Uptake of botulinum neurotoxin into cultured neurons.
Botulinum neurotoxins (BoNTs) act within the synaptic terminal to block neurotransmitter release. The toxin enters the neuron by binding to neuronal membrane receptor(s), being taken up into an endosome-like compartment, and penetrating the endosome membrane via a pH-dependent translocation process. Once within the synaptic cytoplasm, BoNT serotypes A and E cleave separate sites on the C-terminus of the neuronal protein SNAP-25, one of the SNARE proteins required for synaptic vesicle fusion. In this study, we measured the effect of brief toxin exposure on SNAP-25 proteolysis in neuronal cell cultures as an indicator of toxin translocation. The results indicate that (1) uptake of both BoNT-A and -E is enhanced with synaptic activity induced by K+ depolarization in the presence of Ca2+ and (2) translocation of BoNT-A from the acidic endosomal compartment is slow relative to that of BoNT-E. Polyclonal antisera against each toxin protect cells when applied with the toxin during stimulation but has no effect when added immediately after toxin exposure, indicating that toxin endocytosis occurs with synaptic activity. Both serotypes cleave SNAP-25 at concentrations between 50 pM and 4 nM. IC50 values for SNAP-25 cleavage are approximately 0.5 nM for both serotypes. Inhibition of the pH-dependent translocation process by pretreating cultures with concanamycin A (Con A) prevents cleavage of SNAP-25 with IC50 values of approximately 25 nM. Addition of Con A at times up to 15 min after toxin exposure abrogated BoNT-A action; however, addition of Con A after 40 min was no longer protective. In contrast, Con A inhibited, but did not prevent, translocation of BoNT-E even when added immediately after toxin exposure, indicating that pH-dependent translocation of BoNT-E is rapid relative to that of BoNT-A. This study demonstrates that uptake of both BoNT-A and -E is enhanced with synaptic activity and that translocation of the toxin catalytic moiety into the cytosol occurs at different rates for these two serotypes.
Keller
James E
JE
Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. kellerj@cber.fda.gov
Cai
Fang
F
Neale
Elaine A
EA
D000818:Animals; D001904:Botulinum Antitoxin / Q000494:pharmacology; D001905:Botulinum Toxins / Q000378:metabolism* / Q000494:pharmacology; D019274:Botulinum Toxins, Type A / Q000378:metabolism* / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D005333:Fetus; D006868:Hydrolysis; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D021381:Protein Transport; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25
D001904:Botulinum Antitoxin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D003208:Concanavalin A; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D002118:Calcium; C096326:botulinum toxin type E