Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses

The clostridial neurotoxins responsible for tetanus and botulism are proteins consisting of three domains endowed with different functions: neurospecific binding, membrane translocation and proteolysis for specific components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular junction, is internalized and transported retroaxonally to the spinal cord. The spastic paralysis induced by the toxin is due to the blockade of neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven serotypes of botulinum neurotoxins (BoNTs) act at the periphery by inducing a flaccid paralysis due to the inhibition of acetylcholine release at the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G cleave specifically at single but different peptide bonds, of the vesicle associated membrane protein (VAMP) synaptobrevin, a membrane protein of small synaptic vesicles (SSVs). BoNT types A, C and E cleave SNAP-25 at different sites located within the carboxyl-terminus, while BoNT type C additionally cleaves syntaxin. The remarkable specificity of BoNTs is exploited in the treatment of human diseases characterized by a hyperfunction of cholinergic terminals.     

Mechanism of action of tetanus and botulinum neurotoxins

The clostridial neurotoxins responsible for tetanus and botulism are metallo-proteases that enter nerve cells and block neurotransmitter release via zinc-dependent cleavage of protein components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular Junction and is internalized and transported retroaxonally to the spinal cord. Whilst TeNT causes spastic paralysis by acting on the spinal inhibitory interneurons, the seven serotypes of botullnum neurotoxins (BoNT) induce a flaccid paralysis because they intoxicate the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G specifically cleave VAMP/synaptobrevin, a membrane protein of small synaptic vesicles, at different single peptide bonds. Proteins of the presynaptic membrane are specifically attacked by the other BoNTs: serotypes A and E cleave SNAP-25 at two different sites located within the carboxyl terminus, whereas the specific target of serotype C is syntaxin.     

Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond.     

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 (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.     

SNAP-25 is expressed in islets of Langerhans and is involved in insulin release

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP- 25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.     

Rescue of exocytosis in botulinum toxin A-poisoned chromaffin cells by expression of cleavage-resistant SNAP-25. Identification of the minimal essential C-terminal residues

Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1-197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1-206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxin's protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1-201, 1-202, 1-203 afforded a significant return of exocytosis, unlike shorter forms 1-197, -198, -199, or -200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism.     

The role of the synaptic protein snap-25 in the potency of botulinum neurotoxin type A

Botulinum neurotoxin serotype A (BoNT/A) is distinguished from BoNT/E by longer duration of paralysis and greater potency. The proteolytic activity of BoNT/A in cultures of dissociated spinal cord neurons persists beyond 80 days, whereas BoNT/E activity persists for less than 1 day (Keller, J. E., Neale, E. A. Oyler, G., and Adler, M. (1999) FEBS Lett. 456, 137-142). This single quality of toxin activity can account for the differences observed in the duration of muscle block. In the present work we sought to understand the basis for the apparent greater potency of BoNT/A. BoNT/E cleaves a 26-amino acid fragment from the C terminus of the synaptic protein SNAP-25 whereas BoNT/A removes only nine residues creating a 197-amino acid fragment (P197) that is 95% the length of SNAP-25. We show that inhibition of neurotransmitter release by BoNT/E is equivalent to the damage caused to SNAP-25. However, synaptic blockade by BoNT/A is greater than the extent of SNAP-25 proteolysis. These findings can be explained if P197 produces an inhibitory effect on neurotransmitter release. A mathematical model of the experimentally determined relationship between SNAP-25 damage and blockade of neurotransmission supports this interpretation. Furthermore, neurotransmitter release following complete cleavage of SNAP-25 can be achieved by P197, but with about 5-fold less sensitivity to external Ca(2+). In this case, vesicular release is restored by increasing intracellular Ca(2+). These data demonstrate that P197 competes with intact SNAP-25, but is unable to initiate normal synaptic vesicle fusion in physiological concentrations of Ca(2+).     

