Botulinum Neurotoxin Type A is Internalized and Translocated from Small Synaptic Vesicles at the Neuromuscular Junction

Abstract

Botulinum neurotoxin type A (BoNT/A) is the most frequent cause of human botulism and, at the same time, is largely used in human therapy. Some evidence indicates that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been directly proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscope, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor synaptic vesicle protein 2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle trans-membrane pH gradient, we show that the neurotoxin translocation into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by no more than two neurotoxin molecules.

On the translocation of botulinum and tetanus neurotoxins across the membrane of acidic intracellular compartments

“…Such a binding is preliminary to the following step of internalization inside the nerve. TeNT and BoNT/A1 exploit the endocytosis of SV to penetrate into neurons [19][20][21]. Information on the nature of the endocytic vesicle involved in the uptake of the other neurotoxins is still lacking.…”

“…a protein form with native secondary structure and increased hydrophobicity that renders it capable of inserting and moving across membranes [48,[61][62][63][64]. In this view, the initial, low conductance, transmembrane currents [20,21]. In other words…”

Botulinum toxin A, brain and pain

“…BoNT/A internalization into neurons may be mediated by all isoforms of SV2 (SV2 A-C), with the strongest affinity for SV2C (Dong et al, 2006). After binding to protein acceptors on neuronal membrane, BoNT/A undergoes dynamin-dependent endocytosis into the acidic compartment of small synaptic vesicles (Colasante et al, 2013;Harper et al, 2011). The process of BoNT/A endocytosis is augmented by neuronal activity, which promotes synaptic vesicle recycling (Harper et al, 2011).…”

Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

“…The recent finding that auranofin, a TrxR inhibitor, prevented the action of tetanus neurotoxin in cultured neurons prompted us to investigate the presence of the TrxR-Trx system within nerve terminals and on synaptic vesicles, which are the Trojan horses used by BoNT/A to deliver its L chain in the cytosol (Colasante et al, 2013;Harper et al, 2011). Figure 1A shows that the neuromuscular junction, which is the major site of action of BoNTs, highly expresses both TrxR and Trx, as do primary cultures of neurons (Figure S1A).…”

“…The seven BoNT serotypes exhibit exclusive specificities with respect to the different SNARE proteins and therefore can be used as simple tools to determine the effect of knocking out specific SNAREs in cell physiology (Pantano and Montecucco, 2014). To penetrate into neurons, BoNTs exploit the endocytosis of synaptic vesicles (SVs) (Colasante et al, 2013), and the acidification of the SV lumen induces the H-mediated membrane translocation of L (Fischer and Montal, 2013;Montal, 2010). It has been demonstrated that, once on the cytosolic side, the L metalloprotease remains attached to H via the interchain SS bridge and the reduction of this bond releases the L metalloprotease activity, unblocking at the same time the ion channel formed by H in the membrane (Fischer and Montal, 2007).…”