Reddy, Joseph S.

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often measured by Thal phase and Braak stage, respectively. Frequently, Aβ also deposits in the brain vasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. Assessment of these and related endophenotypes can reveal biological insights into the phenotypic variability in AD. We hypothesize that, amongst AD patients, severity of neuropathology and brain levels of AD‐related proteins are influenced by variation in DNA methylation (DNAm), the identification of which may uncover biological pathways for AD pathogenesis. To test this, we performed epigenome‐wide association studies (EWAS) using DNAm from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathology measures (Braak, Thal, and CAA) and brain levels of five proteins (apoE, Aβ40, Aβ42, tau, and p‐tau).MethodsDNAm from autopsy‐confirmed AD cases available through the Mayo Clinic Brain Bank was measured by reduced representation bisulfite sequencing (RRBS) from 471 brain samples, 200 of which had both TCX and CER measures. Bisulfite converted reads were sequenced on the Illumina High Seq4000 and quality filtered. Neuropathology scoring was performed by an experienced neuropathologist. TCX levels of five AD‐related proteins from three fractions (buffer‐soluble, detergent‐soluble, and insoluble) were measured by ELISA (Liu et al. 2020). Individual CpG methylation ratios were tested for association with each neuropathological and biochemical measure using the R package CpGassoc.ResultsWe identified multiple epigenome‐wide significant CpGs unique to each AD‐related measure. Comparisons between the TCX and CER revealed region specific CpG associations. The effects of sex, age at death, APOE genotype, and presence of genetic variants for each significant CpG association were also identified.ConclusionsAlthough all neuropathological and biochemical measures tested reflect core AD pathologies, our results demonstrate unique associations of epigenetic factors with these individual phenotypes, suggesting that their variability may be governed by distinct epigenetic processes. Discovery of DNAm changes that underlie specific neuropathological or biochemical outcomes can enhance our understanding of AD pathophysiology and may lead to the identification of novel gene or pathway targets for the different facets of this complex condition.

AbstractBackgroundProgressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by cell‐type‐specific tau lesions in neurons and glia. Using bulk brain RNAseq, we previously identified PSP‐associated genes and co‐expression networks that are enriched with cell‐type marker genes. We extended our work to identify cell‐type‐specific expression perturbations in PSP using a systems approach and in vivo validations in a model system.MethodSingle‐nucleus RNAseq (snRNAseq) was performed using 33 frozen brain temporal cortex (TCX) samples from PSP and control brains. After quality control (QC), integration, and clustering of nuclei, each cluster was annotated for its cell type. We identified differentially expressed genes (DEG) in PSP and further investigated them using an independent bulk RNAseq dataset comprising 408 PSP and control TCX samples for replication and assessing co‐expression patterns. Top DEGs were identified and then validated using external bulk RNAseq datasets from a tau transgenic mouse model. Further, the ability of the validated DEGs to rescue tau‐mediated cellular toxicity was evaluated in a tau transgenic Drosophila model.ResultAfter QC, 30 clusters comprised of 27,284 nuclei were obtained, encompassing two neuronal and six glial cell types. DEGs of each cluster revealed cell‐type‐specific PSP associations. Notably, many of the up‐regulated DEGs in the astroglial clusters were members of the bulk RNAseq co‐expression modules that were also up‐regulated in PSP and enriched with astrocytic markers. There were also consistent findings for the up‐regulated DEGs in the endothelial and microglial clusters, and for down‐regulated DEGs in the oligodendroglial clusters. Subsequently, we prioritized 240 DEGs with congruent cell‐type‐specific perturbations in snRNAseq and bulk RNAseq and validated 29 of those using transgenic mouse model data. Lastly, a significant number of the up‐regulated glial genes, when knocked down in a tau transgenic Drosophila model, led to reduced tau‐mediated cellular toxicity, suggesting novel potential therapeutic avenues.ConclusionPSP brains demonstrated robust cell‐type‐specific gene expression perturbations in the glial cells populations that were validated across different datasets. Importantly, inhibiting the up‐regulated expression perturbations rescued tau‐mediated toxicity in a Drosophila model. Our findings highlight the cell‐type‐specific transcriptome changes in PSP and provide potential therapeutic options for primary tauopathies.

AbstractBackgroundInter‐cellular communication within the gliovascular unit (GVU) is critical for cerebral blood flow regulation, maintenance of the blood‐brain‐barrier (BBB) properties, and brain energy metabolism. Although numerous cell biology studies demonstrate the role of the GVU for BBB breakdown in Alzheimer’s disease (AD), the precise molecular changes contributing to its pathophysiology are unclear.MethodWe performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in 24 AD and control brains to understand the transcriptional changes in the cells of GVU. We acquired the snRNAseq profile of 79,751 total brain cells that comprise 6,541 astrocytes and 2,210 vascular cells. The latter formed three distinct vascular clusters characterized as activated pericytes, endothelia and resting pericytes. We identified differentially expressed genes (DEGs) and their enriched pathways in these clusters and detected the most transcriptional changes within activated pericytes. Using our data and a knowledge‐based predictive algorithm, we discovered and prioritized molecular interactions between vascular and astrocyte clusters, the main cell types of the GVU of the BBB.ResultsVascular targets predicted to interact with astrocytic ligands have biological functions in signalling, angiogenesis, amyloid ß metabolism and cytoskeletal structure. Top astrocytic and vascular interacting molecules include both novel and known AD risk genes such as APOE, APP and ECE1. We validated gene expression changes using qPCR. We are in the process of evaluating these gene expression changes in antemortem blood from AD and control participants.ConclusionOur findings provide valuable information into the transcriptional changes in predicted vascular‐astrocytic partners at the GVU, highlighting insights into the molecular mechanisms of BBB breakdown in AD.

