Astrocytes, alcohol, and the extracellular matrix

项目总结- P002 几项全基因组研究，包括PARC前一个资助期的研究，确定了 细胞外基质（ECM）和星形胶质细胞参与了乙醇的风险和影响 喝酒脑实质中的细胞被细胞外间隙隔开，约占 20%的成熟啮齿动物大脑的总体积，并含有高度组织化的ECM，形成一个 细胞周围的不溶性网络。脑间质ECM对分子信号有显著贡献 调节神经可塑性。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蛋白酶，其参与ECM的重塑。ECM蛋白是 通过糖基化进行后修饰。蛋白聚糖是脑ECM的主要成分， 由与重复二糖形成的糖胺聚糖（GAG）共价结合的核心蛋白组成， 通过硫酸化改性。透明质酸（HA），也是ECM的主要组分，是非硫酸化的GAG， 不与蛋白质共价结合。GAG参与神经元可塑性的调节。总目标 这项建议的一个重要目的是鉴定星形胶质细胞特异性基因的差异表达（主要强调 参与ECM形成和重塑的基因）和ECM GAG相关的差异水平 饮酒后，大脑中两个区域与成瘾有关：内侧前额叶皮层（mPFC;包括 前边缘和下边缘皮质）和丘脑核（NAc）。我们计划实现这一总体目标 通过追求以下两个具体目标：1）在mPFC和NAc中星形胶质细胞翻译组的定义， 未处理和饮用乙醇的Aldh 1 l1-EGFP-Rpl 10a小鼠;通过 在Aldh 111-EGFP-Rpl 10a小鼠中分别通过qPCR和RNA scope; 乙醇初始高乙醇偏好（HP）和低乙醇偏好（LP）中选定的ECM相关基因 小鼠和饮用乙醇的HP小鼠中的RNA。我们将使用翻译核糖体亲和纯化 在Aldh 111-EGFP-Rpl 10a小鼠中使用TRAP技术，所述小鼠表达经修饰的核糖体蛋白Rpl 10a， eGFP标签（EGFP-Rpl 10a）仅在表达星形胶质细胞标志物Aldh 111的细胞中。差异化选择 ECM相关蛋白的翻译RNA将通过qPCR和它们的差异表达和亚表达来确认。 区域定位将由RNAscope确定。选择的ECM相关基因的差异表达 还将在未经乙醇处理的HP与LP小鼠中测定，以研究涉及乙醇风险的基因 饮酒以及饮酒和幼稚HP小鼠，以通过以下方式研究酒精饮酒在该选定系中的影响： RNAscope。2)硫酸软骨素（CS）-、硫酸乙酰肝素（HS）-和HA-GAG二糖的测定 未接触乙醇和饮用乙醇的Aldh 1 l1-EGFP-Rpl 10a和HP小鼠的mPFC和NAc中的水平，以及 幼稚LP小鼠。拟议的研究将描述星形胶质细胞ECM和糖链在风险中的作用。 和酒精饮用的后果，并确定干预的细胞和分子目标。

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.