Search

Insoluble fibrillar tau, the primary constituent of neurofibrillary tangles, has traditionally been thought to be the biologically active, toxic form of tau mediating neurodegeneration in Alzheimer disease. More recent studies have implicated soluble oligomeric tau species, referred to as high molecular weight (HMW) due to its properties on size exclusion chromatography, in tau propagation across neural systems. These two forms of tau have never been directly compared. We prepared sarkosyl insoluble and HMW tau from the frontal cortex of Alzheimer patients and compared their properties using a variety of biophysical and bioactivity assays. Sarkosyl insoluble fibrillar tau is comprised of abundant paired helical filaments (PHF) as quantified by electron microscopy (EM), and is more resistant to proteinase K, compared to HMW tau which is mostly in an oligomeric form. Sarkosyl insoluble and HMW tau are nearly equivalent in potency in a HEK cell bioactivity assay for seeding aggregates and their injection reveals similar local uptake into hippocampal neurons in PS19 Tau transgenic mice. However, the HMW preparation appears to be far more potent in inducing a glial response including Clec7a-positive rod-microglia in the absence of neurodegeneration or synapse loss and promotes more rapid propagation of misfolded tau to distal, anatomically connected regions, such as entorhinal and perirhinal cortices. These data suggest that soluble HMW tau has similar properties to fibrillar sarkosyl insoluble tau with regard to tau seeding potential but may be equal or even more bioactive with respect to propagation across neural systems and activation of glial responses, both relevant tau-related Alzheimer phenotypes.

Deciphering the striatal interneuron diversity is key to understanding the basal ganglia circuit and to untangle the complex neurological and psychiatric diseases affecting this brain structure. We performed single-nucleus RNA-sequencing (snRNA-seq) of postmortem human caudate nucleus (CN) and putamen (Pu) samples to elucidate the diversity and abundance of interneuron populations and their transcriptional structure in the human dorsal striatum. We propose a new taxonomy of striatal interneurons with eight main classes. We provide specific markers for all subclasses and validated some of them with quantitativein situfluorescence hybridization, such as a novel PTHLH-expressing population that exhibits different abundance and gene expression between CN and Pu. For the most abundant interneuron populations in human striatum, PTHLH and TAC3, we found matching known mouse interneuron populations based on key functional genes such as ion channels and synaptic receptors. Remarkably, human TAC3 and mouse Th populations share important similarities including the expression of the neuropeptide tachykinin 3. Finally, we were able to integrate our dataset with several prior smaller human striatal snRNA-seq studies, thus supporting the generalizability of this new harmonized taxonomy.

INTRODUCTIONOmics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer’s disease (AD) but the spatial relationships with plaques and tangles andAPOE-linked differences remain unclear.METHODSWe performed laser capture microdissection of Aβ plaques, the 50μm halo around them, tangles with the 50μm halo around them, and areas distant (>50μm) from plaques and tangles in the temporal cortex of AD and control donors, followed by RNA-sequencing.RESULTSAβ plaques exhibited upregulated microglial (neuroinflammation/phagocytosis) and downregulated neuronal (neurotransmission/energy metabolism) genes, whereas tangles had mostly downregulated neuronal genes. Aβ plaques had more differentially expressed genes than tangles. We identified a gradient Aβ plaque>peri-plaque>tangle>distant for these changes. ADAPOEε4 homozygotes had greater changes thanAPOEε3 across locations, especially within Aβ plaques.DISCUSSIONTranscriptomic changes in AD consist primarily of neuroinflammation and neuronal dysfunction, are spatially associated mainly with Aβ plaques, and are exacerbated by theAPOEε4 allele.

