Allen, Mariet

AbstractBackgroundA complex, multicellular disease with genetic and immunological elements, Alzheimer’s disease (AD) affects millions worldwide. There has been previous research linking AD to the missense variants ABI3‐rs616338‐T and PLCG2‐rs72824905‐G, and the altered expression of these genes has been shown to disrupt microglial function. In our understanding of AD risk and resilience, limited research has been conducted on how these variants affect microglial subtypes and states in AD.MethodsWe previously identified DOCK8 protein as a target for fluorescent activated nuclei sorting (FANS) to enrich microglia nuclei from frozen human brains. Using this enrichment strategy, we generated microglia enriched snRNAseq data from temporal cortex tissue of 30 donors harboring ABI3 or PLCG2 missense mutations, or neither mutation. We introduced PLCG2 variant in either homozygote, and heterozygote forms into an AD patient derived iPSC through CRISPR/Cas9 approach, differentiated these cells into microglia cells (IMGLs) following established protocols, and generated scRNAseq data after treatment with Amyloidβ. We applied standard alignment and quality control pipelines to the snRNAseq and scRNAseq datasets in parallel. Established markers of microglia states and subtypes were utilized to annotate the clusters; analyzed for their hub gene expression using comparisons between cluster differential profiling to determine signature pathways. Differential gene expression was assessed using pseudo bulk and MAST approaches.ResultsWe obtained single nuclei transcriptome profiles of 54,000 frozen human brain nuclei, of which 35,000 are microglia, through snRNAseq, and single cell profiles of 63,000 iMGLs through scRNAseq, using our standard alignment and quality control procedures. Our differential gene expression analysis identified novel genes and pathways which were identified as part of resilience or AD risk networks in microglia subtypes and states. These alterations were validated using orthogonal experimental methods and further explored for their conservation using in vivo external datasets. Pseudotime inference will be completed to probe microglia marker gene expression dynamics, treatment conditions, along with continuous cell‐state changes.ConclusionOur study uncovers microglia subtype specific resilience and risk factors provided by AD related genetic variants using snRNAseq from frozen human brain and scRNAseq from IPSC derived microglia cells. These findings nominate novel targets and pathways with therapeutic potential.

AbstractBackgroundAlzheimer’s disease (AD) is a devastating neurodegenerative disorder with few therapies to treat, mitigate or prevent its onset. Understanding of this disease is predominantly based on research in non‐Hispanic Whites (NHW) although AD disproportionately affects African Americans (AA) and Latin Americans (LA), underrepresented in AD research. To address this knowledge gap, the Accelerating Medicine Partnership for Alzheimer’s Disease (AMP‐AD) Diversity Working Group was launched to generate multi‐omics data from post‐mortem brain tissue from donors of predominantly AA and LA descent. Here, we focus on genomic and transcriptomics measures, made available through the AD Knowledge Portal (ADKP).MethodBrain tissue from dorsolateral prefrontal cortex (DLPFC), caudate nucleus (CN), superior temporal gyrus (STG) and temporal pole (TP) regions of AA (N = 299), LA (N = 344) and NHW (N = 532) donors was obtained through Mayo Clinic, Mt. Sinai School of Medicine, Emory, Columbia, Rush Alzheimer’s Disease Center, Banner Sun Health, and 1Florida. DNA extracted from DLPFC tissue samples (n = 641) was utilized for whole genome sequencing (WGS) at New York Genome Center (NYGC). RNAseq of 2,224 samples was conducted either at Mayo, Rush or NYGC. Sequencing reads were processed through GenomeGPS and MAPRseq pipelines for WGS and RNAseq data, respectively. Following quality control (QC), RNAseq data was normalized and assessed for sources of variation (SOV). Integration of WGS and RNAseq data to perform comparative eQTL analysis across multi‐ethnic populations and brain regions is ongoing.ResultWGS data for 621 donors, and RNAseq data generated at Mayo (n = 1,136 samples), Rush (n = 627), and NYGC (n = 779) sequencing centers, were deposited to the ADKP. Consensus processing of RNAseq data revealed site, sex, race/ethnicity, RIN, AD diagnosis, age at death and/or library batch as SOV across various sites and brain regions. Principal component analysis of gene expression measures revealed no major stratification by disease or brain region. Accompanying clinical metadata of donors have been distributed through ADKP.ConclusionThese multi‐omics data made available to the research community is expected to be an initial step towards bridging our data and knowledge gap to elucidate conserved and distinct AD related pathways across these underrepresented at‐risk populations.

