Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice

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.

F or almost a century it has been known that cutaneous tissue injury elicits a local vascular response, referred to as neurogenic inflammation, that is associated to a wider area of increased sensitivity to mechanical stimuli 1 . A subset of C-fiber primary afferents, which mediate neurogenic inflammation, is the main source of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) 2,3 . In rodents, noxious stimuli such as capsaicin, a pungent agonist of the transient receptor potential vanilloid 1 (TRPV1) channel 4 , evoke the peripheral release of CGRP which induces arteriolar vasodilatation 2 and of SP which elicits plasma protein extravasation 5 , and produce sensory responses, which encompasses acute nociception and prolonged mechanical allodynia 6 . Capsaicin administration to the human skin elicits a similar pattern of responses, consisting of local cutaneous vasodilatation and focal and transient burning pain (min) associated with widespread, sustained mechanical hypersensitivity (hrs) 7 . While CGRP has been identified as the mediator of neurogenic vasodilatation in rodents 2 and humans 8 , the cellular and molecular mechanisms underlying mechanical allodynia associated with neurogenic inflammation are unknown.
Mechanistic studies in animal models and humans have highlighted the role of CGRP in migraine pain 9 . Thus, small molecule antagonists of the CGRP receptor and monoclonal antibodies against CGRP or its receptor can relieve migraine pain 10 . The poor blood-brain barrier penetration of some smallmolecule antagonists 11,12 and of monoclonal antibodies 13,14 suggests a peripheral contribution to CGRP-mediated migraine pain. However, little is known about the proalgesic actions of CGRP in the periphery. In mice, intraplantar injection of CGRP evokes mechanical allodynia 15 and systemic CGRP causes facial grimace 16 . Periorbital CGRP injection, while failing to evoke spontaneous nociceptive behavior, produces sustained (~4 h) periorbital mechanical allodynia (PMA) 17 . CGRP released from trigeminal peripheral terminals mediates PMA in mice 18 evoked by systemic (intraperitoneal) administration of the pro-headache agent glyceryl trinitrate (GTN) 19 . Facial cutaneous allodynia is one component of the migraine attack 20,21 . Although the process that initiates migraine pain may originate in the central nervous system (CNS) 22,23 , the cell type and signaling pathway by which CGRP acts in the periphery to cause pain are unknown.
The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR), a G protein-coupled receptor (GPCR), and receptor activity-modifying protein 1 (RAMP1), a single transmembrane domain CLR chaperone 24 . These two components coexist in cells that mediate the actions of CGRP, for example, vascular myocytes 2 . Satellite glial cells and Schwann cells express CLR/RAMP1 and are closely associated with peptidergic sensory neurons 25 . While the extracellular space between the soma of trigeminal neurons and satellite glial cells is not a recognized locus for neurotransmission, the varicosities of C-fibers and the ensheathing Schwann cells are sites where neuropeptides, including CGRP 26 , are normally released. Schwann cells from rat sciatic nerve respond to CGRP by increasing intracellular cAMP levels 27 and CLR/RAMP1 are expressed by Schwann cells that wrap CGRP + ve terminals of rat nociceptors 25,28,29 . Schwann cells mediate mechanical allodynia in mouse models of neuropathic and cancer pain 30,31 . Cutaneous Schwann cells can also directly activate sensory nerves to promote mechanical nociception 32 . Although GPCRs are usually considered to signal principally from the plasma membrane, GPCR kinases and β-arrestins (βARRs) rapidly terminate this signaling. Persistent endosomal signaling of GPCRs, including CLR/RAMP1, underlies sustained neuronal activation and nociception in the CNS [33][34][35] .
Herein, we hypothesized that mechanical allodynia associated with neurogenic inflammation is mediated by CGRP which targets CLR/RAMP1 in Schwann cells ensheathing peripheral endings of nociceptors. By selective RAMP1 gene deletion in Schwann cells, we reveal that CGRP released from trigeminal terminals causes PMA by paracrine signaling to the surrounding Schwann cells. We also hypothesized that persistent CGRP/CLR/ RAMP1 signaling from endosomes in Schwann cells underlies sustained PMA. By using inhibitors of clathrin-and dynaminmediated endocytosis and stimulus-responsive nanoparticles designed to release CLR/RAMP1 antagonists in acidified endosomes, we found that CLR/RAMP1 endosomal signaling results in a cAMP-dependent release of nitric oxide (NO), which activates transient receptor potential ankyrin 1 (TRPA1), a proalgesic channel and sensor of oxidative stress 36 .

