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(NSCs)神经发生能力下降， 在妊娠期的早期和中期，这些神经元形成了成人大脑的大部分神经元。这一损失 神经源性能力在一定程度上解释了与胎儿酒精相关的神经行为和脑发育缺陷 频谱障碍(FASD)。然而，介导发作性PAE持续效应的机制 胎儿神经干细胞是未知的。我们和其他人最近发现了一种新型的200纳米以下的 由神经干细胞分泌的细胞外小泡(EV)，作为细胞间转移生物分子的一种手段，包括 蛋白质和核酸。我推测EVS由类似内分泌的细胞器组成，这些细胞器传播这种效应 乙醇在整个干细胞微环境中的时空分布。 我的初步和已发表的数据支持这样的假设：乙醇(I)改变了蛋白质的封存 进入胚胎神经干细胞释放的EVS，并以剂量依赖的方式，并通过干扰 EVS，(Ii)导致干细胞更新丧失和神经发生减少。利用质谱学和 转录学工具以及EV功能的细胞和分子生物学分析小鼠模型，计划和 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和相关神经干细胞中的蛋白质和RNA。我的研究表明，来自父母NSCs的电动汽车 暴露于中等剂量乙醇(Mod-EtoHEV)的无稽之谈中的蛋白质显著增加- 介导的衰变(NMD)，而EVS是在NSC暴露于更高水平的乙醇后获得的(Hi-EtoHEV) 过度表达构成危险相关分子模式(mito-Damp)信号的线粒体蛋白。 这些数据将得到验证，并与受乙醇影响的亲代神经干细胞的细胞内蛋白质组进行比较。目标 2将评估Mod-EtoHEV来源的NMD在NSC增殖、分化和死亡中的中介作用， 在基础和乙醇暴露条件下。我们的工作假设是Mod-EtoHEV转移NMD 对受体细胞的成分，以提供代偿神经保护。目标3将评估以下因素的中介作用 Hi-EtoHEV来源的丝裂原抑制NSC的增殖、分化和死亡。我们的工作假设是 高水平酒精暴露时分泌的“危险”信号可触发促炎细胞因子 受体神经干细胞的反应，导致神经发生受损。 我的研究有望揭示调节PAE效应的新的细胞间通讯途径 关于早期神经成熟的研究。这项培训计划将使我掌握技术和研究方法技能，并 掌握蛋白质组和转录组的数据分析和统计方法 评估。该计划的重点是剖析和破坏PAE和FASD之间的联系 我的职业目标是成为一名科学家，专注于早期环境调解人的作用 发育障碍。

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.