Proteolysis of SNAP-25 Isoforms by Botulinum Neurotoxin Types A, C, and E

Abstract : Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg¹⁹⁸ and Ala¹⁹⁹, depends on the presence of regions Asn⁹³ to Glu¹⁴⁵ and Ile¹⁵⁶ to Met²⁰², and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex ( K _D = 1.9 × 10^-7 M ) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met¹⁴⁶-Gln¹⁹⁷, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile¹⁵⁶-Asp¹⁸⁶. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys¹⁸⁵Asp or Pro¹⁸²Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.     

Inhibition of neurotransmitter release by peptides that mimic the N-terminal domain of SNAP-25

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by cleaving the 206-amino-acid SNARE protein, SNAP-25. For each BoNT serotype, cleavage of SNAP-25 results in the loss of intact protein, the production of an N-terminal truncated protein, and the generation of a small C-terminal peptide. Peptides that mimic the C-terminal fragments of SNAP-25 following BoNT/A or BoNT/E cleavage were shown to depress transmitter release in bovine chromaffin cells and in Aplysia buccal ganglion cells. Similarly, the N-terminal–truncated SNAP-25 resulting from BoNT/A or BoNT/E cleavage has been found to inhibit transmitter exocytosis in various systems. With one exception, however, the inhibitory action of truncated SNAP-25 has not been demonstrated at a well-defined cholinergic synapse. The goal of the current study was to determine the level of inhibition of neurotransmitter release by N-terminal BoNT/A- or BoNT/E-truncated SNAP-25 in two different neuronal systems: cholinergically coupled Aplysia neurons and rat hippocampal cell cultures. Both truncated SNAP-25 products inhibited depolarization-dependent glutamate release from hippocampal cultures and depressed synaptic transmission in Aplysia buccal ganglion cells. These results suggest that truncated SNAP-25 can compete with endogenous SNAP-25 for binding with other SNARE proteins involved in transmitter release, thus inhibiting neurotransmitter exocytosis.     

Differential roles of developmentally distinct SNAP-25 isoforms in the neurotransmitter release process

The role of SNAP-25 (synaptosomal associated protein of 25 kDa) isotypes in the neurotransmitter release process was examined by varying their relative abundance during PC12 cell differentiation induced by nerve growth factor (NGF). Norepinephrine release by NGF-differentiated PC12 cells is more sensitive to type A botulinum toxin (BoNT/A) than by nondifferentiated cells, while both differentiated and nondifferentiated PC12 cells are equally sensitive to type E botulinum toxin (BoNT/E). The differential sensitivity to BoNT/A corresponds to an altered susceptibility of SNAP-25 isotypes to BoNT/A cleavage in vitro, whereas both isotypes are equally vulnerable to cleavage by BoNT/E. Using recombinant SNAP-25 preparations, we show that BoNT/A cleaves SNAP-25b (present in differentiated cells) 2-fold more readily than SNAP-25a (present in both differentiated and nondifferentiated cells). Structural studies using far-ultraviolet circular dichroism (UV--CD) and thermal denaturation suggest a difference in the polypeptide folding as the underlying molecular basis for the differential sensitivity of SNAP-25b and SNAP-25a to BoNT/A cleavage. We propose differential roles for SNAP-25b and SNAP-25a in the neurotransmitter release process since our results suggest that BoNT/A inhibits neurotransmitter release by primarily cleaving SNAP-25b.     

Redox properties of the disulfide bond of human Cu,Zn superoxide dismutase and the effects of human glutaredoxin 1

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.     

The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool

The impact of syntaxin and SNAP-25 cleavage on [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca2+-dependent K+-induced [3H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [3H]NA and [3H]DA were differentially affected: both toxins blocked the former, dependent on external Ca2+, but not the latter, internal Ca2+-dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [3H]catecholamine release. More precisely, [3H]NA exocytosis induced by ionomycin was sensitive to toxins in the early phase of release but not later. The Ca2+-independent [3H]NA exocytosis evoked by hypertonic sucrose, thought to release from the readily releasable pool (RRP) of vesicles, was significantly reduced by BoNT/C1. Pre-treating synaptosomes with phorbol-12-myristate-13-acetate, to increase the RRP, enhanced the sensitivity to BoNT/C1 of [3H]NA release elicited by sucrose or ionomycin. Accordingly, cleavage of syntaxin was augmented by the phorbol-ester. To conclude, our results suggest that clostridial toxins selectively target exocytosis involving vesicles set into the RRP.     