Astrocytes play a critical role in brain homeostasis and normal functions but their changes along the spatiotemporal progression of Alzheimer’s disease (AD) neuropathology remain largely unknown. Here we performed single-nucleus RNA-sequencing on brain regions along the stereotypical progression of AD pathology from donors ranging the entire normal aging-AD continuum comprising 628,943 astrocyte nuclei from 32 donors across 5 brain regions. We discovered temporal gene-expression-trajectories with gene sets differentially activated at various disease stages. Surprisingly, a gene set enriched in proteostasis and energy metabolism, was upregulated in late-stage but unexpectedly returned to baseline levels in end-stage, suggesting exhaustion of response in “burnt-out” astrocytes. The spatial gene-expression-trajectories revealed that astrocytic genes of tripartite synapses are dysregulated in parallel to the stereotypical progression of tangle pathology across regions. We identified astrocyte heterogeneity across brain regions with a continuum from homeostatic to reactive cells through “intermediate” transitional states. These findings suggest complex astrocytic dysfunction in AD neurodegeneration.

In Alzheimer’s disease (AD), Amyloid-beta (Aβ) oligomers are considered an appealing therapeutic- and diagnostic target. However, to date, the molecular mechanisms associated with the pathological accumulation or structure of Aβ oligomers remains an enigma to the scientific community. Here we demonstrate the strong seeding properties of unique Aβ fragment signatures and show that the truncated Aβ peptides of residues Aβ1-23, Aβ1-24 and Aβ1-25, rapidly seed to form small, SDS-PAGE stable assemblies of ∼5kDa to ∼14kDa molecular mass range. Mass spectrometry analysis of SDS-PAGE fractionated and gel extracted oligomers revealed that the truncated Aβ isoforms of residues 1-23 to 1-25 form stable entities with low molecular weight (LMW) oligomers, which strongly resemble the regularly reported Aβ entities of putative dimeric or trimeric assemblies found in human post-mortem AD and Tg mouse brain extracts. Furthermore, electrophysiological recordings in the mouse hippocampus indicate that LMW Aβ assemblies formed by fragments Aβ1-23 to Aβ1-25 significantly impair long-term-potentiation (LTP) in the absence of full-length Aβ1-42. Extensive antibody screening highlights the important observation, that the LMW Aβ assemblies formed by these truncated Aβ peptides escape immuno-detection using conventional, conformation specific antibodies but, more importantly, the clinical antibody aducanumab. Our novel findings suggest that there are new Aβ target “loopholes” which can be exploited for the development of therapeutic antibodies with binding properties against stable target hotspots present in Aβ oligomers. We provide here a first example of a new class of monoclonal antibody with unique binding properties against LMW Aβ oligomers, in the absence of binding to large fibrillar Aβ assemblies, or dense amyloid plaques. Our research supports a novel, unparalleled approach for targeting early, pathological Aβ species during the insidious phase of AD and prior to the appearance of large oligomeric or protofibrilar assemblies.

Difficulty achieving complete, specific, and homogenous staining is a major bottleneck preventing the widespread use of tissue clearing techniques to image large volumes of human tissue. In this manuscript, we describe a procedure to rapidly design immunostaining protocols for antibody labeling of cleared brain tissue. We prepared libraries of .5-1.0 mm thick tissue sections that are fixed, pre-treated, and cleared via similar, but different procedures to optimize staining conditions for a panel of antibodies. Results from a library of mouse tissue correlate well with results from a similarly prepared library of human brain tissue, suggesting mouse tissue is an adequate substitute for protocol optimization. These data show that procedural differences do not influence every antibody-antigen pair in the same way, and minor changes can have deleterious effects, therefore, optimization should be conducted for each target. The approach outlined here will help guide researchers to successfully label a variety of targets, thus removing a major hurdle to accessing the rich 3D information available in large, cleared human tissue volumes.

