Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and under... more 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 (H C)-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 H C ; 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.
Introduction Membrane fusion in eukaryotic cells is essential for the transfer of lumenal content... more Introduction Membrane fusion in eukaryotic cells is essential for the transfer of lumenal contents between intracellular compartments and for excoytosis of secretory proteins, hormones and transmitters. Three membrane proteins, synaptobrevin (Sbr), synaptosome-associated protein of Mr 25,000 (SNAP-25) and syntaxin have been identified as key mediators of the exocytosis of synaptic vesicles and large dense-core granules. Each is known to be the substrate for at least one of the seven serotypes (A-G) of botulinum neurotoxin (BoNT) or tetanus toxin (TeTx), bacterial proteases that potently block neurotransmitter release (
Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and... more 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. Botulinum neurotoxin type A (BoNT/A) 1 is being employed successfully for the therapy of dystonias and dysphonias because it potently and selectively inhibits acetylcholine release
Seven types (A-G) of botulinum neurotoxin (BoNT) target peripheral cholinergic neurons where they... more Seven types (A-G) of botulinum neurotoxin (BoNT) target peripheral cholinergic neurons where they selectively proteolyze SNAP-25 (BoNT/A, BoNT/C1, and BoNT/ E), syntaxin1 (BoNT/C1), and synaptobrevin (BoNT/B, BoNT/D, BoNT/F, and BoNT/G), SNARE proteins responsible for transmitter release, to cause neuromuscular paralysis but of different durations. BoNT/A paralysis lasts longest (4-6 months) in humans, hence its widespread clinical use for the treatment of dystonias. Molecular mechanisms underlying these distinct inhibitory patterns were deciphered in rat cerebellar neurons by quantifying the half-life of the effect of each toxin, the speed of replenishment of their substrates, and the degradation of the cleaved products, experiments not readily feasible at motor nerve endings. Correlation of target cleavage with blockade of transmitter release yielded half-lives of inhibition for BoNT/A, BoNT/C1, BoNT/B, BoNT/F, and BoNT/E (> >31, > >25, ϳ10, ϳ2, and ϳ0.8 days, respectively), equivalent to the neuromuscular paralysis times found in mice, with recovery of release coinciding with reappearance of the intact SNAREs. A limiting factor for the short neuroparalytic durations of BoNT/F and BoNT/E is the replenishment of synaptobrevin or SNAP-25, whereas pulse labeling revealed that extended inhibition by BoNT/A, BoNT/B, or BoNT/C1 results from longevity of each protease. These novel findings could aid development of new toxin therapies for patients resistant to BoNT/A and effective treatments for human botulism.
Sympathetic neurons ramify to innervate multiple cells in target tissues. In compartmentalized cu... more Sympathetic neurons ramify to innervate multiple cells in target tissues. In compartmentalized cultures of rat superior cervical ganglion neurons, cleavage of synaptosomal-associated protein of M r = 25 000 (SNAP-25) in neurites exposed to botulinum neurotoxin type A (BoNT/A) arrested their growth and collapsed interstitial branches, but this required large, nonclinical doses. A protease-inactive mutant proved ineffective, confirming involvement of SNAP-25 in neurite extension and arborization. BoNT/C1 acted like BoNT/A, but BoNT/E caused only mild inhibition, likely due to transient SNAP-25 proteolysis. Near-total lack of susceptibility to BoNT/B or BoNT/D revealed that vesicle-associated membrane protein (VAMPs) isoforms 1-3 are not essential. Neurite length was not reduced when either BoNT/A or BoNT/C1 was applied to the somata, with no detrimental effect on neuron viability being observed. Treatments that protect cells from deprivation of nerve growth factor failed to prevent the toxin-induced loss of neurites. Inactivation of SNAP-25 caused the accumulation at neurite branch sites of Golgi-derived organelles labelled with N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sphingosine conjugated to bovine serum albumin, prior to the collapse of arbors. Notably, neurite growth was~1000-fold less susceptible to BoNT/A than cholinergic transmission in these neurons. Accordingly, a BoNT/A acceptor synaptic vesicle protein 2 (SV2) was found to be colocalized with VAMP 1-3, but not with VAMP 7, which is implicated in the growth of neurites. In conclusion, neurites depend on SNAP-25 for extension but this is quite resistant to BoNT/A, possibly, because of a low density of SV2 at growth sites that are distant from the highly susceptible regions of neurotransmitter release.
