Dual effects of botulinum neurotoxin A on the secretory stages of chromaffin cells

The changes that SNAREs undergo during exocytosis were studied in permeabilised chromaffin cells treated with Ca2+, MgATP or botulinum neurotoxin A. High-resolution 2D SDS-PAGE revealed multiple SDS-resistant SNARE complexes having a wide range of sizes and in which SNAP-25 and syntaxin predominate over synaptobrevin. Their formation increased upon Ca2+-stimulated exocytosis; notably, the 2D protocol proved much superior to 1D SDS-PAGE for the detection of large complexes and revealed that for forms with relative molecular mass greater than 100,000 stimulated induction was more significant than for smaller species. MgATP enhanced Ca2+-triggered catecholamine release but reduced the content of complexes. By contrast, botulinum neurotoxin type A inhibited exocytosis and altered the stoichiometry of the SNAP-25:syntaxin binary association, without lowering its abundance. The individual SNAREs were protected against trypsin proteolysis to varying extents in binary and ternary complexes of different sizes, suggestive of distinct folding intermediates. Our data suggest that Ca2+ triggers an early stage of SNARE complex formation causing an accumulation of partially folded intermediates, especially of binary forms, as well as their maturation into smaller, more protease resistant states. In addition, botulinum neurotoxin A inhibits exocytosis by perturbing the syntaxin:SNAP-25 ratio in binary intermediates.

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Identification of novel inhibitors of botulinum neurotoxin A

Planta Medica ◽

10.1055/s-0035-1556290 ◽

2015 ◽

Vol 81 (11) ◽

Author(s):

C Yalamanchili ◽

VK Manda ◽

AG Chittiboyina ◽

WA Harrell Jr ◽

RP Webb ◽

...

Keyword(s):

Botulinum Neurotoxin ◽

Botulinum Neurotoxin A

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Botulinumtoxin zur Behandlung von neuropathischen Schmerzen

Nervenheilkunde ◽

10.1055/s-0038-1627027 ◽

2017 ◽

Vol 36 (05) ◽

pp. 315-323

Author(s):

N. Üçeyler ◽

C. Sommer

Keyword(s):

Botulinum Neurotoxin ◽

Neuropathische Schmerzen ◽

Botulinum Neurotoxin A ◽

Topische Therapien

ZusammenfassungDie Behandlung neuropathischer Schmerzen mit systemisch wirksamen oral verabreichten Pharmaka ist bei vielen Patienten wirksam, kann jedoch zu zentralnervösen unerwünschten Wirkungen wie Müdigkeit oder Schwindel führen. Daher sind in den letzten Jahren topische Therapien in das Zentrum der Aufmerksamkeit gerückt. Botulinumtoxin, etabliert in der Therapie von Dystonien und Spastik, wurde zunehmend bei Schmerzerkrankungen getestet, hierbei ist Botulinum-Neurotoxin A der am besten untersuchte Serotyp. Die häufigsten Indikationen waren Schmerzen im Trigeminusversorgungsbereich und periphere neuropathische Schmerzen. Bei den meisten Studien war Botulinum-Neurotoxin A Placebo deutlich überlegen. Präklinische Studien zum Wirkmechanismus erbrachten die Erkenntnis, dass neben dem erwarteten peripheren Effekt sehr wahrscheinlich auch eine zentrale Reduktion der Ausschüttung von exzitatorischen Neurotransmittern an der Wirkung beteiligt ist.

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Komplexproteinfreies Botulinum Neurotoxin A - Blepharospasmus und Torticollis

psychoneuro ◽

10.1055/s-2007-991580 ◽

2007 ◽

Vol 33 (9) ◽

pp. 376-376

Keyword(s):

Botulinum Neurotoxin ◽

Botulinum Neurotoxin A

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Primary Cultures of Embryonic Chicken Neurons for Sensitive Cell-Based Assay of Botulinum Neurotoxin: Implications for Therapeutic Discovery

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057106299163 ◽

2007 ◽

Vol 12 (3) ◽

pp. 370-377 ◽

Cited By ~ 29

Author(s):

Andrea M. Stahl ◽

Gordon Ruthel ◽

Edna Torres-Melendez ◽

Tara A. Kenny ◽

Rekha G. Panchal ◽

...

Keyword(s):

Nervous System ◽

Botulinum Toxin ◽

Neutralizing Antibodies ◽

Botulinum Neurotoxin ◽

Potent Inhibitor ◽

Primary Cultures ◽

Embryonic Chick ◽

Sensitive Cell ◽

Botulinum Neurotoxin A ◽

Therapeutic Compounds

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. ( Journal of Biomolecular Screening 2007:370-377)

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Intratracheal inoculation of AHc vaccine induces protection against aerosolized botulinum neurotoxin A challenge in mice

npj Vaccines ◽

10.1038/s41541-021-00349-w ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Changjiao Gan ◽

Wenbo Luo ◽

Yunzhou Yu ◽

Zhouguang Jiao ◽

Sha Li ◽

...

Keyword(s):

Bronchoalveolar Lavage ◽

Clostridium Botulinum ◽

Freeze Drying ◽

Botulinum Neurotoxin ◽

Secretory Iga ◽

Immune Protection ◽

Dry Powder ◽

Botulinum Neurotoxin A ◽

Spray Freeze Drying

AbstractBotulinum neurotoxin (BoNT), produced by Clostridium botulinum, is generally known to be the most poisonous of all biological toxins. In this study, we evaluate the protection conferred by intratracheal (i.t.) inoculation immunization with recombinant Hc subunit (AHc) vaccines against aerosolized BoNT/A intoxication. Three AHc vaccine formulations, i.e., conventional liquid, dry powder produced by spray freeze drying, and AHc dry powder reconstituted in water are prepared, and mice are immunized via i.t. inoculation or subcutaneous (s.c.) injection. Compared with s.c.-AHc-immunized mice, i.t.-AHc-immunized mice exhibit a slightly stronger protection against a challenge with 30,000× LD50 aerosolized BoNT/A. Of note, only i.t.-AHc induces a significantly higher level of toxin-neutralizing mucosal secretory IgA (SIgA) production in the bronchoalveolar lavage of mice. In conclusion, our study demonstrates that the immune protection conferred by the three formulations of AHc is comparable, while i.t. immunization of AHc is superior to s.c. immunization against aerosolized BoNT/A intoxication.

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