Epigenome-wide association studies (EWAS) assessing the link between DNA methylation (DNAm) and phenotypes related to structural brain measures, cognitive function, and neurodegenerative diseases are becoming increasingly more popular. Due to the inaccessibility of brain tissue in humans, several studies use peripheral tissues such as blood, buccal swabs, and saliva as surrogates. To aid the functional interpretation of EWAS findings in such settings, there is a need to assess the correlation of DNAm variability across tissues in the same individuals. In this study, we performed a correlation analysis between DNAm data of a total of n = 120 matched post-mortem buccal and prefrontal cortex samples. We identified nearly 25,000 (3% of approximately 730,000) cytosine-phosphate-guanine (CpG) sites showing significant (false discovery rate q

Primary age-related tauopathy (PART) is a neurodegenerative tauopathy with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-β (Aβ) deposition in plaques. The pathogenesis of PART is unknown, but evidence suggests it is associated with genes that promote tau pathology as well as others that protect from Aβ toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n=647) using Braak neurofibrillary tangle stage as a quantitative trait adjusting for sex, age, genotyping platform, and principal components. We found significant associations with some candidate loci associated with AD and progressive supranuclear palsy, a primary tauopathy (SLC24A4, MS4A6A, HS3ST1, MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brain from tauopathies containing isoforms with four microtubule-binding domain repeats (4R) and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation revealed a direct and specific binding of JADE1 protein to tau containing four (4R) and no N-terminal inserts (0N4R) in post-mortem human PART brain tissue. Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a mediator of neurofibrillary degeneration.

Transposable elements comprise almost half of the mammalian genome. A growing body of evidence suggests that transposable element dysregulation accompanies brain aging and neurodegenerative disorders, and that transposable element activation is neurotoxic. Recent studies have identified links between pathogenic forms of tau, a protein that accumulates in Alzheimer disease and related tauopathies, and transposable element-induced neurotoxicity. Starting with transcriptomic analyses, we find that age- and tau-induced transposable element activation occurs in the mouse brain. Among transposable elements that are activated at the RNA level in the context of brain aging and tauopathy, we find that the endogenous retrovirus (ERV) class of retrotransposons is particularly enriched. We show that protein encoded by Intracisternal A-particle, a highly active mouse ERV, is elevated in brains of tau transgenic mice. We further demonstrate that brains of tau transgenic mice contain increased DNA copy number of transposable elements, raising the possibility that these elements actively retrotranspose in the context of tauopathy. Taken together, our study lays the groundwork for future mechanistic studies focused on transposable element regulation in the aging mouse brain and in mouse models of tauopathy, while providing support for therapeutic approaches targeting transposable element activation for Alzheimer disease and related tauopathies.

Although the molecular mechanisms underlying amyotrophic lateral sclerosis (ALS) are not yet fully understood, recent studies have described alterations in tau protein in both sporadic and familial ALS. However, it is unclear whether alterations in tau contribute to ALS pathogenesis. Here, we leveraged the ALS Knowledge Portal and Project MinE data sets and identified specific genetic variants clustering within the microtubule-binding domain of MAPT, which were unique to ALS cases. Furthermore, our analysis in a large post-mortem cohort of ALS and control motor cortex demonstrates that although there was no significant difference in the presence of phosphorylated tau (pTau) neuropil threads and neurofibrillary tangles between the two groups, pTau-S396 and pTau-S404 mis-localized to the nucleus and synapses in ALS. This was specific to the C-terminus phosphorylation sites as there was a significant decrease in pTau-T181 in ALS synaptoneurosomes compared to controls. Lastly, while there was no change in total tau or pTau-T181 in ALS CSF, there was a decrease in pTau-T181:tau ratio in ALS CSF, as previously reported. Importantly, CSF tau levels were increased in ALS patients diagnosed with bulbar onset ALS, while pTau-T181:tau ratio was decreased in ALS patients diagnosed with both bulbar and limb onset. Additionally, there was an inverse correlation between tau levels in the CSF and the revised ALS functional rating scale (ALSFRS-R) as well as a correlation between pTau-T181:tau ratio and ALSFRS-R. While there were no longitudinal alterations in tau, pTau-T181 and pTau-T181:tau ratio, there was an increase in the rate of ALSFRS-R decline per month associated with increases in tau levels. This decline was also inversely correlated with increases in pTau-T181 in relation to tau levels. Taken together, our findings demonstrate that, like Alzheimer’s disease, hyperphosphorylated tau is mis-localized in ALS and that decreases in CSF pTau-T181 may serve as a biomarker in ALS.