Primary cultures of embryonic chicken neurons for sensitive cell-based assay of botulinum neurotoxin: implications for therapeutic discovery

Botulinum toxin is an exceedingly potent inhibitor of neurotransmission across the neuromuscular junction, causing flaccid paralysis and death. The potential for misuse of this deadly poison as a bioweapon has added a greater urgency to the search for effective therapeutics. The development of sensitive and efficient cell-based assays for the evaluation of toxin antagonists is crucial to the rapid and successful identification of therapeutic compounds. The authors evaluated the sensitivity of primary cultures from 4 distinct regions of the embryonic chick nervous system to botulinum neurotoxin A (BoNT/A) cleavage of synaptosomal-associated protein of 25 kD (SNAP-25). Although differences in sensitivity were apparent, SNAP-25 cleavage was detectable in neuronal cells from each of the 4 regions within 3 h at BoNT/A concentrations of 1 nM or lower. Co-incubation of chick neurons with BoNT/A and toxin-neutralizing antibodies inhibited SNAP-25 cleavage, demonstrating the utility of these cultures for the assay of BoNT/A antagonists.     

Retention of cleaved synaptosome-associated protein of 25 kDa (SNAP-25) in neuromuscular junctions: a new hypothesis to explain persistence of botulinum A poisoning

Botulinum neurotoxins can block neurotransmitter release for several months. The molecular mechanism of these toxins' action is known, but the persistence of neuromuscular paralysis that they cause is unexplained. At frog neuromuscular junctions, application of botulinum toxin type A caused paralysis and reduced the C-terminus immunoreactivity of SNAP-25, but not that of the remaining N-terminus fragment. Botulinum toxin type C caused paralysis and reduced syntaxin immunoreactivity without affecting that of SNAP-25. Co-application of botulinum A and C reduced syntaxin immunoreactivity, and that of both C and N termini of SNAP-25. Application of hydroxylamine to de-palmitoylate SNAP-25 resulted in a slight reduction of the immunoreactivity of SNAP-25 N terminus, while it had no effect on immunoreactivity of botulinum A cleaved SNAP-25. In contrast, application of hydroxylamine to nerve terminals where syntaxin had been cleaved by botulinum C caused a considerable reduction in SNAP-25 N-terminus immunoreactivity. Hence the retention of immunoreactive SNAP-25 at the neuromuscular junction depends on its interactions with syntaxin and plasma membrane. Persistence of cleaved SNAP-25 in nerve terminals may prevent insertion of new SNAP-25 molecules, thereby contributing to the longevity of botulinum A effects.     

Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.     

Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin

A targeted drug carrier (TDC) is described for transferring functional proteins or peptides into motor nerve terminals, a pivotal locus for therapeutics to treat neuromuscular disorders. It exploits the pronounced selectivity of botulinum neurotoxin type B (BoNT/B) for interacting with acceptors on these cholinergic nerve endings and becoming internalized. The gene encoding an innocuous BoNT/B protease-inactive mutant (BoTIM) was fused to that for core streptavidin, expressed in Escherichia coli and the purified protein was conjugated to surface-biotinylated liposomes. Such decorated liposomes, loaded with fluorescein as traceable cargo, acquired pronounced specificity for motor nerve terminals in isolated mouse hemidiaphragms and facilitated the intraneuronal transfer of the fluor, as revealed by confocal microscopy. Delivery of the protease light chain of botulinum neurotoxin type A (BoNT/A) via this TDC accelerated the onset of neuromuscular paralysis, indicative of improved translocation of this enzyme into the presynaptic cytosol with subsequent proteolytic inactivation of synaptosomal-associated protein of molecular mass 25 kDa (SNAP-25), an exocytotic soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) essential for neurotransmitter release. BoTIM-coupled liposomes, loaded with peptide inhibitors of proteases, yielded considerable attenuation of the neuroparalytic effects of BoNT/A or BoNT/F as a result of their cytosolic transfer, the first in situ demonstration of the ability of designer antiproteases to suppress the symptoms of botulism ex vivo. Delivery of the BoNT/A inhibitor by liposomes targeted with the full-length BoTIM proved more effective than that mediated by its C-terminal neuroacceptor-binding domain. This demonstrated versatility of TDC for nonviral cargo transfer into cholinergic nerve endings has unveiled its potential for direct delivery of functional targets into motor nerve endings.     

