High-content high-throughput functional genomics in retinal ganglion cells

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Project Summary/Abstract Vision loss and blindness from glaucoma, regardless of the initiating event, are ultimately the result of dysfunction and death of retinal ganglion cells (RGCs). One approach to preserving vision in glaucoma is to find ways to protect retinal ganglion cells and promote their health and survival. As a current fellow in the Wilmer Ophthalmological Institute's Advanced Specialty Training Program in Glaucoma, I have begun working with Dr. Donald Zack on RGC biology. His lab, through a high content screening approach, identified sunitinib as a potent promoter of RGC survival both in vitro and in vivo, following stresses such as axonal injury and glutamate excitotoxicity. Sunitinib is a broadly selective protein kinase inhibitor that is FDA approved for the treatment of a variety of cancers. Given that sunitinib blocks neurotrophin receptor signaling and promotes apoptosis in cancer cells, its neuroprotective activity on RGCs is somewhat surprising. Understanding sunitinib's molecular mechanism could provide important insights into the pathways mediating RGC survival, but deciphering its mechanism has been challenging because sunitinib is active against many different kinases. We hypothesize that some subset of kinases are involved in RGC death and that they can be identified using RNA interference (RNAi)-based screens. To this end, we have adapted RNAi for use in RGCs both in vitro and in vivo. In Specific Aim 1, we will use short-hairpin RNA (shRNA) to knockdown candidate kinases in arrayed cultured murine RGCs coupled with fluorescence-based imaging to identify shRNAs that provide a survival advantage. In Specific Aim 2, we propose an in vivo screen of pooled, virally delivered shRNAs targeting the murine kinome to identify those kinases that mediate RGC death in response to optic nerve transection. I have decided to pursue a career as a university-based clinician scientist, actively providing patient care and educating residents, but with a majority of my effort being directed towards overseeing an active laboratory research program in RGC neuroprotection. To develop the necessary skills and knowledge to conduct competitive research, I am participating in a multi-year mentored research project here at Wilmer with faculty that have expertise in high- content, high-throughput screening, RGC signal transduction and neuroprotection, and animal models of glaucoma and other optic neuropathies.