Project Summary Central nervous system (CNS) neurons exhibit a limited capacity to survive and regenerate axons following injury, resulting in functional failure in the brain (e.g., stroke or trauma), spinal cord (e.g., paralysis), or the optic pathway (e.g., blindness). However, 5 to 10% of CNS neurons do survive injury and only 10 to 15% of these surviving CNS neurons regrow axons. Critical to understanding the molecular mechanisms of neuronal survival and regeneration is isolating regenerating from non-regenerating neurons and deciphering their unique gene expression and regulatory profiles following injury. We developed a unique method of purifying regenerated from non-regenerated retinal ganglion cells (RGCs) following injury and identified, using bulk whole-genome methylation sequencing, differentially methylated regions throughout the rat genome capable of distinguishing regenerated from non-regenerated RGCs. Bulk RNA-sequencing of these same two RGC populations revealed differentially expressed genes similarly capable of distinguishing regenerating from non-regenerating RGCs, and some of these differentially expressed genes were also differentially methylated. DNA methylation levels are established and maintained by the folate biochemical pathway; thus, discerning specific DNA methylation alterations that correlate with RGC regeneration supports our previous observations that folic acid supplementation improves the ability of RGCs to regenerate axons following injury. Recent reports showed that 46 different RGC subtypes exist and some of which exhibit particular resilience or susceptibility to neurodegeneration. These findings suggest that select RGC subtypes contribute to axon regeneration. Accordingly, using our published optic nerve transection model and established high-throughput single-cell methylomic and transcriptomic protocols, we will determine the molecular profiles of purified regenerating RGCs to identify specific RGC subtypes that contribute to axon regeneration. Moreover, we hypothesize that folic acid alters select regenerating and non-regenerating RGC subtypes to enhance folic acid-dependent axon regeneration. An improved understanding of how different RGC subtypes contribute to axon regeneration can be leveraged to reveal novel neuroprotective mechanisms for a vista of new research opportunities across various neurological disorders of the brain, spinal cord, and optic pathway.