PROJECT SUMMARY Alzheimer's disease (AD) is the most common form of dementia and is characterized by cognitive impairment and progressive neurodegeneration. Genome-wide association studies of AD have identified more than 30 risk loci; however, a major challenge in the field is that the majority of these risk factors are harbored within non- coding regions where their impact on AD pathogenesis has been difficult to establish. Therefore, the molecular basis of AD development and progression remains elusive and, so far, reliable treatments have not been found. AD-associated changes in the regulation of the epigenome can result from primary genetic and non-genetic causal factors and epiphenomena, including changes secondary to disease progression. Thus, the epigenome is a strong proxy marker to study late-onset diseases such as AD, where there can be a gap of multiple decades between initiation of disease progression and the appearance of symptoms. In the original R01 grant titled “Higher Order Chromatin and Genetic Risk for Alzheimer's Disease”, we expanded the panel of molecular markers in the Mount Sinai Brain Bank AD cohort by generating cell type-specific (neurons and non-neurons) ATACseq in the entorhinal cortex and superior temporal gyrus of AD cases and controls. By doing so, we identified shared and distinct regulatory genomic signatures associated with clinical dementia and neuropathological lesions and with early and late stages of AD. Overlap with AD common risk variants identified primary chromatin accessibility perturbations that are driven by genetic variation, compared to secondary changes with no apparent genetic origin. The overarching goal of this proposal is to address the limitations of our previous research by examining and validating AD-related changes on chromatin accessibility and the 3D genome at the single cell level. Based on recent data from our group and others, we hypothesize that genotype-phenotype associations in AD are causally mediated by cell type-specific alterations in the regulatory mechanisms of gene expression. To test our hypothesis, we propose the following Specific Aims: (1) perform multimodal (i.e., within cell) profiling of the chromatin accessibility and transcriptome at the single cell level to identify cell type-specific AD-related changes on the 3D genome; (2) fine-map AD risk loci to identify causal variants, regulatory regions and genes; (3) functionally validate putative causal variants and regulatory sequences using novel approaches that combine massively parallel reporter assays, CRISPR and single cell assays in neurons and microglia derived from induced pluripotent stem cells; and (4) develop and maintain a community workspace that provides for the rapid dissemination and open evaluation of data, analyses, and outcomes. Overall, our multidisciplinary computational and experimental approach will provide a compendium of functionally and causally validated AD risk loci that has the potential to l...