4/11 Neuroimmune and extracellular matrix interactions in alcohol consumption

慢性饮酒导致编码结构成分的基因转录变化 和细胞外基质(ECM)的调节剂。这些变化背后的分子机制和 在过度过度的情况下，ECM基因表达变化的功能含义还没有得到很好的理解 酗酒。我们假设免疫反应相关转录因子STAT3在 星形胶质细胞导致编码ECM成分的基因转录变化，导致 细胞外基质的结构和功能，突触的变化，以及酒精摄入量的增加。拟议中的实验将 用三个具体目标来解释这一假设。在具体目标1中，我们将不偏不倚地 确定长期进食的小鼠大脑皮质细胞外基质蛋白质组的变化 酒精类饮酒法(在黑暗中饮用)，为期6周。尖端蛋白质组学和 将进行血糖测定，以定量测量皮质细胞外基质的变化。这些结果将被比较 到现有和新的INIA转录组数据集中ECM相关基因的变化和分析的基因 启动子中的STAT3结合基序。在特定的目标2中，我们将确定星形胶质细胞表达的作用 通过有条件地敲除星形胶质细胞中的STAT3在酒精消费中的STAT3 星形胶质细胞特异性Cre系ALDH1L1-CreerT2。小鼠将接受类似狂欢的酒精消费测试，并 依赖导致酒精摄入量增加。活化的STAT3与基因启动子的相关性 用染色质免疫沉淀法测定慢性狂饮后的前额叶皮质 用磷酸化的STAT3抗体，然后进行全基因组DNA测序(CHIP-SEQ)。最后， 我们将确定特定的ECM基因和假定的STAT3靶标Brivican(Bcan)在酒精中的作用 消费，通过有条件地敲除星形胶质细胞中的bcan，如上所述。我们还将降低 利用病毒介导的shRNA检测bcan在小鼠前额叶皮质中的表达 入口处。Brivican定位于突触周围，调节突触的可塑性。以确定Brivican是否更多 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，我们将用短链抗体和抗体进行超分辨显微镜检查 慢性暴饮性饮酒后小鼠兴奋性和抑制性突触标志物。在每个特定的目标中， 我们将与INIA-神经免疫和INIA-压力研究人员整合并合作。完成 这些特定的目标将有助于通过以下方式了解分子和细胞机制 哪些长期饮酒会改变细胞外基质，并可能提供新的细胞靶点来减少过量饮酒 饮酒。

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.