Competing roles of microRNAs and RNA-binding proteins in drug addiction

描述(由申请人提供)：控制可卡因的动机是可卡因成瘾长期治疗成功的目标，这可能需要逆转药物诱导的基因表达变化。尽管转录后机制在基因表达控制中起着至关重要的作用，但它们在成瘾行为中的作用却鲜有人关注。RNA结合蛋白和microRNAs是控制基因表达的主开关，据估计，mRNA的稳定性控制了大约20%的脑表达基因。我们的研究表明，RNA结合蛋白HUD和微RNA miR-495在成瘾相关基因的表达和行为控制中扮演着相反的角色：1)它们被预测与mRNAs中富含GU的序列结合；2)它们的结合位点在成瘾相关基因(ARG)数据库的转录本中过度表达；3)它们在成瘾相关脑区表现出不同的调控，miR-495下调和HUD上调；3)体外操作这些分子会对它们的两个靶基因BDNF和Arc的表达产生相反的影响；5)最重要的是，在体内对这些分子的操纵对可卡因的动机显示出截然不同的效果。基于这些结果，我们假设HUD和miR-495通过转录后竞争结合相同的序列和控制相反方向的Args表达在药物滥用中发挥作用。为了验证这一假设，我们将：1)测试HUD和miR-495之间的功能竞争：a)特定的mRNA结合位点和ARG基因表达的控制，以及b)在前脑神经元中过度表达HUD的小鼠的可卡因条件性位置偏爱；2)用以下3种可卡因刺激模型确定病毒介导的miR-495和3)HUD对大鼠伏隔核外壳的影响：i)可卡因强化递增比率的断点，ii)寻求可卡因行为的消失，以及iii)寻求可卡因行为消失的恢复；4)检测大鼠体内miR-495和HUD水平的变化，以及所选择的靶基因，包括BDNF和ARC，这些大鼠已经被操纵以表达不同程度的可卡因动机。这项拟议的工作将Perrone-Bizzozero博士在mRNA稳定性、HUD功能和靶标分析方面的专业知识与Neisewander博士在成瘾动物模型和涉及的神经回路方面的专业知识协同结合在一起。这项工作的结果将为调控成瘾相关基因表达的转录后机制提供新的知识，这是神经科学研究的一个令人兴奋的新领域。更好地了解这些调节机制是将这些新工具应用于成瘾研究并最终用于治疗这种疾病的先决条件。

DESCRIPTION (provided by applicant): Controlling motivation for cocaine is the goal of long-term treatment success of cocaine addiction, which may require reversal of drug-induced changes in gene expression. Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in addictive behaviors has received little attention. RNA binding proteins and microRNAs serve as master switches controlling gene expression, with mRNA stability estimated to control about 20% of brain-expressed genes. Our research suggests that the RNA binding protein HuD and the microRNA miR-495 play opposite roles in the control of addiction-related gene expression and behavior: 1) They are predicted to bind the same GU-rich sequence in mRNAs; 2) Their binding sites are overrepresented in transcripts from an addiction-related gene (ARG) database; 3) They show differential regulation by cocaine in addiction-related brain regions, with miR-495 being downregulated and HuD upregulated; 3) In vitro manipulations of these molecules result in opposite effects on the expression of two of their target genes, BDNF and arc; 5) Most importantly, in vivo manipulations of these molecules show contrasting effects on motivation for cocaine. Based upon these results, we hypothesize that HuD and miR- 495 play a role in drug abuse by post-transcriptionally competing for binding to the same sequences and controlling the expression of ARGs in opposing directions. To test this hypothesis, we will: 1) test the functional competition between HuD and miR-495 for a) specific mRNA binding sites and the control of ARG gene expression, and b) cocaine conditioned place preference in mice overexpressing HuD in forebrain neurons; 2) determine the effects of viral-mediated gene transfer of miR- 495 and 3) HuD to the nucleus accumbens shell of rats using the following 3 models of motivation for cocaine: i) break point on a progressive ratio schedule of cocaine reinforcement, ii) extinction of cocaine-seeking behavior, and iii) reinstatement of extinguished cocaine-seeking behavior; 4) examine changes in the levels of miR-495 and HuD and selected target genes, including BDNF and arc in rats which have been manipulated to express varying degrees of motivation for cocaine. The proposed work synergistically combines the expertise of Dr. Perrone-Bizzozero in mRNA stability, HuD function, and target analyses and Dr. Neisewander in animal models of addiction and the neurocircuitry involved. The outcome of this work will provide new knowledge about the post-transcriptional mechanisms regulating addiction-related gene expression, an exciting new area of neuroscience research. A better understanding of these regulatory mechanisms is a pre-requisite for the application of these new tools in addiction research and ultimately in the treatment of this disorder.