Nr4a1 and GPR88 as novel gene targets for alcoholism: mouse genetic approaches

描述（由申请人提供）：我们假设Nr 4a 1和GprBB主要在扩展杏仁核（EA）水平调节酒精摄入和依赖，并代表治疗酒精中毒的潜在靶点。我们将使用常规和EA条件性敲除小鼠对这两个候选基因以及被认为是酒精研究中有效靶点的已知基因（μ阿片受体）进行测试。这些独特的突变系将在行为（目标1）和成像（目标2）水平进行研究，以表征这三个基因在过度饮酒中的作用。在目标1中，我们将检查我们实验室中可用的常规敲除小鼠的（i）慢性间歇暴露于酒精蒸气（CIE）后的酒精戒断和（ii）自愿饮酒沿着暴露于空气/酒精蒸气（CIE/TBC）的历史，导致娱乐性/过量饮酒（目标1a）。在Aim Ib中，我们将通过杂交floxed小鼠和Wfsl-Cre小鼠来产生和表征所有三种基因的EA条件小鼠，所有这些小鼠都是在过去的资助期内产生的。在目标1c中，我们将检查如目标1a中的条件突变株系。传统基因敲除小鼠的表型将确定靶基因在酒精摄入中的功能作用，而EA条件突变小鼠的表型将揭示EA回路在这些行为中的作用。在目标2中，我们将使用最先进的DT-MRI和纤维跟踪，并在小鼠中实施FcMRI（J. Hennig，弗赖堡，德国），以确定经历过度饮酒史的小鼠的结构和连接重塑。将在野生型小鼠中优化实验条件（目标2a和d），然后在纵向实验中，在慢性暴露（目标2b）或CIE/TBC范例（目标2c）中的自愿饮酒条件下，将其应用于具有最强行为表型（目标1的数据）的突变小鼠。目标2将扩大我们的知识，基因功能的动态解剖尺寸，并提供一个框架，从啮齿动物到人类的翻译研究。总之，该提案整合了独特的小鼠遗传工具和成像方法，在分子和系统水平上对酒精依赖的新基因进行功能研究。与INIA合作伙伴的互动包括分享材料（小鼠品系，AAV-shRNA）和知识（成像，小鼠和病毒）。 公共卫生相关性：INIA基因表达研究已经确定了酒精研究的新候选基因。我们选择了两个基因，在几项研究中受到酒精暴露的调节。Nr 4a 1编码作为转录因子的孤儿核受体，Gpr 88编码具有未知功能的孤儿G蛋白偶联受体。这两种受体都是在成人大脑中表达的药物靶点。对它们在大脑功能中的作用的研究才刚刚开始，它们在酒精成瘾中的潜在参与还没有得到研究。

DESCRIPTION (provided by applicant): We hypothesize that Nr4a1 and GprBB regulate alcohol intake and dependence, mainly at the level of the extended amygdala (EA), and represent potential targets to treat alcoholism. We will test this hypothesis using conventional and EA-conditional knockout mice for the two candidate genes, as well as for a known gene considered a valid target in alcohol research (the mu opioid receptor). These unique mutant lines will be studied at behavioral (Aim 1) and imaging (Aim 2) levels to characterize implication of the three genes in excessive alcohol drinking. In Aim 1 we will examine conventional knockout mice available in our laboratory for (i) alcohol withdrawal after chronic intermittent exposure to alcohol vapors (CIE) and (ii) voluntary drinking along a history of exposure to air / alcohol vapors (CIE/TBC) leading to recreational / excessive drinking (Aim la). In Aim lb we will produce and characterize EA-conditional mice for all three genes by crossing floxed mice and Wfsl-Cre mice, all of which were generated in the past funding period. In Aim 1c we will examine conditional mutant lines as in Aim la. Phenotypes in conventional knockout mice will establish a functional role of targeted genes in alcohol intake, and phenotypes in EA-conditional mutant mice will uncover implication of EA circuitry in those behaviors. In Aim 2, we will use cutting-edge DT-MRI and fiber tracking, and implement FcMRI in mice (coll. J. Hennig, Freiburg, Germany) to identify structural and connectivity remodeling in mice undergoing a history of excessive drinking. Experimental conditions will be optimized in wild-type mice (Aims 2a and d), then applied to mutant mice with strongest behavioral phenotype (data from Aim 1) under conditions of chronic exposure (Aim 2b) or voluntary drinking in the CIE/TBC paradigm (Aim 2c) in longitudinal experiments. Aim 2 will expand our knowledge of gene function with a dynamic anatomical dimension and provide a framework for translational studies from rodents to humans. Together, the proposal integrates unique mouse genetic tools and imaging methodology, to functionally study novel genes in alcohol dependence at molecular and system levels. Interactions with INIA partners involve sharing of material (mouse lines, AAV-shRNAs) and knowledge (imaging, mice and viruses). PUBLIC HEALTH RELEVANCE: INIA gene expression studies have identified novel candidate genes for alcohol research. We have selected two genes, regulated by alcohol exposure in several studies. Nr4a1 encodes an orphan nuclear receptor that acts as a transcription factor, and Gpr88 encodes an orphan G protein coupled receptor with unknown function. Both receptors are druggable targets expressed in the adult brain. Study of their role in brain function is only beginning and their potential involvement in alcohol addiction has not been examined.