PROJECT SUMMARY Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized by cognitive decline and an accumulation of amyloid pathology. A strong genetically-driven innate immune component is thought to play a pathogenic role in AD, implicating a central role for microglial dysfunction. Microglia are long-lived resident innate immune cells in the central nervous system. Activation of their immune and metabolic pathways lead to innate immune memory (IIM), a functional reprogramming process in which the response to an initial stimulus shapes long-lasting epigenetic modifications which inform the response to subsequent stimuli. IIM may result in enhanced activation (training) or suppression (tolerance) based on the identity of the initial inflammatory stimulus. IIM has been shown to alter pathology in AD mouse models, as a consequence of a sustained alteration in microglial functioning. Several common AD risk variants, including CD33, converge to suppress microglial activation, by decreasing inflammatory signaling or increasing its inhibition. Accordingly, my overall hypothesis is that altered microglial IIM as a result of suppressive genetic AD risk variants is a critical mechanism underlying the observed microglial dysfunction in AD. More specifically, I hypothesize that the suppressive CD33 AD-risk variant will reduce epigenetic and metabolic rewiring that occurs upon cellular activation, impairing microglial IIM. This sustained alteration of responsiveness at the epigenetic level may contribute to microglial failure as a pathogenic mechanism in AD. To address these hypotheses, I propose to examine the mechanisms of human microglial IIM in response to AD associated inflammatory stimuli, including amyloid and tau, and the effect of the CD33 risk variant on this imprinting process. In Aim 1, I will investigate human microglial IIM phenotypic outcomes in HMC3s (a human microglial cell line) edited with CRISPR to carry the CD33 AD risk allele. I will test whether a range of AD-associated inflammatory stimuli produce trained or tolerized IIM responses, and how CD33 genotype affects these outcomes. In Aim 2, I will investigate the epigenetic and metabolic mechanisms underlying IIM in HMC3s carrying the CD33 risk or protective alleles. These studies will provide mechanistic insight into longitudinal interactions between microglia and local brain pathology, as well as how the CD33 risk variant mediates microglial dysfunction and ultimately AD susceptibility. Importantly, they will also advance our understanding of IIM in human microglia, of which little is known, and the IIM phenotypes they adopt in response to AD pathologies. Their successful conclusion may thus open novel avenues for the development of potential therapeutics to target microglial dysregulation in AD, and to ultimately improve patient outcomes.