Figure 19

Potassium-evoked glycine release is inhibited by 40 percent in cultures exposed to BoNT A under the conditions of this experiment. Sister cultures maintained in the drug fumonisin B1 are ganglioside-depleted and are almost totally protected from the effects of BoNT A on glycine release. C; control; B; BoNT A; F; fumonisin; F+B; fumonisin + BoNT A.

In both cases, the addition of exogenous gangliosides to ganglioside-depleted neurons restores toxin action. In this regard, GQ1b and GT1b are approximately equivalent while GD1b is less effective in restoring the action of BoNT A. High concentrations of gangliosides enhance toxin effect even beyond that in control cultures. These findings indicate that neuronal membrane gangliosides are a critical component of the receptor required for delivery of BoNT A to the neuronal cytosol.

Figure 20

Sequential additions of BoNT A and E to demonstrate persistent action of BoNT A. Cultures were exposed to 0.4 pM BoNT A for 72 hours. Of the three bands labeled SNAP-25, the highest kDa represents intact SNAP-25; the next lower, BoNT A-cleaved SNAP-25 (missing nine amino acids from the C-terminus); and the lowest, BoNT E-cleaved SNAP25 (missing 26 amino acids from the C-terminus). Lane 1 shows that about half of SNAP-25 is cleaved 25 days after BoNT A exposure when cultures were exposed to 250 pM BoNT E for 24 hours. Both intact and BoNT A-truncated SNAP-25 were converted into the BoNT E-altered form (lane 2). During the ensuing 18 days, increases were observed in intact and BoNT A-truncated SNAP-25 while the BoNT E-truncated SNAP-25 gradually disappeared (lanes 3 through 6). (From Keller et al. FEBS Lett 1999;456:137)

Both A and E serotypes of BoNT cleave the neuronal protein SNAP-25, although BoNT A is more potent and its effects are of longer duration, suggesting differential trafficking of the toxins. We found the long duration of BoNT A blockade to be attributable to the persistence of toxin activity in contrast to a more rapid cessation of BoNT E action. We assessed the effect of endosomal pH on the translocation of BoNTs A and E to the cytosol. Titration of cultures with the antibiotic bafilomycin A1, which blocks acidification of endosomes, shows that BoNT A is more dependent on pH than is BoNT E. This characteristic may be a primary factor underlying distinct trafficking of these two toxins within neurons. Furthermore, whereas the ensuing blockade of neurotransmitter release is consistent with the amount of BoNT E-damaged SNAP-25, block of release is almost twice as great as that expected from the levels of BoNT A-damaged SNAP-25. We have obtained evidence that the BoNT A cleavage product of SNAP-25 acts as a competitive inhibitor of intact SNAP-25 and blocks synaptic vesicle fusion in intact neurons at physiological concentrations of calcium.

Synaptic function depends on a supply of synaptic vesicles, maintained in the nerve terminal by recycling of vesicle membrane through tightly coupled exo- and endocytosis.

Figure 21

BoNT A effects on neurotransmitter release and SNAP-25 cleavage. Potassium-evoked glycine release was measured with 0.15 (squares), 0.25 (triangles), or 2 mM (circles) Ca2+. Level of SNAP-25 cleavage as determined by densitometric analysis of Western blots is also plotted (diamonds). (From Keller and Neale, J Biol Chem 2001;276:13476)

We had shown that blockade of neurotransmitter release by TeNT or BoNT A is correlated with increased numbers of synaptic vesicles jammed up at the presynaptic membrane, consistent with toxin damage to proteins required for vesicle fusion and neurotransmitter release. However, in cultures treated with concentrations of BoNT A sufficient to block vesicle exocytosis, K+ stimulation in the presence of Ca2+ induced synaptic vesicle endocytosis, suggesting that BoNT A can “uncouple” components of the recycling mechanism. Current studies using fluorescence and electron microscopy are aimed at defining the relationship, in individual synaptic terminals, between synaptic vesicle exo- and endocytosis, particularly as modified by BoNT A.

Figure 22

Endocytosis of horseradish peroxidase by K+ depolarization. a. Control. Recycled synaptic vesicles contain peroxidase reaction product. b.. TeNT. Only occasional synaptic vesicles contain reaction product, indicating the failure of synaptic vesicles to undergo recycling. c. BoNT A. A large number of synaptic vesicles contain reaction product, evidence that the vesicles were formed from surface membrane. Insets. Reaction product labeling of clathrin-coated vesicles implicates these structures in the process of vesicle recycling. (From Neale et al. J Cell Biol 1999;147:1249)