Project Summary Possession of the ɛ4 allele of apolipoprotein E (APOE) is the major genetic risk factor for late onset Alzheimer’s disease (AD), although the direct cause remains a source of debate. While much research has focused on the association between APOE4 and Aβ pathology, there is significant evidence that APOE4 expression effects a wide-array of important pathways in the brain that are independent of Aβ, including my own work showing the effects of APOE4 expression on neuronal hyperactivity, endosomal-lysosomal dysregulation and bioenergetics deficits in AD-vulnerable brain regions. In order to expand on this work and to gain a more comprehensive picture of APOE4’s effects in individual cell populations in both mice and humans, I propose to perform single-nucleus RNA-sequencing on entorhinal cortex (EC) tissues from middle-aged mice and humans expressing APOE4 vs. APOE3. Data generated from this sequencing strategy will be robustly analyzed using modern bioinformatics techniques, and gene and pathway hits will be further validating using molecular biology approaches. As APOE4 is the most important genetic determinant of late-onset AD, discovery of cell-type specific effects of APOE4 on previously identified or novel pathways will greatly increase our knowledge of how late-onset AD develops and has the potential to uncover new therapeutic strategies for preventing or treating AD, especially among APOE4 carriers.