Both the replication of protein aggregates and their spreading throughout the brain are implicated in the progression of Alzheimer’s disease (AD). However, the rates of these processes are unknown and the identity of the rate-determining process in humans has therefore remained elusive. By bringing together chemical kinetics with measurements of tau seeds and aggregates across brain regions, we are able to quantify their replication rate in human brains. Remarkably, we obtain comparable rates in several different datasets, with 5 different methods of tau quantification, from seed amplification assaysin vitroto tau PET studies in living patients. Our results suggest that the overall rate of accumulation of tau in neocortical regions is limited not by spreading between brain regions but by local replication, which doubles the number of seeds every ~5 years. Thus, we propose that limiting local replication constitutes the most promising strategy to control tau accumulation during AD.

SummaryNeuropathological and experimental evidence suggests that the cell-to-cell transfer of a-synuclein has an important role in the pathogenesis of Parkinson’s disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook an siRNA, genome-wide high throughput screen to identify genes regulating the cell-to-cell transfer of a-synuclein. We transiently transfected HEK cells stably overexpressing a-synuclein with a construct encoding GFP-2a-aSynuclein-RFP. The cells expressing a-synuclein-RFP through transfection were double positive for GFP and RFP fluorescence, whereas the cells receiving it through transfer were positive only for RFP fluorescence. The amount of a-synuclein transfer was quantified by high content microscopy. A series of unbiased screens confirmed the involvement of 38 genes in the regulation of a-synuclein-RFP transfer. One of those hits was ITGA8, a candidate gene recently identified through a large PD genome wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) showed that the hits clustered in networks that included known PD Mendelian and GWAS risk genes more frequently than expected than random chance. Given the genetic overlap between a-synuclein transfer and PD, those findings provide supporting evidence for the importance of the cell-to-cell transfer of a-synuclein in the pathogenesis of PD, and expand our understanding of the mechanism of a-synuclein spread.

Deposits of Aβ peptides (plaques) and tau protein (neurofibrillary tangles (NFTs)) are ubiquitous features of brain tissue in Alzheimer’s disease. Their contribution to disease etiology remains controversial. The molecular-to-nano-scale organization of fibrillar species in these protein aggregates remains uncertain, but may contain clues as to the contributions of these structures to disease. Whether or not all plaques are the same structure, and all tangles are the same, has implications for current hypotheses about polymorphic templated misfolding of their constituent proteins, Aβ and tau. Here we use x-ray microdiffraction in the small-angle regime (SAXS) to probe the molecular organization of these deposits. Using unstained histological sections of human brain tissue, we demonstrate that SAXS can characterize Aβ fibrils and tau filaments in situ. Aβ fibrils have a cross-sectional radius of gyration (Rxc) of ~45 Å, and larger (Rxc >150 Å) aggregates appear to represent Aβ fibrils that have coalesced side-to-side with one another to create fibrillar bundles or macrofibrillar aggregates. Tau fibrils exhibit an Rxc of ~55 Å with little sign of coalescence into larger structure. The in situ mapping of these structures revealed subtle variation in Aβ structure across different brain areas and different cases.

Each additional copy of the apolipoprotein E4 (APOE4) allele is associated with a higher risk of Alzheimer’s dementia, such that APOE4 homozygotes have a particularly high risk. While the APOE2 allele is associated with a lower risk of Alzheimer’s dementia, it is not yet known whether APOE2 homozygotes have a particularly low risk. We generated Alzheimer’s dementia odds ratios and other findings in more than 5,000 clinically characterized and neuropathologically characterized Alzheimer’s dementia cases and controls. APOE2/2 was associated with exceptionally low Alzheimer’s dementia odds ratios compared to APOE2/3, 3/3 and 4/4, and the impact of APOE2 and APOE4 gene dose was significantly greater in the neuropathologically confirmed group than in more than 24,000 neuropathologically unconfirmed cases and controls. Finding and targeting the factors by which APOE and its variants influence Alzheimer’s disease could have a major impact on the understanding, treatment and prevention of this terrible disease.

