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Levi Todd, Wesley Jenkins, Marcus Hooper, Connor Finkbeiner, Thomas Reh; Inducing regeneration of retinal ganglion-like cells in vivo from Muller glia in the adult mouse using transcription factors.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1871.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is a leading cause of blindness affecting millions of people worldwide. Glaucoma is characterized by neurodegeneration of retinal ganglion cells (RGCs), the projection neurons of the retina. The mammalian retina lacks an endogenous regenerative capacity so loss of RGCs results in permanent blindness. Therefore, an impetus exists to develop strategies to replace lost neurons to offer therapeutic hope to patients. In this study, we provide evidence that Muller glia (MG) cells in the adult mouse retina can be stimulated to regenerate RGC-like cells.
To induce retinal regeneration we previously developed a transgenic mouse model that allowed for MG-specific inducible expression of the proneural transcription factor Ascl1 (Glast-CreER/LNL-tTA/TetO-Ascl1-GFP). We developed a regeneration paradigm in which Ascl1 induction followed by NMDA-induced neural toxicity and histone de-acetylase inhibition stimulated MG-mediated regeneration of bipolar neurons in the adult mouse retina. In this project, we sought to regenerate retinal ganglion cells by overexpressing RGC-fate determining factors in combination with Ascl1. We generated a mouse to overexpress the RGC-factors Pou4f2 (Brn3b) and Islet1 in MG in vivo (Glast-CreER/LNL-tTA/TetO-Ascl1-GFP/TetO-Pou4f2-Islet1). We then used our previously established in vivo regeneration paradigm to test whether overexpression of Pou4f2 and Islet1 in Ascl1-expressing MG could alter the fate of regenerated neurons towards the RGC-lineage. We used immunohistochemistry, scRNA-seq, and scATAC-seq to characterize the regenerated neurons.
Following our in vivo regeneration paradigm, immunohistochemistry analysis of MG-lineage traced cells revealed that the combination of Pou4f2/Islet/Ascl1 stimulated around 25% of MG to regenerate neurons expressing RGC markers (Pou4f2, Satb1, HuC/D). This neural phenotype was not observed with Ascl1-alone. Follow up characterization using scRNA-seq, reveal these regenerated neurons express many genes associated with developing RGCs and axon outgrowth. scATAC analysis confirms that Pou4f2/Islet1 shifts the chromatin accessibility of Ascl1-induced neurons to a state more akin to endogenous RGCs
This work demonstrates a strategy to induce endogenous regeneration of RGC-like cells from MG.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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