Molecular genetic mechanisms of opioid receptor signaling

阿片受体信号传导的分子遗传学机制

基本信息

批准号：
10754689
负责人：
Brock Grill
金额：
$ 14.28万
依托单位：
SEATTLE CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-31 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10754689
关键词：
Acute Affect Agonist American Analgesics Animal Model Animals Behavior Behavioral Biological Biological Assay Biology CRISPR/Cas technology Caenorhabditis elegans Categories Cell model Cell physiology Cells Chronic Clinic Clustered Regularly Interspaced Short Palindromic Repeats Cultured Cells Data Dependence Development Engineering Exhibits FDA approved Fentanyl G-Protein-Coupled Receptors Genes Genetic Genetic Screening Genetic Variation Goals Human Hypersensitivity Impairment In Vitro Knock-out Knowledge L-Type Calcium Channels Link Locomotion Mammalian Cell Mammals Maps Medicine Modeling Molecular Molecular Genetics Monitor Morphine Mus Nematoda Nervous System Neurons Opioid Opioid Receptor Organism Orphan Orthologous Gene Pain Pain management Peripheral Pharmaceutical Preparations Pharmacogenomics Pharmacology Phenotype Play Population Receptor Signaling Regulation Research Rewards Rodent Model Signal Transduction Single Nucleotide Polymorphism Map System Testing Transgenic Organisms Validation Variant addiction antagonist behavioral response candidate identification chronic pain management clinically relevant desensitization design drug action exome sequencing forward genetics genome sequencing in vivo insight mutant nonsynonymous mutation novel opioid exposure opioid use opioid use disorder prescription pain reliever programs response side effect stem therapeutic opioid whole genome

项目摘要

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.

概括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用途。（MOR）是阿片类药物镇痛和奖励作用的主要目标，因此，努力的目标是。开发更安全、更有效的阿片类药物治疗需要更深入地了解 MOR 发信号。我们的长期目标是使用无偏见的正向遗传学来剖析细胞的分子组织 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。为了实现这一目标，我们开发了一种转基因MOR模型（tgMOR），其中表达哺乳动物MOR 我们发现 tgMOR 动物获得了对线虫的神经系统做出反应的能力。阿片类药物，并表现出在高等生物中观察到的阿片类药物反应的所有主要行为特征我们进一步证明了已知的关键作用，包括急性抑郁作用、脱敏作用和耐受性。控制哺乳动物阿片类药物反应的分子角色在 tgMOR 蠕虫中发挥保守功能。利用这个模型，我们完成了对行为修饰因素的公正的、正向的遗传筛选。阿片类药物敏感性，并分离出大量具有改变阿片类药物反应的突变体。使用以下组合来发现、鉴定和验证负责表型的基因的管道我们利用这种方法进行全基因组测序、作图和靶向 CRISPR/Cas9 基因编辑。发现了几个调节蠕虫阿片类药物反应的已知和新基因，并证实了它们的使用培养的哺乳动物细胞进行基于细胞的测定对 MOR 信号传导的影响。我们的研究结果表明，存在一个复杂的、很大程度上未知的参与者网络来监管 MOR 因此，该项目的主要工作重点是通过以下方式识别和表征这些参与者。分析从我们公正的正向遗传筛选中分离出的 tgMOR 突变体，我们的首要目标是鉴定。导致 1) 过敏、2) 过敏和 3) 耐受性受损的基因每个表型类别的突变体子集在第二个目标中，我们将验证并执行。使用综合平台对已识别的、保守的 MOR 信号调节因子进行机制研究第三个目标将集中于探索 MOR 信号传导的各个方面。 MOR 影响行为的药物基因组学为此，我们分析了遗传 MOR 之间的相互作用。发现了人群中自然存在的变异、FDA 批准的阿片类药物以及不同的遗传基因使用人源化 tgMOR 线虫平台的背景预计这些研究将会取得进展。我们对阿片类药物如何发挥作用的理解为开发更安全的阿片类药物疗法铺平了道路。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Axon development is regulated at genetic and proteomic interfaces between the integrin adhesome and the RPM-1 ubiquitin ligase signaling hub.

轴突发育在整合素粘附体和 RPM-1 泛素连接酶信号中枢之间的遗传和蛋白质组界面上受到调节。

DOI：
发表时间：
2023-11-15
期刊：
影响因子：
0
作者：
Amezquita, Jonathan;Desbois, Muriel;Opperman, Karla J;Pak, Joseph S;Christensen, Elyse L;Nguyen, Nikki T;Diaz;Borgen, Melissa A;Grill, Brock
通讯作者：
Grill, Brock