Large-scale Phenotypic Drug Screen Identifies Neuroprotectants in Zebrafish and Mouse Models of Retinitis Pigmentosa

Abstract

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in zebrafish and mouse RP models, reasoning drugs effective across species may translate better clinically. We first performed a large-scale phenotypic drug screen using a larval zebrafish model of inducible RP. 2,934 compounds, mostly humanapproved drugs, were tested across six concentrations. Statistically, 113 compounds achieved "hit" status. Secondary tests of 42 high-priority hits confirmed eleven lead compounds. Nine leads were then evaluated in mouse RP models, with six exhibiting neuroprotective effects. An analysis of potential mechanisms of action suggested complementary activities. Paired lead compound assays in zebrafish showed additive neuroprotective effects for the majority. These results highlight the value of crossspecies phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for patients with RP and IRDs.

NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

“…In dose-response tests (five-fold dilution series, from 5 mM -0.32 μM, 48 h treatment), the NTR 1.0-expressing line produced an EC50 of 540 μM MTZ ( Fig. 4a), in keeping with our prior observation that rod photoreceptors can be ablated with relatively lower MTZ concentrations (2.5 mM) 17 . The NTR 2.0-expressing line, however, had an EC50 of 3 μM MTZ, equating to ~180fold improvement in ablation efficacy ( Fig.…”

“…On the face of it, the artificial nature of the system suggests there may be limited relevance to degenerative disease mechanisms. However, our recent data suggests NTR/MTZ cell ablation is mediated by DNA-damage induced cell death pathways broadly implicated in neurodegenerative disease 17 . Specifically, inhibition of Poly(ADP) ribose polymerase (PARP) protects cells from NTR/MTZ-induced ablation.…”

“…Importantly, fusion proteins between NTR and fluorescent reporters retain MTZinducible cell-specific ablation activity 10 . We therefore adapted the NTR/MTZ ablation system to zebrafish 11 to expand studies of cellular regeneration 12 , reasoning that coexpression of NTR with reporters would enable visualization 13,14 and quantification [14][15][16][17] of MTZ-induced cell loss, and subsequent cell replacement, in vivo. In general, the NTR/MTZ system has proven useful for targeted cell ablation, and has had widespread uptake 12 .…”

Dendrimer-targeted immunosuppression of microglia reactivity super-accelerates photoreceptor regeneration kinetics in the zebrafish retina

“…To control for the possibility that Dex treatment stimulates rod cell survival rather than promotes regeneration, i.e., acted as a neuroprotectant, we modified the protocol and measured YFP levels at 7 dpf instead of 9 dpf. At this time point, maximal loss of YFP signal is observed in Mtz-only controls (17), facilitating tests for neuroprotective effects, as per a large-scale screen we recently performed to identify compounds promoting rod cell survival (17). Here, compared to larvae treated with Mtz alone, treatment with Dex 24 h after induction of cell death had no effect on rod cell survival (Supp.…”

“…Using a selective rod photoreceptor ablation model (9,17), we first investigated the effects of post-ablation Dex treatments on microglia reactivity in the degenerating retina. Adaptive optical lattice light-sheet microscopy (AO-LLSM) based in vivo time-lapse imaging (18), was used to follow microglia dynamics at high spatial and temporal resolution following induction of rod cell loss.…”