Stimulation of functional neuronal regeneration from Müller glia in adult mice

Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage in zebrafish and is necessary for regeneration. Although Ascl1 is not expressed in mammalian MG after injury, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro and in vivo after NMDA (N-methyl-d-aspartate) damage in young mice. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases. Three ANT-treated Glast-CreER animals had their retinas pooled and were dissociated for FACS purification. Live purified cells were then pelleted at 300 g for 10 minutes at 4 şC and resuspended in 0.04% BSA in PBS at a concentration of 2,000 cells per uL. Cells were then loaded onto the Single Cell 3 Chip with a targeted cell recovery of 4000 cells. For control animals, two Rlbp-CreER: tdTomato animals had their retinas pooled and were run in parallel at equal cell concentrations. GEM generation and barcoding, RT, cleanup, cDNA amplification, and library construction were performed according to the Chromium Single Cell 3’ Reagent Kits User Guide. Library QC was determined by TapeStation (Agilent Technologies, Inc., Santa Clara, CA). Single Cell libraries were sequenced on the Illumina NextSeq 500/550 v2 kit. Reads were processed in Cell Ranger (10x Genomics) and aligned to mm10.