Deciphering the Transcriptional Regulatory Network Controlling RGC Axon Growth to Promote RGC Axon Regeneration and Cell Survival after Axonal Injury

项目摘要 在过去的十年里，恢复成熟神经元固有的轴突生长能力取得了令人振奋的结果 促进中枢神经系统(CNS)的轴突再生。然而，到目前为止，轴突再生 导致中枢神经系统功能的成功恢复实际上仍然是不可能的，主要是由于 再生距离和再生轴突数量较少。以前的研究和我的初步数据 已经表明，许多调节固有轴突生长能力的基因在不同的 神经元的发育阶段，表明神经元成熟过程中基因表达水平的变化是一种 内源性轴突生长能力减弱的重要因素。然而，改变后的基因是如何 表达程序是否受到调控仍在很大程度上不得而知。转录因子(TF)在细胞周期中发挥重要作用。 神经元发育，塑造时空基因表达图景以控制细胞活动 包括轴突延长。因此，理解复杂的转录调控网络编排 发育过程中的轴突生长对于解决哺乳动物中枢神经系统轴突再生的挑战至关重要。在……里面 在这项拟议的研究中，我将对纯化的视网膜神经节细胞(RGC)进行平行的RNA-SEQ和ATAC-SEQ 多个发育时间点，并使用先进的综合生物信息学分析来获得 调控轴突伸长功能的转录调控网络综观 RGC发育，并确定作为轴突生长核心调节因子的关键因子。已确定的TF将 在小鼠视神经再生模型中进行功能测试，以验证它们在RGC轴突中是否发挥重要作用 再生和细胞存活。RGC由40多种不同的分子亚型组成。不同 RGC亚型对轴突损伤的易感性不同，对基因调节有不同的反应。这就做 视神经损伤后2周行单细胞RNA-seq(scRNA-seq)检测。 操纵组以获取每个RGC亚型在最终群体中的频率，并确定 通过比较RGC的频率来操作特定的Tf来保护特定的RGC亚型 对照组和TF操纵组之间的亚型。其手法被发现可提高存活率的TFS 在不同的RGC子类型中，将在下一步中组合以确定是否同时操纵这些 TFS可保护多种RGC亚型免受损伤诱导的细胞死亡，并诱导协同作用 促进RGC轴突再生的作用。此外，我还会将这些TF的操作与 视网膜节细胞中非肌肉肌球蛋白IIA/B缺失，通过修饰细胞骨架产生轴突再生 损伤轴突生长锥体的动力学，以找出这种组合方法是否会导致 史无前例的远距离轴突再生。

Project Summary In the past decade, restoring the intrinsic axon growth ability of mature neurons has received promising results in promoting axon regeneration in the central nervous system (CNS). However, to date, axon regeneration that leads to successful functional recovery in the CNS is still practically impossible, primarily due to the inadequate distance of regeneration and the low number of regenerating axons. Previous studies and my preliminary data have shown that many genes mediating the intrinsic axon growth ability are differentially expressed at different developmental stages in neurons, indicating the altered gene expression level during neuronal maturation is an important factor underlying the diminished intrinsic axon growth capacity. However, how the altered gene expression program is regulated remains largely unknown. Transcription factors (TFs) play important roles during neuronal development, shaping the spatiotemporal gene expression landscape to control cellular activities including axon elongation. Thus, understanding the intricate transcriptional regulatory network orchestrating axon growth during development is critical for solving the challenge of mammalian CNS axon regeneration. In this proposed study, I will perform parallel RNA-seq and ATAC-seq of purified retinal ganglion cells (RGCs) at multiple developmental time points, and use advanced integrative bioinformatics analysis to obtain a comprehensive view of the transcriptional regulatory network controlling the axon elongation function during RGC development, and identify key TFs that function as core regulators of axon growth. The identified TFs will be functionally tested in mouse optic nerve regeneration model to verify if they play important roles in RGC axon regeneration and cell survival. RGCs are comprised of more than forty molecular distinct subtypes. Different RGC subtypes vary in vulnerability to axonal injury and have distinct responses toward gene modulations. I will conduct single-cell RNA-seq (scRNA-seq) in RGCs 2 weeks after optic nerve crush from control and TF- manipulated groups to acquire the frequency of each RGC subtype in the final population, and determine what specific RGC subtypes are protected by the manipulation of a specific TF by comparing the frequencies of RGC subtypes between control and TF-manipulated groups. TFs whose manipulations are found to improve survival in distinct RGC subtypes will be combined in the next step to determine if simultaneously manipulating these TFs could protect a wide variety of RGC subtypes from injury-induced cell death and induce synergistic promoting effect on RGC axon regeneration. In addition, I will also combine the manipulations of these TFs with non-muscle myosin IIA/B deletion in RGCs, which produces axon regeneration by modifying cytoskeletal dynamics in the growth cone of injured axons, to find out if this combinatory approach could lead to unprecedented long-distance axon regeneration.