title: Control of autophagosome axonal retrograde flux by presynaptic activity unveiled using botulinum neurotoxin type a.
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.
authors: Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute.
authors: Tong
authors: T
authors: Wang
authors: Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute.
authors: Sally
authors: http://orcid.org/0000-0001-9294-5404
authors: S
authors: Martin
authors: Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute.
authors: Andreas
authors: A
authors: Papadopulos
authors: Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute.
authors: Callista B
authors: CB
authors: Harper
authors: University of Wisconsin Medical School, Madison, Wisconsin 53706.
authors: Timur A
authors: TA
authors: Mavlyutov
authors: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
authors: Dhevahi
authors: D
authors: Niranjan
authors: Australian Institute for Bioengineering and Nanotechnology, and.
authors: Nick R
authors: NR
authors: Glass
authors: Australian Institute for Bioengineering and Nanotechnology, and School of Chemical Engineering, University of Queensland, Brisbane, Queensland 4072, Australia, Materials Science and Engineering Division, Commonwealth Scientific and Industrial Research Organization, Clayton, Victoria 3169, Australia.
authors: Justin J
authors: JJ
authors: Cooper-White
authors: University of Bordeaux, Interdisciplinary Institute for Neuroscience, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5297, Bordeaux, France.
authors: Jean-Baptiste
authors: JB
authors: Sibarita
authors: University of Bordeaux, Interdisciplinary Institute for Neuroscience, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5297, Bordeaux, France, Bordeaux Imaging Center, Unité Mixte de Service 3420, Centre National de la Recherche Scientifique, US4 INSERM, University of Bordeaux, France, and.
authors: Daniel
authors: D
authors: Choquet
authors: Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, United Kingdom.
authors: Bazbek
authors: B
authors: Davletov
authors: Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, f.meunier@uq.edu.au.
authors: Frédéric A
authors: FA
authors: Meunier
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D001370:Axonal Transport / Q000187:drug effects / Q000502:physiology; D019274:Botulinum Toxins, Type A / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006624:Hippocampus / Q000166:cytology*; D066298:In Vitro Techniques; D018942:Macrolides / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D009498:Neurotoxins / Q000378:metabolism* / Q000494:pharmacology; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D004791:Enzyme Inhibitors; D018942:Macrolides; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D009498:Neurotoxins; D017475:Receptors, Nerve Growth Factor; D050825:Synaptosomal-Associated Protein 25; C517634:Ngfr protein, mouse; C040929:bafilomycin A1; D019274:Botulinum Toxins, Type A; D000077191:Wortmannin
keywords: Botulinum neurotoxin; autophagosome; axon; presynaptic; retrograde transport; synaptic vesicle