Identifying asymptomatic older individuals at elevated risk for developing Alzheimer’s disease (AD) is of clinical importance. Among 1,081 asymptomatic older adults, a recently validated polygenic hazard score (PHS) significantly predicted time to AD dementia and steeper longitudinal cognitive decline, even after controlling for APOE ε4 carrier status. Older individuals in the highest PHS percentiles showed the highest AD incidence rates. PHS predicted longitudinal clinical decline among older individuals with moderate to high CERAD (amyloid) and Braak (tau) scores at autopsy, even among APOE ε4 non-carriers. Beyond APOE, PHS may help identify asymptomatic individuals at highest risk for developing Alzheimer’s neurodegeneration.

ImportanceIdentifying individuals at risk for developing Alzheimer’s disease (AD) is of utmost importance. Although genetic studies have identified APOE and other AD associated single nucleotide polymorphisms (SNPs), genetic information has not been integrated into an epidemiological framework for personalized risk prediction.ObjectiveTo develop, replicate and validate a novel polygenic hazard score for predicting age-specific risk for AD.SettingMulti-center, multi-cohort genetic and clinical data.ParticipantsWe assessed genetic data from 17,008 AD patients and 37,154 controls from the International Genetics of Alzheimer’s Project (IGAP), and 6,409 AD patients and 9,386 older controls from Phase 1 Alzheimer’s Disease Genetics Consortium (ADGC). As independent replication and validation cohorts, we also evaluated genetic, neuroimaging, neuropathologic, CSF and clinical data from ADGC Phase 2, National Institute of Aging Alzheimer’s Disease Center (NIA ADC) and Alzheimer’s Disease Neuroimaging Initiative (ADNI) (total n = 20,680)Main Outcome(s) and Measure(s)Use the IGAP cohort to first identify AD associated SNPs (at p < 10-5). Next, integrate these AD associated SNPs into a Cox proportional hazards model using ADGC phase 1 genetic data, providing a polygenic hazard score (PHS) for each participant. Combine population based incidence rates, and genotype-derived PHS for each individual to derive estimates of instantaneous risk for developing AD, based on genotype and age. Finally, assess replication and validation of PHS in independent cohorts.ResultsIndividuals in the highest PHS quantiles developed AD at a considerably lower age and had the highest yearly AD incidence rate. Among APOE ε3/3 individuals, PHS modified expected age of AD onset by more than 10 years between the lowest and highest deciles. In independent cohorts, PHS strongly predicted empirical age of AD onset (p = 1.1 x 10-26), longitudinal progression from normal aging to AD (p = 1.54 x 10-10) and associated with markers of AD neurodegeneration.ConclusionsWe developed, replicated and validated a clinically usable PHS for quantifying individual differences in age-specific risk of AD. Beyond APOE, polygenic architecture plays an important role in modifying AD risk. Precise quantification of AD genetic risk will be useful for early diagnosis and therapeutic strategies.

The neuropathological hallmarks of Alzheimer disease (AD) include "positive" lesions such as amyloid plaques and cerebral amyloid angiopathy, neurofibrillary tangles, and glial responses, and "negative" lesions such as neuronal and synaptic loss. Despite their inherently cross-sectional nature, postmortem studies have enabled the staging of the progression of both amyloid and tangle pathologies, and, consequently, the development of diagnostic criteria that are now used worldwide. In addition, clinicopathological correlation studies have been crucial to generate hypotheses about the pathophysiology of the disease, by establishing that there is a continuum between "normal" aging and AD dementia, and that the amyloid plaque build-up occurs primarily before the onset of cognitive deficits, while neurofibrillary tangles, neuron loss, and particularly synaptic loss, parallel the progression of cognitive decline. Importantly, these cross-sectional neuropathological data have been largely validated by longitudinal in vivo studies using modern imaging biomarkers such as amyloid PET and volumetric MRI.