Numerous naturally-occurring toxins can perturb biological systems when they invade susceptible c... more Numerous naturally-occurring toxins can perturb biological systems when they invade susceptible cells. Coupling of pertinent targeting ligands to the active domains of such proteins provides a strategy for directing these to particular cellular populations implicated in disease. A novel approach described herein involved fusion of one mutated immunoglobulin G (IgG) binding moiety of staphylococcal protein A to the SNARE protease and translocation domain of botulinum neurotoxin A (BoNT/A). This chimera could be monovalently coupled to IgG or via its Fc region to recombinant targeting ligands. The utility of the resulting conjugates is demonstrated by the delivery of a SNARE protease into a cell line expressing tropomyosin receptor kinase A (TrkA) through coupling to anti-TrkA IgG or a fusion of Fc and nerve-growth factor. Thus, this is a versitile and innovative technology for conjugating toxins to diverse ligands for retargeted cell delivery of potential therapeutics.
Inhibition of regulated exocytosis by botulinum toxins type A and B was studied in chromaffin cel... more Inhibition of regulated exocytosis by botulinum toxins type A and B was studied in chromaffin cells. Both virtually abolished catecholamine release triggered from intact cells by depolarising stimuli, whereas the blockade by type A, but not B, was only partial after cell permeabilisation and direct stimulation of exocytosis by Ca 2'. Botulinum toxin A did not alter the [Ca 2' ]-dependency of exocytosis in permeabilised cells but, rather, proportionally reduced the amount of release at each concentration tested. Likewise, this toxin decreased the extents of Ca 2'-induced structural changes in SNAP-25, synaptobrevin and syntaxin (known collectively as SNAREs), whilst leaving their [Ca 2' ]-sensitivity unaltered. Thus, botulinum toxin A does not reduce the Ca 2'-sensitivity of the exocytosis sensor, but hinders transmission of the signal to the SNAREs which mediate fusion.
The utility of botulinum neurotoxin type A (BoNT/A) for treating overactive muscles and endocrine... more The utility of botulinum neurotoxin type A (BoNT/A) for treating overactive muscles and endocrine glands is attributable to a unique conflation of properties honed to exploit and inactivate synaptic transmission. Specific, high-affinity coincident binding to gangliosides plus an intraluminal loop of synaptic vesicle protein 2 (SV2) by the heavy chain (HC) of BoNT/A confers selectivity for presynaptic nerve terminals and subsequent uptake by endocytosis. Upon vesicle acidification, the HC forms a channel for transmembrane transfer of the light chain to the cytosol, as observed by single channel recordings. The light chain is a Zn(2+) -dependent endoprotease that cleaves and inactivates SNAP-25, thereby blocking exocytotic release of transmitters, a discovery that revealed the pivotal role of the latter in synaptic vesicle fusion. A di-leucine motif in BoNT/A light chain stabilizes this protease, contributing to its longevity inside nerves. The ubiquity of SV2 and SNAP-25 has prompted re-evaluation of the nerve types susceptible to BoNT/A. In urology, there is emerging evidence that BoNT/A blocks neuropeptide release from afferent nerves, exocytosis of acetylcholine and purines from efferent nerves, and possibly ATP release from the urothelium. Suppression by BoNT/A of the surface expression of nociceptor channels on bladder afferents might also contribute to its improvement of urological sensory symptoms.
