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

描述(申请人提供)：过度饮酒是一个巨大的公共健康负担，需要更激进的治疗。与过度饮酒有关的大多数健康问题是由两种类型的饮酒引起的：(一)酗酒(两小时内酗酒0.08克)和(二)故态复萌(在一次或多次禁酒后持续酗酒至少两天)。此前，我们发现酗酒(P)大鼠表达Alpha1、Alpha2和Toll样受体4(TLR4)先天免疫受体的GABAA亚单位水平升高，并使用特定的siRNA载体注入中央杏仁核(CEA)，证明该部位对酗酒的调节是由GABAA a2调节的TLR4(A2/TLR4轴)介导的。虽然α1亚单位也被证明可以调节酗酒，但它与腹侧苍白球(VP)相关，并且独立于TLR4(Liu等人，PNAS，2011)。在目前的拨款中，我们建议更好地阐明TLR4效应的机制，重点是趋化因子单核细胞趋化蛋白-1(MCP-1)和多巴胺限速酶酪氨酸羟基酶(TH)--这与上次提交申请后获得的数据有关--以及调节酒精成瘾循环不同领域的大脑部位。工作假设是，神经元TLR4通过激活细胞类型特异的转录因子来诱导MCP-1的表达，并反过来作为神经递质来刺激多巴胺的释放和选定奖赏基因的兴奋性(由TH推断)。研究的具体目标为：目的1.明确P与NP大鼠酒精奖赏基因GABAA a2/A1、TLR4、MCP-1和/或TH的表达。目的II.确定包含MCP-1和/或TH的A2/TLR4轴在冲动性酗酒中所起的作用。目的III.明确A1、A2、TLR4及其下游靶基因(MCP-1和/或TH)在强迫性复饮性饮酒中对酒精奖赏基因的贡献。目的利用PHSVsiMCP-1扩增子，通过下游信号上调MCP-1和TH的表达，明确TLR4介导的酗酒调控机制。更好地了解趋化因子在神经传递中的作用以及对不同的(即。多巴胺能神经元将有助于拓宽目前神经免疫交流的概念，以及它们在过度饮酒中的作用。这一极具创新性的提议将在理解影响酒精成瘾的先天免疫信号和神经元反应之间的关系方面创造一个范式转变。使用无毒的单纯疱疹病毒(HSV)-siRNA构建物在特定的大脑位置抑制相关基因，将确定治疗基因靶点，并测试针对酗酒和复饮者的基因治疗方法的潜力。

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.