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

酒精对胎儿神经干细胞来源的细胞外囊泡蛋白质组和转录组的影响：酒精致畸机制

• 批准号: 10292946
• 负责人: Dae Chung
• 金额: $ 3.45万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10292946
• 关键词: Address; Adult; Affect; Alcohol abuse; Alcoholism; Alcohols; Biological; Biological Assay; Brain; Cells; Cessation of life; Competence; Data; Data Analytics; Developmental Disabilities; Differentiation and Growth; Dose; Endocrine; Ethanol; Eukaryotic Cell; Exhibits; Exposure to; Family; Fetal Alcohol Exposure; Fetal Alcohol Spectrum Disorder; Fetal Development; Fetal alcohol effects; Gene Activation; Gene Expression; Goals; Growth; Human; Immunoprecipitation; Impairment; Inflammation; Inflammatory; Inflammatory Response; Inflammatory Response Pathway; Institutes; Intervention; Liquid Chromatography; Mediating; Mediator of activation protein; Messenger RNA; MicroRNAs; Mission; Mitochondrial Proteins; Molecular; Molecular Chaperones; Multivariate Analysis; Mus; NFIA gene; Neurons; Nonsense Codon; Nonsense-Mediated Decay; Nucleic Acids; Organelles; Parents; Pathway interactions; Pattern; Pattern recognition receptor; Pregnancy; Prevention; Proteins; Proteome; Publishing; RNA; RNA-Binding Proteins; Research; Research Methodology; Role; Scientist; Second Pregnancy Trimester; Sex Differences; Signal Transduction; Stress; Time; Training; Transcript; Translating; Translation Initiation; Translations; alcohol effect; alcohol exposure; alcohol related problem; alcohol response; career; cytokine; data analysis pipeline; disability; extracellular vesicles; fetal; inhibitor/antagonist; intercellular communication; interest; mouse model; nanoscale; nerve stem cell; neurobehavioral; neurodevelopment; neurogenesis; neuromechanism; neuroprotection; non-genetic; novel; overexpression; protein complex; protein expression; receptor; relating to nervous system; response; sex; skills; stem cell division; stem cell growth; stem cell proliferation; stem cell survival; stem cells; tandem mass spectrometry; teratogenesis; tool; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; vesicular release

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.

项目概要 产前酒精暴露（PAE）会导致神经干细胞（NSC）的神经生成能力下降， 在妊娠早期到中期，它产生了成年大脑的大多数神经元。这种损失 神经源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，介导偶发性 PAE 持续影响的机制 胎儿 NSC 未知。我们和其他人最近发现了一类新型的亚 200 纳米尺寸的 NSC 分泌的细胞外囊泡 (EV)，作为生物分子细胞间转移的手段，包括 蛋白质和核酸。我假设 EV 构成了类似内分泌的细胞器，可以传播效应 乙醇在时间和空间上遍布干细胞微环境。 我的初步和已发表的数据支持乙醇 (i) 改变蛋白质隔离的假设 以剂量依赖的方式进入胎儿 NSC 释放的 EV，并且通过干扰 EVs，(ii) 导致干细胞更新丧失和神经发生减少。使用质谱和 小鼠模型中 EV 功能的转录组工具以及细胞和分子生物学分析，计划和 已完成的研究致力于以下具体目标： 目标 1 旨在确定和验证酒精的影响 EV 和相关 NSC 中蛋白质和 RNA 的暴露。我的研究表明，来自父代 NSC 的 EV 暴露于中等剂量的乙醇（Mod-EtoHEV）中，Nonsense-蛋白质显着增加 介导衰变 (NMD)，而 NSC 暴露于较高水平的乙醇后获得的 EV (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，并与亲本 NSC 受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 传输 NMD 受体细胞的成分以赋予代偿性神经保护作用。目标 3 将评估中介作用 Hi-EtoHEV 衍生的 mito-DAMP 对 NSC 增殖、分化和死亡的影响。我们的工作假设是 暴露于高浓度乙醇时分泌“危险”信号会触发促炎细胞因子 受体 NSC 的反应，导致神经发生受损。 我的研究有望揭示介导 PAE 影响的新型细胞间通讯途径 关于早期神经成熟。该培训计划将使我具备技术和研究方法技能， 培养蛋白质组和转录组数据分析和统计方法的具体能力 评估。该计划的重点是剖析和破坏 PAE 与 FASD 之间的联系，整合 我的职业目标是成为一名专注于早期环境调节剂作用的科学家 发育障碍。