Astrocyte gene expression and translation in an in vivo FASD mouse model

体内 FASD 小鼠模型中的星形胶质细胞基因表达和翻译

• 批准号: 10471310
• 负责人: Marina Guizzetti
• 金额: $ 32.6万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10471310
• 关键词: Address; Adult; Affect; Affinity Chromatography; Alcohols; Animal Model; Astrocytes; Behavior; Behavioral; Biological Process; Brain; Brain region; Cell Nucleus; Cell Separation; Cells; Clinical; Data; Development; Disease; Disease Outcome; Ethanol; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fetal Alcohol Exposure; Fetal Alcohol Spectrum Disorder; Fluorescence; Fluorescent in Situ Hybridization; Functional disorder; Gene Expression; Genes; Genetic Transcription; Heterogeneity; Hippocampus (Brain); Immunohistochemistry; Inflammatory Response; Knowledge; Lead; Mediating; Mental disorders; Messenger RNA; Methods; Mission; Molecular; Molecular Target; Mus; NIH Program Announcements; National Institute on Alcohol Abuse and Alcoholism; Neonatal Alcohol Exposure; Nuclear; Nuclear RNA; Pathway Analysis; Play; Prefrontal Cortex; Process; Proteins; Public Health; Publishing; RNA; Reporting; Research; Ribosomal Proteins; Ribosomes; Role; Sorting - Cell Movement; Specificity; Synapses; System; Therapeutic Intervention; Translating; Translations; United States National Institutes of Health; Western Blotting; alcohol effect; alcohol exposure; base; brain cell; cell type; cellular targeting; disability; fetal; in vivo; insight; interest; male; mouse model; neuron development; new technology; novel strategies; promoter; response; sex; synaptogenesis; transcriptome; transcriptome sequencing; translatome

PROJECT SUMMARY The effects of developmental alcohol exposure on astrocytes remains largely unknown, despite the extensive and growing evidence of the roles played by these cells both in the developing and adult brain. This gap in knowledge is due in part to the challenges of studying these cells in vivo. We propose to employ new technologies allowing the study of astrocytes in vivo to gain mechanistic insights into astrocytic functions altered by developmental alcohol exposure to advance the pace of our discoveries of the roles played by astrocytes in Fetal Alcohol Spectrum Disorders (FASD). We propose to use the Aldh1l1-EGFP-Rpl10a mice that allow for the selective pull down of actively translating RNA from astrocytes by the translating ribosome affinity purification (TRAP) method and for the isolation of astrocyte-specific nuclei by Fluorescent-Activated Cell Sorting (FACS). Hence, this system allows to analyze changes in both astrocyte-specific nuclear RNA expression and astrocyte-specific RNA translation by RNA-seq. We hypothesize that neonatal alcohol exposure induces extensive changes in the translation of genes involved in several astrocyte-mediated processes in vivo and on molecular stuctures mainly contributed by astrocytes, such as the extracellular matrix (ECM) that modulate some of these processes. We also hypothesize that changes in translation are in part driven by changes in transcription and in part independent from transcription. Additionally, the proposed studies will assess astrocyte heterogeneity in their response to developmental alcohol exposure across developmental stages, sexes, and brain regions. The cell type-specificity of the proposed studies will help to disentangle astrocyte function and dysfunction in FASD from contributions of other cell types. We are particularly interested in alterations involving the ECM as we have reported that several proteins of the ECM play important roles in neuronal development and are dysregulated by ethanol. We expect the results of our proposed Aims to be very impactful to the FASD field. We will study the prefrontal cortex (PFC) and hippocampus (HPC) of developing (PD7) and adult (PD90) female and male Aldh1l1-EGFP-Rpl10a mice. Aim 1: To identify changes in the astrocyte nuclear transcriptome induced by neonatal ethanol exposure by FACS sorting of astrocyte nuclei followed by RNA-seq and pathway analysis. Aim 2: To identify changes in the astrocyte translatome induced by neonatal ethanol exposure by TRAP-RNA-seq and integrate these findings with transcriptome data. Aim 3: To explore the dysregulation of the astrocyte ECM network that underlies some of the developmental effects of ethanol by TRAP-qPCR, Fluorescence In Situ Hybridization (FISH)-RNAscope, Western blot, and immunohistochemistry to validate at both mRNA and protein levels ethanol-induced changes in ECM proteins. The proposed studies address NIH/NIAA priorities as they will provide mechanistic insights into astrocyte functions altered by developmental alcohol exposure in the developing and adult brain which are likely involved in behavioral abnormalities and mental illnesses developed by adults with FASD.

项目摘要 尽管广泛 以及越来越多的证据表明这些细胞在发育中和成人大脑中所扮演的角色。这个差距 知识部分是由于在体内研究这些细胞的挑战。我们建议雇用新的 允许研究体内星形胶质细胞的技术，从 随着发育性酒精暴露的改变，以提高我们对角色的发现的步伐 胎儿酒精谱系障碍（FASD）中的星形胶质细胞。我们建议使用AldH1L1-EGFP-RPL10A小鼠 这可以通过翻译核糖体从星形胶质细胞中积极地翻译RNA的选择性下降 亲和力纯化（TRAP）方法和用于通过荧光激活的星形胶质细胞特异性核分离 细胞分类（FACS）。因此，该系统允许分析两个星形胶质细胞特异性核RNA的变化 RNA-Seq的表达和星形胶质细胞特异性RNA翻译。我们假设新生儿酒精 暴露会导致几个星形胶质细胞介导的基因的翻译变化很大 体内和分子构的过程主要由星形胶质细胞贡献，例如细胞外基质 （ECM）调节其中一些过程。我们还假设翻译的变化部分是 由转录变化和部分独立于转录的驱动。另外，提议 研究将评估星形胶质细胞异质性在对整个饮酒的反应中 发展阶段，性别和大脑区域。拟议研究的细胞类型特异性将有助于 除其他细胞类型的贡献，FASD中的星形胶质细胞功能和功能障碍。我们是 我们对涉及ECM的更改特别感兴趣，因为我们报告了ECM的几种蛋白质 在神经元发展中起重要作用，并被乙醇失调。我们期望我们的结果 拟议的目标是对FASD领域非常有影响力。我们将研究前额叶皮层（PFC）和 发展中心（PD7）和成人（PD90）的海马（HPC）女性和雄性AldH1L1-EGFP-RPL10A小鼠。目的 1：确定FACS新生儿乙醇诱导的星形胶质细胞核转录组的变化 星形胶质细胞核的排序，然后进行RNA-seq和途径分析。目标2：确定变化 由陷阱-RNA-seq暴露于新生儿乙醇引起的星形胶质细胞翻译体并整合了这些发现 使用转录组数据。目标3：探索构成一些基础的星形胶质细胞ECM网络的失调 乙醇通过陷阱-QPCR的发育作用，荧光原位杂交（FISH） - rnascope， 蛋白质印迹和免疫组织化学在mRNA和蛋白质水平乙醇诱导的变化下验证 在ECM蛋白中。拟议的研究涉及NIH/NIAA的优先事项，因为它们将提供机械见解 在发育中的大脑和成人大脑中，发生的星形胶质细胞功能改变了 FASD成年人开发的行为异常和精神疾病可能涉及。