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

项目总结/摘要 大多数帕金森病（PD）病例没有可识别的遗传，并且以散发形式发生。 流行病学研究发现，PD的风险增加与暴露于环境有关， 降低与咖啡消费相关的PD风险。最近的单神经元测序研究提供了直接的 有证据表明，体细胞脑突变的年龄依赖性增加，表明成年晚期 分化的神经元可以获得体细胞突变，这可能是由环境因素促成的。鉴于 大量相关证据描述了DNA损伤/反应水平 最关键的问题是这种DNA的重要性在多大程度上 不稳定性与神经变性的发病机制有关。一条线索来自一个保护性的 PD的环境因素，咖啡因。它的抗帕金森病作用归因于有效抑制 共济失调-毛细血管扩张突变（ATM）通路。ATM是氧化应激和DNA的关键协调者 损伤响应（DDR）。稳健的DDR与多种神经退行性疾病密切相关。 我们假设暴露于PD相关的环境毒物会损害基因组稳定性， 异常激活DNA损伤反应（集中在ATM激活），这起着核心作用。 致病作用，导致神经变性的最终消亡。衰减异常激活的ATM信令 有神经保护作用采用我们新开发的镶嵌与重复移码（MORF）策略，我们 证明了超变重复序列（占人类基因组的3%）的不稳定性可能是 利用稀疏和随机可视化神经变性和基因组不稳定性在衰老，中风， 神经变性目的一是研制一种AAV介导的定量比率传感器， 整合了不同长度的单核苷酸重复序列、不相容的LoxP位点和 膜系多色荧光蛋白。我们将使用AAV和转基因MORF策略， 追踪环境毒物暴露后DNA不稳定的单个神经元， 后果在目标2中，我们将确定是否在体细胞中DNA损伤反应的遗传减少， DNA不稳定性可以挽救DA神经元变性。我们的项目将开发一种新型的基因传感器和执行器 来研究躯体大脑的镶嵌现象本研究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，从而改变了目前的范式， 环境毒物暴露在散发性PD中的致病作用。 1

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