Category: Chemical Biology
Blinding diseases often involve the loss of a single cell type. In retinitis pigmentosa (RP), rod photoreceptors are lost. Strategies for replacing lost cells are sought as transformative therapies for such conditions. Enhancing the regenerative capacity of endogenous retinal stem cells, i.e., Müller glia (MG) would be ideal. Unfortunately, human MG has limited inherent regenerative capacities. However, MG functions as robustly regenerative stem cells in the zebrafish retina. We hypothesize that identifying factors which alter retinal regeneration kinetics in zebrafish will inform processes aimed at enhancing the regenerative capacity of the human retina.
Accordingly, we have developed whole-organism drug screening platform, automated reporter quantification in vivo (ARQiv), which enables large-scale in vivo drug screening. We are using this platform to identify drugs that alter rod cell regeneration kinetics in a zebrafish RP model. To create an inducible model of RP in fish, a transgenic line expressing a yellow fluorescent protein-nitroreductase (YFP-NTR) fusion protein specifically in rod photoreceptors was established. When YFP-NTR fish are exposed to the NTR prodrug metronidazole (Mtz), rod cell death is induced. For the screen, YFP-NTR larvae are exposed to Mtz for 24 hours to induce rod cell ablation. After Mtz removal, larvae are exposed to either drug or DMSO over three days of recovery and YFP-reporter levels (i.e., rod cells) are quantified using a fluorescent plate reader. Non- parametric multiple group comparisons and strictly standardized mean difference (SSMD) scores are used to identify drugs that enhance the kinetics of rod cell regeneration.
We identified 2 hit compounds in a pilot screen that enhance rod cell regeneration. One, dexamethasone, implicates neuroinflammation as a key regulator of the regenerative process and is being used as a positive control compound for an ongoing ‘repurposing’ screen of ~3,300 FDA-approved drugs. Preliminary results from the ongoing screen demonstrate that the ARQiv-based whole-organism screening platform can successfully identify chemical modulators of retinal neuron regeneration, implicating novel signaling pathways in the regulation of retinal regeneration and thereby providing useful insights toward stimulating reparative processes in the human eye.
Sumitra Sengupta– Postdoctoral fellow, Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, MD