International Journal of Molecular Sciences, Mar 4, 2021
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synapt... more Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCVreleasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.-Meng, J., Wang, J., Lawrence, G. W., Dolly, J. O. Molecular components required for resting and stimulated endo-cytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons. FASEB J. 27, 3167-3180 (2013). www.fasebj.org Key Words: amphiphysin ⅐ AP2 ⅐ clathrin ⅐ dynamin ⅐ large dense-core vesicles ⅐ small clear synaptic vesicles There is a great need to decipher the uptake processes in different types of neurons for the 7 serotypes of botulinum neurotoxin (BoNT) due to these being the most potent poison known, causing life-threatening botulism by intracellularly inhibiting transmitter release (reviewed in refs. 1, 2). Furthermore, clinical use of innocuous, tiny amounts of the toxin for treating neuronal disorders has kindled interest in the molecular machinery responsible for their trafficking. Type A BoNT (BoNT/A) has proved successful in alleviating the symptoms of Ͼ100 conditions arising from nerve overactivity, e.g., dystonias, spasticity, autonomic hypersecretory disorders, and overactive bladder (reviewed in ref. 3). Also, it can reduce the frequency of chronic migraine and chronic daily headaches (4), although not all types of chronic pain are responsive (5). BoNTs consist of a proteolytic light chain linked via a disulfide and noncovalent bonds to a heavy chain (HC). These neurotoxic proteins bind to intravesicular regions of synaptic vesicle protein 2 (SV2; BoNT/A, /D, /E, and /F) or synaptotagmin (BoNT/B and /G) that become exposed on exocytosis and, thereby, enter neurons by exploiting the recycling of small clear synaptic vesicles (SCSVs; refs. 6-15). Hence, BoNT uptake is accelerated by nerve stimulation (16-18). In comparison, little is known regarding the mode of BoNT entry into nerves that mainly secrete neuropeptides from large dense-core vesicles (LDCVs), such as sensory fibers. Study of the toxins' entry into different neuron types is warranted due to the prospect of yielding insights into differences and commonalities
Botulinum neurotoxin (BoNT) types A and B are ZnZ '-requiring endoproteases which potently block ... more Botulinum neurotoxin (BoNT) types A and B are ZnZ '-requiring endoproteases which potently block neurotransmitter release by cleavage of a 25-kDa synaptosomal-associated protein (SNAP-25) and synaptobrevin, respectively. Synaptobrevin is important for the exocytosis of catecholamines from dense-core granules and evidence is presented here for the involvement of SNAP-25 in this process in neuroendocrine cells. The effects of BoNT/A and BoNT/B on regulated secretion were compared in intact bovine chromaffin cells to investigate the consequences of cleavage of the different targets. Catecholamine secretion elicited by BaZ+, by elevated K+ concentrations or by nicotine was prevented by each toxin. A very good correlation was observed between the extents of SNAP-25 cleavage or synaptobrevin cleavage and inhibition of secretion by BoNT/A or BoNT/B, respectively, which indicates the importance of SNAP-25 and synaptobrevin in regulated exocytosis. Despite truncation of almost the entire SNAP-25 pool by exposure of the cells to BoNT/A, a residual fraction of secretion persisted that was induced by 20 pM CaZ+ (and to a lesser extent by 1 mM BaZ+) following permeabilisation. Addition of more BoNT/A failed to reduce this level of secretion. Inclusion of Mg. ATP, which greatly enhanced secretion from permeabilised cells, was required for Ca2+-stimulated or Ba*+-stirnulated BoNT/A-resistant secretion. Furthermore, synaptobrevin is essential for this response because the response was not observed in BoNT/B treated cells. In view of the ability of BoNTE to abolish secretion from permeabilised cells and to delete 26 amino acids from the C-terminus of SNAP-25, it can be deduced that cleavage of only nine residues by BoNT/A does not prevent the resultant truncated form exhibiting attenuated activity under the conditions created by permeabilisation. This identification of a novel component of secretion from permeabilised cells should facilitate investigation of the functional interaction of SNAP-25 with other proteins involved in regulated exocytosis.
Objective: To compare the need for single-event multilevel surgery (SEMLS) in children with cereb... more Objective: To compare the need for single-event multilevel surgery (SEMLS) in children with cerebral palsy (CP) with and without botulinum neurotoxin type A (BoNT-A) injections in a comprehensive rehabilitation program. Methods: We conducted a clinical observation of 149 children (78 boys and 71 girls age 2-14 years) with spastic forms of CP (Gross Motor Function Classification System Levels II-IV [GMFCS II-IV]). The study group included 108 children who had received BoNT-A injections in a comprehensive rehabilitation program. The control group consisted of 41 children who had similar comprehensive treatment (standard therapy) without BoNT-A injections. BoNT-A (Dуsport ® [abobotulinumtoxinA]) was administered intramuscularly. The total dose varied from 225-750 U, 14-30 U/kg (mean dose 28±4 U/kg). Maximum permissible dose was calculated based on body weight and dose distribution, depending on the target muscles. The interval between abobotulinumtoxinA injections ranged from 180-200 days in 70% of the children. The comprehensive rehabilitation program included massage therapy, exercise therapy, dynamic proprioceptive correction (orthopedic costumes "Adeli" and "Graviton"), correct positioning, orthosis, electrical stimulation of weakened muscles, reflexotherapy, hydrotherapy (underwater massage treatment, whirlpool baths for arms and legs), and serial casting. Results: Children in the CP group had received their first surgical correction by the age of 10.5±2.8 years; of these, 5 (4.6%) required repeated operations by the age of 14 years. Children in the control group received their first operation at the age of 6.7±2.1 years; of these, 39 (95%) required repeated operations during the active growth period (10-14 years of age). Conclusions: Our study demonstrated that in children with CP, long-term treatment with BoNT-A (Dуsport ®), compared with standard treatment without BoNT-A, allowed us to postpone surgical corrections and resulted in a 20-fold decrease in repeated single-event orthopedic operation rates.
