Vascular contributions to dementia and Alzheimer’s disease (AD) are increasingly recognized. These findings have led to the “neurovascular hypothesis” of AD, which holds that cerebrovascular dysfunction contributes to cognitive decline and dementia in AD. Apolipoprotein E4 (APOE4), the major AD susceptibility gene, exerts strong cerebrovascular toxic effects, including accelerated blood-brain barrier (BBB) breakdown and degeneration of pericytes, the BBB-associated cells that maintain BBB integrity. Recent studies have suggested that BBB breakdown is an early biomarker of human cognitive dysfunction, including the early clinical stages of AD. Our pilot data show that APOE4 compared with APOE3 leads to an early disruption of the BBB molecular composition followed by dysregulation in multiple signaling mechanisms, which precedes synaptic and neuronal dysfunction and behavioral changes in mice. However, how neurovascular APOE derived from the BBB-associated cells—astrocytes and pericytes—regulates the BBB, synaptic and neuronal functions at the cellular, molecular and systems levels, and in an isoform-specific and gender-specific fashion, remains largely unknown. We also do not have an effective APOE-based therapy for AD targeting the cerebrovascular system. To address these questions, we propose to use humanized APOE4 and APOE3 (control genotype) mouse models generated by Cure Alzheimer’s Fund that allow cell-specific deletion of APOE from the BBB-associated cells astrocytes and pericytes. We will use state-of-the-art molecular analyses (e.g., single nuclear RNA sequencing and novel proteomics analysis) of different vascular cell types and neuronal cells to understand at the cellular and molecular levels how APOE4 affects BBB, synaptic and neuronal functions. We will cross the APOE4 and APOE3 mouse lines with and without APOE in astrocytes and pericytes to mice that model amyloid (APP/PS-21 mice) and tau (P301S mice) AD pathology. This will allow us to evaluate how neurovascular APOE influences BBB integrity in relation to AD pathology using the same molecular methods as above. Finally, we propose to evaluate whether treatment with the novel drug 3K3A-activated protein, which exerts large-scale protective molecular changes in endothelial barriers and neurons, can alleviate harmful effects of APOE4 on cerebral vasculature and synaptic and neuronal function in mice with or without AD-like pathology. If successful, we expect that the proposed studies will advance our understanding of the pathogenesis of AD at the cellular and molecular levels and in an APOE isoform-specific and sex-specific fashion, and will possibly lead to development of new therapeutic approaches for AD.