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

CGRP 在胃肠动力中的亚型和性别特异性功能

• 批准号: 10635765
• 负责人: Meenakshi Rao
• 金额: $ 67.42万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10635765
• 关键词: Ablation; Address; Affect; Afferent Neurons; Amino Acids; Biology; Breeding; Calcitonin Gene-Related Peptide; Calcitonin-Gene Related Peptide Receptor; Cells; Chromosomes; Clinical; Colon; Complex; Constipation; Data; Digestive System Disorders; Disease; Diverticulosis; Drug Targeting; Enteral; Enteric Nervous System; Enzyme-Linked Immunosorbent Assay; Feces; Female; Frequencies; Gastrointestinal Diseases; Gastrointestinal Motility; Gastrointestinal Transit; Gastrointestinal tract structure; Gene Expression; Genes; Genetic; Goals; Gut Mucosa; Health; Heterogeneity; Homeostasis; Human; Immune response; Immunity; Immunohistochemistry; In Vitro; Intestines; Knock-in Mouse; Knock-out; Knowledge; Label; Link; Migraine; Modeling; Molecular; Mucosal Immunity; Mus; Neuroglia; Neurons; Neuropeptides; Nociception; Pathway interactions; Patients; Pattern; Peptide Signal Sequences; Pharmaceutical Preparations; Play; Protein Isoforms; Proteins; Regulation; Relaxation; Reporter; Reporting; Role; Series; Sex Differences; Signal Pathway; Signal Transduction; Smooth Muscle; Source; Symptoms; Testing; Transcript; Vasodilation; Visceral pain; Work; calcium indicator; cell motility; cell type; experience; gastrointestinal; genome wide association study; gut health; in vivo; in vivo evaluation; intestinal homeostasis; male; migraine treatment; molecular phenotype; motility disorder; neuromuscular function; novel; pharmacologic; preservation; receptor; sex; side effect; signal recognition particle receptor; tool; virtual

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.

项目摘要 降钙素基因相关肽（CGRP）由神经元分泌，对血管舒张很重要， 伤害感受和免疫反应。以CGRP为靶点的药物已经成为治疗 偏头痛越来越多的临床证据表明，CGRP信号也可能参与 胃肠道（GI）健康和疾病。例如，便秘是最常见的副作用之一， 新的抗CGRP疗法，暗示这种信号通路对正常的肠道运动很重要。 与偏头痛患者的真实经历相一致，三项全基因组关联研究 最近鉴定出CALCB基因座与大便频率和憩室病密切相关。CALCB 编码CGRP的β-同种型，其在人和小鼠之间高度保守。功能 然而，肠中CALCB的重要性尚未确定。本提案的总体目标是 是确定分泌CALCB的肠神经元，并确定哪些方面的胃肠道运动， 信号传导是体内所必需的。 CALCB和CALCA（CGRP的α-同种型）仅相差3个氨基酸，这使得其具有挑战性， 通过免疫组织化学或ELISA在蛋白水平上区分它们。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水平上容易区分。先前 基因表达研究表明，虽然CALCA是身体大部分部位的主要CGRP同种型，但CALCB 在肠道中占主导地位。尽管这种优势，几乎所有的研究，探讨了作用， CGRP在肠道免疫和内脏痛中的作用已证实CALCA起源于肠道外源性传入神经元 作为关键的同种型，并发现CALCB是不稳定的，其基本功能尚不清楚。利用 临床使用的抗CGRP药物以及我们已经产生的新的遗传工具， 标记和操纵肠神经系统（ENS）中的CALCB神经元，我们将完成三个 目标.第一，确定胃肠道的哪些部分需要CALCB，主要的ENS衍生CGRP， 小鼠的正常运动性。我们的初步数据表明，这些要求是性别特异性的，这表明， 肠中CGRP途径的细胞-分子布线在男性和女性中可能不同。第二， 利用基因编码的报告基因、钙指示剂和化学发生蛋白，我们将定义神经元 在男性和女性ENS中，释放CALCB，以及肠道运动如何通过改变其活性而受到影响。 第三，鉴于越来越多的证据表明CGRP参与胃肠道内稳态，我们将确定 调节这两种亚型的肠道水平的机制。除了推进基本的 了解肠道神经肌肉功能，这项工作的影响将是解释如何广泛 使用的药物类别会导致不良GI效应，相反，如果CGRP的亚型或ENS特异性靶向 信号传导可能有益于治疗GI运动障碍。