Project Summary During peripheral nerve development, reciprocal interactions between axons and Schwann cells (SCs) regulate the striking morphological and phenotypic changes in both cells required for the assembly and function of myelinated axons in action potential propagation. Axons drive SCs to differentiate and form myelin sheaths whereas myelinating SCs drive the re-organization of axons into polarized domains essential for saltatory conduction. While much is known about the instructive role that neurons play in the maturation of SCs during myelination, surprisingly little is known about how SCs regulate the phenotypic changes that axons undergo as a consequence of myelination. The latter requires profound changes in the expression and distribution of cell adhesion molecules, ion channels, and an expansion of axon diameter due to alterations in the local axonal cytoskeleton, and a new reliance on SCs for trophic and metabolic support. The extent to which these phenotypic changes are mediated by transcriptional changes, translational changes, or post-translational changes, remains poorly understood. Recently there has been a growing appreciation for the role of translation as a key regulatory node in gene expression. This can be explored through analysis of the translatome, which refers to all mRNAs recruited to ribosomes for protein synthesis. Given the striking effects that SCs have on the local structure and function of the axons they myelinate, and given the important role of local translation in regulating protein expression, I hypothesize that myelination significantly alters neuronal protein expression at the level of the translatome. To test this directly, I employed translating ribosome affinity purification (TRAP)-based translatome profiling as a way to isolate pools of actively translating mRNAs in neurons. I generated PVCre; Rpl22HA mice, which express hemagglutinin (HA)-tagged ribosomal protein (Rpl22) in parvalbumin (PV)+ neurons, enabling isolation of ribosome-associated transcripts specifically in PV+ dorsal root ganglion (DRG) neurons with minimal contamination from glial or other transcripts. Using this approach, I carried out translatome profiling of DRG neurons from myelinated and amyelinated mice. My preliminary data has revealed there are distinct pools of transcripts enriched in the translatomes of myelinated vs. amyelinated neurons, and furthermore that the translatomes of amyelinated neurons may be depleted in genes associated with mitochondria and metabolism, thus raising the possibility that SC myelination may regulate mitochondrial biogenesis and function. My proposed research plan aims to (1) elucidate the extent to which these observed myelination-dependent translatome changes result from transcriptional regulation vs. translational regulation, and (2) determine what effect myelination has on mitochondrial biogenesis and function. This work would provide a major, new insight into how axo-glial interactions may re...