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.     

A late phase of exocytosis from synaptosomes induced by elevated [Ca2+]i is not blocked by Clostridial neurotoxins

Treatment of rat cerebrocortical synaptosomes with botulinum toxin types E and C1 or tetanus toxin removed the majority of intact SNAP-25, syntaxin 1A/1B, and synaptobrevin and diminished Ca(2+)-dependent K+ depolarization-induced noradrenaline secretion. With botulinum toxin type E, <10% of intact SNAP-25 remained and K(+)-evoked release of glutamate and GABA was inhibited. The large component of noradrenaline release evoked within 120 s by inclusion of the Ca2+ ionophore A23187 with the K+ stimulus was also attenuated by these toxins; additionally, botulinium neurotoxin type E blocked the first 60 s of ionophore-induced GABA and glutamate exocytosis. However, exposure to A23187 for longer periods induced a phase of secretion nonsusceptible to any of these toxins (>120 s for noradrenaline; >60 s for glutamate or GABA). Most of this late phase of release represented exocytosis because of its Ca2+ dependency, ATP requirement, and sensitivity to a phosphatidylinositol 4-kinase inhibitor. Based on these collective findings, we suggest that the ionophore-induced elevation of [Ca2+]i culminates in the disassembly of complexes containing nonproteolyzed SNAP receptors protected from the toxins that can then contribute to neuroexocytosis.     

Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor

The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A–G (BoNT/A–G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.     

Botulinum neurotoxins: from paralysis to recovery of functional neuromuscular transmission.

The neuromuscular junction is one of the most accessible mammalian synapses which offers a useful model to study long-term synaptic modifications occurring throughout life. It is also the natural target of botulinum neurotoxins (BoNTs) causing a selective blockade of the regulated exocytosis of acetylcholine thereby triggering a profound albeit transitory muscular paralysis. The scope of this review is to describe the principal steps implicated in botulinum toxin intoxication from the early events leading to a paralysis to the cellular response implementing an impressive synaptic remodelling culminating in the functional recovery of neuromuscular transmission. BoNT/A treatment promotes extensive sprouting emanating from intoxicated motor nerve terminals and the distal portion of motor axons. The current view is that sprouts have the ability to form functional synapses as they display a number of key proteins required for exocytosis: SNAP-25, VAMP/synaptobrevin, syntaxin-I, synaptotagmin-II, synaptophysin, and voltage-activated Na+, Ca2+ and Ca2+-activated K+ channels. Exo-endocytosis was demonstrated (using the styryl dye FM1-43) to occur only in the sprouts in vivo, at the time of functional recovery emphasising the direct role of nerve terminal outgrowth in implementing the restoration of functional neurotransmitter release (at a time when nerve stimulation again elicited muscle contraction). Interestingly, sprouts are only transitory since a second distinct phase of the rehabilitation process occurs with a return of synaptic activity to the original nerve terminals. This is accompanied by the elimination of the dispensable sprouts. The growth or elimination of these nerve processes appears to be strongly correlated with the level of synaptic activity at the parent terminal. The BoNT/A-induced extension and later removal of "functional" sprouts indicate their fundamental importance in the rehabilitation of paralysed endplates, a finding with ramifications for the vital process of nerve regeneration.