PROJECT SUMMARY/ABSTRACT Loss of synapses is the strongest neuropathological correlate for clinical cognitive decline in Alzheimer’s Disease (AD) patients. In AD mouse models, synapse loss is dependent on neuroinflammatory glial cells and their phagocytic cell surface receptors, many of have been found to interact with extracellularly exposed phosphatidylserine (PS). PS is a plasma membrane phospholipid that is maintained on the cytosolic leaflet but can be flipped to the extracellular leaflet following cellular stress. These interactions led to the hypothesis that glial synapse removal in AD may require synaptic PS exposure. Recent studies have shown that disruption of glial receptor-PS interactions ameliorate synapse loss, electrophysiological deficits, and behavioral phenotypes across several models. While there have been numerous investigations into the mechanisms regulating glial phagocytic receptors, there has yet to be a systematic investigation into mechanisms regulating synaptic PS exposure. I have developed a novel pooled CRISPRi screening platform to systematically elucidate mechanisms of synaptic PS exposure. This initial screen has revealed potential roles for IL-6 and IL-11 signaling in regulating synaptic PS exposure. In my first aim, I propose to determine how the IL-6 and IL-11 signaling pathways differentially affect synaptic PS exposure, glial synaptic engulfment, and in vivo synaptic function both at baseline and within AD model systems using a combination of pharmacological, genetic, and electrophysiological approaches. In my second aim, I propose to identify other novel genetic regulatory mechanisms of synaptic PS exposure in AD. To do this, I will expand my initial functional genomics screen to cover the entire protein-coding genome and conduct differential phosphoproteomic analysis on synapses exposing high and low levels of PS both at baseline and within the context of an in vitro AD model. Finally, I will validate these newly discovered regulators using similar pharmacological, genetic, and electrophysiological approaches as I propose to use in my first aim. Finally, I will study how these validated pathways are differentially expressed within AD patient brains using immunohistochemical techniques. My sponsor, Dr. Martin Kampmann, who co-developed the CRISPRi screening technology, and my cosponsor, Dr. Robert Edwards, who is a physician-scientist and an expert in synaptic biology and neurotransmitter release, are ideally positioned to support my proposed research. In addition to my two sponsors, I will also receive clinical mentorship from Dr. Bruce Miller, a behavioral neurologist who specializes and the diagnosis and management of neurodegenerative diseases with a particular focus on AD and other tauopathies. Overall, the proposed work will uncover novel mechanisms regulating an understudied aspect of AD pathophysiology and identify new therapeutic targets for the treatment of AD. Furthermore, this work will provide me...