Results
CGRP evokes PMA by activating Schwann cell CLR/RAMP1. We detected CLR and RAMP1 mRNA and immunoreactivity in primary cultures of human Schwann cells (HSCs) or mouse Schwann cells (MSCs) taken from the sciatic or trigeminal nerve (Fig. 1a, Supplementary Fig. 1a). The S100 + ve mouse Schwann cell line (IMS32) recapitulated features of primary MSCs, including expression of CLR and RAMP1 mRNA and immunoreactivity ( Supplementary Fig. 1a, b) and TRPA1-dependent Ca 2+ response to allyl isothiocyanate ( Supplementary Fig. 1c). Immunoreactive CLR and RAMP1 were also detected in S100 + ve Schwann cells in nerve bundles in biopsies of human abdominal and mouse periorbital skin (Fig. 1b).
Intravenous CGRP provokes delayed headache attacks in patients 37 . Intraperitoneal CGRP caused PMA and paw allodynia in male and female C57BL/6 J mice without gender difference (Fig. 1e, Supplementary Fig. 1g). In Plp-Cre ERT+ ;Ramp1 fl/fl mice treated with periorbital 4-OHT PMA, but not paw allodynia, was similarly reduced in males and females in response to intraperitoneal CGRP (Fig. 1f, g and Supplementary Fig. 1h, i). Systemic (intraperitoneal) 4-OHT reduced both PMA and paw allodynia by intraperitoneal CGRP (Supplementary Fig. 1j, k). These results reveal an essential role for CLR/RAMP1 of Schwann cells surrounding periorbital trigeminal endings in PMA elicited by local and systemic CGRP.
To investigate the contribution of endocytosis to the activation of Gα proteins and βARRs in endosomes, we preincubated cells with hypertonic sucrose. CGRP increased EbBRET between hCLR-Rluc8 and tdRGFP-Rab5a in HEK-hCLR/RAMP1 cells, consistent with CLR endocytosis (Fig. 5i). Hypertonic sucrose inhibited these changes, which indicates an inhibition of endocytosis (Fig. 5i). Hypertonic sucrose caused a delayed yet more sustained activation of Rluc8-mGα s , Rluc8-mGα sq , Rluc8-mGα si , and Rluc2-βARR2 at the plasma membrane (Supplementary Fig. 4a-f), and an almost complete inhibition of activation of Rluc8-mGα s , Rluc8-mGα sq , Rluc8-mGα si and Rluc2-βARR2 in endosomes (Fig. 5j, k). Sucrose similarly delayed CGRP-induced recruitment of Rluc8-mGα s , Rluc8-mGα sq , Rluc8-mGα si and To examine the contribution of endosomal CLR/RAMP1 signaling to CGRP-induced cAMP formation, we preincubated HSCs expressing the cADDis cAMP reporter with sucrose or vehicle. In vehicletreated cells, CGRP stimulated a rapid (1 min) increase in cAMP formation that was sustained for 30 min (Fig. 5n, o). Sucrose reduced but did not abolish the initial response, yet strongly inhibited the sustained phase of CGRP-stimulated cAMP formation (Fig. 5n, o). Thus, CGRP initially activates Gα and βARR at the plasma membrane, which is followed by sustained activation of Gα and βARR in early endosomes. Endocytosis is necessary for the recruitment of Gα and βARR to endosomes. Gα s continues to signal in endosomes, leading to sustained cAMP formation.
CLR/RAMP1 activation in Schwann cells releases NO, which initiates but does not sustain PMA. We investigated the mechanisms that sustain PMA following CLR/RAMP1 activation and endocytosis in Schwann cells. Pre-but not post-treatment (60 min after CGRP or capsaicin) with CLR/RAMP1 antagonists, olcegepant or CGRP8-37, attenuated PMA evoked by capsaicin and in accordance with previous studies 17, 18 PMA evoked by CGRP ( Supplementary Fig. 5a-f). Similarly, inhibitors of clathrin-and dynamin-mediated endocytosis had no effect when administered  Although NO can release CGRP with proalgesic functions, the contribution of NO to CGRP-evoked allodynia is uncertain. Pretreatment with an NO synthase (NOS) inhibitor (L-NAME) or an NO scavenger (cPTIO) (Fig. 6a), abrogated CGRP-evoked PMA (Fig. 6b, c). L-NAME and cPTIO pretreatment also attenuated capsaicin-evoked PMA (Fig. 6d, e). However, L-NAME and cPTIO did not affect PMA when administered 60 min after CGRP (Supplementary Fig. 5o, p) or capsaicin ( Supplementary Fig. 5q, r). Thus, PKA-dependent NO release 39 is necessary to initiate, but is not sufficient to sustain, CGRP-evoked allodynia.
