Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism

Harper, Callista B., Martin, Sally, Nguyen, Tam H., Daniels, Shari J., Lavidis, Nickolas A., Popoff, Michel R., Hadzic, Gordana, Mariana, Anna, Chau, Ngoc, McCluskey, Adam, Robinson, Phillip J. and Meunier, Frederic A. (2011) Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. Journal of Biological Chemistry, 286 41: 35966-35976. doi:10.1074/jbc.M111.283879

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Author Harper, Callista B.
Martin, Sally
Nguyen, Tam H.
Daniels, Shari J.
Lavidis, Nickolas A.
Popoff, Michel R.
Hadzic, Gordana
Mariana, Anna
Chau, Ngoc
McCluskey, Adam
Robinson, Phillip J.
Meunier, Frederic A.
Title Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
Publication date 2011-10-01
Year available 2011
Sub-type Article (original research)
DOI 10.1074/jbc.M111.283879
Open Access Status File (Publisher version)
Volume 286
Issue 41
Start page 35966
End page 35976
Total pages 11
Place of publication Bethesda, MD, United States
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Subject 1303 Biochemistry
1312 Molecular Biology
1307 Cell Biology
Abstract Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity.
Formatted abstract
The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies.Wefound that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4aTM, was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of>30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.
Keyword Botulinum neurotoxin
retrograde transport
synaptic vesicle
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID MC_U105178791
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Queensland Brain Institute Publications
Official 2012 Collection
School of Biomedical Sciences Publications
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Citation counts: TR Web of Science Citation Count  Cited 80 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 84 times in Scopus Article | Citations
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Created: Sat, 15 Oct 2011, 03:39:07 EST by Mr Tam Nguyen on behalf of Queensland Brain Institute