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，也是滥用最广泛的一类 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， 严重限制了它们用于长期疼痛管理的效用。μ-阿片受体（莫尔）是的主要靶点 阿片类药物的镇痛和奖励作用。因此，旨在开发更安全和更有效的阿片类药物的努力 治疗将需要对莫尔信号传导有更深入的了解。 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。朝向 为此，我们开发了转基因莫尔模型（tg莫尔），其中哺乳动物莫尔在哺乳动物中表达。 线虫C.优雅我们发现tgMOR动物获得了对阿片类药物反应的能力， 并表现出在高等生物中看到的阿片类反应的所有主要行为特征， 抗过敏、脱敏和耐受。我们进一步证明了关键的已知分子， 在哺乳动物中控制阿片样物质应答在tgMOR蠕虫中发挥保守的功能。使用这种新颖的tgMOR 模型，我们已经完成了一个公正的，向前的遗传筛选行为阿片类药物敏感性的修饰剂， 并分离出大量具有改变的阿片样物质反应的突变体。我们已经开发了一个管道， 发现、鉴定和验证负责表型的基因， 基因组测序、作图和靶向CRISPR/Cas9基因编辑。通过这种方法，我们发现 几个已知的和新的基因，调节阿片类药物的反应性蠕虫，并证实他们的影响， 使用培养的哺乳动物细胞的基于细胞的测定的莫尔信号传导。 我们的研究结果表明，一个复杂的，在很大程度上是未知的，网络的球员存在调节莫尔信号。 因此，本项目的主要工作集中在通过分析tgMOR来识别和表征这些参与者 从我们无偏见的遗传筛查中分离出的突变体我们的第一个目标是找出 负责1）超敏反应，2）敏感性减退，3）通过追求阿片类药物的耐受性受损， 来自每个表型类别的突变体的子集。在第二个目标中，我们将验证并执行机械化 使用基于细胞的综合平台对莫尔信号转导的已鉴定的保守调节因子进行研究 监测莫尔信号传导的各个方面的测定。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，以及不同的遗传背景 使用人源化tgMORC. elegans平台预计这些研究将促进我们的理解 阿片类药物如何发挥作用，从而为开发更安全的阿片类药物治疗方法铺平道路。

Summary Opioid drugs are the most widely used analgesics in the 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 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. Using this novel tgMOR model, we have 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 to opioid drugs by pursuing a subset 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 for the development of safer opioid therapeutics.