Altered Hippocampal Neurogenesis and Cognition via Maneb-mediated Changes in the Thiol Redox Proteome.

这项提案的总体目标是阐明改变神经发育的硫醇氧化还原机制， 这会加重唐氏综合征（DS）的认知功能障碍。唐氏综合症（DS）是最 智力残疾的常见遗传原因。重要的是，智力残疾的程度是高度可变的， 受影响个体的父母通常在怀孕期间意识到DS。这就创造了一个机会之窗 通过识别环境因素和相关机制， 发展在DS。我们实验室和其他实验室的初步数据和报告表明，来自DS的细胞 个体表现出扭曲的蛋白质稳态、增强的氧化应激和改变的代谢。我们假设 这些是通过非典型的巯基氧化还原系统在DS细胞连接。这增加了对 巯基反应性外源性物质通过中枢碳代谢失调，改变干细胞命运 通过改变Wnt/β-catenin信号传导的调节来决定认知功能 功能障碍我们以前的研究表明，环境毒物，代森锰（MB），神经毒性二硫代氨基甲酸酯， 杀真菌剂，损害蛋白质稳态，增加氧化应激，并显示出更大的毒性，在DS细胞相比， 整倍体对照。MB还改变线粒体功能，中心碳代谢和我们的新的初步研究。 数据显示DS细胞在Wnt信号通路中显示出显著的基线改变。Wnt信号是一种 重要的细胞信号传导管道对干细胞维持和神经发育至关重要。因此，阐明 影响DS发展的环境机制，例如Wnt信号传导和氧化应激，将提供 一个基金会，优先考虑环境化学监测在DS神经发育。细胞分裂 巯基氧化还原系统，例如巯基氧化还原蛋白质组，是氧化应激的关键特征。这种机制也是至关重要的 在胚胎发育中，胚胎来源的活性氧（ROS）触发干细胞， 区分。因此，下文详述的方法将包括硫醇反应性毒物（TRT）作为一种创新手段 研究影响特殊人群神经发育的基因-环境相互作用，DS。这项建议 涉及三个特定的目的，并利用一个强大的整倍体和21三体诱导多能文库， 干细胞（iPSC）和定向分化方案，以研究巯基氧化还原信号传导在影响 TRT对来自DS个体的干细胞的作用，以及这些暴露如何改变特定的途径（Wnt和 中枢碳代谢）。在具体目标1中，我们将确定21三体介导的 TRT暴露加剧了Wnt功能障碍，导致异常iPSC分化。蛋白质印迹法， qRT-PCR、化学工具和单细胞转录组学将用于研究这一目标。具体目标2是 旨在研究TRT暴露对巯基氧化还原蛋白质组的影响，并确定机制靶点 参与了异常分化在具体目标3中，我们将利用代谢方法，如代谢组学， 将Wnt功能障碍和氧化还原蛋白质组学改变与代谢改变相关联的细胞外通量分析 和发展成果。总之，这里提出的新研究将填补知识的关键空白 关于DS中硫醇反应性毒物的神经发育影响以及这些暴露如何改变 神经发生，可能导致认知变异。最后，这里描述的研究是一个创新的 和数据驱动的扩展我资助的研究进一步探索新的机制，例如Wnt和硫醇 氧化还原信号，通过暴露可以改变DS中的关键神经发育过程。

The overall goal of this proposal is to elucidate the thiol redox mechanisms that alter neurodevelopment, which can exacerbate cognitive dysfunction in Down syndrome (DS). Down syndrome (DS) is the most common genetic cause of intellectual disability. Importantly, the extent of intellectual disability is highly variable, and parents of affected individuals are often aware of DS during pregnancy. This creates a window of opportunity to improve outcomes through identification of environmental factors and associated mechanisms impacting early development in DS. Preliminary data and reports from our laboratory and others demonstrate that cells from DS individuals exhibit distorted proteostasis, enhanced oxidative stress, and altered metabolism. We hypothesize that these are linked through atypical thiol redox systems in DS cells. This increases vulnerability to thiol-reactive xenobiotics through dysregulation of central carbon metabolism, modifying stem cell fate decisions via altered regulation of Wnt/β-catenin signaling as a mechanism contributing to cognitive dysfunction. Our prior results show that the environmental toxicant, maneb (MB), a neurotoxic dithiocarbamate fungicide, impairs proteostasis, increases oxidative stress and displays greater toxicity in DS cells compared to euploid controls. MB also modifies mitochondrial function, central carbon metabolism and our new preliminary data show that DS cells display significant baseline alterations in the Wnt signaling pathway. Wnt signaling is a vital cell signaling conduit critical for both stem cell maintenance and neurodevelopment. Therefore, elucidating the environmental mechanisms impacting DS development, e.g. Wnt signaling and oxidative stress, will provide a foundation to prioritize environmental chemical surveillance in DS neurodevelopment. Disruption of cellular thiol redox systems, e.g. thiol redox proteome, is a key feature of oxidative stress. This mechanism is also critical for embryonic development, where mitochondrially-derived reactive oxygen species (ROS) trigger stem cells to differentiate. Thus, the approach detailed below will include thiol-reactive toxicants (TRT) as an innovative means to study Gene-Environment interactions affecting neurodevelopment in a special population, DS. This proposal involves three Specific Aims and makes use of a powerful library of euploid and trisomy 21 induced pluripotent stem cells (iPSC) and directed differentiation protocols to investigate the role of thiol redox signaling in the effects of TRT on stem cells derived from DS individuals, and how these exposures alter specific pathways (Wnt and central carbon metabolism) during neurodevelopment. In Specific Aim 1 we will determine if trisomy 21-mediated Wnt dysfunction is exacerbated by TRT exposure, resulting in aberrant iPSC differentiation. Western blotting, qRT-PCR, chemical tools and single cell transcriptomics will be used to interrogate this aim. Specific Aim 2 is designed to study the impact of TRT exposure on the thiol redox proteome and identify mechanistic targets involved in aberrant differentiation. In Specific Aim 3 we will utilize metabolic approaches like metabolomics and extracellular flux analyses to correlate Wnt dysfunction and redox proteomic alterations to metabolic alterations and developmental outcomes. Together, the novel research proposed here will fill a critical gap in knowledge with regard to neurodevelopmental impacts of thiol reactive toxicants in DS and how these exposures can alter neurogenesis, potentially contributing to cognitive variability. Finally, the studies described here are an innovative and data-driven extension of my funded research exploring further into new mechanisms, e.g. Wnt and thiol redox signaling, by which exposures can alter critical neurodevelopmental processes in DS.