NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

Abstract

Nerve growth factor (NGF) is known to intensify pain in various ways, so perturbing pertinent effects without negating its essential influences on neuronal functions could help the search for much-needed analgesics. Towards this goal, cultured neurons from neonatal rat trigeminal ganglia—a locus for craniofacial sensory nerves—were used to examine how NGF affects the Ca2+-dependent release of a pain mediator, calcitonin gene-related peptide (CGRP), that is triggered by activating a key signal transducer, transient receptor potential vanilloid 1 (TRPV1) with capsaicin (CAP). Measurements utilised neurons fed with or deprived of NGF for 2 days. Acute re-introduction of NGF induced Ca2+-dependent CGRP exocytosis that was inhibited by botulinum neurotoxin type A (BoNT/A) or a chimera of/E and/A (/EA), which truncated SNAP-25 (synaptosomal-associated protein with Mr = 25 k) at distinct sites. NGF additionally caused a Ca2+-independent enhancement of the neuropeptide release evoked by low concentrations (<100 nM) of CAP, but only marginally increased the peak response to ≥100 nM. Notably, BoNT/A inhibited CGRP exocytosis evoked by low but not high CAP concentrations, whereas/EA effectively reduced responses up to 1 µM CAP and inhibited to a greater extent its enhancement by NGF. In addition to establishing that sensitisation of sensory neurons to CAP by NGF is dependent on SNARE-mediated membrane fusion, insights were gleaned into the differential ability of two regions in the C-terminus of SNAP-25 (181–197 and 198–206) to support CAP-evoked Ca2+-dependent exocytosis at different intensities of stimulation.

Botulinum toxin A for the treatment of first bite syndrome—a systematic review

“…However, we postulate BTA may improve resolution of the disease secondary to decreases in salivary gland weight, cholinergic output, secretory capacity, cellular size, and myoepithelial function after injection ( 74 - 76 ). Additionally, nerve growth factor (NGF) expression can be increased by BTA ( 77 ), which may lead to improved parotid gland sympathetic reinnervation ( 78 ). This may explain the potential long-term improvements some patients experience.…”

OnabotulinumtoxinA: Still the Present for Chronic Migraine

“…This may explain the long-term persistence of BT-A action in nerve terminals, which is up to 1 year in cultured neurons and about 5 months in vivo [ 51 ]. The BT-A light chain is a Zn 2+ dependent metalloprotease that cleaves the synaptosomal-associated protein of 25 kDa (SNAP-25), thus forming SNAP-25 (1–197), which is inactive and forms heterotrimers with other SNARE proteins to create inactive complexes [ 53 ]. The cleavage of SNAP-25 inhibits the exocytosis processes, thus limiting neuropeptides’ and neurotransmitters’ exocytosis and reducing the presentation of receptors on the plasma membrane [ 54 ].…”

Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins

“…Similar to NGF, TNFα mobilises intracellular stores of TRPV1 to the plasma membrane of TGNs in vitro [ 26 , 27 ]. Accordingly, pre-treating sensory neurons with TNFα or NGF enhances the currents elicited by CAP (a TRPV1 activator), intensifies the resultant increases of intracellular Ca 2+ concentration ([Ca 2+ ] i ), as well as augmenting CAP-evoked CGRP release [ 26 , 27 , 28 ]. The surface transfer of TRPV1 can be inhibited by pre-treatment of the TGNs with botulinum neurotoxin type A (BoNT/A) [ 26 ], a bacterial protease that enters nerve endings and cleaves 9 C-terminal residues off a SNARE protein essential for membrane fusion called synaptosomal-associated protein of Mr = 25k (SNAP-25) (reviewed by [ 29 ]).…”

“…In animal pain models, the neurotoxin also inhibits the release of other neuropeptides, such as substance P and pituitary adenylate cyclase-activating peptide (PACAP), and neurotransmitters including glutamate and acetylcholine; notably, the precise sites and mechanisms of action in relation to its migraine relief remain unresolved [ 34 ]. Whilst BoNT/A in vitro blocks the CGRP release from TGNs evoked by relatively mild stimulation with a low concentration of CAP [CAP] (0.02 µM), it is progressively less effective against increasing [CAP] and a rather feeble inhibitor of CGRP release induced by 1 µM CAP [ 28 , 35 ]. The latter has been attributed to the recovered functionality of BoNT/A-truncated SNAP-25 in the presence of sustained large increases in [Ca 2+ ] i [ 36 ].…”

“…Evidence for the transfer of TRPA1 to the cell surface in neurons sensitised with TNFα has been accrued from its inhibition by SNAP-25 inactivating BoNT/A [ 26 ] or a recombinant chimera LC/E-BoNT/A [ 27 ], as well as knock-down of another SNARE called vesicle-associated membrane protein 1 (VAMP1) [ 26 ]. Compared with extensive knowledge of TRPV1 [ 23 , 28 , 38 , 39 ], the relationships between the activation of TRPA1 with different [AITC], the enhancement of their cell excitability by NGF, its potentiation of CGRP release, and susceptibility to BoNTs that inactivate VAMP or SNAP-25, are poorly defined. Therefore, these were investigated herein in cultures of TGNs isolated from neonatal rats using Ca 2+ -imaging of neurons loaded with fluo-4-acetoxymethyl ester (Fluo-4 AM) by time-lapse confocal microscopy, in combination with enzyme-linked immuno-sorbent assay (ELISA) for CGRP and Western blotting for SNAP-25 or VAMP isoforms 1/2/3.…”