Molecular genetic mechanisms of opioid receptor signaling

总结 阿片类药物是临床上使用最广泛的镇痛药，也是滥用最广泛的药物之一 物质.这些药物的不良反应，包括外周副作用、依赖性和耐受性， 严重限制了它们作为处方镇痛药用于长期疼痛管理的效用。µ-阿片受体 (MOR)是阿片类药物镇痛和奖赏作用的主要靶点。因此，旨在 开发更安全、更有效的阿片类药物治疗需要对莫尔有更深入的了解 信号 我们的长期目标是使用无偏见的正向遗传学来剖析基因组的分子组织。 莫尔信号网络使用整个动物对阿片类药物的行为反应作为表型读数。 为此，我们开发了转基因莫尔模型（tg莫尔），其中表达哺乳动物莫尔 在线虫C.优雅我们发现，tgMOR动物获得了对 阿片类药物，并表现出所有的阿片类药物反应的主要行为特征，在高等生物体中可见 包括急性过敏反应、脱敏和耐受性。我们进一步证明了已知的关键 在哺乳动物中控制阿片样物质应答的分子参与者在tgMOR蠕虫中发挥保守的功能。 利用这个模型，我们完成了一个无偏见的，前向遗传筛选的行为修饰剂， 阿片敏感性，并分离出大量的突变体与改变阿片类药物的反应。我们已经开发出一种 用于发现、鉴定和验证负责表型的基因的管道， 全基因组测序、作图和靶向CRISPR/Cas9基因编辑。使用这种方法，我们 发现了几个已知的和新的基因，调节阿片类药物的反应，在蠕虫，并证实了他们的。 对莫尔信号传导的影响。 我们的研究结果表明，存在一个复杂的、基本上未知的参与者网络来调节莫尔 发信号。因此，本项目的主要工作重点是通过以下方式识别和描述这些参与者： 分析从我们的无偏正向遗传筛选中分离的tgMOR突变体。我们的首要目标是找出 基因负责1）超敏反应，2）低敏感性，和3）受损的耐受性，通过追求 来自每个表型类别的突变体的子集。在第二个目标中，我们将验证和执行 使用一个综合的平台，对莫尔信号转导的已鉴定的保守调节因子进行机制研究， 监测莫尔信号传导的各个方面的基于细胞的测定。第三个目标将侧重于探索 莫尔影响行为的药物基因组学。为此，我们分析了遗传莫尔与 在人群中自然发现的变异，FDA批准的阿片类药物，以及不同的遗传 使用人源化tgMORC. elegans平台预计这些研究将取得进展， 我们对阿片类药物作用方式的理解，从而为开发更安全的阿片类药物治疗方法铺平了道路。

Summary Opioid drugs are the most widely used analgesics in clinic, and are also some of the most widely abused substances. The adverse actions of these drugs, including peripheral side effects, dependence and tolerance, severely limit their utility as prescription analgesics for long term pain management. The µ-opioid receptor (MOR) is the primary target of the analgesic and rewarding effects of opioids. Thus, efforts aimed at developing safer and more effective opioid treatments will require a much deeper understanding of MOR signaling. Our long-term goal is to use unbiased forward genetics to dissect the molecular organization of the MOR signaling network using whole-animal behavioral responses to opioids as a phenotypic readout. Towards this goal, we developed a transgenic MOR model (tgMOR), in which mammalian MOR is expressed in the nervous system of the nematode C. elegans. We found that tgMOR animals gain the ability to respond to opioids, and exhibit all the cardinal behavioral hallmarks of opioid responses seen in higher organisms including acute depressant effects, desensitization and tolerance. We further demonstrated key known molecular players that control opioid responsiveness in mammals play conserved functions in tgMOR worms. Taking advantage of this model, we completed an unbiased, forward genetic screen for modifiers of behavioral opioid sensitivity, and isolated a large number of mutants with altered opioid responses. We have developed a pipeline for discovery, identification and validation of genes responsible for phenotypes using a combination of whole genome sequencing, mapping and targeted CRISPR/Cas9 gene editing. Using this approach, we uncovered several known and novel genes that regulate opioid responsiveness in worms, and confirmed their effects on MOR signaling using cell-based assays with cultured mammalian cells. Our findings suggest an elaborate, largely unknown, network of players exists to regulate MOR signaling. Thus, the main effort of this project focuses on identifying and characterizing these players by analyzing tgMOR mutants isolated from our unbiased, forward genetic screen. Our first aim will be to identify the genes responsible for 1) hypersensitivity, 2) hyposensitivity, and 3) impaired tolerance by pursuing a subsets of mutants from each phenotypic category. In the second aim, we will validate and perform mechanistic studies on identified, conserved regulators of MOR signaling using a comprehensive platform of cell-based assays that monitor various aspects of MOR signaling. The third aim will focus on exploring the pharmacogenomics by which MOR impacts behavior. To do so, we analyze interactions between genetic MOR variants found naturally in the human population, FDA-approved opioid drugs, and different genetic backgrounds using a humanized tgMOR C. elegans platform. It is anticipated that these studies will advance our understanding of how opioids act thereby paving the way to the development of safer opioid therapeutics.