Drug Screening for Neuroprotectants in Novel Zebrafish Models of Retinitis Pigmentosa

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PROJECT SUMMARY Retinitis pigmentosa (RP), an inherited retinal degeneration (IRD) disease, is characterized by primary rod photoreceptor death followed by cone cell death; this results in irreversible vision loss. The rate of cell death in RP is relatively slow, providing a protracted time window for therapeutic intervention. However, pharmacological intervention capable of promoting photoreceptor survival remains an unmet need. While many mouse RP models have been established, accelerating drug discovery by conducting large-scale drug screens in mice is impractical. Zebrafish, however, are an established system for modeling human disease and testing therapeutic compounds. In particular, our previous studies have shown zebrafish are especially valuable for large-scale phenotypic drug screens. Thus, we generated genetic zebrafish RP models by knocking out RP associated genes (e.g. rho, pde6a, and pde6b) with fluorescently labeled rod photoreceptors for easy phenotype monitoring. We identified five models which exhibit various rates and severity of rod cell loss, a key feature of RP. In addition, while the disease etiology is unclear, our preliminary data and recent studies suggest that poly-ADP ribose polymerase (PARP) signaling, a key regulator of the parthanatos cell death pathway, plays a significant role. Therefore, we propose to utilize new zebrafish RP models to investigate photoreceptor cell death mechanism(s), and perform an in vivo phenotypic drug re-purposing screen to identify small molecules that preserve degenerating photoreceptors. In Aim 1, we will measure cellular changes and rod photoreceptor degeneration in the new RP models. In addition, we will evaluate the effect of suppressing poly-PARP signaling, a hall mark of PARP1 activation, to determine if photoreceptor degeneration is ameliorated. This will be accomplished using PARP inhibitors and knocking out PARP1. We will assess how the extent of rod degeneration affects visual function by conducting visual behavior assays with the new RP models. In Aim 2, we will use a phenotypic drug screening platform we developed to identify compounds promoting rod photoreceptors survival in our RP models. We previously screened ~3,000 drugs in over 350,000 zebrafish larvae with inducible rod degeneration and successfully identified retinal neuroprotectants showing efficacy in both fish and mouse models. Here, we will screen ~200 hit drugs, mostly human-approved, from that project. In Aim 3, we will validate the identified neuroprotectants in three RP genetic mouse models. This cross-species drug discover approach will accelerate the translation of identified drug for clinical trial, an approach which can applied to a number of degenerative disease models. Therefore, completion of this project will provide insight into the molecular basis of RP pathogenesis and identify compounds for RP clinical trials.