ABSTRACT The most debilitating forms of nervous system injury are those affecting the central nervous system (CNS). Neuronal circuits damaged by CNS injury exhibit very limited regeneration capacity, leading to enduring neurological deficits. Retinal ganglion cells (RGCs), the projection neurons of the retina, transmit visual information via the optic nerve tracts to the brain. In rodent models, optic nerve crush (ONC) induces RGC death, and surviving RGCs fail to extend axons beyond the site of injury. An established method to promote RGC repair following ONC is lens injury (LI). Recent research has revealed that a conditioning lens injury (cLI), applied several days before ONC, results in significantly enhanced RGC protection and regeneration compared to LI applied concurrently with ONC. However, the cellular and molecular basis underlying cLI-induced RGC repair remains elusive and requires further investigation. In my preliminary studies, I employed single-cell RNA- sequencing (scRNA-seq) on ocular tissues (retina, lens, and vitreous) under regenerative (cLI) and non- regenerative (sham-operated) conditions, which led to the identification of a-Crystallins (encoded by Cryaa and Cryab) as top candidates that are remarkedly upregulated in lens fibroblasts, lens epithelial cells, astrocytes, and Müller glia. Immunofluorescence labeling of the retina demonstrated that a-Crystallins are enriched in the optic nerve fiber layer following cLI. Furthermore, the application of recombinant a-Crystallins to primary RGCs stimulated axon outgrowth in vitro. Importantly, we found post-translational modifications on a-Crystallins that significantly enhance neurite outgrowth of RGCs. In Aim 1, I will conduct a-Crystallin loss-of-function studies (Cryaa-/- and Cryab-/- mice) and gain-of-function studies with recombinant and AAV2-transduced wild type and constitutively active a-Crystallins to test the hypothesis that a-Crystallins are necessary and sufficient for cLI- induced RGC protection and axon regeneration. In Aim 2, I will focus on RGC intrinsic mechanisms of cLI- induced axon regeneration. To this end, I have established methods for isolating RGC nuclei from mice subjected to cLI and sham operation. These RGC nuclei will be used for multi-omics analyses (snRNA-seq and snATAC- seq) to assess changes in chromatin accessibility and to identify core cLI-induced gene regulatory networks (GRNs) associated with RGC protection and axon regeneration. By utilizing in silico approaches, I will identify candidate signaling pathways and GRNS that promote axon outgrowth of injured RGCs. As an intermediary step, I will evaluate the growth potential of candidate genes and signaling pathways in primary RGC cultures. The most promising candidates from these in vitro investigations will then undergo testing for their ability to promote RGC regeneration in vivo. The proposed studies aim to elucidate both extrinsic and intrinsic mechanisms underlying the pro-regenerative eff...