumors revealed alterations in genes involved in the MAPK pathway. Additionally, immunohistochemical staining for phosphorylated-ERK was positive in the majority of tumors. Increased awareness of the neoplastic entity and identification of targetable mutations have the potential to decrease the morbidity associated with resection of pituicytomas.

Abstract Introduction In primary progressive aphasia (PPA) patients with autopsy‐confirmed Alzheimer's disease (AD) or frontotemporal lobar degeneration (FLTD), we tested how the core clinical features of logopenic PPA—naming and repetition—change over time and relate to pathologic burden. Methods In PPA with AD (n = 13) or FTLD (n = 16) pathology, Boston Naming Test and Forward Digit Span measured longitudinal naming and repetition; as reference, Mini‐Mental State Examination (MMSE) measured global cognition. Pathologic burden in left peri‐Sylvian regions was related to longitudinal cognitive decline. Results PPA with AD showed greater decline in naming (P = 0.021) and repetition (P = 0.020), compared to FTLD; there was no difference in MMSE decline (P = 0.99). Across all PPA, declining naming (P = 0.0084) and repetition (P = 0.011) were associated with angular, superior‐middle temporal (naming P = 0.014; repetition P = 0.011) and middle frontal (naming P = 0.041; repetition P = 0.030) pathologic burden. Discussion Unique longitudinal profiles of naming and repetition performance in PPA with AD are related to left peri‐Sylvian pathology.

Cell-to-cell transmission of TDP43 occurs in cell cultures and may contribute to pathological TDP43 propagation in FTLD-TDP. In this study, the authors demonstrate using mouse models that a single intracerebral injection of human brain-derived pathological TDP43 from FTLD-TDP cases initiates the process of seeding and spreading of TDP43 pathology in a spatio-temporal dependent manner in the brain.

Abstract Aging-related tau astrogliopathy (ARTAG) describes tau pathology in astrocytes in different locations and anatomical regions. In the present study we addressed the question of whether sequential distribution patterns can be recognized for ARTAG or astroglial tau pathologies in both primary FTLD-tauopathies and non-FTLD-tauopathy cases. By evaluating 687 postmortem brains with diverse disorders we identified ARTAG in 455. We evaluated frequencies and hierarchical clustering of anatomical involvement and used conditional probability and logistic regression to model the sequential distribution of ARTAG and astroglial tau pathologies across different brain regions. For subpial and white matter ARTAG we recognize three and two patterns, respectively, each with three stages initiated or ending in the amygdala. Subependymal ARTAG does not show a clear sequential pattern. For grey matter (GM) ARTAG we recognize four stages including a striatal pathway of spreading towards the cortex and/or amygdala, and the brainstem, and an amygdala pathway, which precedes the involvement of the striatum and/or cortex and proceeds towards the brainstem. GM ARTAG and astrocytic plaque pathology in corticobasal degeneration follows a predominantly frontal-parietal cortical to temporal-occipital cortical, to subcortical, to brainstem pathway (four stages). GM ARTAG and tufted astrocyte pathology in progressive supranuclear palsy shows a striatum to frontal-parietal cortical to temporal to occipital, to amygdala, and to brainstem sequence (four stages). In Pick’s disease cases with astroglial tau pathology an overlapping pattern with PSP can be appreciated. We conclude that tau-astrogliopathy type-specific sequential patterns cannot be simplified as neuron-based staging systems. The proposed cytopathological and hierarchical stages provide a conceptual approach to identify the initial steps of the pathogenesis of tau pathologies in ARTAG and primary FTLD-tauopathies.

