Molecular genetic mechanisms of opioid receptor signaling

摘要 阿片类药物是临床上使用最广泛的止痛药，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止痛剂用于长期止痛的用途。微阿片受体 (MOR)是阿片类药物止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将需要对MOR有更深入的了解 发信号。 我们的长期目标是使用无偏见的前向遗传学来剖析 MOR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，我们开发了一种转基因MOR模型(TgMOR)，在该模型中哺乳动物的MOR得到了表达 在线虫的神经系统中。我们发现，tgMOR动物获得了对 阿片类药物，并表现出高等生物体中阿片类药物反应的所有基本行为特征 包括急性抑郁作用、脱敏和耐受性。我们进一步展示了已知的关键 在哺乳动物中控制阿片类药物反应的分子分子在tgMOR蠕虫中发挥着保守的功能。 利用这一模型，我们完成了对行为修饰物的无偏见、正向遗传筛选 阿片类药物敏感性，并分离出大量阿片反应改变的突变体。我们已经开发出一种 用于发现、鉴定和验证与表型有关的基因的流水线 全基因组测序、作图和有针对性的CRISPR/Cas9基因编辑。使用这种方法，我们 发现了几个调节蠕虫中阿片类药物反应性的已知和新基因，并证实了它们的 用培养的哺乳动物细胞进行基于细胞的分析对MOR信号的影响。 我们的发现表明，存在一个精心设计的、基本上不为人所知的玩家网络，以监管更多 发信号。因此，该项目的主要工作集中在通过以下方式识别和描述这些参与者 分析从我们的无偏见、正向遗传筛查中分离出来的tgMOR突变。我们的首要目标是找出 导致1)过敏症、2)过敏症和3)耐受性受损的基因 来自每个表型类别的突变子集。在第二个目标中，我们将验证和执行 利用综合平台对识别的、保守的MOR信号调节因子进行机制研究 基于细胞的检测，监测MOR信号的各个方面。第三个目标将集中在探索 MOR影响行为的药物基因组学。为此，我们分析了遗传More之间的相互作用 在人类群体中自然发现的变异，FDA批准的阿片类药物，以及不同的基因 背景使用人性化的tgmor秀丽线虫平台。预计这些研究将取得进展。 我们对阿片类药物如何发挥作用的理解，从而为开发更安全的阿片类药物疗法铺平了道路。

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.