Atık, Merve

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.

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.

AbstractBackgroundDementia with Lewy bodies (DLB), the second most prevalent form of degenerative dementia, is characterized by pathological α‐synuclein (αSyn) protein aggregation in neurons. Pathological αSyn shows a protein‐seeding phenomenon that may result in aggregation years before clinical symptoms. Studies using Real‐time quaking‐induced conversion (RT‐QuIC) assay show αSyn seeding from peripheral tissue including skin and cerebrospinal Fluid (CSF). Identifying early biomarkers for DLB is essential for accurate diagnosis, and developing effective treatments to slow progression. Herein, we investigate αSyn seeding activities in CSF from patients with mild cognitive impairment with Lewy bodies (MCI‐LB) phenoconverting to DLB.MethodCSF samples were obtained from 3 MCI‐LB and 2 DLB patients at baseline and two‐year follow‐up appointments. CSF samples were tested for their potential to seed α‐synuclein aggregation using an RT‐QuIC assay. CSF samples were tested by seeding 2 μl of CSF in 98 ul of RT‐QuIC reaction mixture with human recombinant αSyn protein, analyzed on a FLUOstar Omega plate reader. Results were recorded every 30 minutes over a period of 45 hours analyzed based on the reaction kinetics to determine the seeding ability of the α‐synuclein in the CSF samples.ResultWe observed seed amplification at first visit for both DLB patients and one MCI‐LB patient. The absolute quantification of aggregation increased at second visit (2 years apart) for one DLB patient and one MCI‐LB. The other two MCI‐LB patients showed no amplification at baseline visit but showed signal at two‐year visit, by which time all three had phenoconverted to DLB. Interestingly the second DLB patient showed a decline at two‐year follow‐up. The medical and neuroimaging records of that patient indicate that the patient’s cognitive decline began as early‐onset disease (first symptoms at age 50 years).ConclusionWe demonstrate that measures of seed aggregation in CSF with RT‐QuIC are informative in MCI‐LB and DLB, and may also reflect changes in disease progression and dynamics.

AbstractBackgroundCerebral amyloid angiopathy (CAA), characterized by the accumulation of amyloid‐beta in the cerebrovasculature, effects blood vessel integrity leading to brain hemorrhages and an accelerated cognitive decline in Alzheimer’s disease (AD) patients. In a previous genome‐wide association study (GWAS) of 853 neuropathologically confirmed AD cases, we identified a LINC‐PINT splice variant that associated with lower CAA levels amongst individuals that did not have a copy of the APOEe4 AD risk allele (Reddy. J et al 2021). In this study, we perform GWAS to identify further genetic risk factors for CAA and AD neuropathology (Braak stage, Thal phase) in additional AD cases, and participants lacking significant AD pathology (non‐AD).MethodWe expanded the original AD cohort (N = 853) with 400 additional AD cases genotyped using the Infinium Omni2.5+Exome array. We also identified 110 AD and 502 non‐AD donors from the Mayo Clinic Brain Bank, with existing CAA scores and genome‐wide genotypes. We performed QC and imputation of these genotypes to TOPMed. We will conduct GWAS in AD only (N = 1,363), non‐AD only, as well as the combined cohort (N = 1,865). We will test imputed variant dosages for association with square root transformed CAA, Braak and Thal, using linear regression in PLINK, adjusting for relevant covariates. We will perform interaction analysis of CAA‐associated variants with APOEe4 and sex; and pursue stratified analyses. Summary statistics will be utilized to perform SNP‐based gene set enrichment analysis to identify biological pathways implicated in CAA.ResultWe expect that the increased power of this expanded dataset will lead to the identification of additional novel genetic risk factors for CAA and AD neuropathology. Genetic interaction analysis with sex and APOEe4 may further elucidate the role of these risk factors. The addition of 1,102 participants will enable assessment of the LINC‐PINT locus for association with CAA in an independent dataset.ConclusionWe expect this study will provide further insights into the genetic architecture underlying risk for CAA, both in the context of significant AD pathology, and without. Characterization of genetic variants in the context of neuropathology may lead to new avenues of research aimed at identifying biomarkers and therapies to treat CAA, and AD.

AbstractBackgroundAlzheimer’s disease (AD) is a complex, multicellular disease with immunological and genetic components. Previous studies demonstrated associations between AD and the missense variants ABI3‐rs616338‐T and PLCG2‐rs72824905‐G. The perturbed expression of these genes has been demonstrated to disrupt microglial function. To date, the transcriptomic profiles of variant carriers in the context of microglial subtype and state have not been explored in human brain. This represents a knowledge gap in our understanding of AD risk and resilience. We have developed and optimized a single nucleus RNAseq (snRNAseq) approach to study the impact of ABI3 and PLCG2 variants on microglial enriched nuclei from frozen human brain.MethodDOCK8 was identified as a nuclear microglial marker based on previous brain snRNAseq data. DOCK8 based microglial enrichment using fluorescent activated nuclei sorting (FANS) was validated using RT‐qPCR and snRNAseq in human cerebellum and temporal cortex (TCX), respectively. We are applying this enrichment and snRNAseq approach to TCX tissue from a cohort of 30 donors harboring ABI3 or PLCG2 missense mutations, or neither. Standard snRNAseq alignment and quality control pipelines will be applied, and nuclei annotated based on putative microglial markers. Differential gene expression will be assessed using pseudo bulk and MAST approaches, for association with ABI3 and PLCG2 genotype, diagnosis groups, risk factors, and pathology scores.ResultDOCK8 has several commercially available antibodies, making it an ideal microglia‐selection marker. Using RT‐qPCR in microglia enriched nuclei, we demonstrated an enrichment of microglia marker genes and depletion of other brain cell type markers. This is further supported by snRNA‐seq data from DOCK8‐sorted TCX tissue that yielded a 5x increase in microglia proportion (56% vs. 11% in unselected nuclei), enabling analyses of microglia sub‐populations. Leveraging this approach, we will investigate affected genes and pathways and uncover the resilience and risk mechanisms within microglia sub‐populations in ABI3 and PLCG2 variant‐carriers.ConclusionOur study presents a novel method for enriching microglial nuclei from frozen human brains and enables transcriptomic profiling of microglial subpopulations in ABI3 and PLCG2 missense variant‐carriers. We expect these insights will reveal novel genes and pathways that may play a key role in microglial function in AD.