PROJECT SUMMARY Disorders of the peripheral nervous system, particularly those that affect motor function and pain sensitivity are major clinical challenges. Current medications are not curative and only address secondary symptoms of disease. To restore peripheral nerve function after injury, Schwann cells adapt to counteract pathological insults by undergoing a dramatic transformation to generate repair cells. This complex process, termed transdifferentiation, requires coordination between multiple signaling and gene regulation programs. During transdifferentiation Schwann cell myelination, metabolism, adhesion, phagocytic properties and the ability to activate the immune system are adapted to preserve neuronal survival and function. Transcriptional studies of nerve injury models have revealed that several canonical signaling pathways are altered after injury, including: NF-kB, Ras, TGFβ, WNT, and MAPK. Despite the clear upregulation of WNT signaling components, how WNT signaling contributes to the generation of the repair cell is unclear. Poor understanding of the molecular systems controlling repair cell formation and function is a critical barrier towards identifying therapeutic targets to treat nerve injury. We now have the genetic, sequencing and viral tools to dissect how signaling pathways individually contribute to each of the hallmark repair cell phenotypes. We hypothesize that WNT signaling mediates repair cell formation by promoting transdifferentiation and the epithelial-to-mesenchymal transition of repair cells. This grant will 1) provide functional tests of the role of WNT receptors and ligands in the control of repair cell formation 2) determine whether reduction of a WNT antagonist can restore peripheral nerve regeneration after injury. To accomplish these goals, I will leverage my training in disease models of peripheral neuropathy to gain experience in four critical areas: (1) viral vector design of CRISPR/Cas9 tools, and (2) RNA detection and quantification techniques, (3) mechanisms of cellular reprogramming, and (4) professional development. To achieve these training goals, I have assembled an exceptional group of mentors and scientific advisors with specialties in understanding the roles that glia play during development, injury and neurodegeneration, virus-mediated CRISPR/Cas9 delivery, and transcriptomics of myelinated tissues. With expert guidance from this group, the proposed experiments will address the cellular mechanisms of repair cell induction. The results from this application will serve as the basis for an R01 application to dissect how WNT signaling components orchestrate the changes in transcription required to reprogram mature Schwann cells into repair cells and test whether WNT intersects with other relevant intracellular pathways (e.g. MAPK, ERK) during Schwann cell reprogramming. .