In vitro findings recapitulated in vivo results. HSCs, MSCs, and IMS32 cells predominantly expressed NOS3 (eNOS) mRNA, with little or no expression of NOS1 and NOS2 (nNOS and iNOS, respectively) mRNA (Fig. 6f, g; Supplementary Fig. 5s). In both HSCs and IMS32 cells CGRP elicited a transient increase in NOS3 phosphorylation (i.e., activation), consistent with NO generation, which peaked at 5-10 min and declined within 30-60 min (Fig. 6h), and a cAMP increase that was prevented by olcegepant, CGRP8-37 and an adenylyl cyclase inhibitor (SQ22536), but not by L-NAME (Fig. 6i). The increase in cAMP evoked by CGRP but not that elicited by forskolin was reduced in cultured MSCs obtained from Plp-Cre ERT+ ;Ramp1 fl/fl mice as compared to Control mice treated with intraperitoneal 4-OHT (Fig. 6j). In contrast, the CGRP-evoked increase in NO was attenuated by all these interventions, including NOS inhibition (Fig. 6k). The cAMP increase evoked by forskolin was unaffected by CLR/RAMP1 antagonism and NOS inhibition, and olcegepant failed to inhibit NO release by the NO donor NONOate ( Supplementary Fig. 5t, u), indicating selectivity. NO release evoked by CGRP, but not that evoked by NONOate was inhibited by PS2 and Dy4, but not their inactive analogs ( Supplementary  Fig. 5v), further supporting selectivity. These results suggest that clathrin-and dynamin-dependent endocytosis and endosomal CLR/RAMP1 signaling evoke NOS activation and NO generation in Schwann cells.
Schwann cell TRPA1 mediates CGRP-evoked PMA. NO belongs to a series of reactive oxygen species (ROS) that target TRPA1 40 . TRPA1 is coexpressed with TRPV1 and CGRP in a subpopulation of primary sensory neurons 41 . TRPA1 is expressed in Schwann cells of nerve bundles of human skin and mouse sciatic nerve, where it mediates mechanical allodynia in rodent models of pain 30,42 . Immunoreactive TRPA1 was coexpressed with RAMP1 in S100 + ve Schwann cells in human abdominal and mouse periorbital cutaneous nerves bundles (Fig. 7a). Thus, CLR/RAMP1 might engage signaling pathways that activate TRPA1 in trigeminal Schwann cells to initiate allodynia (Fig. 7b). This hypothesis was supported by the observation that both CGRP-and capsaicin-evoked PMA were reduced in Trpa1 −/− mice and in mice with sensory neuron-specific deletion of TRPA1 (Adv-Cre + ;Trpa1 fl/fl ) (Fig. 7c, d and Supplementary Fig. 6a, b). We next investigated the signaling pathway by which the CLR/ RAMP1 activates TRPA1. In HSCs and IMS32 cells, CGRP stimulated a slowly developing yet sustained increase in Ca 2+ response (Fig. 7e, f) and increased H 2 O 2 levels ( Supplementary  Fig. 6c). Olcegepant, CGRP8-37, SQ22536, H89, L-NAME, Ca 2+free medium, a ROS scavenger (PBN) or a NOX1 inhibitor (ML171) attenuated Ca 2+ responses (Fig. 7e, f) and H 2 O 2 levels ( Supplementary Fig. 6c). A TRPA1 antagonist (A967079) inhibited CGRP-stimulated Ca 2+ (Fig. 7e, f) and H 2 O 2 responses ( Supplementary Fig. 6c) but did not affect CGRP-stimulated NO formation (Fig. 7g). CGRP-evoked Ca 2+ responses were reduced in Schwann cells from Trpa1 −/− mice ( Supplementary Fig. 6d). These results support the hypothesis that CGRP liberates NO, which activates Schwann cell TRPA1; activated TRPA1 promotes a Ca 2+ -dependent H 2 O 2 generation that sustains a feed-forward mechanism comprising TRPA1 channel engagement and ROS release.
In vivo results corroborated this hypothesis. Whereas CLR/ RAMP1 antagonists or NO inhibitors attenuated PMA only if given before CGRP or capsaicin, both pre-and post-treatment with a TRPA1 antagonist, a ROS scavenger and a NOX1 inhibitor reduced PMA (Fig. 7h-j; Supplementary Fig. 6e-g). Although pretreatment with TRPA1 or ROS inhibitors did not affect the acute nociception, they inhibited capsaicin-evoked PMA (Supplementary Fig. 6h-j). Post-treatment also attenuated capsaicinevoked PMA (Supplementary Fig. 6h-j). These findings highlight the mechanistic differences between acute nociception and delayed PMA. After an initial and transient NO-dependent phase, PMA is sustained by persistent ROS liberation, which targets TRPA1 in Schwann cells. This hypothesis is robustly supported by the observation that PMA evoked by CGRP or capsaicin was markedly attenuated in mice with selective deletion of TRPA1 in Schwann cells (Plp-Cre ERT+ ;Trpa1 fl/fl ) ( Fig. 7k;  Supplementary Fig. 6k).
