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调控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作为控制基因表达的主开关，mRNA稳定性估计控制约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起着相反的作用：1）它们被预测与mRNA中相同的富含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）可卡因对成瘾相关脑区的调节存在差异，miR-495下调，HuD上调; 3）在体外对这些分子的操作导致对它们的两个靶基因BDNF和arc的表达的相反作用; 5）最重要的是，这些分子的体内操作显示出对可卡因动机的对比效应。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序列并以相反方向控制ARG的表达在药物滥用中发挥作用。为了验证这一假设，我们将：1）在前脑神经元中过表达HuD的小鼠中，测试HuD和miR-495之间对a）特异性mRNA结合位点和ARG基因表达的控制以及B）可卡因条件性位置偏爱的功能竞争; 2）确定病毒介导的miR- 495基因转移的作用，和3）使用以下3种可卡因动机模型对大鼠的延髓核壳进行HuD：i）可卡因强化的渐进比率时间表上的断点，ii）可卡因寻求行为的消退，和iii）恢复消失的可卡因寻求行为; 4）检查大鼠中miR-495和HuD以及所选靶基因（包括BDNF和arc）水平的变化，所述大鼠已被操纵以表达不同程度的可卡因动机。这项拟议的工作协同结合了Perrone-Bizzozero博士在mRNA稳定性、HuD功能和靶点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有关转录后机制调节成瘾相关基因表达的新知识，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治疗这种疾病的先决条件。 公共卫生相关性：虽然转录后机制在基因表达的控制中起着至关重要的作用，但它们在成瘾行为建立中的作用却很少受到关注。因此，表征mR-495和HuD在控制成瘾相关基因中的竞争作用将揭示药物成瘾期间突触可塑性适应不良变化的新机制，并提供潜在的新干预靶点。

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.