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引起的基因表达变化。尽管转录后机制在基因表达的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很少受到关注。RNA结合蛋白和microrna是控制基因表达的主开关，据估计，mRNA的稳定性控制着约20%的脑表达基因。我们的研究表明，RNA结合蛋白HuD和microRNA miR-495在控制成瘾相关基因的表达和行为中发挥相反的作用：1)预计它们在mrna中结合相同的富含gu的序列；2)它们的结合位点在成瘾相关基因（ARG）数据库的转录本中被过度代表；3)可卡因对成瘾相关脑区有差异调节，miR-495下调，HuD上调；3)在体外操作这些分子会对其两个靶基因BDNF和arc的表达产生相反的影响；最重要的是，对这些分子的体内操作显示出对可卡因动机的不同影响。基于这些结果，我们假设HuD和miR- 495在药物滥用中发挥作用，通过转录后竞争结合到相同的序列，并在相反的方向上控制ARGs的表达。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)特异性mRNA结合位点和ARG基因表达的控制，以及b)可卡因条件下前脑神经元中HuD过表达小鼠的位置偏好；2)利用3种可卡因动机模型(1)可卡因强化递进比率表的断点，2)可卡因寻求行为的消失，3)已消失的可卡因寻求行为的恢复)，确定病毒介导的miR- 495基因转移和3)HuD对大鼠伏隔核壳的影响；4)检测miR-495和HuD水平的变化，以及选择的靶基因，包括BDNF和arc，在被操纵表达不同程度的可卡因动机的大鼠中。拟议的工作协同结合了Perrone-Bizzozero博士在mRNA稳定性、HuD功能和目标分析方面的专业知识，以及Neisewander博士在成瘾动物模型和相关神经回路方面的专业知识。这项工作的结果将提供关于调节成瘾相关基因表达的转录后机制的新知识，这是神经科学研究的一个令人兴奋的新领域。更好地理解这些调节机制是在成瘾研究中应用这些新工具并最终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. PUBLIC HEALTH RELEVANCE: Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in the establishment of addictive behaviors has received very little attention. Therefore, characterizing the competing roles of mR-495 and HuD in the control of addiction-related genes will unveil a new mechanism underlying the maladaptive changes in synaptic plasticity during drug addiction and provide potential new targets for intervention.