5/13 ALK and Midkine as Novel Neuroimmune Regulators of Alcohol Consumption

酒精中毒综合神经科学倡议（INIA）的目标之一-神经免疫 联盟是了解大脑免疫信号系统及其在病因和治疗中的作用， 酒精依赖作为INIA的一部分，我们已经确定了受体酪氨酸激酶，间变性淋巴瘤， 激酶（ALK）及其假定的分泌配体中期因子（MDK），作为可能参与 酒精使用障碍已知ALK和MDK通过体内免疫应答途径发出信号， 但ALK和MDK在免疫信号传导中的作用，特别是在大脑中对乙醇的反应， 被检查过了本项目第一个具体目标的目标是鉴定乙醇响应性MDK-和 使用MDK和ALK抑制剂的脑中ALK依赖性神经免疫信号传导通路。为此将 通过给小鼠注射单剂量的乙醇或让小鼠饮用酒精。磷酸化 参与神经免疫信号传导途径的蛋白质将在第一个实验中测量。在第二 在本实验中，将测试编码细胞因子和趋化因子的基因的表达。在第三个实验中， 激活小胶质细胞，大脑中的常驻免疫细胞，和星形胶质细胞，它们被激活， 炎症过程，将被检查。 靶向ALK的小分子抑制剂可有效减少小鼠的狂饮，这表明 ALK是治疗酒精使用障碍的潜在治疗靶点。机制通过 ALK调节过量饮酒的作用尚不清楚。我们的INIA工作证据表明 ALK可能在杏仁核中央核（CeA）中起作用，这是一个与乙醇有关的关键大脑区域 依赖，调节过量的乙醇消耗和GABA神经传递。的目标 本项目的第二个具体目的是测试CeA中MDK和ALK操作对这两种细胞的影响。 参数将进行三个实验。第一个涉及病毒传递短发夹RNA 将ALK或MDK直接靶向至CeA。在第二和第三个实验中，我们将注入ALK和MDK 抑制剂，或重组MDK蛋白，进入CeA。老鼠将被测试酒精的摄入量 以及输注后通过电生理学测定GABA介导的抑制电流。 INIA-Neuroimmune的另一个目标是使用新技术来研究参与神经系统的神经回路。 过度饮酒的发展。为此，第三个具体目标的目标是发展和 为INIA研究人员生产用于选择性敲除、敲除或过表达的尖端病毒载体 基因。这些载体将提供给INIA研究人员，用于递送到参与以下研究的特定大脑区域： 酒精依赖这将允许鉴定INIA候选基因在其中表达的神经通路。 控制饮酒的功能。该提案涉及与INIA-神经免疫的许多相互作用 调查员，并将协助实现联合体的目标。

One of the goals of the Integrative Neuroscience Initiative on Alcoholism (INIA)-Neuroimmune consortium is to understand brain immune signaling systems and their role in the causes and treatments of alcohol dependence. As part of INIA, we have identified the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and its putative secreted ligand midkine (MDK), as novel signaling proteins that may be involved in alcohol use disorders. ALK and MDK are known to signal through immune response pathways in the body, but the roles of ALK and MDK in immune signaling specifically in the brain in response to ethanol have not been examined. The objective of the first Specific Aim of this project is to identify ethanol-responsive MDK- and ALK-dependent neuroimmune signaling pathways in the brain using MDK and ALK inhibitors. This will be done by treating mice either with a single dose of ethanol or allowing mice to drink alcohol. Phosphorylation of proteins involved in neuroimmune signaling pathways will be measured in the first experiment. In the second experiment, expression of genes encoding cytokines and chemokines will be tested. In the third experiment, activation of microglia, the resident immune cells in the brain, and astrocytes, which are activated by inflammatory processes, will be examined. Small-molecule inhibitors targeting ALK are effective in reducing binge-like drinking in mice, suggesting that ALK is a potential therapeutic target for the treatment of alcohol use disorders. The mechanism(s) through which ALK acts to regulate excessive drinking are not well understood. Evidence from our INIA work suggests that ALK might act in the central nucleus of the amygdala (CeA), a key brain region involved in ethanol dependence, to regulate excessive ethanol consumption and GABA neurotransmission. The goal of the second Specific Aim of this project is to test for effects of MDK and ALK manipulation in the CeA on these two parameters. Three experiments will be performed. The first involves viral delivery of short hairpin RNAs targeting ALK or MDK directly into the CeA. In the second and third experiments, we will infuse ALK and MDK inhibitors, or recombinant MDK protein, into the CeA. Mice will be tested for binge-like ethanol consumption and for GABA-mediated inhibitory currents by electrophysiology after infusion. Another goal of INIA-Neuroimmune is to use new technologies to study neural circuits involved in the development of excessive alcohol drinking. To this end, the objective of the third Specific Aim is to develop and produce cutting-edge viral vectors for INIA investigators for selective knockdown, knockout, or overexpression of genes. These vectors will be provided to INIA investigators for delivery to specific brain regions involved in alcohol dependence. This will allow for the identification of the neural pathways in which INIA candidate genes function to control alcohol drinking. This proposal involves numerous interactions with INIA-Neuroimmune investigators and will assist in the achievement of the goals of the consortium.