MicroRNA-mediated mechanisms of heroin drug seeking

 描述(申请人提供)：流行病学趋势表明，近年来，高度成瘾的阿片类海洛因的滥用和过量发生率增加了两倍多。然而，缺乏有效的治疗选择，这反映在阿片类药物复发和过量的高比率上。这个问题的核心是在阿片类药物戒毒后很长一段时间内药物渴望持续甚至增强的能力。近年来，这种“渴望的孵化”效应的行为机制已经得到了很好的刻画，但需要更多的分子洞察来确定大脑中在长期戒毒后持续寻求药物的可用药靶点。为此，该项目的目标是在microRNA(MiRNA)介导的翻译机制有助于长期戒毒后继续寻求海洛因的假设的指导下，确定维持强烈寻求药物的机制。虽然它们在寻找阿片类药物中的作用尚未被描述，但miRNAs是本研究的理想焦点，因为每个miRNA可以靶向数百个基因，使单个miRNA具有高度的机械灵活性和广泛的基因组范围，能够协调复杂的行为。事实上，对它们在成瘾中的作用的有限探索已经产生了令人兴奋和有希望的结果，例如阿片类激动剂的调节和酒精和可卡因寻求的修饰。为了实现该项目的目标，赠款的指导阶段将用于获得海洛因自我给药、蛋白质测序和生物信息学工具方面的培训，以确定miRNA-蛋白质靶标相互作用。目前，候选人正在研究miRNAs在创伤后应激障碍(PTSD)小鼠模型创伤记忆存储中的作用，并在初步数据中使用小RNA测序(miRNA-Seq)来测量创伤学习经历后持续的miRNA表达(&gt；30天)。在指导的K99期间，当前项目的范围将扩大到执行蛋白质组分析，并将其与现有的miRNA-Seq数据集进行比对，从而能够识别将在PTSD模型中进行功能测试的miRNA通路，以确定其对创伤应激记忆的支持。在R00期间，候选人将应用获得的miRNAs和阿片类药物药理学的技能和知识，利用海洛因自我给药的啮齿动物模型研究miRNA在海洛因渴求孵化中的机制。着眼于参与阿片类药物渴求孵化的两个大脑区域，中央杏仁核和眶前皮质，将在体内操纵miRNAs的表达及其途径，以从功能上识别在海洛因戒断30天后支持药物寻找的miRNA机制。该项目的完成将确定在无毒大脑中寻找阿片类药物的新机制，这是防止药物使用复发的关键一步。

 DESCRIPTION (provided by applicant): Epidemiological trends indicate that the incidence of abuse and overdose of the highly addictive opioid heroin have more than tripled in recent years. However, effective treatment options are lacking and this is reflected in the high rates of opioid relapse and overdose. At the center of this issue lies the ability of drug cravings to persist and even strengthen long after opioid detoxification. The behavioral mechanics of this "incubation of craving" effect have become well-characterized in recent years, but more molecular insight is required to identify druggable targets within the brain that sustain drug seeking after prolonged abstinence. To that end, the goal of this project is to identify mechanisms that sustain strong drug seeking, guided by the hypothesis that microRNA (miRNA)-mediated translational mechanisms contribute to continued heroin seeking after prolonged abstinence. While their role in opioid seeking has not been described, miRNAs are an ideal focus for this study because each miRNA can target hundreds of genes, giving a single miRNA a high degree of mechanistic flexibility and a wide genomic range, capable of orchestrating complex behaviors. Indeed, limited exploration into their role in addiction has yielded exciting and promising results, such a regulation by opioid agonists and modification of alcohol and cocaine seeking. To address the goal of the project, the mentored phase of the grant will be used to gain training in heroin self-administration, protein sequencing and bioinformatic tools to identify miRNA-protein target interactions. At present, the candidate is studying the role of miRNAs in traumatic memory storage in a mouse model of post-traumatic stress disorder (PTSD) and in preliminary data employed small RNA sequencing (miRNA-Seq) to measure lasting miRNA expression (>30 days) after a traumatic learning experience. During the mentored K99 period, the scope of the current project will be extended to perform proteomic analysis and align it with the existing miRNA-Seq dataset, enabling the identification of miRNA pathways that will be functionally tested for their support of traumatic stress memories in the PTSD model. In the R00 period, the candidate will then apply the acquired skills and knowledge of miRNAs and opioid pharmacology to study miRNA mechanisms in the incubation of heroin craving using a rodent model of heroin self-administration. Focusing on two brain regions involved in the incubation of opioid craving, the central amygdala and orbitofrontal cortex, expression of miRNAs and their pathways will be manipulated in vivo to functionally identify miRNA mechanisms that sustain drug seeking after 30 days of abstinence from heroin. Completion of this project will identify new mechanisms of opioid seeking in the drug- free brain, which represents a critical step towards preventing substance use relapse.