Targeting endosomal CGRP signaling provides superior relief of CGRP-and capsaicin-evoked PMA. The finding that persistent GPCR signaling from endosomes mediates pain transmission suggests that GPCRs in endosomes rather than at the plasma membrane are a valid and perhaps superior target for the treatment of pain [33][34][35] . Nanoparticles have been used to deliver chemotherapeutics to tumor, where endocytosis and endosomal escape are necessary for drug delivery to cytosolic and nuclear targets 43 . The realization that GPCRs within endosomes are a therapeutic target, raises the possibility of exploiting the acid microenvironment of endosomes as a stimulus for nanoparticle disassembly and release of antagonist cargo 34 .
To determine whether DIPMA-MK-3207 can antagonize CLR in endosomes, we measured CGRP-stimulated cAMP formation using the CAMYEL cAMP BRET sensor, which detects total cellular cAMP. HEK293T cells expressing rat CLR/RAMP1 (HEK-rCRL/ RAMP1) were preincubated with graded concentrations of DIPMA-MK-3207 or free MK-3207, DIPMA-Ø or vehicle (control) for 30 min. Beginning at 0 min, baseline BRET was measured for 5 min, and cells were then challenged with CGRP. At 10 min, cells were washed to remove extracellular CGRP, and BRET was measured up to 35 min. In vehicle-treated cells, CGRP stimulated a prompt increase in cAMP formation (1 st phase, 6-10 min) that gradually declined after agonist removal from the extracellular fluid (2 nd phase, 11-35 min) (Fig. 8e). DIPMA-Ø did not affect this response. Free MK-3207 and DIPMA-MK-3207 (100, 316 nM) both inhibited CGRP-evoked cAMP in the 1 st phase to a similar extent (Fig. 8f). During the 2 nd phase, free MK-3207 was inactive at all concentrations whereas DIPMA-MK3207 (31.6, 100, 316 nM) strongly inhibited responses (Fig. 8g). The results suggest that To assess the antagonism of the pain signaling pathway in HSCs, we measured CGRP-evoked changes in Ca 2+ response, which depend on endosomal CGRP signaling and activation of TRPA1. HSCs were preincubated with graded concentrations of DIPMA-MK-3207 or MK-3207 for 20 min to allow the accumulation in endosomes, and washed to remove extracellular compounds. At 10 min after washing, cells were challenged with CGRP and Ca 2+ response was measured as an index of TRPA1 activity. DIPMA-MK-3207 inhibited CGRP-evoked increase in Ca 2+    (Fig. 8i). DIPMA-Ø had no effect. Thus, endosomal targeting enhances the efficacy of a CLR/RAMP1 antagonist in a preclinical model of migraine pain.

Discussion
The major findings of the present study are that CGRP causes PMA by activating CLR/RAMP1 of Schwann cells, CLR/ RAMP1 signals from endosomes of Schwann cells to activate pain pathways, and endosomal CLR/RAMP1 can be targeted using nanoparticles and endocytosis inhibitors to relieve CGRP-evoked PMA. CLR/RAMP1 stimulation and trafficking to endosomes results in a persistent cAMP-dependent NOS activation and generation of NO, a mediator of migraine pain 19 . The role of NO in PMA is crucial, yet transient, as it is temporally limited to the engagement of TRPA1/NOX1, which releases ROS with a dual function. On one hand, ROS target TRPA1/NOX1 of Schwann cells to maintain ROS generation by a feed-forward mechanism.
On the other hand, as suggested by experiments with selective TRPA1 deletion in primary sensory neurons, ROS target TRPA1 on nociceptors to signal allodynia to the CNS. Periorbital capsaicin injection elicited acute nociception mediated by TRPV1 excitation and ensuing afferent discharge, which signals pain to the CNS. In a larger cutaneous area, capsaicin evoked delayed and prolonged PMA. While the acute pain response is most likely dependent on ion influx associated with TRPV1 activation, the mechanism underlying mechanical hypersensitivity 7,44 has remained elusive. Our findings support the existence of a paracrine mechanism that underlies PMA associated with neurogenic inflammation. We suggest that capsaicin locally activates TRPV1 + ve nerve fibers to generate action potentials that propagate antidromically into collateral fibers which release CGRP in a broader area, thus eliciting widespread PMA. PMA depends on the interaction between peptidergic nerve fibers, surrounding Schwann cells and nociceptive neurons that convey allodynic signals to the CNS. CGRP liberated from the varicosities of trigeminal TRPV1 + ve nerve fibers binds to CLR/ RAMP1 of adjacent Schwann cells.
