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.
Highlights
Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that acts at the level of presynaptic motor nerve terminals, binding both gangliosides and a protein acceptor in order to be endocytosed[1,2,3,4]
We first tested whether BoNT/A was internalized into endocytic synaptic vesicles distinct from those that undergo local recycling by characterizing the trafficking of BoNT/A in cultured hippocampal neurons that were incubated with recombinant BoNT/A-Hc1 labeled with horseradish peroxidase (HRP) at 37 °C
Given that fewer than half of the synaptic vesicles in the nerve terminal contained BoNT/A-Hc, we examined whether this population differed from canonical synaptic vesicles that undergo regulated exocytosis
Summary
Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that acts at the level of presynaptic motor nerve terminals, binding both gangliosides and a protein acceptor in order to be endocytosed[1,2,3,4]. Given that BoNT/A is mainly internalized in synaptic vesicles, it is possible that at least some of the BoNT/A-containing vesicles might not recycle but rather comprise a specific pool of synaptic vesicles with a retrograde trafficking fate. Whether all synaptic vesicles have similar protein content has recently been challenged[15] This raises the possibility that functional heterogeneity may exist within synaptic vesicle pools. As a portion of BoNT/A follows a retrograde route, we hypothesized that it could be internalized into a selective pool of synaptic vesicles whose fate is not to recycle but to undergo retrograde trafficking. Our results demonstrate that BoNT/A-Hc-containing synaptic vesicles are non-recyclable, suggestive of a distinct functional role. They could represent precursors whose fate is to generate retrograde carriers such as autophagosomes
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