Tetanus And Botulinum Neurotoxins And Neuronal Cell Biol

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Clostridial neurotoxins (tetanus and seven serotypes of botulinum neurotoxin) each block vesicular neurotransmitter release by cleaving specific proteins implicated in synaptic vesicle fusion with the presynaptic membrane. These toxins are important therapeutic agents for a number of neurologic disorders including cerebral palsy and are valuable tools for understanding neurotransmitter release, membrane trafficking, and protein sorting, transport, and targeting. We study the presynaptic action of the toxins using biochemical and morphologic techniques in primary cell cultures of fetal mouse spinal cord. We have found that the drug fumonisin B1, which blocks ganglioside synthesis and depletes membranes of gangliosides, protects neurons against the action of botulinum neurotoxin A (BoNT A). Addition of exogenous gangliosides to ganglioside-depleted neurons restores toxin action. In this regard, GQ1b and GT1b are approximately equivalent and GD1b is less effective. 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. Both A and E serotypes of BoNT cleave the neuronal protein SNAP-25, although BoNT A remains catalytically active within the synaptic terminal for a much longer time, suggesting differential trafficking of these toxins. We assessed the effect of endosomal pH on the translocation of BoNT's A and E to the cytosol. Titrating cultures with the antibiotic bafilomycin A1, which blocks acidification of endosomes, shows a greater dependence of BoNT A on pH than of 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. This finding is consistent with the greater potency of BoNT A relative to BoNT E. Current studies using fluorescence and electron microscopy are aimed at defining the relationship between synaptic vesicle exo- and endocytosis particularly as modified by BoNT A.