Calcitonin gene-related peptide (CGRP) in trigeminal ganglion neurons (TGNs) contributes to neurogenic inflammation and chronic migraine (CM). Trafficking of transient receptor potential (TRP) cation channels to plasmalemma has been implicated in TGN sensitisation by inflammatory agents, such as nerve growth factor (NGF), and could potentiate CGRP exocytosis. Its Ca2+-dependent release from sensory fibres is mediated by synaptosomal-associated protein with Mr=25 k (SNAP-25), syntaxin-1, and vesicle-associated membrane protein isoforms 1/2/3 (VAMP1/2/3). Hence, moderation of CGRP release and TRP trafficking by botulinum neurotoxins (BoNTs) is a potential therapeutic strategy for CM.
Cultured TGNs from neonatal rats were exposed to activators of TRP subfamilies V1 and A1; capsaicin (CAP) and allyl isothiocyanate (AITC). This caused dose-dependent increases of intracellular Ca2+ [Ca2+]i, revealed by confocal fluorescence monitoring in Fluo-4 loaded cells, and CGRP secretion quantified by ELISA. However, AITC differed from CAP by inducing a complex bi-phasic response. BoNT type A (/A) cleaved SNAP-25 and reduced neuropeptide exocytosis evoked by low [CAP], but not high concentrations that elevated [Ca2+]i more extensively. The toxin partially reduced CGRP release stimulated by AITC, but raising the concentration of this agonist failed to induce the high [Ca2+]i necessary to fully overcome BoNT/A blockade. Chimeras created by gene recombination, BoNT/EA and LC/E-BoNT/A that both removed a larger fragment of SNAP-25 then /A, partially blocked CGRP secretion evoked by AITC, but extensively inhibited the release evoked by low and high [CAP]. VAMP1/2/3-cleaving BoNT/DA effectively blocked CGRP release evoked by all [CAP] and [AITC].
Herein, it was discovered that NGF provokes Ca2+-dependent CGRP exocytosis that was susceptible to blockade by SNAP-25 and VAMP1/2/3 cleaving proteases. In addition, the neurotrophin enhanced CAP- and AITC-stimulated release, although this effect was more pronounced at low [agonist]. The NGF-induced enhancement of stimulated exocytosis did not require extracellular Ca2+. BoNT/EA was a more effective inhibitor of NGF-enhanced CGRP secretion stimulated by high [CAP] than BoNT/A. On the other hand, all the BoNTs inhibited AITC-stimulated secretion enhanced by NGF to similar extents. BoNTs with distinct protease activities might expand their therapeutic utility for CM in addition to currently used BoNT/A.