CNS perturbations may target the trigeminovascular system and initiate the migraine attack 22,23 . These central mechanisms may underlie the delayed facial allodynia associated with migraine 20,21 . However, the beneficial effect of anti-CGRP medicines that do not cross the blood-brain barrier suggests that CGRP acts in the periphery to elicit pain. The peripheral site of the algesic action of CGRP released from peptidergic C-fibers has been proposed as the CLR/RAMP1 on adjacent non-peptidergic Aδ-fibers 45 and more precisely at the level of the node of Ranvier 28 . The present results in Adv-Cre + ;Ramp1 fl/fl mice suggest that CGRP does not act on trigeminal nociceptors to cause PMA in mice. This is consistent with failure of CGRP administration to elicit any itch, pain or axon reflex responses in humans 46 . Instead, our results support the hypothesis that CGRP released from trigeminal nociceptors targets CLR/PAMP1 on Schwann cells that wrap their terminals to evoke PMA. A limitation of our study is that we only assessed PMA that mimics one component of migraine pain. We cannot exclude the possibility that central mechanisms contribute to other pain symptoms of migraine, and that some of the locally administered antagonists used in the present study penetrated the CNS, where they could also influence pain transmission. Although human Schwann cells express CLR/RAMP1 and show functional responses to CGRP that can account for allodynia in mice, further work is needed to understand whether similar mechanisms account for migraine pain in humans.
Another limitation of the present study is that we cannot pinpoint which type of neuron conveys the signals that underlie mechanical allodynia in the trigeminal region. Specifically, we were unable to distinguish between TRPV1-expressing nerve fibers that release CGRP and TRPA1-expressing nerve fibers that are targeted by Schwann cell ROS and convey allodynic signals centrally, since TRPV1 and TRPA1 may coexist in the same population of CGRP-expressing Aδ-or C-fiber primary sensory neurons 2,41 . Most Schwann cells in Remak bundles contain multiple unmyelinated axons from C-fiber nociceptors, including CGRP + ve fibers, which release the bulk of CGRP 2 , and nonpeptidergic isolectin B4 + ve fibers 47 . Thus, CGRP-evoked release of ROS from Schwann cells could induce allodynia by targeting TRPA1 on three neuronal subtypes, including the same Aδ-or C-fiber that releases CGRP, a different C-fiber of the same Remak bundle, or a different adjacent Aδ-fiber. The observation that both C-fiber and Aδ-fiber nociceptors contribute to capsaicinevoked hypersensitivity in humans 48 supports the hypothesis that both types of neurons 28,45 are implicated in CGRP-mediated allodynia, thus highlighting the complex neural transmission of mechanical allodynia associated with neurogenic inflammation.
CLR/RAMP1 signals from endosomes by G-protein-mediated mechanisms that activate a subset of compartmentalized signals, including cytosolic protein kinase C and nuclear extracellular signal-regulated kinase; these kinases regulate excitation of spinal neurons and pain transmission 35 . Our results show that CLR/ RAMP1 activates Gα s , Gα q and Gα i and recruits βARRs in endosomes of Schwann cells, determined by EbBRET. Inhibitors of clathrin-and dynamin-mediated endocytosis blocked the recruitment of CLR/RAMP1, Gα and βARR to endosomes, which presumably requires CLR/RAMP1 endocytosis. GPCR/Gα signaling complexes have also been detected in endosomes by using conformationally selective nanobodies 49 . The observation that endocytosis inhibitors attenuated CGRP-stimulated cAMP formation and activation of NOS and TRPA1 reveals a central role for CLR/RAMP1 signaling in endosomes of Schwann cells in CGRP-evoked periorbital pain. Endocytosis of other Gs-coupled GPCRs is also necessary for the full repertoire of cAMP-mediated signaling outcomes, which entails endosomal recruitment of adenylyl cyclase 9 50 and assembly of metastable accumulations of PKA 51 . We found that a nanoparticle-encapsulated CLR/RAMP1 antagonist, which targeted CLR/RAMP1 in endosomes and released cargo in the acidified endosomal microenvironment 34 , also attenuated CGRP-stimulated cAMP formation and blunted TRPA1 activation.
The observation that periorbital injection of inhibitors of clathrin and dynamin and of DIPMA-MK-3207 prevented CGRPand capsaicin-evoked PMA provides the evidence for a prominent role of endosomal CGRP signaling of pain from a peripheral site. The finding that nanoparticle encapsulation enhanced the potency of a CGRP antagonist for inhibition of endosomal signaling and resultant nociception supports the hypothesis that CLR/RAMP1 in endosomes mediates facial allodynia which contributes to migraine pain. Nanoparticle encapsulation similarly boosts the efficacy of an NK 1 receptor antagonist in preclinical models of inflammatory and neuropathic pain 34 . An antagonist of CLR/RAMP1 conjugated to a membrane lipid cholestanol also accumulates in endosomes and provides superior relief from pain 35 , which reinforces the importance of CLR/ RAMP1 endosomal signaling for pain transmission.