In search of a basis for the impressive potency of an endoprotease that cleaves SNAP-25, botulinu... more In search of a basis for the impressive potency of an endoprotease that cleaves SNAP-25, botulinum neurotoxin type A (BoNT/A), in treating numerous diseases due to hyper-active autonomic nerves, truncation of its target and inhibition of neurotransmission were studied in rat sympathetic neurons. Tetrodotoxin-sensitive spontaneous cholinergic neurotransmission was blocked >80% by 1 pM BoNT/A despite cleaving <20% of the SNAP-25. A maximum cleavage of ∼60% SNAP-25 could be achieved with >1 nM BoNT/A, despite an absence of non-cleavable SNAP-25 in the detergent-solubilised neurons. In contrast, BoNT/E (100 nM) truncated nearly all the SNAP-25 in the intact cells, but was unable to block neurotransmission at low concentrations like BoNT/A. Chimeras created by inserting the acceptor-binding HC domain of BoNT/A into BoNT/E still cleaved all the SNAP-25, indicating ubiquitous expression of BoNT/A acceptors. Accordingly, SV2 and SNAP-25 were found to be co-expressed and broadly co-localised in neurons, but absent from non-neuronal cells. On the other hand, partial cleavage by the BoNT/A protease persisted upon replacing its HC with counterparts from BoNT/E or BoNT/B. Moreover, limited cleavage of SNAP-25 was conferred onto the protease from BoNT/E when fused to the N-terminus of BoNT/A. Thus, the BoNT/A protease is uniquely well-adapted for selectively inactivating the SNAP-25 directly involved in neurotransmission; this together with the toxin's acceptor and its target being localised on the peri-somatic boutons likely contribute to its exceptional therapeutic utility in the clinic.
Botulinum neurotoxin type B blocks transmitter release via a selective endoproteolysis of the sma... more Botulinum neurotoxin type B blocks transmitter release via a selective endoproteolysis of the small clear vesicle membrane protein synaptobrevin that is essential for neuro-exocytosis. In view of the distinct characteristics of exocytosis of adrenochromaffin granules and considering the controversy over the presence of synaptobrevin on the latter, this study aimed to determine the molecular basis of the inhibition by this toxin of secretion from chromaffin cells. Thus, affinity-purified antibodies against a synaptobrevin synthetic peptide were used to quantify its concentrations in subcellular fractions of bovine adrenal medulla. The latter, as well as density gradient centrifugation and size-exclusion chromatography, showed that > 70% of the protein copurifies with the granules and their marker, dopamine beta-hydroxylase. Notably, much lower concentrations of synaptobrevin and synaptophysin were found in chromaffin granules than in synaptic small clear vesicles (approximately 9% and approximately 2%, respectively); however, isolated granule membranes exhibited greater enrichments (approximately 35% and approximately 9%). A second immunoreactive protein was colocalized with synaptobrevin on chromaffin granules; in view of its susceptibility to the toxin and lower M(r), it is assumed to be cellubrevin and, also, because of its high homology. Involvement of synaptobrevin and cellubrevin in Ca(2+)-triggered granule exocytosis was established by the demonstrated correlation between the extent of botulinum neurotoxin B-induced inhibition of secretion and their selective proteolysis following introduction of the toxin into intact chromaffin cells. On the basis of these collective findings, it is concluded that these proteins occur on chromaffin granules and one or both are essential for exocytosis.
Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and under... more 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 (H C)-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 H C ; 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.
Introduction Membrane fusion in eukaryotic cells is essential for the transfer of lumenal content... more Introduction Membrane fusion in eukaryotic cells is essential for the transfer of lumenal contents between intracellular compartments and for excoytosis of secretory proteins, hormones and transmitters. Three membrane proteins, synaptobrevin (Sbr), synaptosome-associated protein of Mr 25,000 (SNAP-25) and syntaxin have been identified as key mediators of the exocytosis of synaptic vesicles and large dense-core granules. Each is known to be the substrate for at least one of the seven serotypes (A-G) of botulinum neurotoxin (BoNT) or tetanus toxin (TeTx), bacterial proteases that potently block neurotransmitter release (
Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and... more 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. Botulinum neurotoxin type A (BoNT/A) 1 is being employed successfully for the therapy of dystonias and dysphonias because it potently and selectively inhibits acetylcholine release
Seven types (A-G) of botulinum neurotoxin (BoNT) target peripheral cholinergic neurons where they... more Seven types (A-G) of botulinum neurotoxin (BoNT) target peripheral cholinergic neurons where they selectively proteolyze SNAP-25 (BoNT/A, BoNT/C1, and BoNT/ E), syntaxin1 (BoNT/C1), and synaptobrevin (BoNT/B, BoNT/D, BoNT/F, and BoNT/G), SNARE proteins responsible for transmitter release, to cause neuromuscular paralysis but of different durations. BoNT/A paralysis lasts longest (4-6 months) in humans, hence its widespread clinical use for the treatment of dystonias. Molecular mechanisms underlying these distinct inhibitory patterns were deciphered in rat cerebellar neurons by quantifying the half-life of the effect of each toxin, the speed of replenishment of their substrates, and the degradation of the cleaved products, experiments not readily feasible at motor nerve endings. Correlation of target cleavage with blockade of transmitter release yielded half-lives of inhibition for BoNT/A, BoNT/C1, BoNT/B, BoNT/F, and BoNT/E (> >31, > >25, ϳ10, ϳ2, and ϳ0.8 days, respectively), equivalent to the neuromuscular paralysis times found in mice, with recovery of release coinciding with reappearance of the intact SNAREs. A limiting factor for the short neuroparalytic durations of BoNT/F and BoNT/E is the replenishment of synaptobrevin or SNAP-25, whereas pulse labeling revealed that extended inhibition by BoNT/A, BoNT/B, or BoNT/C1 results from longevity of each protease. These novel findings could aid development of new toxin therapies for patients resistant to BoNT/A and effective treatments for human botulism.
Sympathetic neurons ramify to innervate multiple cells in target tissues. In compartmentalized cu... more Sympathetic neurons ramify to innervate multiple cells in target tissues. In compartmentalized cultures of rat superior cervical ganglion neurons, cleavage of synaptosomal-associated protein of M r = 25 000 (SNAP-25) in neurites exposed to botulinum neurotoxin type A (BoNT/A) arrested their growth and collapsed interstitial branches, but this required large, nonclinical doses. A protease-inactive mutant proved ineffective, confirming involvement of SNAP-25 in neurite extension and arborization. BoNT/C1 acted like BoNT/A, but BoNT/E caused only mild inhibition, likely due to transient SNAP-25 proteolysis. Near-total lack of susceptibility to BoNT/B or BoNT/D revealed that vesicle-associated membrane protein (VAMPs) isoforms 1-3 are not essential. Neurite length was not reduced when either BoNT/A or BoNT/C1 was applied to the somata, with no detrimental effect on neuron viability being observed. Treatments that protect cells from deprivation of nerve growth factor failed to prevent the toxin-induced loss of neurites. Inactivation of SNAP-25 caused the accumulation at neurite branch sites of Golgi-derived organelles labelled with N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sphingosine conjugated to bovine serum albumin, prior to the collapse of arbors. Notably, neurite growth was~1000-fold less susceptible to BoNT/A than cholinergic transmission in these neurons. Accordingly, a BoNT/A acceptor synaptic vesicle protein 2 (SV2) was found to be colocalized with VAMP 1-3, but not with VAMP 7, which is implicated in the growth of neurites. In conclusion, neurites depend on SNAP-25 for extension but this is quite resistant to BoNT/A, possibly, because of a low density of SV2 at growth sites that are distant from the highly susceptible regions of neurotransmitter release.
