Abstract
Purpose :
Inherited retinal degenerative diseases can lead to permanent vision loss due to the inability to protect patients from gradual photoreceptor cell death. The purpose of our study was to identify molecules promoting the survival of rod photoreceptors in an inducible zebrafish model of retinitis pigmentosa (RP). To do so, we developed a whole-organism drug screening platform, termed automated reporter quantification in vivo (ARQiv) which enables expansion of in vivo compound testing to true high-throughput capacities.
Methods :
Transgenic zebrafish expressing yellow fluorescent protein (YFP) fused with nitroreductase (NTR) exclusively in rod cells, Tg(rho:YFP-Eco.NsfB), facilitate prodrug-inducible apoptosis of rod photoreceptors. At day 5 of development, zebrafish larvae were dispensed into individual wells of a 96-well plate containing either drug or DMSO. Each drug was tested across six concentrations, a 2-fold dilution series from 4µM to 125nM, at 16 fish per condition. After 4 hours incubation, larvae were treated with 2.5mM metronidazole (Mtz) to induce rod cell ablation. Surviving rod cell numbers were estimated by quantifying YFP reporter expression levels via a fluorescent microplate reader (ARQiv) at day 7. Nonparametric multiple group comparison (Kruskal Wallis test), and strictly standardized mean difference (SSMD) scores were calculated to prioritize hit compounds.
Results :
In preliminary tests, 27 previously implicated neuroprotectants were evaluated. Three drugs, norgestrel, vorinostat, and XL-880, produced an SSMD ≥1.3 indicating a moderate hit. Confirmation of preserved YFP expression levels suggested these drugs significantly protect rod photoreceptors from cell death in vivo. XL-880 was chosen as the positive control compound for an ongoing ‘repurposing’ screen of ~3,300 FDA-approved drugs that, in the end, will involve >500,000 transgenic zebrafish models of RP.
Conclusions :
ARQiv-based whole-organisms HTS drug screening successfully identified neuroprotective compounds in living zebrafish models of RP. Results from the ongoing screen suggest that as many as 25 existing drugs will be identified which have the potential to be repurposed as neuroprotective agents for RP.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.