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

破译控制 RGC 轴突生长的转录调控网络，以促进轴突损伤后 RGC 轴突再生和细胞存活

• 批准号: 10805158
• 负责人: Xuewei Wang
• 金额: $ 1.89万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10805158
• 关键词: ATAC-seq; Axon; Bioinformatics; Cell Death Induction; Cell Survival; Central Nervous System; Complex; Cytoskeleton; Data; Development; Frequencies; Gene Expression; Genes; Glaucoma; Growth Cones; Injury; Mediating; Molecular; Mus; Natural regeneration; Nerve Crush; Neurodegenerative Disorders; Neurons; Nonmuscle Myosin Type IIA; Optic Nerve; Play; Population; Recovery of Function; Retinal Ganglion Cells; Role; Shapes; Testing; Time; axon growth; axon injury; axon regeneration; differential expression; gene regulatory network; improved; multiple omics; nerve injury; neuron development; new therapeutic target; non-muscle myosin; optic nerve regeneration; programs; regeneration model; response; single-cell RNA sequencing; spatiotemporal; transcription factor; transcription regulatory network; transcriptome sequencing

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.

项目摘要 在过去的十年中，恢复成熟神经元的固有轴突生长能力已获得有希望的结果 在促进中枢神经系统（CNS）中的轴突再生中。但是，迄今为止，轴突再生 导致中枢神经系统中成功的功能恢复实际上是不可能的，这主要是由于不足 再生的距离和再生轴突数量少。先前的研究和我的初步数据 已经表明，许多介导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改变是一种 固有轴突生长能力下降的重要因素。但是，如何改变基因 表达程序受到调节，在很大程度上未知。转录因子（TFS）在期间起重要作用 神经元发育，塑造时空基因表达景观以控制细胞活性 包括轴突伸长。因此，了解复杂的转录调节网络编排 发育过程中的轴突生长对于解决哺乳动物CNS轴突再生的挑战至关重要。在 这项拟议的研究，我将在纯化的视网膜神经节细胞（RGC）上进行平行的RNA-seq和Atac-seq。 多个发育时间点，并使用先进的集成生物信息学分析来获得 在控制轴突伸长函数的转录调节网络的全面视图 RGC开发并确定功能充当轴突生长的核心调节剂的关键TF。确定的TF将 在鼠标视神经再生模型中进行功能测试，以验证它们是否在RGC轴突中起重要作用 再生和细胞存活。 RGC由40多个分子不同的亚型组成。不同的 RGC亚型因轴突损伤的脆弱性而异，并且对基因调节有明显的反应。我会 视神经挤压对照和TF-进行2周后，在RGC中进行单细胞RNA-SEQ（SCRNA-SEQ） 操纵群体以获取最终人群中每个RGC亚型的频率，并确定什么 特定的RGC亚型通过比较RGC的频率来保护特定TF的保护 对照组和TF操纵组之间的亚型。发现操纵可以改善生存的TF 在不同的RGC亚型中，将在下一步中合并，以确定是否同时操纵这些 TF可以保护各种RGC亚型免受损伤诱导的细胞死亡的影响并诱导协同作用 促进对RGC轴突再生的影响。此外，我还将将这些TF的操作与 RGC中的非肌肉肌球蛋白IIA/B缺失，通过修饰细胞骨架来产生轴突再生 受伤轴突生长锥的动力学，以找出这种组合方法是否会导致 前所未有的长距离轴突再生。

项目成果

Synchronized cluster firing, a distinct form of sensory neuron activation, drives spontaneous pain.

• DOI: 10.1016/j.neuron.2021.10.019
• 发表时间: 2022-01-19
• 期刊: Neuron
• 影响因子: 16.2
• 作者: Zheng Q;Xie W;Lückemeyer DD;Lay M;Wang XW;Dong X;Limjunyawong N;Ye Y;Zhou FQ;Strong JA;Zhang JM;Dong X
• 通讯作者: Dong X