Fig 2: 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 Ad-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.

Fig 3: DIPMA-MK-3207 nanoparticles target endosomal CLR/RAMP1 signaling and provide superior relief from CGRP-evoked PMA.a pH-responsive DIPMA-MK-3207. b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e–g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f, g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP (n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca2+ response to CGRP in HSCs (DIPMA-MK-3207: -9M, n = 145; -8M, n = 361; -7M, n = 213: -6.5 M, n = 150; or free MK-3207: -8M, n = 83; -6M, n = 106; -5M, n = 87; -4M, n = 127: -3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle (n = 8 mice per group). Mean±SEM. ***P < 0.001 vs. DIPMA-Ø/Veh, ###P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f, g, i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.

Fig 4: Functional CLR/RAMP1 is expressed by HSCs and undergoes clathrin- and dynamin-mediated endocytosis, which underlies nociception.a, b Effects of graded concentrations of CGRP on cAMP formation (n = 5 independent experiments). c, d Effects of graded concentrations of olcegepant on CGRP (100 nM)-evoked cAMP formation (n = 4 independent experiments). e Pharmacological targets. f Representative images of HSCs expressing Rab5a-GFP at 30 min after incubation with TAMRA-CGRP (100 nM). Arrows denote colocalization of TAMRA-CGRP and Rab5a-GFP. Arrowheads denote retention of a weak TAMRA-CGRP signal at the plasma membrane. Cells were preincubated with vehicle, Dyngo-4a (Dy4), Pitstop 2 (PS2), inactive analogs (PS2 and Dy4 inact) (all 30 µM) or sucrose (0.45 M) (n = 4 independent experiments). Scale, 10 µm. g Quantification of localization of TAMRA-CGRP in endosomes (data represent n = 949 veh 0 min, n = 1209 veh 30 min, n = 1111 Dy4 30 min, n = 1016 Dy4 inact 30 min, n = 1700 PS2 30 min, n = 714 PS2 inact 30 min, n = 1896 sucrose) and (h) quantification of the number of TAMRA-CGRP+ve endosomes (data represent n = 5 veh 0 min, n = 7 veh 30 min, n = 7 Dy4 30 min, n = 5 Dy4 inact 30 min, n = 7 PS2 30 min, n = 5 PS2 inact 30 min, n = 5 sucrose). i, j PMA induced by periorbital CGRP (1.5 nmol) or vehicle in C57BL/6 J male mice pretreated (0.5 h) with PS2, Dy4, PS2 or Dy4 inact (all 500 pmol) (n = 8 mice per group). k, l PMA induced by periorbital capsaicin (CPS, 50 pmol) or vehicle in C57BL/6 J male mice pretreated (0.5 h) with PS2, Dy4, PS2 or Dy4 inact (all 500 pmol) (n = 8 mice per group). Mean±SEM. ***P < 0.001 vs. Veh 0 min, and Veh/Veh; §§P < 0.01, §§§P < 0.001 vs. Veh 30 min, PS2 30 min, Dy4 30 min, CGRP/PS2 inact, CGRP/Dy4 inact, CPS/PS2 inact, CPS/Dy4 inact. 1-way (g, h) or 2-way (i–l) ANOVA, Bonferroni correction. Source data are provided as a Source Data file.

Fig 5: Capsaicin induces PMA via CGRP and CLR/RAMP1 in Schwann cells.a Acute nociception and (b) PMA after periorbital injection of capsaicin (CPS) or vehicle in C57BL/6 J mice. c Acute nociception and (d) PMA after CPS (50 pmol) or vehicle in Trpv1+/+ and Trpv1-/- mice. e Acute nociception and (f) PMA after CPS (50 pmol) or vehicle in C57BL/6 J mice pretreated with capsazepine (CPZ, 100 pmol) or vehicle. g, h PMA after periorbital SP (3.5 nmol), CPS (50 pmol) or vehicle in C57BL/6 J mice pretreated (0.5 h) with L-733,060 (20 nmol) or vehicle. i, j PMA after CPS (50 pmol) or vehicle in C57BL/6 J mice pretreated (0.5 h) with olcegepant (1 nmol) or CGRP8-37 (10 nmol) or vehicle. k PMA after CPS (50 pmol) or vehicle in Plp1-CreERT+/Ramp1fl/fl or Control mice treated with periorbital 4-OHT or vehicle. l PMA after periorbital CGRP (1.5 nmol) or vehicle and (m) acute nociceptive response and PMA after periorbital CPS (50 pmol) or vehicle in Adv-Cre+/Ramp1fl/fl or Control mice. n PMA induced by intraperitoneal (i.p.) CGRP (0.1 mg/kg) or vehicle in Adv-Cre+/Ramp1fl/fl or Control mice. Mean±SEM., n = 8 mice per group. **P < 0.01, ***P < 0.001 vs. Veh/Veh, Trpv1+/+-Veh and Control-Veh; §P < 0.05, §§P < 0.01, §§§P < 0.001 vs. Trpv1+/+-CPS, CPS/Veh, SP/Veh, Control-CPS, Control-CGRP. 1-way (a, c, e and m left panel) or 2-way (b, d, f–l, m right panel and n) ANOVA, Bonferroni correction. Source data are provided as a Source Data file.