A landscape of the genetic and cellular heterogeneity in Alzheimer disease

Background Alzheimer disease (AD) has substantial genetic, molecular, and cellular heterogeneity associated with its etiology. Much of our current understanding of the main AD molecular events associated with the amyloid hypothesis ( APP, PSEN1 and PSEN2 ) and neuroimmune modulation ( TREM2 and MS4A ) is based on genetic studies including GWAS. However, the functional genes, downstream transcriptional ramifications, and the cell-type-specific effects of many GWAS loci remain poorly understood. Understanding these effects can point us to the cellular processes involved in AD and uncover potential therapeutic targets. Methods We applied a genetic-based approach to our sample selection; our cohort included carriers of AD pathogenic mutations ( APP, PSEN1) , risk variants in TREM2 , and the resilience variant (rs1582763) in the MS4A cluster associated with cerebrospinal fluid (CSF) soluble TREM2 levels. We performed single-nucleus RNA-sequencing (snRNA-seq) of 1,102,459 nuclei from the human parietal cortex of these carriers. Following initial unbiased clustering and cell-type annotation, we performed deep subclustering analysis per cell type to identify unique cellular transcriptional states associated with these genetic variants. We identified differentially expressed genes between cell states and genetic variant carriers/controls, and performed differential cell proportion analyses to determine key differences among these carriers. We analyzed sequencing data from human dorsolateral prefrontal cortex and mouse models to replicate the enrichment of unique cell states in genetic variant carriers. Finally, we leveraged these cell-state differential expression results to link genes in AD GWAS loci to their functional cell types. Findings We identified cell-specific expression states influenced by AD genetic factors for neurons and glia. Autosomal dominant AD (ADAD) brains exhibited unique transcriptional states in all cell types. TREM2 variant carrier brains were also enriched for specific microglia and oligodendrocyte subpopulations. Carriers of the resilience MS4A variant were enriched for an altered activated-microglia expression state. We mapped AD GWAS genes to their potential functional cell types, and some, including PLCG2 and SORL1 , were expressed in a broader range of brain cell types than previously reported. Interpretation AD pathogenic, risk, or resilience variants are sufficient to alter the transcriptional and cellular landscape of human brains. Overall, our results suggest that the genetic architecture contributes to the cortical cellular heterogeneity associated with disease status, which is a critical factor to consider when designing drug trials and selecting the treatment program for AD patients. Our findings suggest that integrating genetic and single-cell molecular data facilitates our understanding of the heterogeneity of pathways, biological processes and cell types modulated by genetic risk factors for AD. Funding US National Institutes of Health, Hope Center, Archer foundation, Alzheimer Association, CZI. ### Competing Interest Statement Carlos Cruchaga (CC) receives research support from: Biogen, EISAI, Alector and Parabon. The funders of the study had no role in the collection, analysis, or interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. CC is a member of the advisory board of Vivid genetics, Halia Therapeutics and ADx Healthcare. ### Funding Statement This work was possible thanks to the following governmental grants from the National institute of Health: NIA R01AG057777 (OH), R56AG067764 (OH) P30AG066444 (JCM), P01AGO26276 (JCM), U19AG032438 (RJB), R01AG044546 (CC), P01AG003991 (JCM), RF1AG053303 (CC), RF1AG058501 (CC), U01AG058922 (CC), NINDS R01NS118146 (BAB), RFNS110809 (CMK), R01AG062734 (CMK), U01AG072464 (OH, CMK), the BrightFocus Foundation (CMK), and the Chan Zuckerberg Initiative (CZI). O.H. is an Archer Foundation Research Scientist. This work was supported by access to equipment made possible by the Hope Center for Neurological Disorders, and the Departments of Neurology and Psychiatry at Washington University School of Medicine. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Human Research Protective Office of Washington University in St. Louis gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The single nucleus data from the Knight ADRC is publicly available by request from the National Institute on Aging Genetics of Alzheimer's Disease Data Storage Site (NIAGADS) with accession number [NG00108][1] (). To access the data from the DIAN brain bank, special request must be made using this URL: . The 5xFAD mouse microglia data are located in the Gene Expression Omnibus (GEO database) under the accession number GSE141917 (7). The ROSMAP single nucleus RNA sequencing data is available at Synapse under Synapse ID syn21125841 (). Custom code used to analyze the snRNA-seq data and datasets generated and/or analyzed in the current study are available from the corresponding authors upon request. [1]: /lookup/external-ref?link_type=GEN&access_num=NG00108&atom=%2Fmedrxiv%2Fearly%2F2022%2F09%2F02%2F2021.11.30.21267072.atom