Numerous naturally-occurring toxins can perturb biological systems when they invade susceptible c... more Numerous naturally-occurring toxins can perturb biological systems when they invade susceptible cells. Coupling of pertinent targeting ligands to the active domains of such proteins provides a strategy for directing these to particular cellular populations implicated in disease. A novel approach described herein involved fusion of one mutated immunoglobulin G (IgG) binding moiety of staphylococcal protein A to the SNARE protease and translocation domain of botulinum neurotoxin A (BoNT/A). This chimera could be monovalently coupled to IgG or via its Fc region to recombinant targeting ligands. The utility of the resulting conjugates is demonstrated by the delivery of a SNARE protease into a cell line expressing tropomyosin receptor kinase A (TrkA) through coupling to anti-TrkA IgG or a fusion of Fc and nerve-growth factor. Thus, this is a versitile and innovative technology for conjugating toxins to diverse ligands for retargeted cell delivery of potential therapeutics.
Inhibition of regulated exocytosis by botulinum toxins type A and B was studied in chromaffin cel... more Inhibition of regulated exocytosis by botulinum toxins type A and B was studied in chromaffin cells. Both virtually abolished catecholamine release triggered from intact cells by depolarising stimuli, whereas the blockade by type A, but not B, was only partial after cell permeabilisation and direct stimulation of exocytosis by Ca 2'. Botulinum toxin A did not alter the [Ca 2' ]-dependency of exocytosis in permeabilised cells but, rather, proportionally reduced the amount of release at each concentration tested. Likewise, this toxin decreased the extents of Ca 2'-induced structural changes in SNAP-25, synaptobrevin and syntaxin (known collectively as SNAREs), whilst leaving their [Ca 2' ]-sensitivity unaltered. Thus, botulinum toxin A does not reduce the Ca 2'-sensitivity of the exocytosis sensor, but hinders transmission of the signal to the SNAREs which mediate fusion.
The utility of botulinum neurotoxin type A (BoNT/A) for treating overactive muscles and endocrine... more The utility of botulinum neurotoxin type A (BoNT/A) for treating overactive muscles and endocrine glands is attributable to a unique conflation of properties honed to exploit and inactivate synaptic transmission. Specific, high-affinity coincident binding to gangliosides plus an intraluminal loop of synaptic vesicle protein 2 (SV2) by the heavy chain (HC) of BoNT/A confers selectivity for presynaptic nerve terminals and subsequent uptake by endocytosis. Upon vesicle acidification, the HC forms a channel for transmembrane transfer of the light chain to the cytosol, as observed by single channel recordings. The light chain is a Zn(2+) -dependent endoprotease that cleaves and inactivates SNAP-25, thereby blocking exocytotic release of transmitters, a discovery that revealed the pivotal role of the latter in synaptic vesicle fusion. A di-leucine motif in BoNT/A light chain stabilizes this protease, contributing to its longevity inside nerves. The ubiquity of SV2 and SNAP-25 has prompted re-evaluation of the nerve types susceptible to BoNT/A. In urology, there is emerging evidence that BoNT/A blocks neuropeptide release from afferent nerves, exocytosis of acetylcholine and purines from efferent nerves, and possibly ATP release from the urothelium. Suppression by BoNT/A of the surface expression of nociceptor channels on bladder afferents might also contribute to its improvement of urological sensory symptoms.
