Role of Gastrointestinal GCPII in Visceral Pain Signaling

项目总结 迫切需要开发非成瘾性止痛药来治疗腹痛，这是 常见于胃肠道疾病，如肠易激综合征(IBS)和炎症性肠病 (IBD)。谷氨酸羧肽酶II(GCPII)是一种金属多肽酶，表达于中枢和中枢神经。 外周神经系统(分别为CNS和PNS)，在那里它调节谷氨酸的神经传递。 谷氨酸是体内含量最丰富的兴奋性神经递质，被认为是 伤害性感受和疼痛信号。事实上，小分子GCPII抑制剂之前已经被证明 在炎症性、神经性和肌肉骨骼的多种啮齿动物模型中发挥有效的镇痛作用 疼痛。虽然GCPII在中枢和三叉神经痛通路中的作用得到了很好的描述，但GCPII的表达和功能 目前还没有在肠道神经系统中进行研究。我们最近发现GCPII是存在的 在整个ENS中，在回肠和结肠的肠内分泌细胞(EEC)中有显著表达。EECS 是位于整个胃肠道上皮中负责感觉的一群细胞 腔刺激，分泌各种激素和神经递质，据报道参与 迷走神经传入信号与肠脑轴双向通讯。在这里，我们将研究GCPII的生物学 在EEC相关的内脏疼痛信号中。我们的中心假设是GCPII调节EEC谷氨酸 分泌，改变随后的迷走神经传入激活和内脏痛信号。在目标1中，我们将开发一个 谷氨酸生物传感器试验评估GCPII作为EEC谷氨酸分泌调节剂的作用，包括 小分子GCPII抑制剂CRISPR/Cas9基因敲除及外源性给药的评价 GCPII底物。在目标2中，我们将表征GCPII在已建立的啮齿动物中随时间的表达变化 并将表征表达GCPII的EEC亚群的细胞标志物。在……里面 目的#3，我们将创造一种在EECS中靶向缺失GCPII的新型小鼠，并将客观地测量 厌恶的结直肠扩张刺激引起的内脏疼痛反应以研究GCPII在EEC中的作用 体内模型系统中的相关内脏疼痛信号。总的来说，这项研究将扩大我们的 了解GCPII在内脏痛中的表达和功能，为未来的临床前奠定基础 研究开发腹痛的新疗法。

PROJECT SUMMARY There is an urgent need to develop non-addictive analgesic agents for the treatment of abdominal pain which is common in gastrointestinal disorders, such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Glutamate carboxypeptidase II (GCPII) is a metallopeptidase that is expressed throughout the central and peripheral nervous systems (CNS, PNS respectively), where it regulates glutamate neurotransmission. Glutamate is the most abundant excitatory neurotransmitter in the body and is known to be a key player in nociception and pain signaling. In fact, small molecule GCPII inhibitors have been previously demonstrated to exert potent analgesic effects in numerous rodent models of inflammatory, neuropathic and musculoskeletal pain. While GCPII’s role in CNS and PNS pain pathways are well characterized, GCPII’s expression and function have not yet been studied in the enteric nervous system. We have recently discovered that GCPII is present throughout the ENS, with prominent expression in enteroendocrine cells (EEC) of the ileum and colon. EECs are a population of cells located throughout the gastrointestinal epithelium that are responsible for sensing luminal stimuli, secreting various hormones and neurotransmitters, and that have been reported to participate in vagal afferent signaling and bi-directional gut brain axis communication. Here, we will study the biology of GCPII in EEC-associated visceral pain signaling. Our central hypothesis is that GCPII regulates EEC glutamate secretion, altering subsequent vagal afferent activation and visceral pain signaling. In Aim #1, we will develop a glutamate biosensor assay to assess GCPII’s role as a modulator of EEC glutamate secretion, including evaluation of a small molecule GCPII inhibitor, CRISPR/Cas9 knockdown and administration of an exogenous GCPII substrate. In Aim #2, we will characterize GCPII expression changes over time in an established rodent abdominal pain model and will characterize the cellular markers of EEC subpopulations expressing GCPII. In Aim #3, we will create a novel mouse with targeted deletion of GCPII in EECs, and will objectively measure visceral pain reaction in response to aversive colorectal distension stimulation to study GCPII’s role in EEC- associated visceral pain signaling in an in vivo model system. Collectively, this research will expand our knowledge regarding GCPII expression and function in visceral pain, laying the preclinical foundation for future studies to develop novel therapeutics for abdominal pain.