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

项目总结 发育期酒精暴露对星形胶质细胞的影响仍然很大程度上是未知的，尽管有广泛的 越来越多的证据表明，这些细胞在发育中的大脑和成年大脑中都扮演着重要角色。这一差距在 这些知识在一定程度上是由于在体内研究这些细胞所面临的挑战。我们建议雇用新的 允许在体内研究星形胶质细胞以获得对星形胶质细胞功能的机械性见解的技术 被发育中的酒精暴露所改变，以加快我们发现 胎儿酒精光谱障碍(FASD)中的星形胶质细胞我们建议使用ALDH1L1-EGFP-Rpl10a小鼠 这允许通过翻译核糖体选择性地下拉星形胶质细胞主动翻译的RNA 亲和纯化(TRAP)方法及荧光激活分离星形胶质细胞特异性核 细胞分选(FACS)。因此，该系统允许分析星形胶质细胞特有的核RNA的变化 RNA-seq表达及星形胶质细胞特异性RNA翻译。我们假设新生儿酒精 暴露诱导几种星形胶质细胞介导的基因翻译的广泛变化 主要由星形胶质细胞参与的体内过程和分子结构，如细胞外基质 (ECM)调节其中一些过程。我们还假设，翻译中的变化在一定程度上 由转录中的变化驱动，并且部分独立于转录。此外，建议的 研究将评估星形胶质细胞对发育期酒精暴露反应的异质性 发育阶段、性别和大脑区域。拟议研究的细胞类型特异性将有助于 将FASD中星形胶质细胞的功能和功能障碍与其他细胞类型的贡献区分开来。我们是 对涉及细胞外基质的改变特别感兴趣，因为我们已经报道了细胞外基质的几种蛋白质 在神经元发育中发挥重要作用，并受乙醇的调节失调。我们期待着我们的结果 提出的建议旨在对FASD领域产生很大影响。我们将研究前额叶皮质(PFC)和 发育期(PD7)和成年(PD90)雌雄ALDH1L1-EGFP-Rpl10a小鼠的海马区(HPC)。目标 1.应用流式细胞仪检测乙醇暴露对星形胶质细胞核转录组的影响 对星形胶质细胞核进行分类，然后进行rna-seq和通路分析。目标2：确定 TRAP-RNA-SEQ检测新生儿酒精暴露诱导的星形胶质细胞转录组及其整合研究 转录组数据。目的3：探讨星形胶质细胞ECM网络的失调。 用TRAP-qPCR、荧光原位杂交(FISH)-RNAScope、 Western印迹和免疫组织化学在mRNA和蛋白水平验证乙醇诱导的变化 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性的见解 在发育中的和成年的大脑中，由于发育中的酒精暴露而改变的星形胶质细胞功能 可能与患有FASD的成年人的行为异常和精神疾病有关。

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.