Astrocytes, alcohol, and the extracellular matrix

项目总结--P002 几项全基因组研究，包括前一次PARC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 喝酒。脑实质中的细胞由细胞外空间隔开，约占 占成熟啮齿动物脑总体积的20%，并含有高度组织化的细胞外基质，形成 无法溶解的细胞周围网络。脑间质ECM对分子信号有重要贡献 调节神经元的可塑性。最近的研究表明，星形胶质细胞是大脑的主要制造者 间质细胞外基质和细胞外基质蛋白水解酶参与细胞外基质的重塑。ECM蛋白是 翻译后通过糖基化修饰。蛋白多糖是脑细胞外基质的主要成分， 由一种核心蛋白共价结合到由重复二糖形成的糖胺多聚糖(GAG)上， 通过硫酸盐化而改变的。透明质酸(HA)也是ECM的主要成分，是一种非硫酸盐化的GAG，是 不与蛋白质共价结合的。GAG参与了神经元可塑性的调节。总目标 这一建议的目的是确定星形胶质细胞特异性基因的差异表达(主要强调 参与细胞外基质的形成和重塑的基因)和相关的细胞外基质GAG的差异水平 饮酒与成瘾有关的两个大脑区域：内侧前额叶皮质(mPFC；包括 前额叶和下缘皮质)和伏隔核(NAC)。我们计划实现这一首要目标 通过追求以下两个具体目标：1)在mPFC和NAC中定义星形胶质细胞翻译组 幼稚和饮酒的ALDH1L1-EGFP-Rpl10a小鼠；差异翻译和表达的验证 在ALDH1L1-EGFP-Rpl10a小鼠中分别通过qPCR和RNA显微镜检测； 乙醇天真高偏好(HP)和低偏好(LP)中ECM相关基因的筛选 用RNAScope对饮酒的幽门螺杆菌小鼠进行了检测。我们将使用翻译核糖体亲和纯化 (TRAP)技术在ALDH1L1-EGFP-Rpl10a小鼠中表达修饰的核糖体蛋白Rpl10a EGFP标记(EGFP-Rpl10a)仅在表达星形细胞标记ALDH1L1的细胞中表达。差异化选择 ECM相关蛋白的翻译RNA将通过qPCR及其差异表达和亚 地区本地化将由RNAScope决定。部分细胞外基质相关基因的差异表达 也将在乙醇幼稚的HP和LP小鼠中确定，以研究与乙醇风险相关的基因 在饮酒和饮酒中，以及幼稚的HP小鼠中，通过以下方式研究酒精饮酒的影响 RNAScope。2)硫酸软骨素(CS)、硫酸乙酰肝素(HS)和HA-GAG双糖的测定 单纯酒精和饮酒ALDH1L1-EGFP-Rpl10a和HP小鼠及正常小鼠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。 和酒精饮酒的后果，并确定干预的细胞和分子靶点。

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.