Abstract Background: Alterations in innate immunity are pathologically associated with and genetically implicated in Alzheimer’s disease (AD). In the whole exome sequence (WES) dataset generated by the Alzheimer’s Disease Sequencing Project (ADSP), only the previously identified p.R47H variant in the innate immunity gene, TREM2, shows study-wide association with risk of AD. Using a novel approach, we searched the ADSP WES data to identify additional immune pathway genes with deleterious variants that, like TREM2.pR47H, show strong association with AD. Methods: Using polygenic risk scores (PRS) to analyze association with AD, we evaluated deleterious variants (CADD Phred-scaled score > 20) with a minor allele count of 20 or more in 228 genes comprising an immune co-expression network containing TREM2 (CENTREM2). A significant polygenic component composed of deleterious stop-gain and non-synonymous variants was identified, and false discovery rates were determined for the variants in this component. In genes harboring a significant variant, PRS for all variants in the genes were then analyzed. Results: The PRS for the 182 deleterious variants in CENTREM2 showed significant association with AD that was driven by 142 deleterious variants (136 non-synonymous, 6 stop-gain). In the 142 variant polygenic component, four variants had significant AD risk association: TREM2.pR47H, two deleterious stop-gain variants (FCGR1A.pR92X, and LILRB1.pY331X) in novel AD genes and 1 non-synonymous variant (ATP8B4.pG395S). Remarkably, PRS for the 36 additional variants in these four genes also showed significant association with AD. The PRS for all 40 variants in the 4 genes, showed significant, replicable association with AD and 3 additional variants in this polygenic component had significant false discovery rates: ATP8B4.pR1059Q, LILRB1.pP7P, and LILRB1.pY327Y. Conclusions: Here, we identify 3 immune pathway genes (ATP8B4, LILRB1, and FCGR1A) with a variant that associates with AD. Like TREM2.pR47H, each of the variants has a minor allele frequency less than 1% and is a deleterious, protein altering variant with a strong effect that increases or decreases (LILRB1.pY331X) risk of AD. Additional variants in these genes also alter risk of AD. The variants identified here are ideally suited for studies aimed at understanding how the innate immune system may be modulated to alter risk of AD.

AbstractBackgroundAfrican Americans (AA) remain underrepresented in Alzheimer’s disease (AD) research, despite the prevalence of AD being ∼2X higher in this population compared to non‐Hispanic whites. To address this disparity, our group has established the Florida Consortium for African American Alzheimer’s Disease Studies (FCA3DS), focusing primarily on the identification of genetic risk factors and novel plasma biomarkers.MethodUtilizing plasma samples from FCA3DS AD cases (n=158) and cognitively unimpaired elderly controls (n=272), we have performed differential gene expression (DEG) and expression quantitative trait locus (eQTL) analyses on 50 transcripts measured with a custom NanoString® panel that we have designed to measure transcript levels in plasma of genes relevant to AD. Transcript counts were determined using the NanoString® nCounter system, following manufacturer’s protocols. Transcript counts from 43 of the 50 customs assays passed pre‐established quality control thresholds and were utilized for the DEG and eQTL analyses. Our published FCA3DS whole exome sequence (WES) data was utilized for the eQTL analyses. Linear mixed models adjusted for relevant biological and technical covariates were implemented with the lme4 package in R software (v3.6.2) for the DEG and eQTL analyses.ResultAn association with higher plasma CLU in controls compared to AD remained significant after Bonferroni correction for the 43 transcripts tested. The eQTL analysis revealed significant association (FDR q‐value<0.05) with 105 WES variants in cis with 22 of 43 genes tested, including variants in genes previously associated with AD risk in AA. ConclusionThe results from this AA plasma eQTL analysis revealed ABCA7 variants that are significantly associated with lower plasma ABCA7 transcript levels. One of the significant plasma ABCA7 eQTLs was previously shown by our group to be associated with increased AD risk in AA, suggesting that this may be a functional eQTL underlying the risk of AD in AA. The association of higher levels of CLU transcript in plasma from controls compared to AD is in line with results published by others which showed that higher baseline concentration of clusterin protein in plasma was associated with slower rates of brain atrophy. These results support plasma transcripts as novel endophenotypes in AD biomarker studies.