Limitations of the present study include uncertainty about the nature of the CLR/RAMP1 signaling complex in endosomes of Schwann cells, which warrants further investigation by proteomics approaches. Although some of the pharmacological inhibitors used to dissect the signaling pathway can have nonspecific actions, we bolstered confidence in selectivity by using inhibitors of the same pathway and by genetic deletion of GPCRs and TRP channels. Our findings reveal a prominent role for CLR/ RAMP1 in Schwann cells for CGRP-evoked periorbital pain. Future studies will investigate the role of this pathway in preclinical models of migraine pain.
Monoclonal antibodies to CGRP, although beneficial, are not effective in all patients 10 . While non-CGRP-dependent mechanisms might explain this failure 52 , monoclonal antibodies likely do not inhibit CGRP signaling in endosomes. The small molecule CLR/RAMP1 antagonist, rimegepant, was found to resolve migraine attacks in patients treated with the anti-CLR/RAMP1 monoclonal antibody, erenumab 53 . This unexpected result was interpreted by the inherent membrane permeability of the lipophilic antagonist rimegepant 54 that might favor inhibition of CGRP signaling in endosomes 53 , while neither receptor-targeted nor ligand-targeted monoclonal antibodies internalized with CLR/RAMP1 activated by CGRP 55 . Our results showing a superior inhibition of CGRP signaling in Schwann cells and of PMA by DIPMA-MK-3207, which selectively targets receptor activity in endosomes, reveal a better approach to control allodynia.
In 1936, Sir Thomas Lewis postulated 1 that in human skin action potentials are carried antidromically from the injured nerve terminal to collateral branches from where a chemical substance is released that produces the flare and increases the sensitivity of other fibers responsible for pain. CGRP has been previously identified as the mediator of neurogenic vasodilatation in rodents 2 , and in humans 8 . Herein, we propose that CGRP is the 'chemical substance' that, via the essential role of endosomal CLR/RAMP1, TRPA1/NOX1 and oxidative stress of surrounding Schwann cells, sustains the enhanced sensitivity of primary sensory neurons associated with neurogenic inflammation (Fig. 9).
The present results suggest that peripherally acting anti-CGRP medicines reduce migraine pain in part by targeting the facial allodynia that originates from CGRP-mediated endosomal signaling in Schwann cells. The group size of n = 8 animals for behavioral experiments was determined by sample size estimation using G*Power (v3.1) 60 to detect size effect in a post-hoc test with type 1 and 2 error rates of 5 and 20%, respectively. Mice were allocated to vehicle or treatment groups using a randomization procedure (http://www.randomizer.org/). Four independent and blinded investigators performed the treatments, behavioral experiments, genotyping and data analysis, respectively. No animals were excluded from experiments.

Methods
The behavioral studies followed the animal research reporting in vivo experiment (ARRIVE) guidelines 61 . Mice were housed in a temperature (20 ± 2°C) and humidity (50 ± 10%) controlled vivarium (12 h dark/light cycle, free access to food and water, five animals per cage). At least 1 h before behavioral experiments, mice were acclimatized to the experimental room and behavior was evaluated between 9:00 am and 5:00 pm. All the procedures were conducted following the current guidelines for laboratory animal care and the ethical guidelines for investigations of experimental pain in conscious animals set by the International Association for the Study of Pain 62 . Animals were anesthetized with a mixture of ketamine and xylazine (90 mg/kg and 3 mg/kg, respectively, i.p.) and euthanized with inhaled CO 2 plus 10-50% O 2 . Behavioral experiments Treatment protocol. Subcutaneous injections were made in the periorbital area 2-3 mm from the external eyelid corner 17 . Briefly, the mouse was lifted by the base of the tail and placed on a solid surface with one hand and the tail was pulled back. Then, it was quickly and firmly picked up by the scruff of the neck with the thumb and index finger of the other hand. The injection was made rapidly by a single operator with minimal animal restraint. Mice received unilateral (right side) injections (10 μl/site) of CGRP (1.5 nmol in 0.9% NaCl), SP (3.5 nmol in 0.9% NaCl), capsaicin (10, 50, 100 pmol in 0.1% dimethyl sulfoxide, DMSO), or vehicles (control). Mice received bilateral injections (10 µl/site, right side same site as stimulus, left side symmetrical to right side) of antagonists and inhibitors. CGRP (1.5 nmol in 0.9% NaCl) or vehicle was also administered by intraplantar (i.pl., 20 μl/site) or systemic (0.1 mg/kg, i.p.) injection. GTN was administered at 10 mg/ kg, i.p. injection.
