Isoform- and Sex-Specific Functions of CGRP in Gastrointestinal Motility

项目总结 降钙素基因相关肽(CGRP)由神经元分泌，对血管扩张有重要作用。 伤害性感受和免疫反应。以降钙素基因相关肽为靶点的药物在治疗高血压方面取得了突破性进展 偏头痛。越来越多的临床证据表明，降钙素基因相关肽信号可能也参与了 胃肠(GI)健康和疾病。例如，便秘是一种最常见的副作用。 新的抗CGRP疗法，暗示这一信号通路对正常的肠道运动很重要。 结合偏头痛患者的真实经历，三项全基因组关联研究 最近发现CALCB基因座与大便频率和憩室病密切相关。CALCB 编码降钙素基因相关肽的β-亚型，在人类和小鼠之间高度保守。功能界别 然而，CALCB在肠道中的意义尚未确定。这项提案的首要目标是 是定义分泌CALCB的肠神经元，并确定CALCB在胃肠道运动的哪些方面 体内信号转导是必需的。 Calcb和calca是降钙素基因相关肽的α异构体，只有3个氨基酸不同，这使得它具有挑战性。 通过免疫组织化学或酶联免疫吸附试验从蛋白水平对其进行鉴别。这两种异构体由 然而，不同的基因座使得它们的表达在转录本水平上很容易区分。上一首 基因表达研究表明，虽然Calca是大多数身体部位的主要CGRP亚型，但CALCB 在肠道中占主导地位。尽管有这种优势，但几乎所有探索这种作用的研究 降钙素基因相关肽在肠道免疫和内脏痛中的作用已确定Calca起源于肠道外源性传入神经元 作为关键的异构体，发现CALCB是可有可无的，其基本功能尚不清楚。利用 临床上使用的抗CGRP药物以及我们已经产生的新的遗传工具，使选择性 肠神经系统(ENS)CALCB神经元的标记和操作，我们将完成三项工作 目标。其一，确定胃肠道的哪些节段需要CALCB，这是主要的ENS来源的CGRP，用于 小鼠正常活动。我们的初步数据显示，这些要求是针对性别的，这表明 肠道中CGRP途径的细胞-分子连接在男性和女性中可能是不同的。二, 使用遗传编码的报告、钙指示剂和化学发生蛋白，我们将定义神经元 在释放CALCB的雄性和雌性EN中，以及改变它们的活动如何影响肠道运动。 第三，鉴于越来越多的证据表明CGRP参与了胃肠道的动态平衡，我们将确定 调节两种异构体肠道水平的机制。除了推进根本的 了解肠道的神经肌肉功能，这项工作的影响将如何广泛地解释 使用的药物会导致不良的胃肠道反应，反之，如果CGRP的异构体或ENS特异性靶向 信号转导可能对胃肠动力障碍的治疗有益。

PROJECT SUMMARY Calcitonin gene-related peptide (CGRP) is secreted by neurons and is important for vasodilation, nociception, and immune responses. Drugs targeting CGRP have been a breakthrough in the treatment of migraine headaches. Increasing clinical evidence suggests that CGRP signaling may also be involved in gastrointestinal (GI) health and disease. For example, constipation is one of the most common side effects of the new anti-CGRP therapies, hinting that this signaling pathway is important for normal gut motility. Converging with the real-world experience of migraine patients, three genome-wide association studies recently identified the CALCB locus as strongly linked to stool frequency and diverticular disease. CALCB encodes the β-isoform of CGRP, which is highly conserved between humans and mice. The functional significance of CALCB in the bowel, however, has not been identified. The overarching goal of this proposal is to define the enteric neurons that secrete CALCB and determine which aspects of GI motility that CALCB signaling is necessary for in vivo. CALCB and CALCA, the α-isoform of CGRP, differ by only 3 amino acids making it challenging to distinguish them at the protein level by immunohistochemistry or ELISA. The two isoforms are encoded by distinct loci, however, enabling their expression to be readily distinguished at the transcript level. Previous gene expression studies showed that while CALCA is the major CGRP isoform in most of the body, CALCB dominates in the intestine. Despite this dominance, virtually all of the studies that have probed the roles of CGRP in gut immunity and visceral pain have identified CALCA originating from gut-extrinsic afferent neurons as the key isoform and found CALCB to be dispensable, leaving its essential functions unclear. Utilizing the anti-CGRP agents in clinical use as well as new genetic tools that we have generated to enable selective labeling and manipulation of CALCB neurons in the enteric nervous system (ENS), we will accomplish three objectives. One, determine which segments of the GI tract require CALCB, the major ENS-derived CGRP, for normal motility in mice. Our preliminary data show that these requirements are sex-specific, suggesting that the cellular-molecular wiring of the CGRP pathway in the intestine may be different in males and females. Two, using genetically encoded reporters, calcium indicators and chemogenetic proteins, we will define the neurons in the male and female ENS that release CALCB and how gut motility is affected by altering their activity. Three, given the accumulating evidence for CGRP involvement in GI homeostasis, we will determine the mechanisms that regulate the intestinal levels of both isoforms. In addition to advancing the fundamental understanding of neuromuscular function in the bowel, the impact of this work will be to explain how a widely used class of drugs causes adverse GI effects and, conversely, if isoform- or ENS-specific targeting of CGRP signaling may be beneficial for treating GI dysmotility.