Jianghui Meng

Synaptobrevin I mediates exocytosis of CGRP from sensory neurons and inhibition by botulinum toxins reflects their anti-nociceptive potential

Calcitonin-gene-related peptide (CGRP), a potent vasodilator that mediates inflammatory pain, is elevated in migraine; nevertheless, little is known about its release from sensory neurons. In this study, CGRP was found to occur in the majority of neurons from rat trigeminal ganglia, together with the three exocytotic SNAREs [SNAP25, syntaxin 1 and the synaptobrevin (Sbr, also known as VAMP) isoforms] and synaptotagmin. Ca2+-dependent CGRP release was evoked with K+-depolarisation and, to lower levels, by capsaicin or bradykinin from neurons that contain the vanilloid receptor 1 and/or bradykinin receptor 2. Botulinum neurotoxin (BoNT) type A cleaved SNAP25 and inhibited release triggered by K+ > bradykinin >> capsaicin. Unlike BoNT type D, BoNT type B did not affect exocytosis, even though the neurons possess its receptor and Sbr II and Sbr III got proteolysed (I is resistant in rat) but, in mouse neurons, it additionally cleaved Sbr I and blocked transmitter release. Sbr I and II were found in CGRP-containing vesicles, and each was shown to separately form a SNARE complex. These new findings, together with punctate staining of Sbr I and CGRP in neurites, implicate isoform Sbr I in exocytosis from large dense-core vesicles together with SNAP25 (also, probably, syntaxin 1 because BoNT type C1 caused partial cleavage and inhibition); this differs from Sbr-II-dependent release of transmitters from small synaptic vesicles. Such use of particular Sbr isoform(s) by different neurons raises the functional implications for other cells previously unrecognised.

Activation of TRPV1 Mediates Calcitonin Gene-Related Peptide Release, Which Excites Trigeminal Sensory Neurons and Is Attenuated by a Retargeted Botulinum Toxin with Anti-Nociceptive Potential

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 of Mr 25,000 (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 (HC) 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+]i with capsaicin. This successful targeting of /EA to nociceptive neurons and inhibition of CGRP release in vitro and in situ highlight its potential as a new therapy for sensory dysmodulation and chronic pain.

Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics

Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A–G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.

Neuro-exocytosis: botulinum toxins as inhibitory probes and versatile therapeutics.

For the fundamental process of quantal neurotransmitter release, a consensus is being reached on the recycling pathways for transmitter-containing, small synaptic vesicles (SSVs), and major inroads have been made into deciphering the multiple steps of regulated exocytosis. These advances arose from the identification of approximately 80 proteins in SSVs, elucidation of the structures of pertinent macromolecular complexes, utilisation of different serotypes (A-G) of botulinum neurotoxin (BoNT) together with transgenic mice lacking key genes. Hence, converging evidence continues to emerge for the sequential formation of complexes between the three SNAREs (SNAP-25, syntaxin and VAMP) and their regulatory proteins (complexins, Munc18), as well as for the Ca2+ triggering of membrane fusion/exocytosis via its sensor, synaptotagmin. Moreover, molecular data gained on BoNTs have been translated into Clinical Medicine with type A now being applied worldwide for effectively treating >100 human conditions due to overactivity of nerves supplying various muscles or glands. A recent advance is the successful engineering of a chimera from two BoNTs to acquire the capability of re-targeting a more active moiety to sensory neurons, with resultant inhibition of the release of a pain mediator. Encouragingly, this novel recombinant protein blocks the exocytotic response triggered by a stimulant (capsaicin) of nociceptive C fibres that activates their vanilloid receptors, a feat not possible for either parental toxin. Reaching this landmark has generated optimism for designing further variants of such a versatile therapeutic for normalising the hyper-activity of particular cell types, especially those underlying the many cases of chronic pain that do not respond to existing drugs.

A dileucine in the protease of botulinum toxin A underlies its long-lived neuroparalysis: transfer of longevity to a novel potential therapeutic.

Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNTA) 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 BoNTA 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 (LCA) 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 BoNTA activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LCE to a BoNTA enzymically inactive mutant (BoTIMA) yielded a novel LCE-BoTIMA protein that targets neurons, and the BoTIMA moiety also delivers and stabilizes the inhibitory LCE, giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNTE. LCE-BoTIMA(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNTE, reaffirming that these residues are critical for the persistent action of LCE-BoTIMA as well as BoNTA. LCE-BoTIMA 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.

E1A-expressing adenoviral E3B mutants act synergistically with chemotherapeutics in immunocompetent tumor models

The majority of clinical trials evaluating replication-selective oncolytic adenoviruses utilized mutants with immunomodulatory E3B genes deleted, likely contributing to the attenuated efficacy. We investigated whether an intact immune response could contribute to the observed improved efficacy in response to combinations with chemotherapeutics. Seven carcinoma cell lines were evaluated by combining viral mutants; dl309 (ΔE3B), dl704 (ΔE3gp19K), dl312 (ΔE1A) or wild-type Ad5 with the commonly used clinical drugs cisplatin and paclitaxel. Synergistic effects on cell death were determined by generation of combination indexes in cultured cells. In vivo tumor growth inhibition was achieved by virotherapy alone and was most efficacious with wild-type virus and least with the ΔE3B mutant. Significantly higher efficacy was observed when the viruses were combined with drugs. The greatest enhancement of tumor inhibition was in combination with the ΔE3B mutant restoring potency to that of Ad5 wild-type levels, observed only in animals with intact immune response. Increases in infectivity, viral gene expression and replication were identified as potential mechanisms contributing to the synergistic effects. Our results suggest that the attenuation of ΔE3B mutants can be overcome by low doses of chemotherapeutics only in the presence of an intact immune response indicating a role for T-cell-mediated functions.