International Journal of Molecular Sciences, Mar 4, 2021
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synapt... more Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCVreleasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.-Meng, J., Wang, J., Lawrence, G. W., Dolly, J. O. Molecular components required for resting and stimulated endo-cytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons. FASEB J. 27, 3167-3180 (2013). www.fasebj.org Key Words: amphiphysin ⅐ AP2 ⅐ clathrin ⅐ dynamin ⅐ large dense-core vesicles ⅐ small clear synaptic vesicles There is a great need to decipher the uptake processes in different types of neurons for the 7 serotypes of botulinum neurotoxin (BoNT) due to these being the most potent poison known, causing life-threatening botulism by intracellularly inhibiting transmitter release (reviewed in refs. 1, 2). Furthermore, clinical use of innocuous, tiny amounts of the toxin for treating neuronal disorders has kindled interest in the molecular machinery responsible for their trafficking. Type A BoNT (BoNT/A) has proved successful in alleviating the symptoms of Ͼ100 conditions arising from nerve overactivity, e.g., dystonias, spasticity, autonomic hypersecretory disorders, and overactive bladder (reviewed in ref. 3). Also, it can reduce the frequency of chronic migraine and chronic daily headaches (4), although not all types of chronic pain are responsive (5). BoNTs consist of a proteolytic light chain linked via a disulfide and noncovalent bonds to a heavy chain (HC). These neurotoxic proteins bind to intravesicular regions of synaptic vesicle protein 2 (SV2; BoNT/A, /D, /E, and /F) or synaptotagmin (BoNT/B and /G) that become exposed on exocytosis and, thereby, enter neurons by exploiting the recycling of small clear synaptic vesicles (SCSVs; refs. 6-15). Hence, BoNT uptake is accelerated by nerve stimulation (16-18). In comparison, little is known regarding the mode of BoNT entry into nerves that mainly secrete neuropeptides from large dense-core vesicles (LDCVs), such as sensory fibers. Study of the toxins' entry into different neuron types is warranted due to the prospect of yielding insights into differences and commonalities
Botulinum neurotoxin (BoNT) types A and B are ZnZ '-requiring endoproteases which potently block ... more Botulinum neurotoxin (BoNT) types A and B are ZnZ '-requiring endoproteases which potently block neurotransmitter release by cleavage of a 25-kDa synaptosomal-associated protein (SNAP-25) and synaptobrevin, respectively. Synaptobrevin is important for the exocytosis of catecholamines from dense-core granules and evidence is presented here for the involvement of SNAP-25 in this process in neuroendocrine cells. The effects of BoNT/A and BoNT/B on regulated secretion were compared in intact bovine chromaffin cells to investigate the consequences of cleavage of the different targets. Catecholamine secretion elicited by BaZ+, by elevated K+ concentrations or by nicotine was prevented by each toxin. A very good correlation was observed between the extents of SNAP-25 cleavage or synaptobrevin cleavage and inhibition of secretion by BoNT/A or BoNT/B, respectively, which indicates the importance of SNAP-25 and synaptobrevin in regulated exocytosis. Despite truncation of almost the entire SNAP-25 pool by exposure of the cells to BoNT/A, a residual fraction of secretion persisted that was induced by 20 pM CaZ+ (and to a lesser extent by 1 mM BaZ+) following permeabilisation. Addition of more BoNT/A failed to reduce this level of secretion. Inclusion of Mg. ATP, which greatly enhanced secretion from permeabilised cells, was required for Ca2+-stimulated or Ba*+-stirnulated BoNT/A-resistant secretion. Furthermore, synaptobrevin is essential for this response because the response was not observed in BoNT/B treated cells. In view of the ability of BoNTE to abolish secretion from permeabilised cells and to delete 26 amino acids from the C-terminus of SNAP-25, it can be deduced that cleavage of only nine residues by BoNT/A does not prevent the resultant truncated form exhibiting attenuated activity under the conditions created by permeabilisation. This identification of a novel component of secretion from permeabilised cells should facilitate investigation of the functional interaction of SNAP-25 with other proteins involved in regulated exocytosis.
Objective: To compare the need for single-event multilevel surgery (SEMLS) in children with cereb... more Objective: To compare the need for single-event multilevel surgery (SEMLS) in children with cerebral palsy (CP) with and without botulinum neurotoxin type A (BoNT-A) injections in a comprehensive rehabilitation program. Methods: We conducted a clinical observation of 149 children (78 boys and 71 girls age 2-14 years) with spastic forms of CP (Gross Motor Function Classification System Levels II-IV [GMFCS II-IV]). The study group included 108 children who had received BoNT-A injections in a comprehensive rehabilitation program. The control group consisted of 41 children who had similar comprehensive treatment (standard therapy) without BoNT-A injections. BoNT-A (Dуsport ® [abobotulinumtoxinA]) was administered intramuscularly. The total dose varied from 225-750 U, 14-30 U/kg (mean dose 28±4 U/kg). Maximum permissible dose was calculated based on body weight and dose distribution, depending on the target muscles. The interval between abobotulinumtoxinA injections ranged from 180-200 days in 70% of the children. The comprehensive rehabilitation program included massage therapy, exercise therapy, dynamic proprioceptive correction (orthopedic costumes "Adeli" and "Graviton"), correct positioning, orthosis, electrical stimulation of weakened muscles, reflexotherapy, hydrotherapy (underwater massage treatment, whirlpool baths for arms and legs), and serial casting. Results: Children in the CP group had received their first surgical correction by the age of 10.5±2.8 years; of these, 5 (4.6%) required repeated operations by the age of 14 years. Children in the control group received their first operation at the age of 6.7±2.1 years; of these, 39 (95%) required repeated operations during the active growth period (10-14 years of age). Conclusions: Our study demonstrated that in children with CP, long-term treatment with BoNT-A (Dуsport ®), compared with standard treatment without BoNT-A, allowed us to postpone surgical corrections and resulted in a 20-fold decrease in repeated single-event orthopedic operation rates.