Abstract Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is associated with the accumulation of pathological neuronal and glial intracytoplasmic inclusions as well as accompanying neuron loss. We explored if cortical neurons detected by NeuN decreased with increasing TDP-43 inclusion pathology in the postmortem brains of 63 patients with sporadic and familial FTLD-TDP. Semi-automated quantitative algorithms to quantify histology in tissue sections stained with antibodies specific for pathological or phosphorylated TDP-43 (pTDP-43) and NeuN were developed and validated in affected (cerebral cortex) and minimally affected (cerebellar cortex) brain regions of FTLD-TDP cases. Immunohistochemistry (IHC) for NeuN and other neuronal markers found numerous neurons lacking reactivity, suggesting NeuN may reflect neuron health rather than neuron loss in FTLD. We found three patterns of NeuN and pTDP-43 reactivity in our sample of cortical tissue representing three intracortical region-specific stages of FTLD-TDP progression: Group 1 showed low levels of pathological pTDP-43 and high levels NeuN, while Group 2 showed increased levels of pTDP-43, and Group 3 tissues were characterized by reduced staining for both pTDP-43 and NeuN. Comparison of non-C9orf72/GRN FTLD-TDP with cases linked to both GRN mutations and C9orf72 expansions showed a significantly increased frequency of Group 3 histopathology in the latter cases, suggesting more advanced cortical disease. Hence, we propose that IHC profiles of pTDP-43 and NeuN reflect the burden of pTDP-43 and its deleterious effects on neuron health.

Aging-related neurodegenerative diseases are progressive and fatal neurological diseases that are characterized by irreversible neuron loss and gliosis. With a growing population of aging individuals, there is a pressing need to better understand the basic biology underlying these diseases. Although diverse disease mechanisms have been implicated in neurodegeneration, a common theme of altered RNA processing has emerged as a unifying contributing factor to neurodegenerative disease. RNA processing includes a series of distinct processes, including RNA splicing, transport and stability, as well as the biogenesis of non-coding RNAs. Here, we highlight how some of these mechanisms are altered in neurodegenerative disease, including the mislocalization of RNA-binding proteins and their sequestration induced by microsatellite repeats, microRNA biogenesis alterations and defective tRNA biogenesis, as well as changes to long-intergenic non-coding RNAs. We also highlight potential therapeutic interventions for each of these mechanisms.

Summary: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While several pathogenic mutations have been identified, the vast majority of ALS cases have no family history of disease. Thus, for most ALS cases, the disease may be a product of multiple pathways contributing to varying degrees in each patient. Using machine learning algorithms, we stratify the transcriptomes of 148 ALS postmortem cortex samples into three distinct molecular subtypes. The largest cluster, identified in 61% of patient samples, displays hallmarks of oxidative and proteotoxic stress. Another 19% of the samples shows predominant signatures of glial activation. Finally, a third group (20%) exhibits high levels of retrotransposon expression and signatures of TARDBP/TDP-43 dysfunction. We further demonstrate that TDP-43 (1) directly binds a subset of retrotransposon transcripts and contributes to their silencing in vitro, and (2) pathological TDP-43 aggregation correlates with retrotransposon de-silencing in vivo. : Tam et al. present transcriptome profiling results from a large set of amyotrophic lateral sclerosis (ALS) patient cortex samples, finding 3 distinct groups. Two ALS subtypes are marked by gene pathways previously associated with ALS disease, while a third group shows elevated retrotransposon expression linked to TDP-43 pathology. Keywords: amyotrophic lateral sclerosis, retrotransposons, transposable elements, TDP-43, neurodegenerative disease, neurodegeneration, genetics and genomics of ALS

