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. 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, 50-60% of Müller glia underwent neuronal reprogramming, whereas Ascl1 alone results in a 30-35% 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.

Prospective purification and characterization of Müller glia in the mouse retina regeneration assay

“…Thus, the temporal expression profile of cell‐cycle regulators indicated cell‐cycle re‐entry and proliferation upon retinal explantation, followed by cell‐cycle inhibition and exit recapitulating the timing of MG proliferation and derived progeny differentiation observed by cell tracking and transgenic lineage tracing (Figure b,c and Löffler et al, ; Ueki et al, ). Further, Ascl1 is required to stimulate retinal regeneration in zebrafish (Fausett, Gumerson, & Goldman, ; Ramachandran, Fausett, & Goldman, ; Ramachandran et al, ; Wan et al, ), is sufficient to reprogram MG to a neurogenic state (Pollak et al, ; Ueki et al, ; Wohl & Reh, ), and is a master regulator gene of retinal stem cells, along with Vsx2 and the neurogenic transcription factors Neurog2 and Notch signaling. Previous studies have shown that MG could be stimulated to endogenously express Ascl1, Neurog2, and Vsx2 at the protein level (Löffler et al, ; Nelson et al, ), which we determined here in isolated and enriched MG at the molecular level.…”

Dual modulation of neuron‐specific microRNAs and the REST complex promotes functional maturation of human adult induced neurons

“…The forced expression of miR124 and another neuron-specific microRNA, miR9/9*, is sufficient for the cells converted from adult fibroblasts to adopt neuronal features [14]. In line with this, it was shown that the combination of miR9/9* and miR124 can accelerate neuronal conversion [23,24]. The effect of short hairpin-mediated knockdown of REST (RESTi) on neuronal conversion efficiency is mediated at least in part via the upregulation of miR9 and miR124 as an effect of RESTi, but it is becoming clear that RESTi also acts via microRNA-independent pathways [22].…”

microRNA expression in the neural retina: Focus on Müller glia

“…Cluster analysis of these data revealed subgroups of miRNAs that showed defined temporal expression profiles. They included, for example, members of the let‐7 family or miR‐124, known to be involved in retinal cell maturation and Müller glia reprogramming (Quintero, Gómez‐Montalvo, & Lamas, ; Wohl & Reh, ; Xia & Ahmad, ) and miR‐17, ‐106, reported to be key regulators of the neurogenic‐to‐gliogenic transition in neural progenitor cells (Naka‐Kaneda et al, ). A specific cluster (C2) that included miR‐29a, ‐29b, ‐29c, ‐143, ‐129‐3p underwent a dramatic decrease of expression as retinal development proceeded (Hackler et al, ).…”

“…Hence, there is evidence that ectopically expressed miR-124, the most abundantly expressed miRNA in neuronal cells (Cheng, Pastrana, Tavazoie, & Doetsch, 2009), induces murine MGDPs to acquire an early neuronal (b-III tubulin-expressing) phenotype (Quintero et al, 2016). Moreover, lentiviral gene transfer-mediated overexpression of miR-124, -9, and -9* leads to upregulation of retinal progenitor genes (Ascl1 and Hes5) and neuronal genes (Neurofilament-M, doublecortin, MAP2, and TUJ1) in PN12 cultured M€ uller glia (Wohl & Reh, 2016b) (Figure 3). The authors also demonstrate that overexpression of miR-124-9-9* enhances the ascl1-mediated reprogramming of M€ uller glia to neurons (Wohl & Reh, 2016b).…”