AbstractBackgroundProgressive supranuclear palsy (PSP) is a devastating primary tauopathy with rapid progression to death. Although several therapies currently in the development pipeline show promising safety profiles and robust target engagement, few demonstrated significant efficacy in patients, underscoring the need to interrogate additional targets with novel therapeutic modalities to expand the potential therapeutic arsenal. To diversify the therapeutic avenues for PSP and related tauopathies (e.g. Alzheimer’s disease), we systematically integrated multi‐omics data from human brains of PSP and control donors with cross‐species validation to nominate high‐confidence therapeutic targets. We plan to translate our findings into safe and effective treatments for PSP using antisense oligonucleotides (ASO).MethodWe analyzed brain gene expression profiles in PSP and control individuals at bulk tissue (N = 408) and single‐cell levels (snRNAseq, N = 36). Cell‐type‐specific expression perturbations were systematically prioritized using a cross‐species validation paradigm, including rTG4510 tau mice and tau‐overexpressing Drosophila models. We conducted in vitro screening of ASO candidates against our prioritized target genes. ASO efficacy and toxicity were measured. RNAseq experiments will be performed to interrogate off‐target effects. The safety and efficacy of the lead ASOs will be assessed in PSP‐patient‐derived iPSC models.ResultWe previously reported discovery of novel genes with significant differential expression in PSP brains, characterized their brain cell‐specificity and validated them using snRNAseq data. We prioritized 21 genes using gene expression data from a mouse tauopathy model. Validation of these 21 high‐priority genes using a Drosophila tau model nominated astrocytic STOM, KANK2, and DDR2 as potential therapeutic targets for PSP. Knocking down their expression in Drosophila significantly rescues tau‐mediated neurodegenerative pathology. In vitro screening identified ASO leads that reduced the target expressions at mRNA and protein levels with low cellular toxicity.ConclusionWe developed a systems biology pipeline and nominated STOM, KANK2, and DDR2 as candidate gene targets for PSP. We identified ASOs that modulate these targets’ expression without cellular toxicity, suggesting they may be suitable as potential therapeutic candidates. Importantly, the shared pathophysiology and molecular aberrations between PSP and other tauopathies, such as Alzheimer’s disease, suggests that such therapeutic candidates may be repurposed for multiple neurodegenerative diseases, further accelerating, and streamlining the therapeutic pipeline.

