Excessive Alcohol Drinking Associated with GABA Alpha 2-Regulated TLR4 Expression

描述（由申请人提供）：过度饮酒是一个巨大的公共卫生负担，需要更彻底的治疗。大多数与过度饮酒有关的健康问题是由两种类型的饮酒引起的：(i)狂欢（2小时内酒精浓度为0.08克）和（ii）复发（在一次或多次戒酒后持续大量饮酒至少2天）。先前，我们发现嗜酒(P)大鼠的GABAA亚基α 1、α 2和toll样受体4 （TLR4）先天免疫受体的表达水平升高，并使用特定的siRNA载体注入中央杏仁核（CeA），证明了GABAA a2调节的TLR4 （a2/TLR4轴）介导了该部位对酗酒的调节。虽然alpha1亚基也被证明可以调节酗酒，但它与腹侧白质（VP）有关，并且独立于TLR4 （Liu et al.， PNAS, 2011）。在目前的拨款中，我们建议更好地阐明TLR4效应的机制，重点关注趋化因子单核细胞趋化蛋白-1 （MCP-1）和多巴胺速率限制酶酪氨酸羟化酶(TH)-与上次提交申请后获得的数据有关-以及调节酒精成瘾周期不同域的大脑部位。工作假设是神经元TLR4通过激活细胞类型特异性转录因子诱导MCP-1表达，反过来，它作为神经递质刺激多巴胺释放和兴奋性（由TH推断）在选择的奖励位点。目的1 .确定P / NP大鼠酒精奖励位点GABAA a2/a1、TLR4、MCP-1和/或TH的表达。目的二世。确定a2/TLR4轴在冲动性狂饮中酒精奖励位点的作用，包括MCP-1和/TH。第三目标。确定a1， a2， TLR4及其下游靶标（MCP-1和/或TH）在酒精奖励位点在强迫饮酒复发中的作用。目的IV.通过使用pHSVsiMCP-1扩增子，重点关注上调MCP-1和TH表达的下游信号，明确tlr4介导的酗酒调节机制。更好地理解趋化因子在神经传递中的作用和不同（即多巴胺能）神经元的调节将有助于拓宽当前神经免疫通讯的概念及其在过度饮酒中的作用。这一高度创新的提议将为理解先天免疫信号与影响酒精成瘾的神经元反应之间的关系创造一个范式转变。使用无毒的单纯疱疹病毒(HSV)- siRNA构建物抑制特定脑位点的相关基因，将确定治疗基因靶点，并测试基因治疗方法治疗酗酒和复发的潜力。

DESCRIPTION (provided by applicant): Excessive alcohol drinking is an enormous public health burden in need of more radical therapy. Most health problems associated with excessive drinking are due to two types of drinking: (i) binge (BALs > 0.08 grams % in a 2-hour period) and (ii) relapse (sustained heavy drinking for at least 2 days following a single or multiple abstinence periods). Previously, we showed that alcohol- preferring (P) rats express elevated levels of the GABAA subunits for alpha1, alpha2 and toll-like receptor 4 (TLR4) innate immunity receptors, and using specific siRNA vectors infused into the central amygdala (CeA), demonstrated that regulation of binge drinking at this site is mediated by GABAA a2 regulated TLR4 (a2/TLR4 axis). While the alpha1 subunit was also shown to regulate binge drinking, it was associated with the ventral pallidum (VP) and was independent of TLR4 (Liu et al., PNAS, 2011). In the current grant, we propose to better elucidate the mechanism responsible for the TLR4 effect, focusing on the chemokine monocyte chemotactic protein-1 (MCP-1) and the dopamine rate- limiting enzyme tyrosine hydroxylase (TH)--implicated by data obtained after the application was last submitted--and on brain sites that regulate different domains of the alcohol addiction cycle. The working hypothesis is that neuronal TLR4 induces MCP-1 expression via activation of cell type- specific transcription factors and it, in turn, functions as a neurotransmitter to stimulate dopamine release and excitability (inferred by TH) in select reward loci. The specific aims are: Aim I. Define the expression of GABAA a2/a1, TLR4, MCP-1 and/or TH at alcohol reward loci from P vs NP rats. Aim II. Define the role played by the a2/TLR4 axis that encompasses MCP-1 and/TH at alcohol reward loci in impulsive binge drinking. Aim III. Define the contribution of a1, a2, TLR4 and its downstream targets (MCP-1 and/or TH) at alcohol reward loci in compulsive relapse drinking. Aim IV. Define the mechanism of TLR4-mediated regulation of binge drinking by focusing on downstream signals that upregulate MCP-1 and TH expression, through the use of the pHSVsiMCP-1 amplicon. Better understanding of the role of chemokines in neurotransmission and the regulation of distinct (viz. dopaminergic) neurons will help broaden current concepts of neuroimmune communication and their roles in excessive drinking. This highly innovative proposal will create a paradigm shift in understanding the relationship between innate immunity signals and neuronal responses that impact alcohol addiction. Use of non-toxic herpes simplex virus (HSV)- siRNA constructs to inhibit relevant genes at specific brain loci will define therapeutic gene targets and test the potential of gene therapy approaches for binge and relapse drinking.