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Liyun Zhang, Conan Chen, Jiang Qian, Hongkai Ji, Donald J Zack, Justin Hanes, David Ackerley, Baerbel Rohrer, Jeff S Mumm; Large-scale Whole-organism Phenotypic Screen Identifies Rod Cell Neuroprotectants Effective in Zebrafish and Mouse Models of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2018;59(9):980. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa (RP) is caused by progressive rod photoreceptor death. We performed a large-scale whole-organism phenotypic screen to identify neuroprotective compounds, which preserve rod cell survival in fish and mouse models of RP.
For the primary screen, a transgenic zebrafish model of RP expressing yellow fluorescence protein (YFP) and nitroreductase (NTR) in rod photoreceptors was used. NTR enabled metronidazole (Mtz)-induced rod cell death, while YFP allowed for quantification of rod cells in vivo. Over 300,000 zebrafish were evaluated in a screen of ~3,000 human-approved compounds tested across six concentrations. Briefly, 5 day post-fertilization (dpf) larvae were dispensed into test compounds or DMSO solution, 4 hours later Mtz was added, the larvae were then incubated for 2 days. Surviving rods were quantified by measuring YFP levels at 7 dpf using a fluorescence plate reader. Initial hits went through a series of orthogonal validation tests, including secondary screening, in vivo confocal microscopy, NTR inhibition, rod genesis and regeneration, and cell death assays. Combinational drug tests were conducted to assess additive effects. Lead candidates were further evaluated in primary mouse retinal cell cultures and Pde6brd1 mouse retinal explants.
114 hits were identified in the primary screen. 42 top-performing drugs underwent secondary and orthogonal validation tests, with 11 compounds being confirmed. Combinatorial assays designed to test complementation between implicated neuroprotective signaling pathways demonstrated additive effects for 7 of 17 tested pairs. Furthermore, two lead candidates protected primary mouse retinal cell cultures from ER stressors, and preserved photoreceptors in Pde6brd1 mouse retinal explants.
Whole-organism phenotypic screening in a zebrafish model of RP identified 2 novel rod cell neuroprotective compounds that were also effective in preserving mouse rod photoreceptors. Combinatorial assays suggest that therapies targeting complementary neuroprotective signaling pathways may provide an improved strategy for RP treatment. Future tests will evaluate extended release formulations of our lead drugs in rd10 (Pde6brd10) mouse retinas. Collectively, these data validate the use of zebrafish retinal disease models for large-scale drug discovery.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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