4/11 Neuroimmune and extracellular matrix interactions in alcohol consumption

慢性酒精消耗诱导编码结构成分的基因的转录变化 和细胞外基质（ECM）的调节剂。这些变化背后的分子机制， ECM基因表达改变的功能意义在过度表达的背景下还没有得到很好的理解。 饮酒。我们假设，免疫应答相关转录因子STAT3的激活可能与免疫应答相关转录因子STAT3的激活有关。 星形胶质细胞导致编码ECM成分的基因的转录变化，导致细胞外基质的改变。 ECM结构和功能，突触变化和乙醇摄入量增加。拟议的实验将 通过三个具体目标来解决这一假设。在具体目标1中，我们将使用无偏见的方法， 确定皮质ECM蛋白质组的变化，或在小鼠中的"基质体"， 乙醇在一个暴饮暴食的协议（在黑暗中饮用）6周。尖端的蛋白质组学和 将进行糖组学以定量测量皮质ECM的变化。这些结果将被比较 在现有的和新的INIA转录组数据集中ECM相关基因的变化，以及分析的基因， STAT3结合基序。在具体目标2中，我们将确定星形胶质细胞表达的 通过使用floxed Stat3小鼠有条件地敲除星形胶质细胞中的Stat3， 星形胶质细胞特异性Cre系Aldh1l1-CreERT2。将测试小鼠的酒精摄入量， 酒精摄入量的依赖性增加。活化的STAT3与肿瘤细胞基因启动子的相关性 还将使用染色质免疫沉淀法在慢性酗酒后确定前额叶皮质 用磷酸化STAT3的抗体，然后进行全基因组DNA测序（ChIP-Seq）。最后， 我们将确定一个特定的ECM基因和假定的STAT3靶点，短蛋白聚糖（Bcan）在酒精中的作用。 通过如上所述有条件地敲除星形胶质细胞中的Bcan，可以抑制Bcan的消耗。我们还将减少 使用病毒递送的shRNA在小鼠前额叶皮层中表达Bcan并测量暴食样乙醇 摄入短蛋白聚糖定位于突触周围并调节突触可塑性。为了确定Brevican是否比 与慢性饮酒后的突触高度相关，如果PFC中的突触结构被改变， 慢性乙醇，我们将进行超分辨率显微镜与短蛋白聚糖抗体和抗体， 兴奋性和抑制性突触标记物在小鼠慢性狂饮后。在每个具体目标中， 我们将与INIA-Neuroimmune和INIA-Stress研究人员进行整合和合作。完成 这些具体目标将有助于分子和细胞机制的基本知识， 慢性饮酒改变了ECM，并可能提供新的细胞靶点，以减少过度的细胞毒性。 酒精消费。

Chronic alcohol consumption induces transcriptional changes in genes encoding structural components and regulators of the extracellular matrix (ECM). The molecular mechanisms underlying these alterations and the functional implications of altered ECM gene expression are not well understood in the context of excessive alcohol drinking. We hypothesize that activation of the immune response-related transcription factor STAT3 in astrocytes results in transcriptional changes in genes encoding ECM components, resulting in alterations in ECM structure and function, synaptic changes, and increased ethanol intake. The proposed experiments will address this hypothesis with three specific aims. In Specific Aim 1, we will use an unbiased approach to determine changes in the cortical ECM proteome, or “matrisome” in mice that have chronically consumed ethanol in a binge-like drinking protocol (drinking in the dark) for 6 weeks. Cutting-edge proteomics and glycomics will be performed to quantitatively measure changes in cortical ECM. These results will be compared to changes in ECM-related genes in existing and new INIA transcriptome datasets and the genes analyzed for STAT3 binding motifs in their promoters. In Specific Aim 2, we will determine the role of astrocyte-expressed STAT3 in alcohol consumption by conditionally knocking out Stat3 in astrocytes using floxed Stat3 mice and the astrocyte-specific Cre line, Aldh1l1-CreERT2. Mice will be tested for binge-like ethanol consumption and dependence-induced escalation of ethanol intake. Association of activated STAT3 with gene promoters in the prefrontal cortex will also be determined after chronic binge-like drinking using chromatin immunoprecipitation with an antibody to phosphorylated STAT3, followed by whole-genome DNA sequencing (ChIP-Seq). Finally, we will determine the role of a specific ECM gene and putative STAT3 target, brevican (Bcan) in alcohol consumption, by conditionally knocking out Bcan in astrocytes as described above. We will also reduce the expression of Bcan in the prefrontal cortex of mice using viral-delivered shRNA and measure binge-like ethanol intake. Brevican is localized perisynaptically and regulates synaptic plasticity. To determine if brevican is more highly associated with synapses after chronic ethanol drinking and if synapse structure in the PFC is altered by chronic ethanol, we will perform super-resolution microscopy with brevican antibody and antibodies to excitatory and inhibitory synaptic markers in mice after chronic binge-like drinking. Within each Specific Aim, we will integrate and collaborate with INIA-Neuroimmune and INIA-Stress investigators. The completion of these Specific Aims will contribute to fundamental knowledge of the molecular and cellular mechanisms by which chronic alcohol drinking alters the ECM and potentially provide new cellular targets to reduce excessive alcohol consumption.