Abstract
Purpose :
Neurodegeneration in the mammalian retina results in permanent blindness. Significant progress has been made in stimulating Muller glia (MG) to regenerate functional neurons in the adult mouse by transgenic expression of the transcription factor Ascl1. While these results showed that MG can serve as an endogenous source of neuronal replacement, the efficacy of this process is limited: only 25% of the MG generate neurons.
To uncover molecules that enhance neurogenic reprogramming of MG, we combined our in vitro MG reprogramming paradigm with sci-Plex to test a library of 96 compounds for their ability to promote neurogenesis using scRNA-seq as a read out. We identified molecules that increase neurogenesis from MG the adult mouse retina.
Methods :
sci-Plex uses combinatorial indexing of nuclei from individual wells “hashed” with DNA oligos that serve as sample-specific barcodes. For in vitro screening, cultures of MG from postnatal day 11 rtTA:Ascl1:GFP mice were treated with 92 molecules from a Tocris library focused on stem cell targets. Doxycycline was used to induce Ascl1 in MG for 5 days, the cells were processed by sci-Plex, and the scRNAseq output was analyzed. In vivo reprogramming of MG was performed on adult mice (Glast-CreER/LNL-tTA/tetO-Ascl1-GFP) of both sexes; Ascl1 is induced specifically in MG by tamoxifen. To induce regeneration of neurons, we make intravitreal injections of NMDA, followed by HDAC-inhibition (TSA) and hits from the in vitro screen; MG neurogenesis was assessed with immunofluorescence and scRNAseq.
Results :
With the sci-Plex output, we assigned cells to clusters and assessed the effects of the treatments, focusing on compounds that increased MG-derived neurons compared to Ascl1 alone. We uncovered small molecules that target the Notch pathway (DBZ), BMP-pathway (DMH-1), TGF-B signaling (ITD-1, RepSox, SB431542), AMPK-signaling (Metformin) and the Lrc and Srk kinases (WH-4-023) that promote the ability of MG to acquire a neurogenic phenotype. Compounds from the in vitro screen were tested in vivo and both DBZ and Metformin significantly boosted neuronal regeneration in the adult mouse retina.
Conclusions :
Our work outlines a strategy using scRNA-seq and plate-based screening assays to test large libraries of compounds that can enhance the ability of MG to produce neurons for cell replacement approaches.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.