AbstractBackgroundAlzheimer’s disease (AD) is neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often quantified by Thal phase and Braak stage, respectively. Aβ also frequently deposits in the cerebrovasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. These and related measures often show high variability within AD suggesting distinct underlying mechanisms. We hypothesize that, within the AD brain, neuropathology and levels of core AD‐related proteins are influenced by variations in DNA methylation (DNAm). To test this, we performed epigenome‐wide association studies (EWAS) using DNAm measures from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathologic measures (Braak, Thal, CAA) and brain biochemical levels of five proteins (apoE, Aβ40, Aβ42, tau, p‐tau).MethodsDNAm from neuropathologically‐confirmed AD cases was measured by reduced representation bisulfite sequencing (RRBS) from 471 TCX samples, 200 of which also had CER RRBS. TCX levels of five AD‐related proteins from three tissue fractions (buffer‐, detergent‐, and in‐soluble) were measured previously by ELISA (Liu 2020). CpG methylation (CpGm) was binned by a 15‐state chromatin model (Kundaje 2015) and averaged into CpGm clusters (rCpGm). rCpGms were tested for association with each AD endophenotype and expression levels of nearby genes through multi‐variable linear regression. Replication of significant rCpGms was performed in two independent datasets with AD‐related endophenotypes (Shireby 2022, De Jager 2014).ResultsOur innovative binning method demonstrated biologically relevant CpGm patterns. We found epigenome‐wide significant associations primarily with tau‐related endophenotypes including 93 that had significant and concordant associations in the replication datasets. These rCpGms also significantly associated with expression of nearby genes showing enrichment in oligodendrocyte marker genes including those related to myelination. In vitro validation of tau effects on oligodendrocyte gene expression through DNAm is ongoing.ConclusionsAlthough all endophenotypes tested are core to AD pathophysiology, our results suggest each has a distinct epigenetic architecture underlying their variability in the AD brain. In particular, we found evidence of DNAm variability associating with brain oligodendrocyte gene expression and TCX tau levels. By discovering AD brain endophenotype‐specific DNAm changes, we can identify core components of complex mechanisms revealing important biological insights into AD pathophysiology.

Abstract INTRODUCTION Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, yet our comprehension predominantly relies on studies within non‐Hispanic White (NHW) populations. Here we provide an extensive survey of the proteomic landscape of AD across diverse racial/ethnic groups. METHODS Two cortical regions, from multiple centers, were harmonized by uniform neuropathological diagnosis. Among 998 unique donors, 273 donors self‐identified as African American, 229 as Latino American, and 434 as NHW. RESULTS While amyloid precursor protein and the microtubule‐associated protein tau demonstrated higher abundance in AD brains, no significant race‐related differences were observed. Further proteome‐wide and focused analyses (specific amyloid beta [Aβ] species and the tau domains) supported the absence of racial differences in these AD pathologies within the brain proteome. DISCUSSION Our findings indicate that the racial differences in AD risk and clinical presentation are not underpinned by dramatically divergent patterns in the brain proteome, suggesting that other determinants account for these clinical disparities. Highlights We present a large‐scale proteome (∼10,000 proteins) of DLPFC (998) and STG (244) across AD cases. About 50% of samples were from racially and ethnically diverse brain donors. Key AD proteins (amyloid and tau) correlated with CERAD and Braak stages. No significant race‐related differences in amyloid and tau protein levels were observed in AD brains. AD‐associated protein changes showed a strong correlation between the brain proteomes of African American and White individuals. This dataset advances understanding of ethnoracial‐specific AD pathways and potential therapies.

Abstract BACKGROUND Metabolic dysregulation is a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD) and progressive supranuclear palsy (PSP). Although metabolic dysregulation is a common link between these two tauopathies, a comprehensive brain metabolic comparison of the diseases has not yet been performed. METHODS We analyzed 342 postmortem brain samples from the Mayo Clinic Brain Bank and examined 658 metabolites in the cerebellar cortex and the temporal cortex between the two tauopathies. RESULTS Our findings indicate that both diseases display oxidative stress associated with lipid metabolism, mitochondrial dysfunction linked to lysine metabolism, and an indication of tau‐induced polyamine stress response. However, specific to AD, we detected glutathione‐related neuroinflammation, deregulations of enzymes tied to purines, and cognitive deficits associated with vitamin B. DISCUSSION Our findings underscore vast alterations in the brain's metabolome, illuminating shared neurodegenerative pathways and disease‐specific traits in AD and PSP. Highlights First high‐throughput metabolic comparison of Alzheimer's diesease (AD) versus progressive supranuclear palsy (PSP) in brain tissue. Cerebellar cortex (CER) shows substantial AD‐related metabolic changes, despite limited proteinopathy. AD impacts both CER and temporal cortex (TCX); PSP's changes are primarily in CER. AD and PSP share metabolic alterations despite major pathological differences.

