Decrypting nociceptive signaling pathways in Caenorhabditis elegans using behavioral analysis and mass spectrometry-based proteomics

我的 NSERC Discovery 研究项目致力于了解与伤害性行为相关的分子过程、突触传递的神经化学，并确定可以调节伤害性处理的目标。 该提案的总体目标是 1) 表征内源性大麻素和大麻素对秀丽隐杆线虫 (C. elegans) 对有害热的伤害反应的影响，并与香草类化合物进行比较，2) 使用特定的秀丽隐杆线虫突变体、药理学操作和蛋白质组学来表征大麻素和香草素系统之间的相互作用； 3) 使用用于研究配体结合时蛋白质的热稳定性的热位移测定的变体，结合无偏见的全蛋白质组方法来鉴定特异性结合蛋白靶点，鉴定秀丽隐杆线虫中内源性大麻素、大麻素和香草素的分子靶标。 线虫是与哺乳动物生理学相关的强大实验模型系统。线虫由 959 个细胞组成，其中 302 个是神经元，这使得该模型对于研究伤害感受很有吸引力，感觉神经系统负责触发对有害刺激的反应。伤害感受器是专门检测潜在伤害的感觉受体，包括温度、机械和化学伤害。瞬时受体电位 (TRP) 通道是有害刺激的重要探测器和传感器。线虫基因组编码多种 TRP 通道，包括瞬时受体电位阳离子通道亚家族 V（TRPV 或香草酸受体）直向同源物（OSM-9 和 OCR1-4）。最近已经确定，线虫 TRP 通道与行为和生理过程（包括感觉转导）相关。许多线虫 TRP 通道与其脊椎动物对应通道具有相似的激活和调节机制。内源性大麻素系统参与伤害感受的调节，最近的证据表明与香草素系统存在相互作用。许多研究确定了秀丽隐杆线虫内源性大麻素系统的组成部分，包括许多内源性大麻素配体。因此，我们相信线虫表达的受体与哺乳动物大麻素受体的功能同源物一样，是研究新的大麻素信号通路及其与香草素系统相互作用的理想实验模型。除了两种主要的大麻素受体（CB1 和 CB2）之外，越来越多的证据表明哺乳动物中还存在其他尚未识别的大麻素受体。 该研究提案将对表征香草素受体介导的热激活信号通路及其与大麻素系统成分的相互作用的基础知识的产生产生重大影响。这是确保实验模型转化成功的基本步骤。此外，这项综合研究可能会发现一种新型大麻素受体。

My NSERC Discovery research program is dedicated to understanding the molecular processes associated with nociceptive behaviors, the neurochemistry of synaptic transmission and identify targets that can regulate nociceptive processing. The overall goal of this proposal will be to 1) characterize the effects of endocannabinoids and cannabinoids on nocifensive response of Caenorhabditis elegans (C. elegans) to noxious heat and compared with vanilloids, 2) characterize the interplay between cannabinoid and vanilloid systems using specific C. elegans mutants, pharmacological manipulations and proteomics; 3) identify the molecular target of endocannabinoids, cannabinoids and vanilloids in C. elegans using a variant of the thermal shift assays used to study thermal stabilization of proteins upon ligand binding combined with an unbiased, proteome-wide approach for the identification of specific-binding protein targets. C. elegans is a powerful experimental model system relevant to mammalian physiology. C. elegans consists of 959 cells, of which 302 are neurons, which makes this model attractive to study nociception, the sensory nervous system's responsible for triggering a response to harmful stimuli. Nociceptors are sensory receptors specialized in detecting potential harm, including temperature, mechanical, and chemical. Transient receptor potential (TRP) channels are essential detectors and transducers of harmful stimuli. C. elegans genome encodes several TRP channels including transient receptor potential cation channel subfamily V (TRPV or vanilloid receptor) orthologs (OSM-9 and OCR1-4). It has been recently established that C. elegans TRP channels are associated with behavioral and physiological processes, including sensory transduction. Many C. elegans TRP channels share similar activation and regulatory mechanisms with their vertebrate counterparts. The endocannabinoid system is involved in modulation of nociception and recent evidence suggests interactions with the vanilloid systems. Many studies identified components of a C. elegans endocannabinoid system including numerous endocannabinoid ligands. Thus, we believe that C. elegans expresses receptors that act as functional homologs of mammalian cannabinoid receptors and is an ideal experimental model to study new cannabinoid signaling pathways and their interplay with the vanilloid systems. In addition to the two main cannabinoid receptors (CB1 and CB2), there is growing evidence that there are other, not yet identified, cannabinoid receptors in mammals. This research proposal will have a significant impact on the production of knowledge fundamental to characterize heat activated signaling pathways mediated by vanilloid receptors and their interaction(s) with the cannabinoid system components. This is a fundamental step to ensure translational success of experimental models. Moreover, this comprehensive study may lead to the discovery of a novel cannabinoid receptor.