Astrocytes, alcohol, and the extracellular matrix

星形胶质细胞、酒精和细胞外基质

• 批准号: 10056067
• 负责人: Marina Guizzetti
• 金额: $ 12.3万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10056067
• 关键词: Accounting; Adult; Affinity Chromatography; Alcohol consumption; Alcohols; Astrocytes; Brain; Brain region; CCL18 gene; Categories; Cell Adhesion Molecules; Cells; Chondroitin Sulfate A; Chondroitin Sulfates; Control Animal; Core Protein; Disaccharides; Discrimination; Ethanol; Extracellular Matrix; Extracellular Matrix Proteins; Extracellular Space; Funding; Genes; Glycosaminoglycans; Goals; Harvest; Heparitin Sulfate; Hyaluronic Acid; Intervention; Linear Models; Medial; Messenger RNA; Methods; Molecular; Molecular Genetics; Molecular Target; Mus; Neuronal Plasticity; Nucleus Accumbens; Peptide Hydrolases; Prefrontal Cortex; Proteins; Proteoglycan; Protocols documentation; RNA; RNA analysis; Research; Ribosomal Proteins; Ribosomes; Risk; Rodent; Role; Signal Transduction; Structure; Sulfate; Techniques; Technology; Therapeutic Intervention; Translating; Translations; Validation; Water; addiction; alcohol availability; alcohol effect; behavioral genomics; brain cell; brain parenchyma; cell type; cellular targeting; differential expression; drinking; extracellular; genome-wide analysis; glycosylation; in vivo; inhibitor/antagonist; interstitial; liquid chromatography mass spectrometry; neuroadaptation; polysulfated glycosaminoglycan; preference; sex; sugar; tool; transcriptome sequencing; translatome

PROJECT SUMMARY – P002 Several genome-wide studies, including studies from the previous funding period of the PARC, identified the extracellular matrix (ECM) and astrocytes as being involved in both the risk for and the effects of ethanol drinking. Cells in the brain parenchyma are separated by the extracellular space, accounting for approximately 20% of the total volume of the mature rodent brain and containing a highly organized ECM that forms an insoluble network around cells. The brain interstitial ECM significantly contributes to the molecular signals regulating neuronal plasticity. Recent research has shown that astrocytes are major producers of the brain interstitial ECM as well as ECM proteases, which are involved in the remodeling of the ECM. ECM proteins are post-translationally modified by glycosylation. Proteoglycans are major components of the brain ECM and consist of a core protein covalently bound to glycosaminoglycans (GAGs) formed by repeating disaccharides, modified by sulfation. Hyaluronic acid (HA), also a major component of the ECM, is a non-sulfated GAG and is not covalently bound to proteins. GAGs are involved in the modulation of neuronal plasticity. The overall goal of this proposal is to identify differential expression of astrocyte-specific genes (with a major emphasis on genes involved in the formation and remodeling of the ECM) and differential levels of ECM GAGs associated with ethanol drinking in two brain regions involved in addiction: the medial prefrontal cortex (mPFC; including the prelimbic and infralimbic cortex) and the nucleus accumbens (NAc). We plan to reach this overarching goal by pursuing the following two specific aims: 1) Definition of the astrocyte translatome in the mPFC and NAc of naïve and ethanol-drinking Aldh1l1-EGFP-Rpl10a mice; validation of differential translation and expression by qPCR and by RNA scope, respectively, in Aldh1l1-EGFP-Rpl10a mice; determination of the expression of selected ECM-related genes in ethanol naïve high ethanol preference (HP) and low ethanol preference (LP) mice, and in ethanol-drinking HP mice by RNAscope. We will use the translating ribosome affinity purification (TRAP) technology in Aldh1l1-EGFP-Rpl10a mice that express a modified ribosomal protein Rpl10a with an eGFP tag (EGFP-Rpl10a) only in cells expressing the astrocytic marker Aldh1l1. Selected differentially translated RNAs for ECM-related proteins will be confirmed by qPCR and their differential expression and sub- regional localization will be determined by RNAscope. Differential expression of selected ECM-related genes will also be determined in ethanol naïve HP vs. LP mice to investigate genes involved in the risk of ethanol drinking and in drinking and naïve HP mice to investigate the effects of ethanol drinking in this selected line by RNAscope. 2) Determination of chondroitin sulfate (CS)-, heparan sulfate (HS)-, and HA-GAG disaccharide levels in the mPFC and NAc of ethanol naïve and ethanol-drinking Aldh1l1-EGFP-Rpl10a and HP mice and in naïve LP mice. The proposed studies will characterize the role of astrocyte ECM and of sugar chains in the risk and consequences of ethanol drinking and identify cellular and molecular targets for intervention.

项目摘要 - P002 几项全基因组研究，包括PARC先前资助期的研究，确定了 细胞外基质（ECM）和星形胶质细胞参与乙醇的风险和影响 喝。大脑实质中的细胞被细胞外空间分开，约占 成熟啮齿动物大脑总体积的20％，并包含形成高度有组织的ECM 细胞周围的不溶性网络。大脑间质性ECM显着有助于分子信号 调节神经元可塑性。最近的研究表明，星形胶质细胞是大脑的主要生产者 间质ECM以及ECM蛋白，与ECM的重塑有关。 ECM蛋白是 通过糖基化对翻译后修饰。蛋白聚糖是大脑ECM的主要组成部分， 由核心蛋白共价结合到与重复二糖，形成的糖胺聚糖（GAG）的组成， 通过硫酸盐修饰。透明质酸（HA）也是ECM的主要组成部分，是一种非硫酸化的GAG，IS 与蛋白质无共价结合。插科打涉及神经元可塑性的调节。总体目标 该建议的是确定星形胶质细胞特异性基因的差异表达（主要强调 与ECM的形成和重塑有关的基因和相关的ECM插科 在涉及成瘾的两个大脑区域中饮用乙醇：内侧前额叶皮层（MPFC；包括 前缘和注射皮层）和伏隔核（NAC）。我们计划达到这个总体目标 通过追求以下两个具体目的：1）MPFC和NAC中星形胶质细胞翻译的定义 幼稚和乙醇饮用的Aldh1l1-EGFP-RPL10A小鼠；通过 QPCR和RNA范围分别在ALDH1L1-EGFP-RPL10A小鼠中；确定表达 乙醇幼稚的高乙醇偏好（HP）和低乙醇偏好（LP）中选定的ECM相关基因 小鼠，以及rnascope的乙醇饮用的HP小鼠。我们将使用翻译核糖体亲和力净化 （TRAP）ALDH1L1-EGFP-RPL10A小鼠中的技术，该小鼠表达具有改性的核糖体蛋白RPL10A EGFP TAG（EGFP-RPL10A）仅在表达星形细胞标记ALDH1L1的细胞中。选择差异 ECM相关蛋白的翻译RNA将通过QPCR及其差异表达和亚 区域定位将由rnascope确定。选定的ECM相关基因的差异表达 还将在乙醇天真的HP与LP小鼠中确定，以研究涉及乙醇风险的基因 喝酒和饮酒和幼稚的HP小鼠，以调查乙醇在这条选定系中的饮用作用 rnascope。 2）测定硫酸软骨素（CS） - 硫酸乙酰肝素（HS） - 和HA-GAG二糖 乙醇和乙醇饮用的Aldh1L1-EGFP-RPL10A和HP小鼠的MPFC和NAC的水平 幼稚的LP小鼠。拟议的研究将表征星形胶质细胞ECM和糖链在风险中的作用 乙醇饮用的后果，并确定干预的细胞和分子靶标。