Acute nociception. Immediately after the p.orb. injection, mice were placed inside a plexiglass chamber and spontaneous nociception was assessed for 10 min by measuring the time (s) that the animal spent rubbing the injected area of the face with its paws 17,65 .
Periorbital mechanical allodynia. PMA was assessed using the up-down paradigm 66,67 in the same mice in which acute nociceptive responses were monitored. Briefly, mice were placed in a restraint apparatus designed for the evaluation of periorbital mechanical thresholds 17 . One day before the first behavioral observation, mice were habituated to the apparatus. PMA was evaluated in the periorbital region over the rostral portion of the eye (i.e., the area of the periorbital region facing the sphenoidal rostrum) 68 before (basal threshold) and after (0.5, 1, 2, 4, 6, 8 h) treatments. On the day of the experiment, after 20 min of adaptation inside the chamber, a series of 7 von Frey filaments in logarithmic increments of force (0.02, 0.04, 0.07, 0.16, 0.4, 0.6, and 1.0 g) were applied to the periorbital area perpendicular to the skin, with sufficient force to cause slight buckling, and held for approximately 5 s to elicit a positive response. Mechanical stimuli were applied homolaterally outside the periorbital area at a distance of 6-8 mm from the site where stimuli were injected. The response was considered positive by the following criteria: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking. Mechanical stimulation started with the 0.16 g filament. The absence of response after 5 s led to the use of a filament with increased force, whereas a positive response led to the use of a weaker (i.e. lighter) filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated from these scores by using a δ value of 0.205, previously determined.
Paw mechanical allodynia. Paw mechanical allodynia was evaluated by measuring the paw withdrawal threshold by using the up-down paradigm 66,67 . Mice were acclimatized (1 h) in individual clear plexiglass boxes on an elevated wire mesh platform, to allow for access to the plantar surfaces of the hind paws. von Frey filaments of increasing stiffness (0.07, 0.16, 0.4, 0.6, and 1.0, 1.4 and 2 g) were applied to the hind paw plantar surfaces of mice with enough pressure to bend the filament. The absence of a paw being lifted after 5 s led to the use of the next filament with an increased force, whereas a lifted paw indicated a positive response, leading to the use of a subsequently weaker filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated.
Primary culture of mouse Schwann cells. Mouse Schwann cells (MSC) were isolated from sciatic or trigeminal nerves of C57BL/6 J, and from sciatic nerve of Trpa1 +/+ and Trpa1 −/− , Plp1-Cre ERT+ ;Ramp1 fl/fl and Plp1-Cre ERT-;Ramp1 fl/fl mice 30,69 . The epineurium was removed, and nerve explants were divided into 1 mm segments and dissociated enzymatically using collagenase (0.05%) and hyaluronidase (0.1%) in Hank's Balanced Salt Solution (HBSS, 2 hr, 37°C). Cells were collected by centrifugation (150xg, 10 min, room temperature) and the pellet was resuspended  Fig. 9 Schematic representation of the pathway that signal prolonged cutaneous allodynia elicited by CGRP released and associated with neurogenic inflammation. The pro-migraine neuropeptide, CGRP, released from trigeminal cutaneous afferents, activates CLR/RAMP1 on Schwann cells. CLR/RAMP1 traffics to endosomes, where sustained G protein signaling increases cAMP and stimulates PKA that results in nitric oxide synthase activation. The ensuing release of nitric oxide targets the oxidant-sensitive channel, TRPA1, in Schwann cells, which elicits persistent ROS generation. ROS triggers TRPA1 on adjacent C-(1) or Aδ-fiber (2) afferents resulting in periorbital allodynia, a hallmark of migraine pain. The inset shows several unmyelinated axons invaginated into a Schwann cell forming a Remak bundle. and cultured in DMEM containing fetal calf serum (10%), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), neuregulin (10 nM) and forskolin (2 μM). Three days later, cytosine arabinoside (Ara-C, 10 mM) was added to remove fibroblasts. Cells were cultured at 37°C in 5% CO 2 and 95% O 2 . The culture medium was replaced every 3 days and cells were used after 15 days of culture.
qRT-PCR. Total RNA was extracted from HSCs, IMS32 and sciatic or trigeminal MSCs cells using the RNeasy Mini kit (Qiagen SpA), according to the manufacturer's protocol. RNA concentration and purity were assessed spectrophotometrically by measuring the absorbance at 260 nm and 280 nm. RNA was reverse transcribed with the Qiagen QuantiTect Reverse Transcription Kit (Qiagen SpA) following the manufacturer's protocol. For mRNA relative quantification, rt-PCR was performed on Rotor Gene® Q (Qiagen SpA, Rotor-Gene® Q-Software Version 2.3.1.49). The relative abundance of mRNA transcripts was calculated using the delta CT method and normalized to GAPDH levels. The sets of primers for human and mouse cells are listed in the Supplementary Table 3.
