Excessive release of inflammatory/pain mediators from peripheral sensory afferents renders nerve endings hyper-responsive, causing central sensitization and chronic pain. Herein, the basal release of proinflammatory calcitonin gene-related peptide (CGRP) was shown to increase the excitability of trigeminal sensory neurons in brainstem slices via CGRP1 receptors because the effect was negated by an antagonist, CGRP8–37. This excitatory action could be prevented by cleaving synaptosomal-associated protein ofM_r25,000 (SNAP-25) with botulinum neurotoxin (BoNT) type A, a potent inhibitor of exocytosis. Strikingly, BoNT/A proved unable to abolish the CGRP1 receptor-mediated effect of capsaicin, a nociceptive TRPV1 stimulant, or its elevation of CGRP release from trigeminal ganglionic neurons (TGNs) in culture. Although the latter was also not susceptible to BoNT/E, apparently attributable to a paucity of its acceptors (glycosylated synaptic vesicle protein 2 A/B), this was overcome by using a recombinant chimera (EA) of BoNT/A and BoNT/E. It bound effectively to the C isoform of SV2 abundantly expressed in TGNs and cleaved SNAP-25, indicating that its /A binding domain (H_C) mediated uptake of the active /E protease. The efficacy of /EA is attributable to removal of 26 C-terminal residues from SNAP-25, precluding formation of SDS-resistant SNARE complexes. In contrast, exocytosis could be evoked after deleting nine of the SNAP-25 residues with /A but only on prolonged elevation of [Ca²⁺]_iwith capsaicin. This successful targeting of /EA to nociceptive neurons and inhibition of CGRP releasein vitroandin situhighlight its potential as a new therapy for sensory dysmodulation and chronic pain.

Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT_A) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT_A was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC_A) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6–7 N terminus residues diminished BoNT_A activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC_E to a BoNT_A enzymically inactive mutant (BoTIM_A) yielded a novel LC_E-BoTIM_A protein that targets neurons, and the BoTIM_A moiety also delivers and stabilizes the inhibitory LC_E, giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT_E. LC_E-BoTIM_A(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT_E, reaffirming that these residues are critical for the persistent action of LC_E-BoTIM_A as well as BoNT_A. LC_E-BoTIM_A inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves.

Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. BoNT_A (botulinum neurotoxin type A) is a protease that binds to peripheral nerve terminals, becomes endocytosed and causes prolonged blockade of transmitter release by cleaving SNAP-25 (synaptosome-associated protein of 25 kDa). Retrograde transport of the toxin has been suggested, but not of the transient muscle relaxant, BoNT_E (botulinum neurotoxin type E). In the present study, dispersal of these proteases in compartmented cultures of rat sympathetic neurons was examined after focal application of BoNT_A or BoNT_E to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. BoNT_E yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. Neurite transection prevented movement of BoNT_A. The BoNT_A protease could be detected only in the supernatants of neurites or cell body lysates, hence these proteases must move along neuronal processes in the axoplasm or are reversibly associated with membranes. Substitution into BoNT_E of the BoNT_A acceptor-binding domain did not alter its potency or mobility. Spontaneous or evoked transmission to cell bodies were not inhibited by retrogradely migrated BoNT_A except with high doses, concurring with the lack of evidence for a direct central action when used clinically.