Cellular signaling in alcoholism

酒精使用障碍(AUD)是退伍军人中以及普通人群中的一个主要健康问题。 退伍军人管理局使命的一个组成部分是为退伍军人管理局的酒精和/或药物患者提供护理。 虐待问题。停止长期饮酒会导致负性戒断 促进饮酒增加并维持酒精成瘾行为的症状。杏仁脑 众所周知，结构在焦虑行为和酒精成瘾中发挥着重要作用。这个 分子机制，特别是非编码microRNAs(miRNAs；21-23nt)在特定情况下的作用 参与酒精期间焦虑样行为发展的大脑神经回路 慢性酒精暴露后的戒断还不是很清楚。利用微阵列分析，我们最近 报道称，急性酒精暴露会在杏仁核产生不同的miRNA表达。此数据 显示急性酒精暴露可降低miRNA-494(miR-494)的表达，其靶点为 基因mRNA水平升高，并参与乙醇抗焦虑作用的调节。这项建议 将扩展这些研究并确定miR-494在染色质和突触调节中的作用 杏仁核重塑，导致酒精中毒的负面情绪状态。此外，这项提议将 确定酒精依赖期间其他miRNAs表达的变化，然后提出实验方案 为了研究miRNAs中的这些变化如何协调靶基因途径的表达变化， 调节慢性酒精暴露后戒断过程中的焦虑行为。我们假设 慢性暴露后停用乙醇会产生几个miRNAs的差异表达，并且 其中之一可能是miR-494。MiR-494的上调将导致靶基因的变化， 调节杏仁核的突触可塑性和树突，这可能是发育的基础 在酒精戒断过程中的焦虑行为。该提案的具体目标是：1)履行 酒精戒断大鼠杏仁核miRNA表达谱的微阵列研究 在长期接触酒精之后。新出现的miRNAs将得到验证，其表达的变化 假定的目标基因也将被检查。2)检查a)miR-494在CEA或 急性酒精刺激将减轻焦虑样行为，即大脑皮质激素受体基因表达(mRNA和蛋白)的缺失 直接靶标(CBP、p300、Cited2)和间接靶标(BDNF、Arc和突触素)以及 慢性酒精戒断大鼠杏仁核内突触和树突 暴露和b)将miR-494的合成模拟物注入对照组大鼠的CEA中会引起焦虑样反应 并减少杏仁核中靶基因的表达。3)检查CEA的影响是否 在对照组大鼠中注射Cited2 siRNA将a)产生焦虑样行为，从而模拟戒断- 与焦虑相关；b)防止对照组大鼠因急性酒精而产生的抗焦虑作用。行为 这些变化是由于Cited2的表达减少以及突触和树突的相关减少所致 大鼠CEA中的脊椎。拟议的研究将提供有关miRNA调控的新信息。 杏仁核中可能参与突触异常的表达及其靶基因 在酒精依赖过程中观察到的可塑性，并将识别新的miRNA靶标 开发治疗AUD的潜在药物。

Alcohol use disorder (AUD) is a major health concern in the VA population as well as in the general population. An integral part of the mission of the VA is to provide care for VA patients suffering from alcohol and/or drug abuse problems. Cessation of chronic ethanol consumption leads to development of negative withdrawal symptoms that promote increased alcohol drinking and maintain alcohol addictive behaviors. Amygdaloid brain structures are known to play an important role in anxiety behaviors as well as in alcohol addiction. The molecular mechanisms, and particularly the role of non-coding microRNAs (miRNAs; 21-23 nt), in specific neural circuits of the brain that are involved in the development of anxiety-like behaviors during ethanol withdrawal after chronic ethanol exposure are not well understood. Using microarray analysis, we recently reported that acute ethanol exposure produces differential miRNA expression in the amygdala. This data revealed that expression of miRNA-494 (miR-494) was decreased by acute ethanol exposure and its target gene mRNA levels were increased and involved in the regulation of anxiolytic effects of ethanol. This proposal will extend these studies and establish the role of miR-494 in the regulation of chromatin and synaptic remodeling in the amygdala, leading to negative affective state of alcoholism. In addition, this proposal will identify changes in the expression of other miRNAs during alcohol dependence and then propose experiments to investigate how these changes in miRNAs coordinate shifts in the expression of target gene pathways that regulate anxiety-like behaviors during withdrawal after chronic ethanol exposure. We hypothesize that ethanol withdrawal after chronic exposure will produce differential expression of several miRNAs, and one of these may be miR-494. The upregulation of miR-494 will induce changes in target genes that regulate synaptic plasticity and dendritic spines in the amygdala which may underlie the development of anxiety-like behaviors during ethanol withdrawal. The specific aims of the proposal are: 1) To perform miRNA expression profiling using a microarray approach in the amygdala of rats during ethanol withdrawal after chronic ethanol exposure. The emerging miRNAs will be validated and changes in the expression of their putative target genes will also be examined. 2) To examine if a) miR-494 antagomir infusion in the CeA or acute ethanol challenge will attenuate anxiety-like behaviors, deficits in the expression (mRNA and protein) of direct targets (Cbp, p300, Cited2) and indirect targets (Bdnf, Arc, and synaptophysin) as well as deficits in synapses and dendritic spines in the amygdala of rats during ethanol withdrawal after chronic ethanol exposure and b) synthetic mimics of miR-494 infused into CeA of control rats will provoke anxiety-like behaviors and reduce the expression of target genes in the amygdala. 3) To examine if the effects of CeA infusion of a Cited2 siRNA in control rats will a) produce anxiety-like behaviors, thus mimicking withdrawal- associated anxiety and b) prevent the anxiolytic effects produced by acute ethanol in control rats. Behavioral changes will be due to decreased expression of Cited2 and associated reductions in synapses and dendritic spines in the CeA of rats. The proposed studies will provide new information on the regulation of miRNA expression and their target genes in the amygdala that may be involved in the abnormal synaptic plasticity observed during alcohol dependence, and will also identify novel miRNA targets for the development of potential drugs for the treatment of AUD.