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Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the G -protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.

Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked peripheral pain remain unclear. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. Our data suggest that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice. The mechanism of CGRP-evoked peripheral pain is unclear. Here, the authors show that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective 1 , 2 . This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury 2 , 3 . Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 – 12 . However, how neuro–immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the Na V 1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro–immune interactions has potential to treat non-healing tissues in which dysregulated neuro–immune interactions impair tissue healing. Experiments in mouse models show that Na V 1.8 +  nociceptors innervate sites of injury and provide wound repair signals to immune cells by releasing calcitonin gene-related peptide (CGRP).

Cancer cells have been shown to exploit neurons to modulate their survival and growth, including through the establishment of neural circuits within the central nervous system 1 , 2 – 3 . Here we report a distinct pattern of cancer–nerve interactions between the peripheral nervous system and gastric cancer. In multiple mouse models of gastric cancer, nociceptive nerves demonstrated the greatest degree of nerve expansion in an NGF-dependent manner. Neural tracing identified CGRP + peptidergic neurons as the primary gastric sensory neurons. Three-dimensional co-culture models showed that sensory neurons directly connect with gastric cancer spheroids. Chemogenetic activation of sensory neurons induced the release of calcium into the cytoplasm of cancer cells, promoting tumour growth and metastasis. Pharmacological ablation of sensory neurons or treatment with CGRP inhibitors suppressed tumour growth and extended survival. Depolarization of gastric tumour membranes through in vivo optogenetic activation led to enhanced calcium flux in jugular nucleus complex and CGRP release, defining a cancer cell–peptidergic neuronal circuit. Together, these findings establish the functional connectivity between cancer and sensory neurons, identifying this pathway as a potential therapeutic target. Functional connectivity between gastric cancer cells and sensory neurons offers a potential therapeutic target.

The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache 1 , 2 . Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year 3 – 5 . How pain and neuroimmune interactions impact meningeal antibacterial host defences are unclear. Here we show that Nav1.8 + nociceptors signal to immune cells in the meninges through the neuropeptide calcitonin gene-related peptide (CGRP) during infection. This neuroimmune axis inhibits host defences and exacerbates bacterial meningitis. Nociceptor neuron ablation reduced meningeal and brain invasion by two bacterial pathogens: Streptococcus pneumoniae and Streptococcus agalactiae . S.   pneumoniae activated nociceptors through its pore-forming toxin pneumolysin to release CGRP from nerve terminals. CGRP acted through receptor activity modifying protein 1 (RAMP1) on meningeal macrophages to polarize their transcriptional responses, suppressing macrophage chemokine expression, neutrophil recruitment and dural antimicrobial defences. Macrophage-specific RAMP1 deficiency or pharmacological blockade of RAMP1 enhanced immune responses and bacterial clearance in the meninges and brain. Therefore, bacteria hijack CGRP–RAMP1 signalling in meningeal macrophages to facilitate brain invasion. Targeting this neuroimmune axis in the meninges can enhance host defences and potentially produce treatments for bacterial meningitis. Two Streptococcus spp. can utilize a neuropeptide (CGRP) and its receptor (RAMP1) on macrophages to promote brain invasion, a finding that may help the development of therapies for bacterial meningitis.

Haematopoietic stem cells (HSCs) reside in specialized microenvironments in the bone marrow—often referred to as ‘niches’—that represent complex regulatory milieux influenced by multiple cellular constituents, including nerves 1 , 2 . Although sympathetic nerves are known to regulate the HSC niche 3 – 6 , the contribution of nociceptive neurons in the bone marrow remains unclear. Here we show that nociceptive nerves are required for enforced HSC mobilization and that they collaborate with sympathetic nerves to maintain HSCs in the bone marrow. Nociceptor neurons drive granulocyte colony-stimulating factor (G-CSF)-induced HSC mobilization via the secretion of calcitonin gene-related peptide (CGRP). Unlike sympathetic nerves, which regulate HSCs indirectly via the niche 3 , 4 , 6 , CGRP acts directly on HSCs via receptor activity modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CALCRL) to promote egress by activating the Gα s /adenylyl cyclase/cAMP pathway. The ingestion of food containing capsaicin—a natural component of chili peppers that can trigger the activation of nociceptive neurons—significantly enhanced HSC mobilization in mice. Targeting the nociceptive nervous system could therefore represent a strategy to improve the yield of HSCs for stem cell-based therapeutic agents. Stimulation of pain-sensing neurons, which can be achieved in mice by the ingestion of capsaicin, promotes the migration of haematopoietic stem cells from the bone marrow into the blood.

Background Different responses in human coronary arteries (HCA) and human middle meningeal arteries (HMMA) were observed for some of the novel CGRP receptor antagonists, the gepants, for inhibiting CGRP-induced relaxation. These differences could be explained by the presence of different receptor populations in the two vascular beds. Here, we aim to elucidate which receptors are involved in the relaxation to calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2) in HCA and HMMA. Methods RNA was isolated from homogenized human arteries (23 HCAs; 12 F, 11 M, age 50 ± 3 years and 26 HMMAs; 14 F, 12 M, age 51 ± 3 years) and qPCR was performed for different receptor subunits. Additionally, relaxation responses to CGRP, AM or AM2 of the human arteries were quantified using a Mulvany myograph system, in the presence or absence of the adrenomedullin 1 receptor antagonist AM 22-52 and/or olcegepant. Results Calcitonin-like receptor (CLR) mRNA was expressed equally in both vascular beds, while calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3) expression was low and could not be detected in all samples. RAMP1 expression was similar in HCA and HMMA, while RAMP2 expression was higher in HMMA. Moreover, receptor component protein (RCP) expression was higher in HMMA than in HCA. Functional experiments showed that olcegepant inhibits relaxation to all three agonists in both vascular beds. In HCA, antagonist AM 22-52 did not inhibit relaxation to any of the agonists, while a trend for blocking relaxation to AM and AM2 could be observed in HMMA. Conclusion Based on the combined results from receptor subunit mRNA expression and the functional responses in both vascular tissues, relaxation of HCA is mainly mediated via the canonical CGRP receptor (CLR-RAMP1), while relaxation of HMMA can be mediated via both the canonical CGRP receptor and the adrenomedullin 1 receptor (CLR-RAMP2). Future research should investigate whether RAMP2 predominance over RAMP1 in the meningeal vasculature results in altered migraine susceptibility or in a different response to anti-migraine medication in these patients. Moreover, the exact role of RCP in CGRP receptor signalling should be elucidated in future research.

