miR-124-9-9* potentiates Ascl1-induced reprogramming of cultured Müller glia.
miR-124-9-9* potentiates Ascl1-induced reprogramming of cultured Müller glia.
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作者：
Wohl SG;Reh TA
miR-124-9-9*的过表达诱导前神经因子Ascl1和其他神经元标志物，包括TUJ1和MAP2的表达。当Ascl1和miR-124-9-9*联合使用时，50-60%的突触神经胶质细胞发生了神经元重编程，而单独使用Ascl1的重编程率为30-35%。对miR-124-9-9*处理的胶质细胞的分析显示，Ctdsp1和Ptbp1的水平降低，这表明REST通路在抑制<s:1> ller胶质细胞神经元基因中起关键作用。我们的数据进一步表明，miR-124-9-9*和REST复合体可能在调节<s:1>勒神经胶质细胞向祖细胞的重编程中发挥作用，这是斑马鱼视网膜再生的基础。鱼类的神经神经胶质为损伤后的神经元再生提供了一个来源，但它们在哺乳动物中不这样做。我们之前的研究表明，慢病毒将转录因子achaet - scre同源物1 （Ascl1/Mash1）转移到小鼠<s:1>神经胶质细胞培养中，导致细胞部分重编程为视网膜祖细胞。microRNAs (miRNAs) miR-124-9-9*促进成纤维细胞的神经元重编程，但其在胶质细胞重编程中的作用尚未报道。本研究的目的是测试1)慢病毒基因转染miR-124-9-9*是否可以将<s:1> ller胶质细胞重编程为视网膜神经元，2)miR-124-9-9*是否可以改善ascl1诱导的重编程。用慢病毒颗粒（即miR-124-9-9*-RFP、nonense -RFP、Ascl1-GFP或gfp对照）转导出生日(P) 11/12小鼠的原代神经胶质瘤培养物。感染后3周内进行基因表达和免疫荧光分析。
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. 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.