Elucidating the role of RNA oxidation on amyotrophic lateral sclerosis onset and progression

项目总结 越来越多的证据表明肌萎缩侧索硬化症(ALS)与氧化应激有关。尽管如此，它还是 目前尚不清楚这是病因、副产品还是疾病的后果。氧化应激对血管紧张素转换酶的影响 由活性氧引起的细胞损伤通常归因于蛋白质和DNA的修饰。 然而，RNA氧化发生的频率是DNA氧化的十倍。重要的是，高浓度的氧化 在ALS患者的相关神经元组织中检测到RNA，而ALS小鼠模型显示 脊髓运动神经元在症状前期早期的RNA氧化。仍然缺乏的是， 然而，是对RNA氧化和ALS发病和功能关系的理解 进步。因此，迫切需要确定哪些运动神经元转录本在早期、早期和早期被氧化。 肌萎缩侧索硬化症的症状阶段以及这种失调如何导致神经元死亡和其他分子 肌萎缩侧索硬化症的特征。我相信有许多尚未发现的RNA结合蛋白(RBP)是 对于控制被氧化的RNA的命运至关重要。我假设RNA氧化驱动运动神经元 肌萎缩侧索硬化症的退化是由于限制性商业惯例对适当的RNA加工的失调。我将通过(1)来检验这一假设 在IPSC来源的运动神经元中阐明RNA靶标和已知限制性商业惯例耗竭的后果 与氧化的RNA相互作用并识别新的氧化RNA；(2)识别和比较IPSC- 建立运动神经元模型；以及(3)在单细胞分辨率下研究RNA氧化对 IPSC衍生的脊髓器质性肌萎缩侧索硬化模型中的转录、翻译和RBP-RNA相互作用。如果成功， 该项目将生成基本方法和见解，以实现早期、症状前阶段 降低高危个体RNA氧化水平的诊断方法和干预措施。 我在DNA损伤修复和单细胞转录学方面的背景以及Yeo实验室的专业知识 关于RNA加工和神经退行性变的研究使我成为完成这项研究的理想人选 上面。这三个目标将作为我独立学术地位的基础，产生 研究RNA损伤效应的方法和见解。加州大学圣迭戈分校的Yeo实验室是一个理想的环境 进行这项研究，并完成我的培训，以追求一个独立的学术教师职位，如 它一直是开发实验和计算方法来表征 RNA加工和RBP调控。此外，YEO实验室的位置靠近卓越 加州大学圣迭戈分校、索尔克研究所和拉霍亚的其他研究机构和生物技术公司的研究人员 将提供充足的机会，在执行研究和开发独立的 研究计划。

PROJECT SUMMARY A growing body of evidence implicates oxidative stress in Amyotrophic Lateral Sclerosis (ALS). Nevertheless, it is currently unknow if it is a cause, a by-product or a consequence of disease. The effects of oxidative stress on cellular damage caused by reactive oxygen species is usually attributed to modifying proteins and DNA. However, RNA oxidation occurs ten times more often than DNA oxidation. Importantly, high levels of oxidized RNA are detected in relevant neuronal tissues of patients with ALS while, mouse models of ALS show increased RNA oxidation in motor neurons of the spinal cord at an early pre-symptomatic stage. What remains lacking, however, is an understanding of the functional relationship between RNA oxidation and ALS onset and progression. Thus, there is a critical need to identify which motor neuron transcripts are oxidized in early, pre- symptomatic stages of ALS and how this dysregulation contributes to neuronal death and other molecular hallmarks of ALS. I believe that there are many, yet to be discovered, RNA Binding Proteins (RBPs) that are crucial for controlling the fate of oxidized RNAs. I hypothesize that RNA oxidation drives motor neuron degeneration in ALS by dysregulating proper RNA processing by RBPs. I will test this hypothesis by (1) elucidating, in iPSC-derived motor neurons, the RNA targets and the consequences of depletion of known RBPs that interact with oxidized RNAs and identify novel ones; (2) identifying and comparing oxidized RNAs in iPSC- derived motor neuron models; and (3) investigating, at a single cell resolution, the effects of RNA oxidation on transcription, translation and RBP-RNA interactions in iPSC-derived spinal organoid ALS models. If successful, this project will generate the foundational methods and insights to enable early, pre-symptomatic-stage diagnostic approaches and interventions to reduce RNA oxidation levels in high-risk individuals. My background in DNA damage and repair and single cell transcriptomics together with the Yeo lab’s expertise on RNA processing and neurodegeneration make me an ideal candidate to accomplish the research proposed above. These three aims will serve as a basis for my independent academic position generating the foundational methods and insights to study the effects of RNA damage. The Yeo lab at UCSD is an ideal environment to perform this research and complete my training towards pursuit of an independent academic faculty position, as it has consistently been a leader in developing both experimental and computational methods to characterize RNA processing and RBP regulation. Additionally, the location of the Yeo lab proximal to outstanding researchers at UCSD, the Salk Institute, and other research institutes and biotechnology companies in La Jolla will provide ample opportunities for mentored training in performing research and developing an independent research program.