Calcitonin gene-related peptide (CGRP) regulates inflammation via signaling through receptor activity-modifying protein (RAMP) 1. Here, we investigated the role of RAMP1 signaling in growth of lymphatic vessels during inflammation. Lymphangiogenesis in the diaphragm of RAMP1-deficient (−/−) mice or their wild-type (WT) counterparts was induced by repeated intraperitoneal injection of lipopolysaccharide (LPS). Compared with WT mice, LPS-induced lymphangiogenesis in RAMP1−/− mice was suppressed. This was accompanied by the reduced expression of vascular endothelial growth factor (VEGF)-C and VEGF-D. The number of CD4+ cells in diaphragm tissue from WT mice was greater than RAMP1−/− mice. Removing CD4+ cells attenuated lymphangiogenesis and expression of VEGF-C and VEGF-D. CD4+ cells isolated from RAMP1−/− mice exhibited reduced expression of VEGF-C and VEGF-D. The number of CD11b+ cells from RAMP1−/− mice was higher than WT mice and was associated with the upregulated expression of genes related to pro-inflammatory macrophage phenotype and downregulation of reparative macrophage phenotype-related expression. When fluorescein isothiocyanate (FITC)-dextran was injected into the peritoneal cavity, the amount of residual FITC-dextran in WT mice was lower than that in RAMP1−/− mice. The present results suggest that RAMP1 signaling in immune cells plays a critical role in inflammation-related lymphangiogenesis; therefore, it represents a novel target for controlling lymphangiogenesis.

Dental pulp stem cells (DPSCs) are responsible for maintaining pulp structure and function after pulp injury. DPSCs migrate directionally to the injury site before differentiating into odontoblast-like cells, which is a prerequisite and a determinant in pulp repair. Increasing evidence suggests that sensory neuron-stem cell crosstalk is critical for maintaining normal physiological functions, and sensory nerves influence stem cells mainly by neuropeptides. However, the role of sensory nerves on DPSC behaviors after pulp injury is largely unexplored. Here, we find that sensory nerves released significant amounts of calcitonin gene-related peptide (CGRP) near the injury site, acting directly on DPSCs via receptor activity modifying protein 1 (RAMP1) to promote collective migration of DPSCs to the injury site, and ultimately promoting pulp repair. Specifically, sensory denervation leads to poor pulp repair and ectopic mineralization, in parallel with that DPSCs failed to be recruited to the injury site. Furthermore, in vitro evidence shows that sensory nerve-deficient microenvironment suppressed DPSC migration prominently among all related behaviors. Mechanistically, the CGRP-Ramp1 axis between sensory neurons and DPSCs was screened by single-cell RNA-seq analysis and immunohistochemical studies confirmed that the expression of CGRP rather than Ramp1 increases substantially near the damaged site. We further demonstrated that CGRP released by sensory nerves binds the receptor Ramp1 on DPSCs to facilitate cell collective migration by an indirect co-culture system using conditioned medium from trigeminal neurons, CGRP recombinant protein and antagonists BIBN4096. The treatment with exogenous CGRP promoted the recruitment of DPSCs, and ultimately enhanced the quality of pulp repair. Targeting the sensory nerve could therefore provide a new strategy for stem cell-based pulp repair and regeneration.

Both enhanced motion-induced nausea and increased static imbalance are observed symptoms in migraine and especially vestibular migraine (VM). Motion-induced nausea and static imbalance were investigated in a mouse model, nestin/hRAMP1, expressing elevated levels of human RAMP1 which enhances CGRP signaling in the nervous system, and compared to non-affected littermate controls. Behavioral surrogates such as the motion-induced thermoregulation and postural sway center of pressure (CoP) assays were used to assess motion sensitivity. Nausea readouts revealed that the nestin/hRAMP1 mouse exhibit an increased sensitivity to CGRP’s effects at lower doses compared to unaffected controls. In addition, the nestin/hRAMP1 mice exhibit a higher dynamic range in postural sway than their wildtype counterparts, along with increased sway observed in nestin/hRAMP1 male mice that was not present in male unaffected controls. Results from migraine blocker experiments were challenging to interpret, but the data suggests that olcegepant is incapable of reversing CGRP-induced or endogenous alterations in the nestin/hRAMP1 mice, while rizatriptan was ineffective in both the nestin/hRAMP1 and control mice. The results indicate that overexpression of hRAMP1 leads to heightened endogenous CGRP signaling. Results also suggest that both olcegepant and rizatriptan are ineffective in reducing nausea and sway in this hypersensitive CGRP mouse model. This study suggests that the hypersensitive nestin/hRAMP1 mouse may serve as a model for difficult to treat cases of migraine that exhibit increased motion sensitivity.