In-cell ELISA assay. HSCs or IMS32 cells were plated in 96-well black wall clear bottom plates (Corning Life Sciences) (5 × 10 5 cells/well) and maintained at 37°C in 5% CO 2 and 95% O 2 for 24 h. HSCs and IMS32 cells were exposed to CGRP (1 and 10 μM, respectively) or its vehicle (phosphate-buffered saline, PBS) for 5, 10, 15, 30 and 60 min, at 37°C, then washed with DMEM pH 2.5 and fixed in 4% paraformaldehyde for 30 min. Cells were then washed with TBST (0.05%) and blocked with donkey serum (5%) for 4 h at room temperature and incubated overnight 4°C with eNOS pS1177 (#ab184154, rabbit polyclonal, 1:100, Abcam, Lot: GR3257047-9). Cells were then washed and incubated with donkey anti-rabbit IgG conjugated with horseradish peroxidase (HRPO, 1:2000, Bethyl Laboratories Inc.) for 2 h at room temperature. Cells were then washed and stained using SIGMA-FAST OPD for 30 min protected from light. After the incubation period, the absorbance was measured at 450 nm. Change in NOS3 phosphorylation was calculated as a percentage of the signal in vehicle-treated cells. cAMP ELISA assay. cAMP level was determined by the CatchPoint™ cyclic-AMP fluorescent assay kit (#R8088, Molecular Device) according to the manufacturer's protocol. Briefly, HSCs or IMS32 cells were plated in 96-well black wall clear bottom plates (Corning Life Sciences) (5 × 10 5 cells/well) and maintained in 5% CO 2 and 95% O 2 (24 h, 37°C). The cultured medium was replaced with HBSS added with olcegepant (100 nM), CGRP8-37 (100 nM), SQ22536 (100 μM), L-NAME (10 μM) or vehicle (0.1% DMSO in HBSS) for 20 min at room temperature. HSCs or IMS32 cells were then stimulated with CGRP (1 and 10 μM, respectively), forskolin (1 μM, positive control) or their vehicles (HBSS) and maintained for 40 min at room temperature protected from light. Signal was detected 60 min after exposure to the stimuli. cAMP level was calculated using cAMP standards and expressed as nmol/1.
Ultra-performance liquid chromatography-mass spectrometry (LC-MS). MK-3207 loading into the core of NPs was assessed by LC-MS using a Waters Micromass Quattro Premier triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC (USA). Freeze-dried DIPMA-MK-3207 (1 ml, 1 mg/ml) were dissolved in a mixture of DMSO and formic acid 0.1% (5:2). The samples were prepared for analysis by mixing an aliquot of each preparation with internal standard solution (diazepam, 5 µg/ml) in a 5:2 proportion and made up to 500 µl with the dilution solvent (acetonitrile 50%: formic acid 0.1%, 1:1). Samples were fractionated on a Supelco Ascentis Express RP Amide column (50 mm by 2.1 mm, 2.7 µm particle size) equipped with a Phenomenex SecurityGuard precolumn fitted with a Synergi Polar cartridge, maintained at 40°C. MK-3207 loading was quantified against MK-3207 standards (0.016-20 µM). Compounds were eluted under gradient conditions with a mobile phase of formic acid (0.05%) and acetonitrile. Mass spectrometry was conducted in positive electrospray ionization conditions and elution of compounds were monitored with multiple-reaction monitoring.
Transmission electron microscopy (TEM). The morphology of NPs was determined by TEM imaging using a Tecnai F20 transmission electron microscope at an accelerating voltage of 120 kV at room temperature. Carbon-coated grids were prepared by plasma discharge (35 s). DIPMA-MK-3207 samples (5 µl, 1 mg/ml) were placed on the grid for 20 s. Samples were negatively stained with uranyl acetate (5 µl, 0.5 wt %, 25 s).
Statistical analysis. Results are expressed as mean ± standard error of the mean (SEM). For multiple comparisons, a one-way analysis of variance (ANOVA) followed by the post-hoc Bonferroni's test or Dunnett's test was used. Two groups were compared using Student's t-test. For behavioral experiments with repeated measures, the two-way mixed model ANOVA followed by the post-hoc Bonferroni's test was used. Statistical analyses were performed on raw data using Graph Pad Prism 8 (GraphPad Software Inc.). IC 50 values and confidence intervals were determined from non-linear regression models using Graph Pad Prism 8 (GraphPad Software Inc.). P values less than 0.05 (P < 0.05) were considered significant. Statistical tests used and the sample size for each analysis are listed in the Fig. legend.