Abstract/Summary This supplement proposal will provide a critical and timely opportunity to take advantage of the wide expertise, diverse experimental systems, substantial emerging datasets, and novel impactful findings made by the FunGen- AD consortium. It will support one part of a multi-group collaborative project across the consortium that was not envisioned when the individual grant applications were made but has emerged through information and data sharing within the consortium as each individual project has progressed. The FunGen-AD external advisory board (EAB) has recognized the value and is fully supportive of this collaboration within the consortium. Substantial emerging data support the critical role of the endolysosomal pathway (ELP) and its role in lipid homeostasis (LH) in the development and progression of Alzheimer’s disease (AD). Studies in human brains have highlighted changes in the ELP and LH among the first changes in AD, suggesting that these functionally integrated pathways might provide a target for early intervention. Genome-wide association studies (GWAS) of AD have already highlighted several strong candidate risk genes that are central to ELP/LH, including APOE, SORL1, and BIN1. Over the course of the functional analyses conducted within the FunGen-AD consortium, the role of other known AD-relevant genes, including PICALM, LRP1, and CD2AP have underscored the importance of the ELP/LH pathway. Studies to date indicate that we need to identify more genes implicated in the ELP/LH and their impact on different neural cell types. The results could be transformative in the search for viable targets to combat AD. The ELP/LH systems play critical yet distinct cell type-specific roles in the brain. Hence, a single variant can disrupt ELP/LH in neurons, glial, and vascular cells in different ways. In Aim 1, we will conduct a CRISPRi screen of c.1,000 candidate AD-risk genes in individual human iPSC-derived cell types: neurons, astrocytes, microglia, vascular endothelial cells (ECs), and pericytes. This will comprehensively identify risk genes important for ELP/LH in each major neural cell type. The data will be integrated with that from other collaborating groups, prioritizing genes for additional screens and in-depth functional analyses. Our group is particularly focused on the role of AD risk genes in vascular pathology, which is a prevalent early contributor to AD. Effective ELP/LH function is essential to maintain vascular health, blood brain barrier integrity, and regulating vascular inflammatory status, thus maintaining neural cell health and function. Hence, in Aim 2, we will examine a prioritized subset of ELP/LH genes in the context of vascular ECs. We will perform in-depth characterization of functional outcomes following knockdown of ELP/LH hits in iPSC-derived 2D vascular and 3D neurovascular models. The CRISPR screen and characterization data will be aligned with and inform collaborative studies of the ELP/LH acro...