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

项目摘要 发育期酒精暴露对星形胶质细胞的影响在很大程度上仍然是未知的，尽管广泛的研究表明， 越来越多的证据表明这些细胞在发育和成年大脑中所起的作用。中的这一空白 这些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见解的技术 通过发展酒精暴露来改变，以推进我们发现的作用的步伐， 胎儿酒精谱系障碍（FASD）中的星形胶质细胞。我们建议使用Aldh 111-EGFP-Rpl 10a小鼠 允许翻译核糖体选择性地从星形胶质细胞中拉下主动翻译的RNA 亲和纯化（TRAP）方法和用于分离星形胶质细胞特异性细胞核的亲和活化 细胞分选（FACS）。因此，该系统允许分析星形胶质细胞特异性核RNA 表达和星形胶质细胞特异性RNA翻译。我们假设新生儿酒精 暴露诱导了参与几种星形胶质细胞介导的细胞凋亡的基因翻译的广泛变化。 主要由星形胶质细胞，如细胞外基质， (ECM)来调节这些过程。我们还假设，翻译中的变化部分是 由转录的变化驱动，部分独立于转录。此外，拟议的 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中的星形胶质细胞功能和功能障碍与其他细胞类型的贡献分开。我们 特别感兴趣的是涉及ECM的改变，因为我们已经报道了ECM的几种蛋白质， 在神经元发育中起重要作用，并且被乙醇失调。我们期待我们的结果 建议的目的是非常有影响力的FASD领域。我们将研究前额叶皮层（PFC）， 发育中（PD 7）和成年（PD 90）雌性和雄性Aldh 111-EGFP-Rpl 10a小鼠的海马（HPC）。目的 1：通过流式细胞术鉴定新生儿乙醇暴露诱导的星形胶质细胞核转录组的变化 星形胶质细胞核的分选，然后进行RNA-seq和途径分析。目标2：确定 通过TRAP-RNA-seq通过新生儿乙醇暴露诱导星形胶质细胞翻译组并整合这些发现 转录组数据。目的3：探索星形胶质细胞ECM网络的失调，这是一些神经胶质细胞损伤的基础。 通过TRAP-qPCR，荧光原位杂交（FISH）-RNAscope， 蛋白质印迹和免疫组织化学在mRNA和蛋白质水平验证乙醇诱导的变化 在ECM蛋白中。拟议的研究解决了NIH/NIAA的优先事项，因为它们将提供机制见解 在发育和成年大脑中， 可能与FASD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.