Molecular genetic mechanisms of opioid receptor signaling

阿片受体信号传导的分子遗传学机制

• 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
• 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

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 阿片类药物是诊所中使用最广泛的镇痛药，也是一些最广泛的滥用 物质。这些药物的不利作用，包括外围副作用，依赖性和耐受性， 严重限制了他们作为长期疼痛管理的处方镇痛药的效用。 µ阿片类接收器 （MOR）是阿片类药物的镇痛和奖励作用的主要目标。那是针对 开发更安全，更有效的阿片类药物治疗将需要更深入地了解MOR 信号。 我们的长期目标是利用公正的前瞻性遗传学来剖析分子组织 MOR信号网络使用对蛋白类药物的全动物行为反应作为表型读数。 为了实现这一目标，我们开发了一个转基因MOR模型（TGMOR），其中表达了哺乳动物的MOR 在线虫C.秀丽隐杆线虫的神经系统中。我们发现TGMOR动物具有回应的能力 卵虫类药物，并表现出在高等生物体中看到的所有基本行为标志 包括急性抑制作用，脱敏和耐受性。我们进一步证明了已知的关键 控制哺乳动物中阿片类药物反应能力的分子玩家在TGMOR蠕虫中发挥了配置的功能。 利用此模型，我们完成了一个无偏向的前向遗传屏幕，以实现行为的修饰符 阿片类药物敏感性，并隔离了大量的阿片类药物反应改变的突变体。我们已经开发了 通过结合的结合，用于发现，识别和验证基因的基因的管道 整个基因组测序，映射和靶向CRISPR/CAS9基因编辑。使用这种方法，我们 发现了几个已知和新颖的基因，这些基因调节了蠕虫的阿片类药物反应性，并确认了它们 使用基于培养的哺乳动物细胞的基于细胞的测定对MOR信号传导的影响。 我们的发现表明，存在一个详尽的，未知的玩家网络来调节MOR 信号。这，该项目的主要努力专注于确定和表征这些参与者 分析从我们无偏向的正向遗传筛选中分离出来的TGMOR突变体。我们的第一个目的是确定 负责1）超敏反应的基因，2）低敏性和3）通过追求耐受性损害 来自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 关于确定的，组成的MOR信号调节器的机械研究，使用一个综合平台 基于细胞的暗示，监测MOR信号的各个方面。第三个目标将着重于探索 MOR会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 在人群中自然发现的变体，FDA批准的阿片类药物和不同的遗传 背景使用人源化的TGMOR C.秀丽隐杆线平台。预计这些研究将进步 我们对阿片类药物的作用的理解，从而为安全阿片类药物疗法开发铺平了道路。

项目成果

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: 10.1101/2023.11.15.566604
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Amezquita,Jonathan;Desbois,Muriel;Opperman,KarlaJ;Pak,JosephS;Christensen,ElyseL;Nguyen,NikkiT;Diaz-Garcia,Karen;Borgen,MelissaA;Grill,Brock
• 通讯作者: Grill,Brock