The role of microRNAs in Müller glia reprogramming into neurons in the postnatal murine retina: cellular analysis in vitro and in vivo

microRNA（miRNAs）是一类小的非编码RNA分子，作为翻译抑制因子，在不同组织的发育和功能中发挥重要作用。最近的研究表明，miRNAs调控视网膜发育和光感受器功能的几个关键步骤。此外，斑马鱼视网膜再生的研究表明miRNAs参与了视网膜再生。这一过程通过一种特殊类型的视网膜神经胶质细胞发生，即缪勒神经胶质细胞。在鱼类视网膜中，Müller胶质细胞可以去分化为祖细胞样细胞，并在损伤后再生受损的神经元。这种现象被称为转分化或调节重编程，但后者更合适，因为转分化意味着一种细胞类型直接转化为另一种细胞类型。这种调节性重编程中的关键调节因子是miRNA。在哺乳动物中，神经元从米勒神经胶质再生的潜力似乎受到抑制，尽管这种抑制的潜在机制仍然未知。迄今为止，还没有研究哺乳动物Müller胶质细胞中miRNAs的报道。因此，该建议的总体目标是揭示小鼠Müller神经胶质细胞表达的一组miRNAs，以1.确定特定的miRNA是否抑制小鼠Müller神经胶质细胞的重编程潜力; 2.分析诱导神经发生所需的miRNA的过表达是否可以将小鼠Müller胶质细胞重编程为视网膜祖细胞和/或成熟神经元。已经描述了用于其他细胞类型的有希望的候选物的功能获得和丧失实验，即作为再生阻遏物的miRNAs let-7和作为神经发生激活剂的miR-124，将通过慢病毒基因转移和RNA转染在出生后第12天（P）小鼠的Müller神经胶质原代细胞培养物中以及在N-甲基-D-天冬氨酸（NMDA）成年小鼠的损伤。将通过RT-qPCR、蛋白质印迹和免疫荧光染色以及随后的共聚焦显微镜进行细胞分析。为了揭示小鼠Müller神经胶质细胞miRNAs的整个集合，并找到Müller神经胶质细胞重编程的新的潜在候选者，将进行下一代RNA测序，这是一种发现特定时间点RNA存在和数量的创新方法。这些拟议的实验将深入了解Müller神经胶质细胞状态的分子调控，以及miRNAs是否可以用作成功重编程小鼠Müller神经胶质细胞以恢复损伤后视网膜组织的工具。

MicroRNAs (miRNAs) are small, non-coding RNA molecules that act as translational repressors and play important roles in the development and function of different tissues. Recent studies have shown that miRNAs regulate several key steps in retinal development and photoreceptor function. In addition, studies of regenerating zebrafish retina have implicated miRNAs in regeneration. This process occurs via a specialized type of retinal glia, the Müller glia. In the fish retina, Müller glia cells can dedifferentiate into progenitor-like cells and regenerate damaged neurons after injury. This phenomenon has been variously called transdifferentiation or regulated reprogramming, but the latter term is more appropriate since transdifferentiation implies direct conversion of one cell type into another. Key regulators in this regulated reprogramming are miRNAs. In mammals, the potential for regeneration of neurons from Müller glia appears to be repressed, although the underlying mechanisms for this repression are still unknown. Until now, there are no reports analyzing miRNAs in mammalian Müller glia. Therefore, the overall goal of this proposal is to reveal the set of miRNAs expressed by murine Müller glia cells in order to 1. Determine if specific miRNA repress the reprogramming potential of murine Müller glia cells and 2. Analyze if overexpression of miRNAs, which are required to induce neurogenesis, can reprogram murine Müller glia into retinal progenitor cells and/or mature neurons. Gain and loss of function experiments of promising candidates which have been described for other cell types, i.e. the miRNAs let-7, as repressor of regeneration, and miR-124, as activator of neurogenesis, will be performed by means of lentiviral gene transfer and RNA transfection in Müller glia primary cell cultures of postnatal day (P) 12 mice as well as in vivo after N-methyl-D-aspartate (NMDA) damage in adult mice. Cellular analysis will be conducted by RT-qPCR, Western Blot, and immunofluorescent stainings with subsequent confocal microscopy. In order to reveal the entire set of murine Müller glia miRNAs, and find new potential candidates for Müller glia reprogramming, next generation RNA-Sequencing will be performed, an innovative method to uncover presence and quantity of RNA at a certain time point. These proposed experiments will provide insight into the molecular regulation of the Müller glia cell state and whether or not miRNAs can be used as a tool to successfully reprogram murine Müller glia cells to restore retinal tissue following damage.