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

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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.