PROJECT SUMMARY/ABSTRACT A functional nervous system requires active maintenance of neuronal structures by supporting cells. Some supporting cells are phagocytes that engulf unnecessary or damaged neuronal parts by detecting “eat-me” signals exposed on neuronal membranes. Phosphatidylserine (PS) has been shown to be a potent neuronal eat-me signal that mediates phagocytosis of neurons in many neurodegenerative events. Normally hidden in the inner leaflet of the plasma membrane, this phospholipid is exposed specifically on the degenerative parts of neurons to induce phagocytosis. However, it is unknown whether heathy neurons expose PS on their surface in ways unrelated to phagocytosis in vivo. Using an in vivo system to visualize PS exposure on peripheral sensory neurons, it was discovered that certain neurons in Drosophila naturally expose PS on their dendrites in spatially defined patterns without inducing phagocytosis. This finding raises two specific and important questions: (1) How is PS exposure spatially maintained on these dendrites? (2) What prevents these PS- exposing dendrites from being engulfed by phagocytes? The overall objectives of this proposal are to investigate the mechanistic causes of this unexpected PS exposure on healthy neurons and to elucidate how these PS-exposing neurons are protected against phagocytosis-induced degeneration. Answering these questions is an important step toward our long-term goal of elucidating neuron-phagocyte interactions in the development, maintenance, and degeneration of the nervous system. To achieve these objectives, the following two aims are proposed: (1) Elucidate the mechanisms underlying natural PS exposure on dendrites. Specifically, the endogenous PS flippase ATP8A will be fluorescently tagged in specific neurons to investigate whether the flippase distribution along dendrites defines the patterns of natural PS exposure. In addition, the roles of dendrite excitation and membrane potential in natural PS exposure will be examined. (2) Define the pathways that regulate susceptibility of dendrites to PS-induced degeneration. A pilot RNAi screen revealed the involvement of a septate junction component and a Deg/ENaC sodium channel in regulating the resistance of PS-exposing dendrites to phagocytosis. Thus, it will be tested whether septate junction proteins protect PS- exposing dendrites from being engulfed and whether neuronal activity promotes PS-related degeneration. Together, these aims will define the molecular basis of natural neuronal PS exposure and reveal new mechanisms of neuroprotection. The knowledge gained in this project will be important for understanding how different neuronal types utilize the same phagocytic signal to interact with phagocytes in diverse ways.