Project Summary/Abstract Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the world and afflicts ~6 million Americans. With no disease-modifying treatments available, this number is expected to double by 2050. Understanding the genetic etiology of AD is critical to inform effective therapies but remains a challenge. Genetic heritability of late-onset AD is ~60–80%, and genome-wide association studies (GWAS) have uncovered dozens of single nucleotide polymorphisms (SNPs) that influence AD risk. Two key challenges for the functional interpretation of AD genetic risk are (1) determining the relevant cell types in which SNPs operate and (2) identifying the genes they dysregulate to drive AD pathogenesis. Single-nucleus sequencing studies of human neurons, microglia, and other brain cell types have begun addressing these challenges. Yet, many AD risk SNPs remain unmapped. One possibility is that they are expressed in other brain cell types missed by current methods. Indeed, though most AD patients exhibit vascular pathology—and vascular cell density approaches total glia density—sequencing studies have lost these cells for unknown reasons. To address this challenge, we invented a new Vessel Isolation and Nuclei Extraction for Sequencing method (VINE-seq) to efficiently capture human brain vascular cell types from postmortem brains for single-nucleus RNA sequencing. Surprisingly, we discovered that 30 of the top 45 nominated AD GWAS genes are enriched in the human brain vasculature. Thus, we hypothesize that AD risk SNPs are active in human brain vascular cell types and that they drive AD genetic risk by functionally dysregulating genes involved in inflammatory and protein transport pathways. Combining VINE-seq with standard single nucleus workflows, we propose to systematically determine the host cell type and target genes for each AD SNP. We will begin by determining the AD GWAS SNPs harbored by brain vascular and parenchymal cell types from high-quality frontal cortical tissue across disease stages (Aim 1). We will then identify the genes disrupted by each AD risk SNP in each brain cell type and define their relationship to AD progression and pathology (Aim 2). Upon the completion of this study, we expect to understand how AD genetic risk variants operate in and dysregulate human brain vascular cells. We will provide an authoritative single nuclei transcriptomic and epigenetic resource to decipher the molecular basis of vascular vulnerability and dysregulation across disease stages, and how they relate to neuronal and glial dysfunction. Given the importance of vascular function for brain health, the insights we reveal here may be critical to understanding and treating AD and mixed pathology dementias.