Single-cell genetic analysis of the pathogenic role of neuronal genomic instability in environmental toxicant exposure

环境毒物暴露中神经元基因组不稳定性致病作用的单细胞遗传学分析

• 批准号: 10267733
• 负责人: Xiaohong Lu
• 金额: $ 18.25万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-22 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10267733
• 关键词: ATM Signaling Pathway; ATM activation; Accounting; Adult; Aging; Air Pollutants; Antiparkinson Agents; Attenuated; Base Excision Repairs; Brain; Brain Diseases; Caffeine; Cells; Coffee; Color; Consumption; DNA; DNA Damage; DNA Repair; DNA analysis; Development; Disease; Environment; Environmental Risk Factor; Exposure to; Fluorescence; Frameshift Mutation; Frequencies; Genetic; Genome Stability; Genomic Instability; Goals; Human; Human Genome; Huntington Disease; Lead; Length; Link; Literature; Mediating; Membrane; Metals; Methods; Microsatellite Instability; Mitochondrial DNA; Modeling; Mosaic Viruses; Mosaicism; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Organic solvent product; Oxidative Stress; Paraquat; Parkinson Disease; Pathogenesis; Pathogenicity; Pathologic; Pathology; Pathway interactions; Pesticides; Pharmacology; Play; Proteins; Recombinant adeno-associated virus (rAAV); Reporter; Reporting; Research; Resolution; Risk; Role; Rotenone; Site; Somatic Cell; Somatic Mutation; Stroke; Toxic Environmental Substances; Toxicant exposure; Transgenic Organisms; Visualization; age related; alpha synuclein; ataxia telangiectasia mutated protein; base; cell type; dopaminergic neuron; druggable target; epidemiology study; gene environment interaction; gene repair; genetic analysis; genetic variant; in vivo; knock-down; mouse model; novel; oxidative DNA damage; pre-formed fibril; ratiometric; repaired; response; sensor; synucleinopathy; targeted treatment; temporal measurement; tool; vector

Project Summary/Abstract The majority of all Parkinson’s disease (PD) cases have no identifiable inheritance and occur in a sporadic form. Epidemiologic studies have found an increased risk of PD associated with exposure to environmental, while the reduced risk of PD associated with coffee consumption. The recent single-neuron sequencing studies offer direct evidence there is an age-dependent increase of somatic brain mutations, suggesting adult terminally differentiated neurons can acquire somatic mutations, possibly precipitated by environmental factors. Given the plethora of correlational evidence describing pathological conditions in which levels of DNA damage/response and mutations were identified, the most critical question is the extent to which the significance of such DNA instability has relevance for the pathogenesis of neurodegeneration. One clue comes from a protective environmental factor of PD, caffeine. Its antiparkinsonian effects have been attributed to the potent inhibition of the Ataxia-Telangiectasia Mutated (ATM) pathway. ATM is the key orchestrator of oxidative stress and DNA damage response (DDR). Robust DDR has been closely connected to multiple neurodegenerative disorders. We hypothesize that the exposure to PD associated environmental toxicants compromises genomic stability to abnormally activate the DNA damage response (converging on ATM activation), which plays a central pathogenic role to lead to the final demise of neurodegeneration. Attenuating abnormally activated ATM signaling is neuroprotective. Employing our newly developed Mosaicism with Repeat Frameshift (MORF) strategy, we demonstrated the instability of a hypermutable repeat sequence (accounting for 3% of human genome) could be harnessed for sparse and stochastic visualize neurodegeneration and genomic instability in aging, stroke, and neurodegeneration. In aim 1, we will develop an AAV mediated quantitative ratiometric sensor of genomic instability that integrates the varying lengths of mononucleotide repeats, incompatible LoxP sites, and membrane-tethered multi-color fluorescence proteins. We will use both AAV and transgenic MORF strategy to track single-neuron with DNA instability after environmental toxicant exposure to determine the pathologic consequence. In aim 2, we will determine if the genetic reduction of DNA damage response in somatic cells with DNA instability can rescue DA neuron degeneration. Our project will develop a novel genetic sensor and actuator to study somatic brain mosaicism. The results will reveal the pathogenic significance of environment-driven neuronal genomic instability and the crucial role of the DDR pathway, thus transforming the current paradigm of the pathogenic role of environmental toxicant exposure in sporadic PD. 1

项目摘要/摘要 大多数帕金森氏病(PD)病例没有可识别的遗传，并以零星形式发生。 流行病学研究发现，暴露在环境中会增加患帕金森病的风险，而 减少与饮用咖啡有关的帕金森病风险。最近的单神经元测序研究提供了直接的 有证据表明，体细胞脑突变随年龄的增加而增加，这表明成年晚期 分化的神经元可以获得体细胞突变，可能是由环境因素引起的。给定 过多的相关证据描述了DNA损伤/反应水平的病理状况 和突变被识别，最关键的问题是这种DNA的意义在多大程度上 不稳定性与神经退行性变的发病机制有关。一条线索来自一种保护性的 帕金森病的环境因素、咖啡因。其抗帕金森症的作用被归因于对 共济失调-毛细血管扩张突变(ATM)途径。ATM是氧化应激和DNA的关键协调者 损害反应(DDR)。强健的DDR与多种神经退行性疾病密切相关。 我们假设暴露在与PD相关的环境毒物中会损害基因组的稳定性 异常激活DNA损伤反应(集中在ATM激活上)，这在中枢 致病作用导致神经退行性变的最终死亡。衰减异常激活的ATM信令 具有神经保护作用。采用我们最新开发的具有重复移码(Morf)策略的马赛克，我们 证明了高度可变的重复序列(占人类基因组的3%)可能是不稳定的 利用稀疏性和随机性可视化老化、中风和 神经退行性变。在目标1中，我们将开发一种aav介导的基因组定量比率传感器。 整合不同长度单核苷酸重复、不相容的loxP位点的不稳定性，以及 膜系留多色荧光蛋白。我们将同时使用AAV和转基因Morf策略来 追踪环境毒物暴露后DNA不稳定的单个神经元以确定病理 后果。在目标2中，我们将确定在体细胞中DNA损伤反应的遗传减少 DNA不稳定可挽救DA神经元变性。我们的项目将开发一种新型的遗传传感器和执行器 研究体细胞脑嵌合体。这一结果将揭示环境驱动的致病意义 神经元基因组的不稳定性和DDR途径的关键作用，从而改变了目前的 环境毒物暴露在散发性帕金森病发病中的作用。 1