Clostridial neurotoxins (tetanus and seven serotypes of botulinum neurotoxin) each block vesicular neurotransmitter release by cleaving specific proteins implicated in synaptic vesicle fusion. 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 have studied the presynaptic action of the toxins using biochemical and morphololgic techniques in primary cell cultures of fetal mouse spinal cord.We have found, using the drug fumonisin B1 to block ganglioside synthesis, that neuronal membrane gangliosides are required for tetanus toxin (TeNT) internalization and are a critical component of the TeNT receptor. Studies of synaptic vesicle membrane recycling demonstrate that TeNT and botulinum neurotoxin A (BoNT A) both block vesicle exocytosis, that vesicle endocytosis can be driven in BoNT A- but not in TeNT- blocked terminals, and that the membrane endocytosis is calcium-dependent. These findings suggest that a reservoir of synaptic vesicle membrane resides on the plasma membrane of the nerve terminal and that BoNT A does not interfere with vesicle membrane retrieval. Finally, both A and E serotypes of BoNT act on the same protein substrate SNAP-25, but neurons in cell culture as in vivo regain function more slowly after exposure to BoNT A than to E. We have shown that the prolonged duration of synaptic blockade after BoNT A is due to persistence of its catalytic activity rather than a failure of SNAP-25 turnover. - tetanus, botulinum, synaptic vesicle recycling, exocytosis, endocytosis, gangliosides, receptor, spinal cord cell cultures
