MicroRNA regulation of addiction-related genes and drug abuse-related behavior

描述(申请人提供)：药物依赖的特点是反复、无法控制地使用药物，即使在长期戒毒后也是如此。这表明，大脑发生了持久的变化，即使在停止使用药物后也可以持续。长期变化背后的一个机制是基因表达的改变。最近的研究主要集中在DNA水平上的表观遗传学改变，然而，对转录后过程对基因表达的影响知之甚少。MicroRNAs(MiRNAs)是一类小的、高度保守的非编码转录本，控制着靶mRNAs的持续时间和表达，并与发育和疾病状态有关。这一令人兴奋的研究领域为揭示药物成瘾的潜在机制开辟了新的领域。我们的实验室与Nora Perrone-Bizzozero博士合作，有初步数据表明，miR-495是一种纹状体表达的miRNA，有几个与成瘾相关的基因作为预测靶点，在急性可卡因反应中，miR-495在伏隔核(NAC)中下调，而它的两个预测靶点BDNF和Arc上调。此外，miR-495在NAC中的病毒过表达降低了BDNF和ARC的水平，更重要的是，减少了大鼠在递增比率(PR)可卡因强化计划中的反应。我们假设，miR-495水平的增加对参与可卡因强化和动机的成瘾相关基因进行了负面调节。为了验证我们的假设，我们将首先检查病毒过表达或下调NAC中miR-495水平对可卡因自我给药低比率和PR强化时间表的影响，并测量中皮质边缘结构中miR-495、BDNF和ARC水平的相关变化。我们希望重复我们之前的发现，即miR-495过表达后PR反应减弱，BDNF和ARC水平下降，我们预测当miR-495下调时会产生相反的效果。此外，我们计划在蔗糖强化上使用相同的操作来测试我们的效果是否对可卡因特有。其次，我们将研究操纵miR-495水平对短期和长期强制戒毒后寻求可卡因行为的影响。由于长期戒断与药物动机的增加(即孵化效应)有关，这些实验将更具体地研究miR-495在药物动机中的作用。我们预测miR-495的过表达将减少这两个时间点的可卡因寻找，并可能阻止孵化效应和相关的BDNF和ARC表达的变化，同时保持蔗糖寻找不变。这一结果可能表明了一种新的方法来转录后调控几个成瘾相关基因，从而为开发药物依赖的治疗提供了新的途径。

DESCRIPTION (provided by applicant): Drug dependence is characterized by recurrent, uncontrollable bouts of drug use, even after prolonged periods of abstinence. This suggests enduring changes occur in the brain that can last even after drug use has discontinued. A mechanism underlying long-lasting changes is altered gene expression. Recent research has focused on epigenetic alterations at the DNA level, however, little is known about the impact of post- transcriptional processes on gene expression. MicroRNAs (miRNAs), a class of small, highly conserved non- coding transcripts, control the duration and expression of target mRNAs and have been implicated in development and disease states. This exciting research area forges new ground into uncovering potential mechanisms of drug addiction. Our laboratory, in collaboration with Dr. Nora Perrone-Bizzozero, has preliminary data demonstrating that miR-495, a striatal-expressed miRNA that has several addiction-related genes as predicted targets, is downregulated in the nucleus accumbens (NAc) in response to acute cocaine, while two of its predicted targets, BDNF and arc, are upregulated. Furthermore, viral overexpression of miR- 495 in the NAc decreases BDNF and arc levels, and more importantly, reduces responding in rats during a progressive ratio (PR) schedule of cocaine reinforcement. We hypothesize that increasing levels of miR-495 negatively regulates addiction-related genes involved in cocaine reinforcement and motivation. To test our hypothesis, we will first examine the effects of virally overexpressing or knocking down levels of miR-495 in the NAc on cocaine self-administration on low ratio and PR schedules of reinforcement, and measure the associated changes in miR-495, BDNF, and arc levels in mesocorticolimbic structures. We expect to replicate our previous finding of attenuated PR responding and decreased levels of BDNF and arc after miR-495 overexpression, and we predict the opposite effect when miR-495 is downregulated. Furthermore, we plan to use the same manipulations on sucrose reinforcement to test whether our effects are specific to cocaine. Second, we will examine the effects of manipulating miR-495 levels on cocaine-seeking behavior following short and long periods of forced abstinence. Because prolonged abstinence is associated with increased motivation for drug (i.e. incubation effect), these experiments will more specifically address the role of miR-495 in drug motivation. We predict overexpression of miR-495 will decrease cocaine seeking at both time points and may prevent the incubation effect and associated changes in BDNF and arc expression, while leaving sucrose seeking unaltered. The results may suggest a novel approach to post-transcriptionally regulate several addiction-related genes thereby providing a new avenue for developing treatment for drug dependence.