Retention of cleaved synaptosome-associated protein of 25 kDa (SNAP-25) in neuromuscular junctions: a new hypothesis to explain persistence of botulinum A poisoning.

Botulinum neurotoxins can block neurotransmitter release for several months. The molecular mechanism of these toxins' action is known, but the persistence of neuromuscular paralysis that they cause is unexplained. At frog neuromuscular junctions, application of botulinum toxin type A caused paralysis and reduced the C-terminus immunoreactivity of SNAP-25, but not that of the remaining N-terminus fragment. Botulinum toxin type C caused paralysis and reduced syntaxin immunoreactivity without affecting that of SNAP-25. Co-application of botulinum A and C reduced syntaxin immunoreactivity, and that of both C and N termini of SNAP-25. Application of hydroxylamine to de-palmitoylate SNAP-25 resulted in a slight reduction of the immunoreactivity of SNAP-25 N terminus, while it had no effect on immunoreactivity of botulinum A cleaved SNAP-25. In contrast, application of hydroxylamine to nerve terminals where syntaxin had been cleaved by botulinum C caused a considerable reduction in SNAP-25 N-terminus immunoreactivity. Hence the retention of immunoreactive SNAP-25 at the neuromuscular junction depends on its interactions with syntaxin and plasma membrane. Persistence of cleaved SNAP-25 in nerve terminals may prevent insertion of new SNAP-25 molecules, thereby contributing to the longevity of botulinum A effects.

Different VAMP/synaptobrevin complexes for spontaneous and evoked transmitter release at the crayfish neuromuscular junction.

Different VAMP/synaptobrevin complexes for spontaneous and evoked transmitter release at the crayfish neuromuscular junction. J. Neurophysiol. 80: 3233-3246, 1998. Although vesicle-associated membrane protein (VAMP/synaptobrevin) is essential for evoked neurotransmitter release, its role in spontaneous transmitter release remains uncertain. For instance, many studies show that tetanus toxin (TeNT), which cleaves VAMP, blocks evoked transmitter release but leaves some spontaneous transmitter release. We used recombinant tetanus and botulinum neurotoxin catalytic light chains (TeNT-LC, BoNT/B-LC, and BoNT/D-LC) to examine the role of VAMP in spontaneous transmitter release at neuromuscular junctions (nmj) of crayfish. Injection of TeNT-LC into presynaptic axons removed most of the VAMP immunoreactivity and blocked evoked transmitter release without affecting nerve action potentials or Ca2+ influx. The frequency of spontaneous transmitter release was little affected by the TeNT-LC when the evoked transmitter release had been blocked by >95%. The spontaneous transmitter release left after TeNT-LC treatment was insensitive to increases in intracellular Ca2+. BoNT/B-LC, which cleaves VAMP at the same site as TeNT-LC but uses a different binding site, also blocked evoked release but had minimal effect on spontaneous release. However, BoNT/D-LC, which cleaves VAMP at a different site from the other two toxins but binds to the same position on VAMP as TeNT, blocked both evoked and spontaneous transmitter release at similar rates. The data indicate that different VAMP complexes are employed for evoked and spontaneous transmitter release; the VAMP used in spontaneous release is not readily cleaved by TeNT or BoNT/B. Because the exocytosis that occurs after the action of TeNT cannot be increased by increased intracellular Ca2+, the final steps in neurotransmitter release are Ca2+ independent.

Presynaptic protein interactions in vivo: evidence from botulinum A, C, D and E action at frog neuromuscular junction.

The present study examines the paralytic action of botulinum neurotoxins at their natural target, the neuromuscular junction. We asked whether syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP/synaptobrevin), the proteins proteolysed by botulinum, are susceptible to cleavage in frog nerve terminals, and whether they form complexes in vivo. In control terminals, the three SNAREs were distributed in broad bands at 1 micrometer intervals, at sites consistent with presynaptic Ca2+ channels. Within 3 h, botulinum A, C, D and E (BoNT/A/C/D/E) blocked nerve-evoked muscle contractions but their effects on substrate immunoreactivity varied. The effect of BoNT/A on either C-terminus or N-terminus immunoreactivity of SNAP-25 was undetectable after 3-h incubation, although C-terminus immunoreactivity was reduced after 24 h; N-terminus immunoreactivity was not affected even after 36 h. BoNT/E reduced C-terminus immunoreactivity of SNAP-25 1.5 h after toxin application when transmitter release was blocked, but required 24 h to reduce N-terminus immunoreactivity. BoNT/C reduced syntaxin immunoreactivity after 24-h incubation but did not affect SNAP-25. BoNT/D reduced VAMP immunoreactivity at 3 h while it increased SNAP-25 C-terminal staining fourfold. BoNT/A and BoNT/C applied together for 24 h reduced syntaxin immunoreactivity and that of both C- and N-terminus of SNAP-25, indicating that retention of SNAP-25 N-terminus after cleavage by BoNT/A depended on intact syntaxin. Therefore, we infer that SNAP-25 interacts with VAMP and with syntaxin in vivo. Neurotoxin action abolished only 40-60% of SNAP-25, VAMP or syntaxin immunoreactivity suggesting that distinct pools of these proteins, not immediately involved in triggered exocytosis, are resistant to proteolysis.