Abstract INTRODUCTION Multi‐omics studies in Alzheimer's disease (AD) revealed many potential disease pathways and therapeutic targets. Despite their promise of precision medicine, these studies lacked Black Americans (BA) and Latin Americans (LA), who are disproportionately affected by AD. METHODS To bridge this gap, Accelerating Medicines Partnership in Alzheimer's Disease (AMP‐AD) expanded brain multi‐omics profiling to multi‐ethnic donors. RESULTS We generated multi‐omics data and curated and harmonized phenotypic data from BA ( n = 306), LA ( n = 326), or BA and LA ( n = 4) brain donors plus non‐Hispanic White ( n = 252) and other ( n = 20) ethnic groups, to establish a foundational dataset enriched for BA and LA participants. This study describes the data available to the research community, including transcriptome from three brain regions, whole genome sequence, and proteome measures. DISCUSSION The inclusion of traditionally underrepresented groups in multi‐omics studies is essential to discovering the full spectrum of precision medicine targets that will be pertinent to all populations affected with AD. Highlights Accelerating Medicines Partnership in Alzheimer's Disease Diversity Initiative led brain tissue profiling in multi‐ethnic populations. Brain multi‐omics data is generated from Black American, Latin American, and non‐Hispanic White donors. RNA, whole genome sequencing and tandem mass tag proteomicsis completed and shared. Multiple brain regions including caudate, temporal and dorsolateral prefrontal cortex were profiled.

AbstractBackgroundA hallmark of Alzheimer’s disease is amyloid‐β (Aβ) deposition in the brain parenchyma as senile plaques which Aß frequently accompanies in the cerebral vasculature as cerebral amyloid angiopathy (CAA). Previous studies demonstrated that the cerebrovascular system is essential for Aβ clearance and interactions exist between cerebrovascular disease and AD. Neuroimaging data, including measures on small vessel disease, amyloid deposits, and microhemorrhages (MCH), can reflect both cerebrovascular disease and AD pathology, thereby serving as intermediate phenotypes of both conditions. We aim to identify genes and co‐expression networks in blood associated with these neuroimaging endophenotypes and the biological pathways enriched in these perturbed transcriptional networks.MethodWe conducted differential gene expression and Weighted Gene Co‐Expression Network analyses (WGCNA) on blood RNA sequencing data of 407 participants from the longitudinally followed Mayo Clinic Study of Aging cohort. The neuroimaging phenotypes include MCH, infarction, and amyloid deposits measured by Pittsburgh compound B positron emission tomography (PiB PET).ResultWGCNA analysis showed a nominally significant association (p<0.05) between several module eigengenes (MEs) and MCH and PiB PET. One module, M42, remained significantly associated with PiB PET after Bonferroni correction for all 43 modules tested. Pathway enrichment analysis of M42 identified two significant pathways (FDR‐adjusted p<0.05) including phosphodiesterase I activity and IgE binding. These pathways contain genes such as MS4A2 encoding the high‐affinity IgE receptor involved in the immune response. Similarly, ENPP2 in the phosphodiesterase I activity pathway represents an additional candidate gene previously implicated in Alzheimer’s disease.ConclusionWe generated co‐expression networks and identified one network module with a statistically significant association between brain amyloid deposits and blood transcriptome data. Further investigations of this network and its enriched pathways are necessary to understand their roles in amyloidosis, CAA, and neurodegeneration. Future studies will include seeking replication of these results in other datasets. Such studies can help elucidate the biological mechanisms of pathogenesis of cerebrovascular damage and amyloid deposits. In addition, in a clinical setting, blood levels of transcripts discovered in this study may serve as predictive or diagnostic biomarkers with mechanistic implications.

