Astrocytes, alcohol, and the extracellular matrix

点击翻译按钮获取中文摘要

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.