PROJECT SUMMARY (APOE U19: PROJECT 2) Project 2 seeks to improve understanding of how apolipoprotein E (apoE) isoforms influence glial lipid metabolism and inflammatory responses underlying immune-mediated damage in the brain in Alzheimer’s disease (AD) and aging-related pathologies. Evidence indicates that the apoE protein not only affects Aβ deposition and structure, it strongly influences microglial-activation, Aβ-induced neuritic dystrophy, and tau- mediated neurodegeneration. ApoE produced by both astrocytes and microglia plays an important role in these effects, and microglia are critical effectors of neurodegeneration. In alignment with the ApoE Cascade Hypothesis of this U19, we anticipate that structural differences and related biochemical properties among the apoE isoforms are likely to mediate the differential effects of apoE on the brain’s innate immune response. Factors such as lipid metabolism and sterol processing, can influence microglial reactivity in ways that are relevant to AD pathogenesis. Recent evidence indicates that apoE and aging critically influence the process of cholesterol and lipid clearance in the brain, specifically in microglia. Accumulation of lipid debris in microglia under different conditions is linked with microglial activation and inflammasome-mediated damage. There is also literature and we have preliminary data that 25-hydroxycholesterol (25HC), a known oxysterol immunomodulator, is produced in the brain by microglia and can modulate cholesterol metabolism as well as the microglial cytokine response in an apoE isoform-dependent manner in vitro. These data lead us to hypothesize that apoE-dependent regulation and clearance of cholesterol esters and other lipids specifically in microglia regulates the microglial inflammatory response in aging and AD. We further hypothesize that this microglial response including 25HC production impacts Aβ-induced local damage, Aβ- induced tau spreading, and tau-mediated neurodegeneration in an apoE, age, and sex-dependent fashion. We propose these aims: Aim 1: Determine the effects of apoE and apoE isoforms on cholesterol, cholesterol esters, and other lipids in astrocytes and microglia during aging, in the setting of Aβ and tau pathology, and with LXR agonist stimulation. Aim 2: Assess the effects of the immunomodulatory oxysterol, 25- hydroxycholesterol (25HC) on Aβ-mediated tau-spreading, tau-mediated neurodegeneration, and age-related brain phenotypes and how these are modified by apoE. Aim 3: Assess the effect of apoE isoform on the lipidomic profile of astrocytes, microglia, and secreted apoE-containing lipoproteins as well as the mechanistic relationship between lipid droplet formation, apoE signaling, and inflammation in vitro. We will also assess the mechanism of the effects of ch25h on apoE-mediated neurodegeneration in vitro. Impact/Integration: Project 2 will work closely with Projects 1, 3, 5, Core B, Core D, Core E, Core F, and Core G on production, purification, and characterization of apoE particles and lipids from mouse glia in vitro and in vivo and iPSC derived glia in vitro. Histological analysis in Project 2 will be done with the Neuropathology Core (Core C).