Project Summary Peripheral nerve injuries (PNI) cause motoneuron (MN) axotomy, endangering MNs to cell death which results in individuals never achieving a full functional recovery. Therefore, identifying the mechanisms which govern selective MN loss following an injury or insult is crucial for therapeutic advancement in MN disease and for improving patient outcomes. Research has shown microglia become activated, proliferate and migrate to injured MN cell bodies after PNI, displaying a range of characteristics from pro-regenerative to degenerative. Currently, it is unknown what types of microglia-MN interactions occur on injured MNs destined for survival or cell death. It is also unknown how microglia monitor MN health status, how this relates to MN fate, or what signaling mechanisms regulate these relationships. Our objective is to identify microglial dynamics and cellular communication with injured MNs that determine MN fate following PNI. We will first visualize microglia-MN interactions using advanced microscopy methods and then probe for signaling mechanisms that may regulate microglia behavior on MNs of various health statuses. Colony Stimulating Factor 1 (CSF1), Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), and Fc Receptors (FcR) have all been independently shown to modulate microglial activation via DNAX activating protein of 12kDa (DAP12) and Spleen Tyrosine Kinase (SYK) activation. Literature has shown CSF1 is necessary for microglia proliferation and migration towards injured MNs. Preliminary data shows FcRs are expressed on all activated microglia, whereas TREM2 has a greater expression on microglia surrounding dying MNs compared to surviving or healthy MNs. Given the previous research and our preliminary findings, I predict that microglia will display different dynamics and uptake of MN content depending on the MN health status and that the switch between protective to death environments is governed by the degree of synergistic activation of DAP12 and SYK; more specifically that TREM2 is acting as a molecular switch. To test this, I will use a PNI mouse model (sciatic axotomy) with microglia genetically labeled with GFP and axotomized MNs retrogradely labeled from muscle. This will allow the investigation of microglial-MN interactions on injured MNs of different health statuses. In aim 1, I will quantitatively characterize microglia-MN dynamics with confocal, two-photon, and super-resolution STED microscopy. This aim will reveal how microglia could change from sampling MN contents through endocytosis or trogocytosis to MN removal by neuronal phagocytosis. In aim 2, I will investigate signaling mechanisms that govern microglial phenotypes and their subsequent effect on MN fate. Specifically, we will develop a TREM2 microglia knock-out mouse model to analyze resulting microglia phenotypes and MN interactions, signaling cascades, and any effects on MN regeneration or cell death. This research will provide foundations for neu...