AbstractIntroductionA few copy number variations (CNVs) have been reported for Alzheimer's disease (AD). However, there is a lack of a systematic investigation of CNVs in AD based on whole genome sequencing (WGS) data.MethodsWe used four methods to identify consensus CNVs from the WGS data of 1,411 individuals and further investigated their functional roles in AD using the matched transcriptomic and clinicopathological data.ResultsWe identified 3,012 rare AD‐specific CNVs whose residing genes are enriched for cellular glucuronidation and neuron projection pathways. Genes whose mRNA expressions are significantly correlated with common CNVs are involved in major histocompatibility complex class II receptor activity. Integration of CNVs, gene expression, and clinical and pathological traits further pinpoints a key CNV that potentially regulates immune response in AD.DiscussionWe identify CNVs as potential genetic regulators of immune response in AD. The identified CNVs and their downstream gene networks reveal novel pathways and targets for AD.

Background/Objective: The aim of this study was to determine if plasma concentrations of 5 surrogate markers of Alzheimer’s disease (AD) pathology and neuroinflammation are associated with disease status in African Americans. Methods: We evaluated 321 African Americans (159 AD, 162 controls) from the Florida Consortium for African-American Alzheimer’s Disease Studies (FCA3DS). Five plasma proteins reflecting AD neuropathology or inflammation (Aβ42, tau, IL6, IL10, TNFα) were tested for associations with AD, age, sex, APOE and MAPT genotypes, and for pairwise correlations. Results: Plasma tau levels were higher in AD when adjusted for biological and technical covariates. APOE ɛ4 was associated with lower plasma Aβ42 and tau levels. Older age was associated with higher plasma Aβ42, tau, and TNFα. Females had lower IL10 levels. Inflammatory proteins had strong pairwise correlations amongst themselves and with Aβ42. Conclusion: We identified effects of demographic and genetic variants on five potential plasma biomarkers in African Americans. Plasma inflammatory biomarkers and Aβ42 may reflect correlated pathologies and elevated plasma tau may be a biomarker of AD in this population.

Abstract Background: Genetic studies have shifted to sequencing-based rare variants discovery after decades of success in identifying common disease variants by Genome-Wide Association Studies using Single Nucleotide Polymorphism chips. Sequencing-based studies require large sample sizes for statistical power but often inadvertently introduce batch effects because samples are typically collected, processed, and sequenced at multiple centers. Conventionally, batch effects are first detected and visualized using Principal Components Analysis and then controlled by including batch covariates in the disease association models. For sequencing-based genetic studies, because all variants included in the association analyses have passed quality control measures, this conventional approach treats every variant as equal and ignores the substantial differences still remaining in variant qualities and characteristics such as genotype quality scores, alternative allele fractions (fraction of reads supporting alternative allele at a variant position) and sequencing depths. Results: In the Alzheimer’s Disease Sequencing Project (ADSP) exome dataset of 9,904 cases and controls, we discovered hidden variant-level differences between sample batches of three sequencing centers and two exome capture kits. Although sequencing centers were included as a covariate in our association models, we observed differences at the variant level in genotype quality and alternative allele fraction between samples processed by different exome capture kits that significantly impacted both the confidence of variant detection and the identification of disease-associated variants. Furthermore, we found that the association signals of a subset of top disease risk variants came exclusively from samples processed by one exome capture kit that was more effective at capturing the alternative alleles compared to the other kit. Conclusions: Our findings highlight the importance of additional variant-level quality control for large sequencing-based genetic studies. More importantly, we demonstrate that automatically filtering out variants with batch differences may lead to false negatives if the batch discordance came largely from quality differences and if the variants from one batch had better quality scores.

AbstractIntroductionMAPT H1 haplotype is implicated as a risk factor for neurodegenerative diseases including Alzheimer's disease (AD).MethodsUsing Alzheimer's Disease Genetics Consortium (ADGC) genome‐wide association study (GWAS) data (n = 18,841), we conducted a MAPT H1/H2 haplotype–stratified association to discover MAPT haplotype–specific AD risk loci.ResultsWe identified 11 loci—5 in H2‐non‐carriers and 6 in H2‐carriers—although none of the MAPT haplotype–specific associations achieved genome‐wide significance. The most significant H2 non‐carrier–specific association was with a NECTIN2 intronic (P = 1.33E‐07) variant, and that for H2 carriers was near NKX6‐1 (P = 1.99E‐06). The GABRG2 locus had the strongest epistasis with MAPT H1/H2 variant rs8070723 (P = 3.91E‐06). Eight of the 12 genes at these loci had transcriptome‐wide significant differential expression in AD versus control temporal cortex (q < 0.05). Six genes were members of the brain transcriptional co‐expression network implicated in “synaptic transmission” (P = 9.85E‐59), which is also enriched for neuronal genes (P = 1.0E‐164), including MAPT.DiscussionThis stratified GWAS identified loci that may confer AD risk in a MAPT haplotype–specific manner. This approach may preferentially enrich for neuronal genes implicated in synaptic transmission.