Vascular contributions to dementia and Alzheimer’s disease (AD) increasingly are being recognized. Recent studies have shown that blood-brain barrier (BBB) breakdown is an early independent biomarker of human cognitive dysfunction, including the early clinical stages of AD. Apolipoprotein E4 (APOE4), the major AD susceptibility gene, exerts strong cerebrovascular toxic effects, including accelerated BBB breakdown and degeneration of BBB-associated cells such as pericytes that maintain BBB integrity. Our recent neuroimaging and biomarker data show that APOE4 leads to early BBB dysfunction predicting human cognitive decline, and to neuronal and synaptic dysfunction in humanized APOE4 transgenic mice, and does so independently of the classical Alzheimer’s amyloid beta and tau pathways. However, how APOE’s effects on BBB and BBB-associated cells contribute to vascular and brain dysfunction remains largely unknown. We also do not have an effective APOE-based therapy for AD targeting the cerebrovascular system. To begin addressing these questions, we propose to use stem cell technology to generate new human BBB models with neurons (BBB on a chip) from APOE4 and APOE3 living donors clinically characterized by cognitive, neuroimaging and biomarker studies. We also will use APOE4 and APOE3 new mouse models generated with Cure Alzheimer’s Fund support that allow cell-specific deletion of APOE from BBB-associated cells. We will use molecular (single cell, nuclear RNA-seq, proteomics) analyses of different vascular and neuronal cell types to understand at the cellular and molecular level how APOE4 effects lead to BBB dysfunction, causing brain dysfunction. We will use bioinformatics tools to establish molecular signatures of each studied cell type; compare BBB with neuronal transcriptomes and BBB with synaptic protein interaction networks; relate molecular to functional findings; and predict candidate master regulators for targeting. We expect to identify and validate key new genes, proteins and pathways at the BBB (and the BBB-associated cell types) to slow down vascular and brain disorders caused by APOE4. Future studies will next determine how the APOE4 BBB pathway(s) interact with Alzheimer’s amyloid beta and tau pathways to influence AD pathogenesis, and will develop potentially new therapeutic approaches for dementia and AD based on DNA designer strategies, gene and cell therapy, and/or pharmacologic approaches targeting key pathogenic and protective genes, proteins and pathways at the BBB and the cerebrovascular system to control brain functions.