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Botulinum Neurotoxin Type A is Internalized and Translocated from Small Synaptic Vesicles at the Neuromuscular Junction

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Abstract

Botulinum neurotoxin type A (BoNT/A) is the most frequent cause of human botulism and, at the same time, is largely used in human therapy. Some evidence indicates that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been directly proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscope, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor synaptic vesicle protein 2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle trans-membrane pH gradient, we show that the neurotoxin translocation into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by no more than two neurotoxin molecules.

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Acknowledgments

The work carried out in the authors' laboratories is supported by grants from the Ministero dell'Università e della Ricerca (Progetto PRIN) and from the University of Padova to OR, from the Progetto Strategico "An in Vivo Approach to the Physiopathology of Signal Transduction" of the University of Padova and from Fondazione CARIPARO "Synaptic Functions and Role of Glial Cells in Brain and Muscle Diseases" to CM, and by a EU VIIth Frame Program grant 241835 to JM.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Laboratoire de Neurobiologie et Développement, Institut de Neurobiologie Alfred Fessard, CNRS-UPR 3294, 91198, Gif-sur-Yvette Cedex, France

    Cesare Colasante & Jordi Molgó

  2. Dipartimento di Scienze Biomediche and Istituto CNR di Neuroscienze, Università di Padova, Viale G. Colombo 3, Padova, 35131, Italy

    Ornella Rossetto, Laura Morbiato, Marco Pirazzini & Cesare Montecucco

Authors
  1. Cesare Colasante
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  2. Ornella Rossetto
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  3. Laura Morbiato
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  4. Marco Pirazzini
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  5. Jordi Molgó
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  6. Cesare Montecucco
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Correspondence to Jordi Molgó or Cesare Montecucco.

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Colasante, C., Rossetto, O., Morbiato, L. et al. Botulinum Neurotoxin Type A is Internalized and Translocated from Small Synaptic Vesicles at the Neuromuscular Junction. Mol Neurobiol 48, 120–127 (2013). https://doi.org/10.1007/s12035-013-8423-9

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  • DOI: https://doi.org/10.1007/s12035-013-8423-9

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