In search of a basis for the impressive potency of an endoprotease that cleaves SNAP-25, botulinu... more In search of a basis for the impressive potency of an endoprotease that cleaves SNAP-25, botulinum neurotoxin type A (BoNT/A), in treating numerous diseases due to hyper-active autonomic nerves, truncation of its target and inhibition of neurotransmission were studied in rat sympathetic neurons. Tetrodotoxin-sensitive spontaneous cholinergic neurotransmission was blocked >80% by 1 pM BoNT/A despite cleaving <20% of the SNAP-25. A maximum cleavage of ∼60% SNAP-25 could be achieved with >1 nM BoNT/A, despite an absence of non-cleavable SNAP-25 in the detergent-solubilised neurons. In contrast, BoNT/E (100 nM) truncated nearly all the SNAP-25 in the intact cells, but was unable to block neurotransmission at low concentrations like BoNT/A. Chimeras created by inserting the acceptor-binding HC domain of BoNT/A into BoNT/E still cleaved all the SNAP-25, indicating ubiquitous expression of BoNT/A acceptors. Accordingly, SV2 and SNAP-25 were found to be co-expressed and broadly co-localised in neurons, but absent from non-neuronal cells. On the other hand, partial cleavage by the BoNT/A protease persisted upon replacing its HC with counterparts from BoNT/E or BoNT/B. Moreover, limited cleavage of SNAP-25 was conferred onto the protease from BoNT/E when fused to the N-terminus of BoNT/A. Thus, the BoNT/A protease is uniquely well-adapted for selectively inactivating the SNAP-25 directly involved in neurotransmission; this together with the toxin's acceptor and its target being localised on the peri-somatic boutons likely contribute to its exceptional therapeutic utility in the clinic.
Botulinum neurotoxin type B blocks transmitter release via a selective endoproteolysis of the sma... more Botulinum neurotoxin type B blocks transmitter release via a selective endoproteolysis of the small clear vesicle membrane protein synaptobrevin that is essential for neuro-exocytosis. In view of the distinct characteristics of exocytosis of adrenochromaffin granules and considering the controversy over the presence of synaptobrevin on the latter, this study aimed to determine the molecular basis of the inhibition by this toxin of secretion from chromaffin cells. Thus, affinity-purified antibodies against a synaptobrevin synthetic peptide were used to quantify its concentrations in subcellular fractions of bovine adrenal medulla. The latter, as well as density gradient centrifugation and size-exclusion chromatography, showed that > 70% of the protein copurifies with the granules and their marker, dopamine beta-hydroxylase. Notably, much lower concentrations of synaptobrevin and synaptophysin were found in chromaffin granules than in synaptic small clear vesicles (approximately 9% and approximately 2%, respectively); however, isolated granule membranes exhibited greater enrichments (approximately 35% and approximately 9%). A second immunoreactive protein was colocalized with synaptobrevin on chromaffin granules; in view of its susceptibility to the toxin and lower M(r), it is assumed to be cellubrevin and, also, because of its high homology. Involvement of synaptobrevin and cellubrevin in Ca(2+)-triggered granule exocytosis was established by the demonstrated correlation between the extent of botulinum neurotoxin B-induced inhibition of secretion and their selective proteolysis following introduction of the toxin into intact chromaffin cells. On the basis of these collective findings, it is concluded that these proteins occur on chromaffin granules and one or both are essential for exocytosis.
Uploads
Papers by Gary Lawrence