Wednesday, December 4, 2024
12/4/2024
Abstract/Project Summary Glaucoma and other optic neuropathies are characterized by retinal ganglion cell (RGC) non-regenerative axon damage and eventual cell death. Optic nerve crush (ONC), which transects all RGC axons, is often used to model this process, and to seek interventions that preserve RGCs and promote regeneration of their axons. Following ONC in mice, ~80% of the RGCs die within 2 weeks, and virtually none of the survivors regenerate axons. We and others have used ONC to identify interventions that lead to increased survival, increased regeneration or both. However, these treatments are only partially effective and many relevant targets are not well-suited for the clinic. It is therefore important to identify additional and improved promoters of survival and regeneration. Kinases are key regulators of cellular responses to insults and stresses. Furthermore, because of their discrete enzymatic activity, kinases are druggable targets. Thus, we performed a preliminary in vivo CRISPR-assisted kinase screen paired with ONC in adult mice to determine which kinases prevent RGC survival and axon regeneration. To our excitement, the first round of screening through 750 kinases identified 17 anti-survival and 5 anti-regenerative hits. Many of these hits represent new mechanistic and translational insights. In the proposed study, we will first examine the mechanistic underpinnings of two novel target kinase pathways (namely LKB1/SNRK and Mkk4/Mkk7) that we have already verified the efficacy of with larger experimental cohorts beyond our preliminary screen. These experiments will allow us to more completely understand their mechanisms of action to formulate ideal therapeutic interventions. Additionally, we will complete the validation of our remaining target kinases with full experimental cohorts in the ONC model. Finally, we will choose the most promising gRNAs with robust neuroprotective effects and test their ability to protect RGCs in our recently developed glaucoma model. At the completion of these studies, we expect to be well-positioned to leverage the knowledge generated here into druggable targets to treat multiple conditions causing blindness.
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