AbstractBackgroundInter‐cellular communication within the gliovascular unit (GVU) is critical for cerebral blood flow regulation, and maintenance of the blood‐brain‐barrier (BBB) properties. The breakdown of BBB in Alzheimer’s disease (AD) is well‐established, but precise underlying molecular changes remain unclear. Additionally, whether GVU molecular alterations observed in AD brains are also detected in blood from living patients is unknown. Further, these GVU molecular perturbations require further investigation in different model systems to identify both human brain‐specific and cross‐species conserved alterations. In this study, we investigated prioritized GVU molecules altered in AD brains for their conservation in blood and cross‐species model systems.MethodsWe performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in AD and control brains. We analyzed this data to detect cell‐specific GVU molecular perturbations and their interactions. We investigated molecular interactions between vascular and astrocyte clusters, the major cell types of the GVU of the BBB. To determine whether GVU transcriptional alterations detected in the brain are preserved in the blood, existing blood expression, genetic, and neuroimaging data from two longitudinal antemortem cohorts were analyzed. Using model systems, including mouse, drosophila, and zebrafish, we evaluated the cross‐species conservation of the top GVU alterations detected in AD brains.ResultsBrain snRNAseq revealed transcriptional profiles of 6,541 astrocytes and 2,210 vascular cells. The latter formed three distinct vascular clusters characterized as pericytes, endothelia and perivascular fibroblasts. We identified differentially expressed genes and their enriched pathways within these clusters and observed the highest levels of transcriptional changes within pericytes. Vascular targets that interact with astrocytic ligands have biological functions in cell signaling, angiogenesis, amyloid ß metabolism, and cytoskeletal architecture. We discovered that genetic variants influencing blood expression levels of some of the prioritized GVU genes were associated with neuroimaging burden of cerebrovascular disease in living human cohorts. Our ongoing studies in model systems revealed conservation of some of the top prioritized molecular perturbations across species.ConclusionOur findings prioritized by multiscale, cross‐tissue human data revealed GVU perturbations within interacting pericyte and astrocyte molecules, which are conserved across multiple cross‐species models. These results nominate new molecular targets and mechanistic insights for BBB disruptions in AD.

AbstractBackgroundNeurodegenerative disorders affect different brain regions at different time points and to varying degrees, although the underlying reasons for this variability in regional susceptibility is unknown. Brain regions have different proportions of cell types. This cell proportion variability may be further influenced by presence and degree of brain disorders, including neurodegenerative diseases. We postulate that genetic and other factors that influence brain cell type proportions may also provide insights into brain regional susceptibility to neurodegenerative diseases.MethodsUtilizing bulk transcriptomics data from 7 brain regions obtained from 3 cohorts and corresponding whole genome sequencing (WGS) data, genetic variants were evaluated for association with cell type proportions across brain regions. Utilizing cell type specific marker genes for deconvolution provided by McKenzie et.al, we estimated surrogate variables for proportion (SVP) of various cell types using BRETIGEA. We then evaluated relationship between SVP and AD as well as Age, Sex, APOE ε4 and neuropathology measures. Genetic variants from WGS were then tested for association with SVP to identify distinct and conserved patterns across 6 cell types and 7 brain regions.ResultsAnalysis of SVP by diagnosis, sex, age, APOE ε4 and neuropathology measures identified significant differences in cell proportions in various cell types and brain regions. GWAS of SVP identified 134 loci that reached genome‐wide significance (5e‐8). After conservative adjustment for multiple testing (6 cell types and 7 brain regions; 42 tests), variants proximal to SYNPR and VENTXP7 remained significant (p‐value<1.19E‐09). Variants proximal to SYNPR were associated with increased proportion of oligodendrocytes in cerebellum whereas variants near VENTXP7 were associated with reduced proportion of astrocytes in DLPFC. Variants in SYNPR have previously been associated with neurofibrillary tangles and diffuse plaque measurements while variants in VENTXP7 have been associated with measurement of neurofibrillary tangles, cognitive decline, unipolar depression, and bipolar disorder.ConclusionsIt is likely that there are genetic variants associated with cell proportion in brain, may vary by cell type and brain region, while others may act more broadly. Identification of genetic variants associated with cell proportions in different brain regions may provide insights into affected cells and regional vulnerabilities.