title: Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism.
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. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a(TM), 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.
authors.affiliation: Queensland Brain Institute, the University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Callista B
authors.initials: CB
authors.lastname: Harper
authors.affiliation: Queensland Brain Institute, the University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Sally
authors.initials: S
authors.lastname: Martin
authors.affiliation: Queensland Brain Institute, the University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Tam H
authors.initials: TH
authors.lastname: Nguyen
authors.affiliation: Queensland Brain Institute, the University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Shari J
authors.initials: SJ
authors.lastname: Daniels
authors.affiliation: School of Biomedical Sciences, the University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Nickolas A
authors.initials: NA
authors.lastname: Lavidis
authors.affiliation: Unité des Bactéries anaérobies et Toxines, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris cedex, France.
authors.forename: Michel R
authors.initials: MR
authors.lastname: Popoff
authors.affiliation: Centre for Chemical Biology, Chemistry Building, the University of Newcastle, Callaghan, New South Wales 2308, Australia.
authors.forename: Gordana
authors.initials: G
authors.lastname: Hadzic
authors.affiliation: Children's Medical Research Institute, the University of Sydney, Sydney, New South Wales 2145, Australia.
authors.forename: Anna
authors.initials: A
authors.lastname: Mariana
authors.affiliation: Children's Medical Research Institute, the University of Sydney, Sydney, New South Wales 2145, Australia.
authors.forename: Ngoc
authors.initials: N
authors.lastname: Chau
authors.affiliation: Centre for Chemical Biology, Chemistry Building, the University of Newcastle, Callaghan, New South Wales 2308, Australia.
authors.forename: Adam
authors.initials: A
authors.lastname: McCluskey
authors.affiliation: Children's Medical Research Institute, the University of Sydney, Sydney, New South Wales 2145, Australia.
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
authors.affiliation: Queensland Brain Institute, the University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: f.meunier@uq.edu.au.
authors.forename: Frederic A
authors.initials: FA
authors.lastname: Meunier
mesh_terms: D000818:Animals; D019274:Botulinum Toxins, Type A / Q000494:pharmacology*; D001906:Botulism / Q000378:metabolism / Q000517:prevention & control*; D002478:Cells, Cultured; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D034281:Dynamins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects*; D006624:Hippocampus / Q000378:metabolism*; D006835:Hydrazones / Q000494:pharmacology; D051379:Mice; D009284:Naphthols / Q000494:pharmacology; D009474:Neurons; D009498:Neurotoxins / Q000494:pharmacology*; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism
chemical_list: D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D009284:Naphthols; D009498:Neurotoxins; C568360:dyngo-4a; D019274:Botulinum Toxins, Type A; D034281:Dynamins