Epigenomic mechanisms regulating RGC survival and axon regeneration

项目总结/摘要 这项拟议中的研究是一项为期五年的职业发展研究计划，重点是解剖表观遗传 视神经夹伤小鼠视网膜神经节细胞变性和再生的调控 和青光眼。候选人目前是F.M.的博士后研究员。柯比神经生物学中心 波士顿儿童医院和哈佛医学院。候选人打算进一步扩展他的专业知识 在表观基因组分析技术，视神经病变的机制和神经再生的发展， 通过整合波士顿儿童医院和哈佛医学中心的何志刚博士的导师团队， 学校，哈佛大学的约书亚萨尼斯博士和哈佛脑科学倡议，杰弗里戈德堡博士在 斯坦福大学和拜尔斯眼科研究所，以及哈佛干细胞和再生医学中心的杰森？ 麻省理工学院和哈佛大学生物学系和布罗德研究所。候选人还招募了丹尼尔博士 Geschwind，他目前和拟议研究的合作者，作为他的顾问，具体的科学和技术 支持.拟议的实验和培训活动将使候选人能够发表顶级 眼科研究工作，并独特地定位他作为一个独立的主要研究者追求新的 用于视网膜疾病如青光眼的治疗剂。 青光眼是美国第二大致盲原因，至少有3，000，000人受到影响。 到2030年，如果没有新的治疗方法，这个数字可能会增加60%。由于无法 RGC死亡导致的视力丧失是不可逆的， 会导致永久性失明我们的初步实验使用基于CRISPR/Cas9的体内正向表达， 遗传筛选已经发现，敲低损伤诱导的表观遗传调节因子，如CCCTC- 结合因子（CTCF）可以有力地促进RGC轴突再生或/和存活。去探索 调节RGC存活和再生的表观遗传机制，拟议的研究将特别追求 以下目的：（1）通过ATAC测序（指导）分析RGCs损伤诱导的染色质重塑 （2）探讨存活和再生调节因子的调节机制 （3）评估表观遗传学的机制和影响， 调节剂在青光眼模型（独立阶段）。在过去两年的K99颁奖典礼上，我 成功完成了Aim 1和Aim 2中的所有实验和目标，并发表了两篇论文。 2022年的神经元（并被选为封面故事）。在我的K奖的延长期内，我将进一步追求 我的方法在青光眼相关小鼠模型中的转化研究。拟议研究的结果 将提供深入和定量的见解，为什么以及如何再生的命运RGC是前， 从表观基因组学的角度确定，这可以直接转化为新的治疗光学 神经病

Project Summary/Abstract The proposed study is a five-year career development research plan that focuses on dissecting the epigenetic regulation of retinal ganglion cell (RGC) degeneration and regeneration in mouse models of optic nerve crush and glaucoma. The candidate is currently a postdoctoral research fellow at F.M. Kirby Neurobiology Center at Boston Children’s Hospital and Harvard Medical School. The candidate intends to further extend his expertise in epigenomic profiling technologies, mechanisms of optic neuropathies and development of neural regenerative therapeutics by integrating the mentor team of Dr. Zhigang He at Boston Children’s Hospital and Harvard Medical School, Dr. Joshua Sanes at Harvard University and Harvard Brain Science Initiative, Dr. Jeffery Goldberg at Stanford University and Byers Eye Institute, and Dr. Jason Buenrostro at Harvard Stem Cell and Regenerative Biology (SCRB) Department and Broad Institute of MIT and Harvard. The candidate has also recruited Dr. Daniel Geschwind, a collaborator of his current and proposed studies, as his advisor for specific scientific and technical support. The proposed experiments and training activities will enable the candidate to publish top-tier ophthalmology research works and uniquely position him as an independent principal investigator pursuing novel therapeutics for retinal disease such as glaucoma. Glaucoma is the second leading cause of blindness in the United States with at least 3,000,000 people affected. This number is likely to increase by 60% by 2030 if no new therapeutics could be developped. Due to the inablity of central nervous system to regenerate after injury, the vision loss resulted from RGC death is irreversible and will lead to permanent blindness. Our preliminary experiments using the CRISPR/Cas9-based in vivo forward genetic screen have discovered that the knockdown of injury induced epigenetic regulators, such as CCCTC- binding factor (CTCF), can robustly promote RGC axonal regeneration or/and survival. To explore the underlying epigenetic mechanisms regulating RGC survival and regeneration, the proposed study will specifically pursue the following aims: (1) To profile injury-induced chromatin remodeling in RGCs by ATAC sequencing (mentored phase); (2) To assess the mechanisms that mediate differential effects of survival and regeneration regulators upon injury (mentored and independent phase); and (3) To assess the mechanisms and effects of epigenetic regulators in a glaucoma model (independent phase). For the past two years during this K99 award, I have successfully accomplished all experiments and goals in Aim 1 and Aim2, with two manuscripts published on Neuron in 2022 (and selected as cover story). For the extended period of my K award, I will further pursue the translational study of my approaches in glaucoma relevant mouse models. The outcome of the proposed study will provide in-depth and quantitative insights into why and how the regenerative fates of RGCs are pre- determined from an epigenomic perspective, which can be directly transformed to new cures for optic neuropathies.