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Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles.

ABSTRACT: Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.

SUBMITTER: Harper CB

PROVIDER: S-EPMC4726273 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles.

Harper Callista B CB Papadopulos Andreas A Martin Sally S Matthews Daniel R DR Morgan Garry P GP Nguyen Tam H TH Wang Tong T Nair Deepak D Choquet Daniel D Meunier Frederic A FA

Scientific reports 20160125

Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, ...[more]

PMID: 26805017

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Project description:Experimental studies have demonstrated that botulinum neurotoxin serotype A (BoNT/A) causes flaccid paralysis by a multi-step mechanism. Following its binding to specific receptors at peripheral cholinergic nerve endings, BoNT/A is internalized by receptor-mediated endocytosis. Subsequently its zinc-dependent catalytic domain translocates into the neuroplasm where it cleaves a vesicle-docking protein, SNAP-25, to block neurally evoked cholinergic neurotransmission. We tested the hypothesis that mathematical models having a minimal number of reactions and reactants can simulate published data concerning the onset of paralysis of skeletal muscles induced by BoNT/A at the isolated rat neuromuscular junction (NMJ) and in other systems. Experimental data from several laboratories were simulated with two different models that were represented by sets of coupled, first-order differential equations. In this study, the 3-step sequential model developed by Simpson (J Pharmacol Exp Ther 212:16-21,1980) was used to estimate upper limits of the times during which anti-toxins and other impermeable inhibitors of BoNT/A can exert an effect. The experimentally determined binding reaction rate was verified to be consistent with published estimates for the rate constants for BoNT/A binding to and dissociating from its receptors. Because this 3-step model was not designed to reproduce temporal changes in paralysis with different toxin concentrations, a new BoNT/A species and rate (k(S)) were added at the beginning of the reaction sequence to create a 4-step scheme. This unbound initial species is transformed at a rate determined by k(S) to a free species that is capable of binding. By systematically adjusting the values of k(S), the 4-step model simulated the rapid decline in NMJ function (k(S) >or= 0.01), the less rapid onset of paralysis in mice following i.m. injections (k (S) = 0.001), and the slow onset of the therapeutic effects of BoNT/A (k(S) < 0.001) in man. This minimal modeling approach was not only verified by simulating experimental results, it helped to quantitatively define the time available for an inhibitor to have some effect (t(inhib)) and the relation between this time and the rate of paralysis onset. The 4-step model predicted that as the rate of paralysis becomes slower, the estimated upper limits of (t(inhib)) for impermeable inhibitors become longer. More generally, this modeling approach may be useful in studying the kinetics of other toxins or viruses that invade host cells by similar mechanisms, e.g., receptor-mediated endocytosis.

Dataset Information

Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles.

Publications

Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles.

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