Alcohol Effects on the Proteome and Transcriptome of Fetal Neural Stem Cell-Derived Extracellular Vesicles: Mechanism for Alcohol Teratogenesis

项目摘要 产前酒精暴露（PAE）可导致神经干细胞（NSC）的神经原性能力降低， 在妊娠早期至中期，这些神经元产生成人大脑的大多数神经元。的这种损失 神经原性能力部分解释了与胎儿酒精相关的神经行为和大脑生长缺陷 谱系障碍（FASD）。然而，介导阵发性PAE持续作用的机制尚不清楚。 胎儿NSC是未知的。我们和其他人最近发现了一种新型的亚200纳米大小的 由NSC分泌的细胞外囊泡（EV），作为生物分子的细胞间转移的手段，包括 蛋白质和核酸。我假设EV构成内分泌样细胞器， 乙醇在整个干细胞微环境中的时间和空间。 我的初步和发表的数据支持的假设，乙醇（i）改变了蛋白质的螯合 以剂量依赖性方式，通过干扰胚胎神经干细胞释放的EV中的蛋白表达， EV，（ii）导致干细胞更新的丧失和神经发生的减少。使用质谱和 转录组学工具以及在鼠模型中EV功能的细胞和分子生物学分析， 已完成的研究涉及以下具体目标：目标1旨在确定和验证酒精的影响 暴露于EV和相关NSC中的蛋白质和RNA。我的研究表明来自母神经干细胞的EV 暴露于中等剂量的乙醇（Mod-EtoHEV）的小鼠表现出无义- 介导衰减（NMD），而在NSC暴露于更高水平的乙醇后获得的EV（Hi-EtoHEV） 过表达构成危险相关分子模式（mito-DAMP）信号的线粒体蛋白。 这些数据将被验证并与亲本NSC的乙醇影响的细胞内蛋白质组进行比较。目的 2将评估Mod-EtoHEV衍生的NMD对NSC增殖、分化和死亡的介导作用， 在基础和乙醇暴露条件下。我们的工作假设是Mod-EtoHEV将NMD 成分到受体细胞，以提供补偿性神经保护。目标3将评估 Hi-EtoHEV衍生的mito-DAMP对NSC增殖、分化和死亡的影响我们的假设是 高水平乙醇暴露后，“危险”信号的分泌会触发促炎细胞因子 受体NSC的反应，导致受损的神经发生。 我的研究有望揭示介导PAE效应的新的细胞间通讯途径 早期神经成熟该培训计划将使我具备技术和研究方法技能， 发展蛋白质组和转录组的数据分析和统计方法的具体能力 考核该计划的重点是解剖和破坏PAE和FASD之间的连接， 我的职业目标是成为一名科学家，专注于早期环境介质的作用， 发育性残疾

Project Summary Prenatal alcohol exposure (PAE) can result in decreased neurogenic capacity of neural stem cells (NSCs), which, during the early-to-mid gestation period, give rise to most neurons of the adult brain. This loss of neurogenic capacity explains, in part, neurobehavioral and brain growth deficits associated with Fetal Alcohol Spectrum Disorders (FASD). However, the mechanisms that mediate the persistent effects of episodic PAE on fetal NSCs are unknown. We and others have recently identified a novel class of sub-200 nanometer-sized extracellular vesicles (EVs) secreted by NSCs, as a means for intercellular transfer of biomolecules including proteins and nucleic acids. I hypothesize that EVs constitute endocrine-like organelles that propagate the effects of ethanol throughout the stem cell microenvironment in time and space. My preliminary and published data support the hypotheses that ethanol (i) alters the sequestration of proteins into EVs released by fetal NSCs, in a dose-dependent manner, and, by interfering with protein expression in EVs, (ii) results in loss of stem cell renewal and diminished neurogenesis. Using mass spectrometric and transcriptomic tools and cell and molecular biological analyses of EV function in a murine model, planned and completed studies address the following specific aims: Aim 1 seeks to identify and validate the effects of alcohol exposure on proteins and RNAs in EVs and associated NSCs. My studies show that EVs from parent NSCs exposed to moderate doses of ethanol (Mod-EtoHEVs) exhibit a significant increase in proteins of the Nonsense- Mediated Decay (NMD), whereas EVs obtained following NSC exposure to higher levels of ethanol (Hi-EtoHEVs) overexpress mitochondrial proteins that constitute a Danger-Associated Molecular Pattern (mito-DAMP) signal. These data will be validated and compared to the ethanol-affected intracellular proteome of parent NSCs. Aim 2 will evaluate the mediating role of Mod-EtoHEV-derived NMD on NSC proliferation, differentiation, and death, under basal and ethanol-exposure conditions. Our working hypothesis is that Mod-EtoHEVs transfer NMD components to recipient cells to confer compensatory neuroprotection. Aim 3 will evaluate the mediating role of Hi-EtoHEV-derived mito-DAMP on NSC proliferation, differentiation, and death. Our working hypothesis is that the secretion of a ‘danger’ signal in response to high levels of ethanol exposure triggers a pro-inflammatory cytokine response in recipient NSCs, resulting in impaired neurogenesis. My studies are expected to uncover novel intercellular communication pathways that mediate the effects of PAE on early neural maturation. This training plan will equip me with technical and research methodology skills and develop specific competency with data analytic and statistical approaches for proteome and transcriptome assessment. The plan’s focus on dissecting and disrupting the connection between PAE and FASD integrates well with my career goal to become a scientist with a focus on the role of early environmental mediators of developmental disability.