Conditional Dicer1 manipulation to study miRNA involvement in opioid addiction

描述（由申请人提供）：阿片类药物在疼痛管理中是无价的。不幸的是，长期阿片类药物给药可导致许多不良后果，如镇痛疗效（耐受性）的进行性下降，难以克服的疼痛和成瘾。因此，阿片类药物极高的治疗价值因长期服用阿片类药物和滥用倾向的有害影响而降低，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，关于与长期阿片类药物给药不良后果相关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经典途径的重要作用，并发现了参与吗啡镇痛耐受和药物自我给药的microRNA（miRNA）。由于其对神经元分化和树突状结构的网络样转录后效应，miRNA表达是协调对慢性药物暴露的复杂反应的强有力的候选者。发现特异性miRNA参与阿片类药物自我给药的作用可能为新的治疗策略打开大门。该CEBRA申请的目的是使用两种新策略来定义miRNA在自我给药中的作用：1）具有限速miRNA加工酶Dicer 1的细胞类型特异性条件性敲低的遗传工程小鼠，和2）miRNA和mRNA表达谱分析以发现参与神经适应性变化的miRNA：mRNA调节配对。我们的假设是，药物摄入量的增加和自我给药习惯的发展是由选择的miRNA表达的协调变化引起的。提出了以下目的：具体目的1：该目的的目的是产生Cre-loxP动物模型，以使得能够以能够区分主动强化和被动药物暴露的方式在多巴胺能和GA-BA能神经元（分别使用Slc 6a 3和Gad 2启动子）中定时和特异性Dicer 1敲低以及表型自我施用行为（和随后的脑组织）。该目标的结果将提供对miRNA的基本作用、所涉及的细胞类型的有价值的洞察，并提供用于目标2的表型锚。具体目标2：在吗啡自我给药后，将在多个脑区域中确定区域miRNA和mRNA表达谱。对吗啡的基因型依赖性行为反应和与miRNA和mRNA表达的性状特异性关联/相关性的表达谱分析的系统过程将鉴定与自我给药特异性相关的候选者。计算方法将有助于确定miRNA：mRNA调控配对的优先级，并提供一个功能框架，将高价值配对置于可靶向的生物学途径中。多层次、多学科方法（行为、分子、生物信息学）旨在探索遗传结构（miRNA）的未知层，以发现用于治疗干预的新分子靶点。 阿片类药物成瘾给个人、家庭和社会带来巨大的情感和经济代价。不幸的是，关于与长期阿片类药物给药不良后果相关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。这项资助申请的目的是整合复杂的行为技术（小鼠中的轭式控制静脉注射自我给药（SA）），先进的基因工程（可诱导的cre-loxP系统删除Dicer 1）和尖端的分子遗传技术（miRNA表达谱），以发现参与成瘾的miRNA候选物，并为真正新颖的治疗干预提供发现平台。

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.