The role of cannabinoid signaling in the nematode Caenorhabditis elegans

描述（申请人提供）：哺乳动物内源性大麻素系统在许多生理功能中发挥作用，是一个重要的治疗靶点。除了两种主要的大麻素受体之外，越来越多的证据表明，人类中还有其他尚未确定的CB受体。大麻素受体已经在许多不同的物种中被鉴定出来，包括一些无脊椎动物，但它们还没有在秀丽隐杆线虫中被鉴定出来。在秀丽隐杆线虫中，一个保守的胰岛素信号通路在发育过程中起作用，调节进行生殖生长或形成大幼虫的决定，大幼虫是一个交替的幼虫阶段，使蠕虫能够在种群过度拥挤和饥饿的时期存活下来。在化学筛选中，我发现合成大麻素受体拮抗剂AM251抑制了胰岛素受体突变体daf-2的水形成。AM251防止水形成的能力被合成大麻素受体激动剂的存在所抑制，这表明存在线虫大麻素受体。通过对蠕虫内源性大麻素系统的成分的鉴定，包括多种内源性大麻素配体和修饰内源性大麻素水平的生物合成酶，进一步加强了这一观点。在这方面，我发现蠕虫含有几种内源性大麻素，它们在结构上与哺乳动物的CB受体配体相关。这些内源性大麻素的水平在抑制线虫同源脂肪酸酰胺水解酶（一种参与内源性大麻素降解和失活的水解酶）后升高。此外，一些内源性大麻素在饥饿期间升高，而另一些则降低，这表明在喂养和对饥饿的反应中起作用。最后，一种蠕虫内源性大麻素，二十碳五烯醇乙醇酰胺，一种哺乳动物内源性大麻素花生四烯醇乙醇酰胺的异构体，以与AM251相同的方式促进daf-2突变体的生殖生长。由于哺乳动物1型和2型大麻素受体在蠕虫中似乎没有同源物，我提出秀丽隐杆线虫表达一种受体，作为哺乳动物大麻素受体的功能同源物。因此，该项目的总体目标是确定蠕虫中大麻素的分子靶点，并更详细地表征线虫内源性大麻素系统。在线虫中发现新的大麻素受体亚型，将使秀丽隐杆线虫作为内源性大麻素生理研究的主要模型系统的优势，并增加我们对大麻素和胰岛素信号通路相互作用的理解。最后，蠕虫大麻素受体的发现可能会导致新的人类CB受体的鉴定，这有可能为大麻素类药物提供新的治疗靶点。公共卫生相关性：线虫线虫秀丽隐杆线虫中内源性大麻素系统的发现和两种主要哺乳动物受体同源物的缺失表明存在一种新的大麻素受体。该受体在蠕虫中的鉴定可能有助于新的哺乳动物大麻素受体的鉴定，并为开发基于大麻素的治疗方法提供新的靶点。

DESCRIPTION (provided by applicant): The endocannabinoid system in mammals plays a role in many physiological functions and is an important therapeutic target. In addition to the two main cannabinoid receptors there is increasing evidence that there are other, as yet unidentified, CB receptors in humans. Cannabinoid receptors have been identified in many different species, including some invertebrates, but they have not been identified in the nematode Caenorhabditis elegans. In C. elegans a conserved insulin signaling pathway acts during development to regulate the decision to proceed with reproductive growth or form the dauer larva, an alternate larval stage that allows the worm to survive periods of population overcrowding and starvation. In a chemical screen, I discovered that the synthetic cannabinoid receptor antagonist AM251 suppressed dauer formation in the insulin receptor mutant daf-2. The ability of AM251 to prevent dauer formation is inhibited by the presence of synthetic cannabinoid receptor agonists, suggesting the presence of a nematode cannabinoid receptor. This idea is further strengthened by the identification of components of a worm endocannabinoid system including multiple endocannabinoid ligands and biosynthetic enzymes that modify endocannabinoid levels. In this respect, I have discovered that the worm contains several endocannabinoids that are structurally related to the CB receptor ligands in mammals. Levels of these endocannabinoids are elevated following inhibition of the worm ortholog of fatty acid amide hydrolase, a hydrolytic enzyme involved in endocannabinoid degradation and inactivation. Furthermore, some of the endocannabinoids are elevated during starvation while others are decreased, suggesting a role in feeding and response to starvation. Finally one of the worm endocannabinoids, eicosapentaenoyl ethanolamide, an isoform of the mammalian endocannabinoid arachidonoyl ethanolamide, acts in the same way as AM251 to promote reproductive growth in daf-2 mutants. Since the mammalian type 1 and type 2 cannabinoid receptors do not appear to have orthologs in worms, I propose that C. elegans expresses a receptor that acts as a functional homolog of mammalian cannabinoid receptors. The overall goal of this project therefore is to identify the molecular target of cannabinoids in worms and to characterize in more detail the nematode endocannabinoid system. The identification of a new cannabinoid receptor subtype in nematodes will bring the strengths of C. elegans as a major model system to the study of endocannabinoid physiology and increase our understanding of how cannabinoid and insulin signaling pathways interact. Finally the discovery of a worm cannabinoid receptor may lead to the identification of a novel human CB receptor, which has the potential to provide a new therapeutic target for cannabinoid based drugs. PUBLIC HEALTH RELEVANCE: The discovery of an endocannabinoid system in the nematode roundworm Caenorhabditis elegans and the absence of orthologs of the two main mammalian receptors suggest the existence of a novel cannabinoid receptor. The identification of this receptor in worms may help in the identification of new mammalian cannabinoid receptors and provide new targets for the development of cannabinoid based therapeutics.