Novel therapeutics based on recombinant botulinum neurotoxins to normalize the release of transmitters and pain mediators

A major unmet clinical need exists for long‐acting neurotherapeutics to alleviate chronic pain in patients unresponsive to available nonaddictive analgesics. Herein, a new strategy is described for the development of potent and specific inhibitors of the neuronal exocytosis of transmitters and pain mediators that exhibit unique antinociceptive activity. This entailed recombinant production in Escherichia coli of two serotypes of botulinum neurotoxin (BoNT) (BoNTA and BoNTE), which are proteins that are known to block the release of transmitters by targeting and entering nerve endings, where their proteases cleave and inactivate a protein, synaptosomal protein of Mr 25 000 (SNAP‐25), that is essential for Ca2+‐regulated exocytosis. Site‐directed mutagenesis of Leu428 and Leu429 in BoNTA revealed that the remarkable longevity of its neuroparalytic action is attributable to a dileucine‐containing motif. BoNTE acts transiently, because it lacks these residues, but is a superior inhibitor of transient receptor potential vanilloid type 1‐mediated release of pain peptides from sensory nerves. The advantageous features of each serotype were harnessed by attaching the BoNTE protease moiety to an enzymically inactive mutant of BoNTA. The resultant purified composite protein could target motoneurons by binding to the BoNTA ectoacceptor and persistently produce BoNTE‐truncated SNAP‐25. As this enzyme lasted for more than 1 month (as compared with 5 days for BoNTE alone), such a dramatic extension in the lifetime of this BoNTE protease is attributable to a stabilizing influence of the BoNTA mutant. Most importantly, injecting this novel biotherapeutic into the foot pads of rats resulted in extended amelioration of inflammatory pain. Thus, a new generation of biotherapeutics has been created with the potential to give long‐term relief of pain.

Longer-acting and highly potent chimaeric inhibitors of excessive exocytosis created with domains from botulinum neurotoxin A and B

Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT (botulinum neurotoxin). The BoNT/A complex is widely used because of its longer-lasting benefits; also, autonomic side-effects are more often reported for BoNT/B. To establish if this distinct effect of BoNT/B could be exploited therapeutically, BoNT/A was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action. The advantageous protease and translocation domain of BoNT/A were recombinantly combined with the acceptor-binding moiety of type B [H(C)/B (C-terminal half of BoNT/B heavy chain)], creating the chimaera AB. This purified protein bound the BoNT/B acceptor, displayed enhanced capability relative to type A for intraneuronally delivering its protease, cleaved SNAP-25 (synaptosome-associated protein of 25 kDa) and induced a more prolonged neuromuscular paralysis than BoNT/A in mice. The BA chimaera, generated by substituting H(C)/A (C-terminal half of BoNT/A heavy chain) into BoNT/B, exhibited an extremely high specific activity, delivered the BoNT/B protease via the BoNT/A acceptor into neurons, or fibroblast-like synoviocytes that lack SNAP-25, cleaving the requisite isoforms of VAMP (vesicle-associated membrane protein). Both chimaeras inhibited neurotransmission in murine bladder smooth muscle. BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the BoNT/A-acceptor and utilising VAMP, but not SNAP-25, in exocytosis.

TNFα induces co-trafficking of TRPV1/TRPA1 in VAMP1-containing vesicles to the plasmalemma via Munc18–1/syntaxin1/SNAP-25 mediated fusion

Transient receptor potential (TRP) A1 and V1 channels relay sensory signals, yet little is known about their transport to the plasmalemma during inflammation. Herein, TRPA1 and TRPV1 were found on vesicles containing calcitonin gene-related peptide (CGRP), accumulated at sites of exo- and endo-cytosis, and co-localised on fibres and cell bodies of cultured sensory neurons expressing both. A proinflammatory cytokine, TNFα, elevated their surface content, and both resided in close proximity, indicating co-trafficking. Syntaxin 1–interacting protein, Munc18–1, proved necessary for the response to TNFα, and for TRPV1-triggered CGRP release. TNFα-induced surface trafficking of TRPV1 and TRPA1 required a synaptic vesicle membrane protein VAMP1 (but not 2/3), which is essential for CGRP exocytosis from large dense-core vesicles. Inactivation of two proteins on the presynaptic plasma membrane, syntaxin-1 or SNAP-25, by botulinum neurotoxin (BoNT)/C1 or /A inhibited the TNFα-elevated delivery. Accordingly, enhancement by TNFα of Ca2+ influx through the upregulated surface-expressed TRPV1 and TRPA1 channels was abolished by BoNT/A. Thus, in addition, the neurotoxins’ known inhibition of the release of pain transmitters, their therapeutic potential is augmented by lowering the exocytotic delivery of transducing channels and the resultant hyper-sensitisation in inflammation.

Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis

Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor‐nerve endings and undergo distinct trafficking; their light‐chain (LC) proteases cleave soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor‐binding moieties (HC)—or in one case, heavy chain (HC)—were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve‐induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or HC; BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP‐25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.—Wang, J., Zurawski, T. H., Meng, J., Lawrence, G. W., Aoki, K. R., Wheeler, L., Dolly, J. O. Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis. FASEB J. 26, 5035–5048 (2012). www.fasebj.org