Project Summary/Abstract Cerebrovascular dysfunction leads to the deposition of amyloid plaques and neurofibrillary tangles in the brain which underlie the development of Alzheimer’s disease (AD); an epidemic that affects more than five million Americans. Brain lipoproteins (BLps) are lipid-protein complexes that are essential for maintaining brain lipid homeostasis and protecting against neurodegeneration. Unfortunately, detailed studies on BLps have been severely limited as their abundance in cerebrospinal fluid (CSF) is 200-fold less than lipoprotein levels in plasma. It is known that BLps are produced independently in the central nervous system and they are thought to emulate compositional and functional features of plasma high-density lipoproteins (HDL). The dominant organizing scaffold on BLps is APOE which has three isoforms—APOE2, APOE3, and APOE4 that differ by cysteine- arginine exchanges at residues 112 and 158. Carriers of APOE4 are at an elevated risk for development and increased severity of AD. We hypothesize that, similar to APOA1 on HDL, APOE acts as a regulatory scaffold on BLps and conformational differences in APOE isoforms result in altered speciation and particle function that underlies the APOE isoform-specific impact on development of AD. We have developed a highly-sensitive lipoprotein profiling technology unique to our laboratory to overcome the BLp abundance hurdle in CSF. Our preliminary work shows that, like plasma HDL, BLps are a widely diverse population of unique particles with distinct protein complements. Moreover, we have developed a protocol for using cryo-electron microscopy to visualize lipid-bound apolipoproteins in unprecedented detail. We will leverage these technologies to 1) determine the impact of APOE isoform on the composition and distribution of BLps and 2) determine the molecular basis of how APOE isoforms structurally regulate BLps. Importantly, we will directly relate differences to clinical AD endpoints in the same individuals. This work will significantly accelerate our understanding of BLp composition, structure, and function and the specific role of APOE in the development of AD.