7/11 Biochemical and Genetic Determinants of Alcohol Consumption

项目摘要 该项目是前一个INIA项目的继续和进一步发展，该项目是基于研究 显示乙醇诱导的动物模型和人类神经免疫基因表达的变化。那些 数据表明，乙醇通过髓样分化失调Toll样受体（TLR）信号传导 第一反应基因88（MyD 88），从而促进过量的乙醇摄入。我们最近的工作 表明通过TIR结构域对TLR信号传导的另一个分支的遗传和药理学操纵， 含有衔接子诱导干扰素-β（TRIF）蛋白的蛋白质也调节乙醇摄入。在探索下游 这些信号中断的作用机制，我们发现，慢性酒精消费增加， 几种TRIF依赖性信号成分，包括1型干扰素（IFN 1）。这些发现使我们 这一更新申请的中心假设是，慢性酒精暴露激活了 导致IFN 1的产生和干扰素刺激基因（ISG）的表达， 消费为了验证这一假设，我们将研究小鼠的酒精摄入量和酒精相关行为 缺乏导致IFN 1产生和信号传导的途径的关键组分， 阻断IFN 1信号传导的化合物。该提案有三个具体目标。具体目标1将研究 在每隔一天进行两瓶选择（EOD-2BC）饮酒的小鼠中， 至少4周。为了减少IFN 1的产生，我们将在基因缺失的小鼠中检查这些行为。 转录因子Irf 3或Irf 7。为了研究IFN 1信号传导的作用，我们将使用Ifnar 1敲除小鼠， 其在IFN 1受体中携带无效突变。我们还将研究野生型小鼠， 激酶TYK 2，其介导IFN 1受体信号传导。具体目标2将确定EOD-2BC是否饮用 通过检测Mx 1GFP小鼠的Fos表达，诱导特定脑区的IFN 1应答， Mx 1基因的干扰素刺激反应元件驱动GFP表达。从这些大脑中提取的数据 影像学研究将与Harsan-Keiffer项目共享，以纳入其多模式 连接体分析。为了确定IFN 1是否调节某些区域的酒精反应，我们将敲除 Ifnar 1使用局部显微注射Cre重组酶在floxed Ifnar小鼠中，并使用Fos-Cre-ER（TRAP 2）小鼠， 在激活的神经元和神经胶质中敲除或敲低Ifnar 1。具体目标3将识别由以下因素引起的ISG： EOD-2BC目标2中确定的大脑区域饮酒。梅菲尔德项目将帮助我们分析 转录组学数据来识别ISG。为了研究因果关系，将使用以下方法击倒ISG组： 与Farris-Homanics项目合作的多重CRISPR干扰。我们预计， INIA项目将确定需要用药理学试剂进行行为测试的其他蛋白质， 转基因小鼠，我们将作为这个联盟的一部分进行。 !

PROJECT SUMMARY This project is a continuation and further development of a previous INIA project, which was based on studies showing ethanol-induced changes in neuroimmune gene expression in animal models and humans. Those data suggested that ethanol dysregulates Toll-like receptor (TLR) signaling through the myeloid differentiation primary response gene 88 (MyD88) and thereby promotes excessive ethanol intake. Our most recent work showed that genetic and pharmacological manipulation of another branch of TLR signaling via the TIR-domain- containing adapter-inducing interferon-β (TRIF) protein also regulates ethanol intake. In exploring downstream mechanisms by which these signaling disruptions act, we found that chronic alcohol consumption increased several TRIF-dependent signaling components, including type 1 interferons (IFN1s). These findings lead us to the central hypothesis of this renewal application, which is that chronic alcohol exposure activates pathways leading to IFN1 production and expression of interferon-stimulated genes (ISGs), which increase alcohol consumption. To test this hypothesis, we will study alcohol intake and alcohol-related behaviors in mice deficient in critical components of pathways leading to IFN1 production and signaling, and in mice administered compounds that block IFN1 signaling. The proposal has three Specific Aims. Specific Aim 1 will study changes in alcohol consumption in mice undergoing every other day two-bottle choice (EOD-2BC) drinking for at least 4 weeks. To reduce IFN1 production, we will examine these behaviors in mice with genetic deletion of the transcription factors Irf3 or Irf7. To examine the role of IFN1 signaling, we will use Ifnar1 knockout mice which carry a null mutation in the IFN1 receptor. We will also study wild-type mice treated with inhibitors of the kinase TYK2, which mediates IFN1 receptor signaling. Specific Aim 2 will determine if EOD-2BC drinking induces IFN1 responses in specific brain regions by detecting Fos expression in Mx1GFP mice in which the interferon-stimulated response element of the Mx1 gene drives GFP expression. Data from these whole brain imaging studies will be shared with the Harsan-Keiffer project to be incorporated into their multimodal connectome analyses. To determine if IFN1 regulates alcohol responses in certain regions, we will knockout Ifnar1 using local microinjection of Cre recombinase in floxed Ifnar mice and use Fos-Cre-ER (TRAP2) mice to knockout or knockdown Ifnar1 in activated neurons and glia. Specific Aim 3 will identify ISGs induced by EOD-2BC alcohol drinking in brain regions identified in Aim 2. The Mayfield project will help us analyze transcriptomic data to identify ISGs. To investigate causality, groups of ISGs will be knocked down using multiplex CRISPR interference in collaboration with the Farris-Homanics project. We anticipate that some other INIA projects will identify additional proteins that require behavioral testing with pharmacological agents or genetically modified mice, which we will carry out as part of this consortium. !