Epigenomic mechanisms regulating RGC survival and axon regeneration

项目摘要/摘要 这项拟议的研究是一个为期五年的职业发展研究计划，重点是解剖表观遗传学 视神经卡压模型小鼠视网膜神经节细胞(RGC)变性和再生的调控 还有青光眼。这位候选人目前是F.M.柯比神经生物学中心的博士后研究员 波士顿儿童医院和哈佛医学院。这位候选人打算进一步扩展他的专业知识 在表观基因组图谱技术中，视神经病变机制和神经再生的发展 整合波士顿儿童医院何志刚博士和哈佛医学院导师团队的治疗 哈佛大学的约书亚·萨尼斯博士和哈佛脑科学倡议的杰弗里·戈德堡博士 斯坦福大学和拜尔斯眼科研究所，以及哈佛干细胞和再生中心的Jason Buenrostro博士 麻省理工学院和哈佛大学生物(SCRB)系和广泛研究所。候选人还招募了丹尼尔博士 Geschind是他当前和拟议研究的合作者，作为他的特定科学和技术顾问 支持。建议的实验和培训活动将使候选人能够发布顶级 眼科研究著作，独一无二的地位，他是追求小说的独立首席研究员 治疗青光眼等视网膜疾病。 青光眼是美国第二大致盲原因，至少有300万人受到影响。 如果不能开发新的治疗方法，到2030年，这一数字可能会增加60%。由于无能力 损伤后中枢神经系统的再生，RGC死亡导致的视力丧失是不可逆转的， 会导致永久性失明。基于CRISPR/CAS9的体内正演实验的初步研究 基因筛查已经发现，伤害的敲除诱导了表观遗传调节，如CCCTC- 结合因子(CTCF)能有力地促进RGC轴突再生或存活。探索潜在的 关于调控RGC存活和再生的表观遗传学机制，拟议的研究将专门探讨 目的如下：(1)用ATAC测序(MENTORED)分析损伤诱导的视网膜节细胞染色质重塑 阶段)；(2)评估调节存活和再生调节剂不同作用的机制 在受伤时(指导和独立阶段)；和(3)评估表观遗传学的机制和影响 青光眼模型中的调节者(独立阶段)。在过去的两年里，在这个K99奖项中，我 成功完成了目标1和目标2中的所有实验和目标，并在上发表了两篇手稿 2022年的神经元(并被选为封面故事)。对于我的K奖的延长期限，我将进一步追求 我的方法在青光眼相关小鼠模型中的翻译研究。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RGC的再生命运是前 从表观基因组学角度确定的，可以直接转化为治疗视神经的新疗法 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.