mi R ‐124‐9‐9* potentiates A scl1‐induced reprogramming of cultured M üller glia

The Müller glia of fish provide a source for neuronal regeneration after injury, but they do not do so in mammals. We previously showed that lentiviral gene transfer of the transcription factor Achaete‐scute homolog 1 (Ascl1/Mash1) in murine Müller glia cultures resulted in partial reprogramming of the cells to retinal progenitors. The microRNAs (miRNAs) miR‐124‐9‐9* facilitate neuronal reprogramming of fibroblasts, but their role in glia reprogramming has not been reported. The aim of this study was to test whether (1) lentiviral gene transfer of miR‐124‐9‐9* can reprogram Müller glia into retinal neurons and (2) miR‐124‐9‐9* can improve Ascl1‐induced reprogramming. Primary Müller glia cultures were generated from postnatal day (P) 11/12 mice, transduced with lentiviral particles, i.e., miR‐124‐9‐9*‐RFP, nonsense‐RFP, Ascl1‐GFP, or GFP‐control. Gene expression and immunofluorescence analyses were performed within 3 weeks after infection. Overexpression of miR‐124‐9‐9* induced the expression of the proneural factor Ascl1 and additional markers of neurons, including TUJ1 and MAP2. When Ascl1 and miR‐124‐9‐9* were combined, 50 to 60% of Müller glia underwent neuronal reprogramming, whereas Ascl1 alone results in a 30 to 35% reprogramming rate. Analysis of the miR‐124‐9‐9* treated glial cells showed a reduction in the level of Ctdsp1 and Ptbp1, indicating a critical role for the REST pathway in the repression of neuronal genes in Müller glia. Our data further suggest that miR‐124‐9‐9* and the REST complex may play a role in regulating the reprogramming of Müller glia to progenitors that underlies retinal regeneration in zebrafish. GLIA 2016;64:743–762