Digital pathology is increasingly prominent in neurodegenerative disease research, but variability in immunohistochemical staining intensity between staining batches prevents large-scale comparative studies. Here we provide a statistically rigorous method to account for staining batch effects in a large sample of brain tissue with frontotemporal lobar degeneration with tau inclusions (FTLD-Tau, N = 39) or TDP-43 inclusions (FTLD-TDP, N = 53). We analyzed the relationship between duplicate measurements of digital pathology, i.e., percent area occupied by pathology (%AO) for grey matter (GM) and white matter (WM), from two distinct staining batches. We found a significant difference in duplicate measurements from distinct staining batches in FTLD-Tau (mean difference: GM = 1.13 ± 0.44, WM = 1.28 ± 0.56; p < 0.001) and FTLD-TDP (GM = 0.95 ± 0.66, WM = 0.90 ± 0.77; p < 0.001), and these measurements were linearly related (R-squared [Rsq]: FTLD-Tau GM = 0.92, WM = 0.92; FTLD-TDP GM = 0.75, WM = 0.78; p < 0.001 all). We therefore used linear regression to transform %AO from distinct staining batches into equivalent values. Using a train-test set design, we examined transformation prerequisites (i.e., Rsq) from linear-modeling in training sets, and we applied equivalence factors (i.e., beta, intercept) to independent testing sets to determine transformation outcomes (i.e., intraclass correlation coefficient [ICC]). First, random iterations (×100) of linear regression showed that smaller training sets (N = 12–24), feasible for prospective use, have acceptable transformation prerequisites (mean Rsq: FTLD-Tau ≥0.9; FTLD-TDP ≥0.7). When cross-validated on independent complementary testing sets, in FTLD-Tau, N = 12 training sets resulted in 100% of GM and WM transformations with optimal transformation outcomes (ICC ≥ 0.8), while in FTLD-TDP N = 24 training sets resulted in optimal ICC in testing sets (GM = 72%, WM = 98%). We therefore propose training sets of N = 12 in FTLD-Tau and N = 24 in FTLD-TDP for prospective transformations. Finally, the transformation enabled us to significantly reduce batch-related difference in duplicate measurements in FTLD-Tau (GM/WM: p < 0.001 both) and FTLD-TDP (GM/WM: p < 0.001 both), and to decrease the necessary sample size estimated in a power analysis in FTLD-Tau (GM:-40%; WM: -34%) and FTLD-TDP (GM: -20%; WM: -30%). Finally, we tested generalizability of our approach using a second, open-source, image analysis platform and found similar results. We concluded that a small sample of tissue stained in duplicate can be used to account for pre-analytical variability such as staining batch effects, thereby improving methods for future studies.

Summary: Loss of the nuclear RNA binding protein TAR DNA binding protein-43 (TDP-43) into cytoplasmic aggregates is the strongest correlate to neurodegeneration in amyotrophic lateral sclerosis and frontotemporal degeneration. The molecular changes associated with the loss of nuclear TDP-43 in human tissues are not entirely known. Using subcellular fractionation and fluorescent-activated cell sorting to enrich for diseased neuronal nuclei without TDP-43 from post-mortem frontotemporal degeneration-amyotrophic lateral sclerosis (FTD-ALS) human brain, we characterized the effects of TDP-43 loss on the transcriptome and chromatin accessibility. Nuclear TDP-43 loss is associated with gene expression changes that affect RNA processing, nucleocytoplasmic transport, histone processing, and DNA damage. Loss of nuclear TDP-43 is also associated with chromatin decondensation around long interspersed nuclear elements (LINEs) and increased LINE1 DNA content. Moreover, loss of TDP-43 leads to increased retrotransposition that can be inhibited with antiretroviral drugs, suggesting that TDP-43 neuropathology is associated with altered chromatin structure including decondensation of LINEs. : Liu et al. fractionated and sorted for diseased neuronal nuclei from post-mortem FTD-ALS human brains and showed that loss of an RNA-binding protein, TDP-43, altered the transcriptome and chromatin accessibility. Their results suggest that loss of nuclear TDP-43 is associated with decondensation of LINE retrotransposons. Keywords: frontotemporal dementia, motor neuron disease, amyotrophic lateral sclerosis, RNA, chromatin, ATAC-seq, autoregulation, splicing, histone, reverse transcriptase

A hexanucleotide G4C2 repeat expansion in C9orf72 is the most common genetic cause of familial and sporadic cases of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). The mutation is associated with a reduction of C9orf72 protein and accumulation of toxic RNA and dipeptide repeat aggregates. The accumulation of toxic RNA has been proposed to sequester RNA binding proteins thereby altering RNA processing, consistent with previous transcriptome studies that have shown that the C9orf72 repeat expansion is linked to abundant splicing alterations and transcriptome changes. Here, we used a subcellular fractionation method and FACS to enrich for neuronal nuclei from C9orf72 repeat expanded post-mortem human ALS/FTD brains, and to remove neuronal nuclei with TDP-43 pathology which are observed in nearly all symptomatic C9orf72 repeat expanded cases. We show that the C9orf72 expansion is associated with relatively mild gene expression changes. Dysregulated genes were enriched for vesicle transport pathways, which is consistent with the known functions of C9orf72 protein. Further analysis suggests that the C9orf72 transcriptome is not driven by toxic RNA but is rather shaped by the depletion of pathologic TDP-43 nuclei and the loss of C9orf72 expression. These findings argue against RNA binding protein sequestration in neurons as a major contributor to C9orf72 mediated toxicity.