Abstract
Chimeras of botulinum neurotoxin (BoNT) serotype A (/A) combined with /E protease might possess improved analgesic properties relative to either parent, due to inheriting the sensory neurotropism of the former with more extensive disabling of SNAP-25 from the latter. Hence, fusions of /E protease light chain (LC) to whole BoNT/A (LC/E-BoNT/A), and of the LC plus translocation domain (HN) of /E with the neuronal acceptor binding moiety (HC) of /A (BoNT/EA), created previously by gene recombination and expression in E. coli., were used. LC/E-BoNT/A (75 units/kg) injected into the whisker pad of rats seemed devoid of systemic toxicity, as reflected by an absence of weight loss, but inhibited the nocifensive behavior (grooming, freezing, and reduced mobility) induced by activating TRPV1 with capsaicin, injected at various days thereafter. No sex-related differences were observed. c-Fos expression was increased five-fold in the trigeminal nucleus caudalis ipsi-lateral to capsaicin injection, relative to the contra-lateral side and vehicle-treated controls, and this increase was virtually prevented by LC/E-BoNT/A. In vitro, LC/E-BoNT/A or /EA diminished CGRP exocytosis from rat neonate trigeminal ganglionic neurons stimulated with up to 1 µM capsaicin, whereas BoNT/A only substantially reduced the release in response to 0.1 µM or less of the stimulant, in accordance with the /E protease being known to prevent fusion of exocytotic vesicles.
Highlights
Serotypes A and E of botulinum neurotoxin (BoNT/A and /E), proteins (Mr~150 k) produced by the requisite Clostridium botulinum and containing a disulphide-linked heavy (HC) and light chain (LC), are exquisite inhibitors of acetylcholine release from peripheral nerves [1]. Such preferential blockade underlies the great success of BoNT/A preparations in the clinical treatment of numerous conditions, due to over-activity of cholinergic nerves supplying various muscles or glands [2]. This selectivity and potent action are aided by the rapid exocytosis and recycling of small clear synaptic vesicles containing fast neurotransmitters [3], because their membrane possesses the high-affinity receptors for BoNT/A and /E, variants of synaptic vesicle protein 2 (SV2) [4,5,6]
Inhibition by BoNT/A of the stimulated release of pain mediators, such as substance P and calcitonin gene-related peptide (CGRP), established that the exocytosis of large dense-core vesicles from rat sensory neurons is susceptible to SNAP-25 cleavage [13,14,15,16,17]
After verifying that LC/E-BoNT/A did not cause any discomfort or pain to the animals, as reflected by their unaltered natural behavior, another experimental cohort was injected with LC/E-BoNT/A (75 units/kg) or vehicle 1 into the right whisker pad, followed by vehicle 2 or capsaicin (2.5 μg in 20 μL) on days 4, 8, 15, and
Summary
Serotypes A and E of botulinum neurotoxin (BoNT/A and /E), proteins (Mr~150 k) produced by the requisite Clostridium botulinum and containing a disulphide-linked heavy (HC) and light chain (LC), are exquisite inhibitors of acetylcholine release from peripheral nerves [1] Such preferential blockade underlies the great success of BoNT/A preparations in the clinical treatment of numerous conditions, due to over-activity of cholinergic nerves supplying various muscles or glands [2]. Subsequent translocation to the cytosol has been attributed to the N-terminal portion of HC, whereas the metalloprotease activity of LC/A and /E, respectively, cleaves off 9 and 26 C-terminal residues from synaptosomal-associated protein with Mr = 25 k (SNAP-25), yielding SNAP-25A or SNAP-25E (reviewed by [10]) Such distinct proteolysis results in blockade of transmitter release because of their substrate being a SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor), required for the exocytosis of all neurotransmitter types [11,12]. Evidence for the involvement of CGRP in the pathology of migraine [18] has generated research interest in the possible potential of BoNT/A as an anti-nociceptive agent (see below)
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