Conditional Dicer1 manipulation to study miRNA involvement in opioid addiction

描述(由申请人提供)：阿片类药物在疼痛治疗中具有无价的价值。不幸的是，长期服用阿片类药物会导致许多不良后果，如止痛效果(耐受性)逐渐下降、无法克服的疼痛和成瘾。因此，阿片类药物极高的治疗价值被长期使用阿片类药物和滥用责任的有害影响所削弱，并可能给个人、家庭和社会造成巨大的情感和经济代价。不幸的是，有关慢性阿片类药物不良后果的神经生物学机制的知识尚未产生干预或替代治疗药物。需要一种新的方法来发现替代治疗策略。我们最近的合作研究揭示了参与神经可塑性的典型通路的重要作用，以及microRNA(MiRNA)参与吗啡镇痛耐受和药物自我给药的发现。MiRNA的表达是协调慢性药物暴露的复杂反应的有力候选者，因为它们在神经元分化和树突结构中具有网络样的转录后效应。发现特定的miRNA参与对阿片类药物自我给药的反应可能会打开新的治疗策略的大门。本CEBRA应用程序的目的是使用两种新策略来确定miRNAs在自我给药中的作用：1)通过对特定细胞类型的miRNA加工酶Dicer1进行条件敲除的基因工程小鼠，以及2)miRNA和mRNA表达谱，以发现参与神经适应性变化的miRNA：mRNA调控配对。我们的假设是，药物摄入量的增加和自我给药习惯的养成是由特定miRNAs表达的协调变化引起的。提出了以下目标：具体目标1：本目标的目的是建立Cre-loxP动物模型，以区分主动强化和被动药物暴露的方式，在多巴胺和GA-B能神经元(分别使用SLC6a3和GAD2启动子)和表型自我给药行为(以及随后的脑组织)中进行定时和特异的Dicer1敲除。这一目标的结果将为深入了解miRNA的基本作用、所涉及的细胞类型提供有价值的见解，并为目标2的使用提供表型锚。具体目标2：在吗啡自我给药后，将确定多个脑区的区域miRNA和mRNA的表达谱。表达谱、对吗啡的依赖于基因型的行为反应以及与miRNA和mRNA表达的特定特征的关联/关联的系统过程将确定与自我给药具体相关的候选者。计算方法将有助于确定miRNA：mRNA调控对的优先顺序，并提供一个功能框架，将高价值对置于靶向生物途径中。这种多层次、多学科的方法(行为、分子、生物信息学)旨在探索遗传结构(MiRNA)的未知层，以发现治疗干预的新分子靶点。 公共卫生相关性：阿片成瘾给个人、家庭和社会造成巨大的情感和经济代价。不幸的是，有关慢性阿片类药物不良后果的神经生物学机制的知识尚未产生干预或替代治疗药物。需要一个新的焦点来发现替代治疗策略--miRNAs提供了一个独特的机会。这项赠款申请的目的是整合复杂的行为技术(枷锁控制静脉注射。这些技术包括基因工程(小鼠的自我给药(SA))、先进的基因工程(删除Dicer1的可诱导cre-loxP系统)和尖端分子遗传技术(miRNA表达谱分析)，以发现与成瘾有关的miRNA候选分子，并为真正新颖的治疗干预措施提供发现平台。

DESCRIPTION (provided by applicant): Opioids are invaluable in pain management. Unfortunately, chronic opioid administration can lead to numerous adverse consequences such as a progressive decline in analgesic efficacy (tolerance), insurmountable pain and addiction. Thus, the extremely high therapeutic value of opioids is diminished by the detrimental impact of chronic opioid administration and abuse liability and can inflict enormous emotional and economic cost to individuals, families and society. Unfortunately, knowledge concerning the neurobiological mechanism associated with adverse consequences of chronic opioid administration has not produced an intervention or alternative treatment medication. A new approach is required to discover alternative treatment strategies. Our recent collaborative investigations have revealed a major role for canonical pathways involved in neuroplasticity and the discovery of microRNA (miRNA) involvement in morphine analgesic tolerance and drug self-administration. MiRNA expression is a strong candidate for coordinating the complex response to chronic drug exposure due to their network-like post-transcriptional effects on neuronal differentiation and dendritic architecture. Discovering the role of specific miRNA involvement in response to opioid self-administration may open the door to novel therapeutic strategies. The purpose of this CEBRA application is to define the role of miRNAs in self-administration using two novel strategies: 1) Genetically engineered mice with cell type-specific conditional knockdown of the rate- limiting miRNA processing enzyme, Dicer1, and 2) miRNA and mRNA expression profiling to discover miRNA:mRNA regulatory pairings involved in neuroadaptive changes. Our hypothesis is that escalated drug intake and the development of the self-administration habit is caused by a coordinated change in expression of select miRNAs. The following aims are proposed: Specific Aim 1: The purpose of this aim is to generate Cre-loxP animal models to enable timed and specific Dicer1 knockdown in dopaminergic and GA- BAergic neurons (using the Slc6a3 and Gad2 promotors, respectively) and phenotype self-administration behavior (and subsequent brain tissue) in a manner that can discriminate between active reinforcement and passive drug exposure. The results of this aim will provide valuable insight into miRNA's essential role, the cell- types involved and provide a phenotypic anchor for use in Aim 2. Specific Aim 2: Regional miRNA and mRNA expression profiles will be determined in multiple brain regions following morphine self-administration. The systematic process of expression profiling genotype-dependent behavioral responses to morphine and trait-specific associations/correlations with miRNA and mRNA expression will identify candidates specifically connected to self-administration. Computational methods will help prioritize miRNA:mRNA regulatory pairings and provide a functional framework to place the high-value pairs into targetable biological pathways. The multi- tiered, multi-disciplinary approach (behavior, molecular, bioinformatics) is designed to probe an unchartered layer of the genetic architecture (miRNA) to discover novel molecular targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Opioid addiction inflicts enormous emotional and economic cost to individuals, families and society. Unfortunately, knowledge concerning the neurobiological mechanism associated with adverse consequences of chronic opioid administration has not produced an intervention or alternative treatment medication. A new focus is required to discover alternative treatment strategies--miRNAs provide a unique opportunity. The purpose of this grant application is to integrate of sophisticated behavioral techniques (yoked-control i.v. self-administration (SA) in the mouse), advanced genetic engineering (inducible cre-loxP system to delete Dicer1) and cutting edge molecular genetic techniques (miRNA expression profiling) to discover miRNA candidates involved in addiction and provide a discovery platform for truly novel treatment interventions.