title: miR-124-9-9* potentiates Ascl1-induced reprogramming of cultured Müller glia.
abstract: 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. 1. Overexpression of miR-124-9-9* induced the expression of the proneural factor Ascl1 and additional markers of neurons, including TUJ1 and MAP2. 2. 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. 3. 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.
authors.affiliation: Department of Biological Structure, University of Washington, Seattle, Washington.
authors.forename: Stefanie Gabriele
authors.initials: SG
authors.lastname: Wohl
authors.affiliation: Department of Biological Structure, University of Washington, Seattle, Washington.
authors.forename: Thomas Andrew
authors.initials: TA
authors.lastname: Reh
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000970:Antineoplastic Agents / Q000494:pharmacology; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D064032:Calbindin 2 / Q000378:metabolism; D064026:Calbindins / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology*; D002478:Cells, Cultured; D063928:Ependymoglial Cells; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010671:Phenylurea Compounds / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D014404:Tubulin / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D051792:Basic Helix-Loop-Helix Proteins; D064032:Calbindin 2; D064026:Calbindins; D035683:MicroRNAs; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D010671:Phenylurea Compounds; D010749:Phosphoprotein Phosphatases; D013629:Tamoxifen; D014404:Tubulin; C065500:ASCL1 protein, human; C540079:Mirn124 microRNA, mouse; C079240:Mtap2 protein, mouse; C016601:afimoxifene; C032766:ethylene diurea; C413483:CTDSP1 protein, human
keywords: Ctdsp1/SCP1; Ptbp1/PTB; REST; microRNA; neuronal differentiation; retina