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.

项目概要 尽管广泛研究，发育过程中酒精暴露对星形胶质细胞的影响仍然很大程度上未知。 越来越多的证据表明这些细胞在发育中和成人大脑中发挥的作用。这个差距在 知识的部分原因是体内研究这些细胞所面临的挑战。我们建议聘用新 允许在体内研究星形胶质细胞以获得星形胶质细胞功能的机制见解的技术 因发育性酒精暴露而改变，以加快我们发现酒精所扮演的角色的步伐 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 允许翻译核糖体选择性地从星形胶质细胞中选择性地拉下积极翻译的RNA 亲和纯化 (TRAP) 方法以及通过荧光激活分离星形胶质细胞特异性细胞核 细胞分选（FACS）。因此，该系统可以分析星形胶质细胞特异性核 RNA 的变化 通过 RNA-seq 进行表达和星形胶质细胞特异性 RNA 翻译。我们假设新生儿酒精 暴露会引起星形胶质细胞介导的一些基因翻译的广泛变化 体内和主要由星形胶质细胞贡献的分子结构上的过程，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于 由转录变化驱动，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。我们是 对涉及 ECM 的改变特别感兴趣，因为我们已经报道了 ECM 的几种蛋白质 在神经元发育中发挥重要作用，并因乙醇而失调。我们期待我们的结果 拟议的目标对 FASD 领域非常有影响。我们将研究前额皮质（PFC）和 发育中 (PD7) 和成年 (PD90) 雌性和雄性 Aldh1l1-EGFP-Rpl10a 小鼠的海马 (HPC)。目的 1：通过 FACS 鉴定新生儿乙醇暴露引起的星形胶质细胞核转录组的变化 星形胶质细胞核分选，然后进行 RNA 测序和通路分析。目标 2：识别变化 通过 TRAP-RNA-seq 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 与转录组数据。目标 3：探索星形胶质细胞 ECM 网络的失调，这是某些 通过 TRAP-qPCR、荧光原位杂交 (FISH)-RNAscope 分析乙醇对发育的影响， 蛋白质印迹和免疫组织化学在 mRNA 和蛋白质水平上验证乙醇诱导的变化 存在于 ECM 蛋白中。拟议的研究涉及 NIH/NIAA 的优先事项，因为它们将提供机制见解 发育中和成年大脑中因发育性酒精暴露而改变的星形胶质细胞功能 可能与 FASD 成人出现的行为异常和精神疾病有关。