PROJECT SUMMARY 300,000 Americans suffer paralysis from spinal cord axon injury. Glia permit axon regeneration by clearing debris, partly through autophagy. Inappropriate release of glial debris can inhibit axon regeneration. To treat axon injuries, it is essential to understand mechanisms of glial recovery after axon injury, permitting glia to facilitate axon regeneration. Larval fly sensory neurons are a genetically-tractable model for axon regeneration in the mammalian peripheral nervous system, with regrowth dependent on wrapping glia. In my postdoctoral experiments, I have discovered that fly sensory glia release large exopher-like vesicles (ELVs) (~3 µm) at the injury site, a phenomenon previously undescribed in glia. Glia also display membrane damage and subsequent repair at the injury site, and loss of wound healing pathway components results in diminished axon regeneration. These data suggest that glia may adapt to the stress of axon injury through ELV release and membrane repair. Exophers, large (~3 µm) vesicles so far documented in C. elegans neurons and mouse cardiomyocytes, sequester oxidatively-stressed mitochondria for release. In mice, the autophagic pathway is required to produce exophers. My preliminary experiments in fly larvae showed loss of an essential autophagy regulator results in smaller glial ELVs and reduced axon regeneration. Together, these data suggest that the wound healing and autophagic pathways are important for glial ELV production and axon regeneration. As previous work in exophers revealed modes of stress recovery, understanding the origins of fly glial ELVs could uncover novel mechanisms of glial recovery from the stress of axotomy, likely necessary for glia to facilitate axon regeneration. This proposal aims to define the origins of this novel glial ELV response and its effects on axon regeneration. Aim1 of this proposal will identify the cell responsible for autophagy-mediated glial ELV release and axon regeneration by utilizing glial- and neuronal-specific RNAi knock-downs. Furthermore, Aim1 will use electron microscopy and fluorescent reporters to determine if ELVs isolate stressed mitochondria, akin to exophers, in an autophagy-dependent manner. Aim 2 of this proposal will identify the cell-responsible for wound-healing mediated axon regeneration, interactions between wound-healing and autophagic pathways, and effects of the wound-healing pathway and extrinsic signals on glial ELV release. This will be achieved via glial- and neuronal- specific RNAi knock-down constructs for components of the wound healing pathway, antibody staining for wound healing components, and a genetic system to simulate axotomy without physical injury. This proposal will uncover novel mechanisms of glial recovery and glial facilitation of axon regeneration. These studies may help inform and improve methods to drive human axon regeneration in the CNS and PNS, aligning with the mission of NINDS. With the expertise of the Song lab...