Clostridial neurotoxins are zinc endopeptidases that block neurotransmission and have been shown to cleave, in vitro, specific proteins involved in synaptic vesicle docking and/or fusion. We have used immunohistochemistry and immunoblotting to demonstrate alterations in toxin substrates in intact neurons under conditions of toxin-induced blockade of neurotransmitter release. Vesicle-associated membrane protein, which colocalizes with synaptophysin, is not detectable in tetanus toxin-blocked cultures. Syntaxin, also concentrated in synaptic sites, is cleaved by botulinum neurotoxin C. Similarly, the carboxyl terminus of the synaptosomal-associated protein of 25 kDa (SNAP-25) is not detectable in botulinum neurotoxin A-treated cultures. Unexpectedly, tetanus toxin exposure causes an increase in SNAP-25 immunofluorescence, reflecting increased accessibility of antibodies to antigenic sites rather than increased expression of the protein. Furthermore, botulinum neurotoxin C causes a marked loss of the carboxyl terminus of SNAP-25 when the toxin is added to living cultures, whereas it has no action on SNAP-25 in vitro preparations. This study is the first to demonstrate in functioning neurons that the physiologic response to these toxins is correlated with the proteolysis of their respective substrates. Furthermore, the data demonstrate that botulinum neurotoxin C, in addition to cleaving syntaxin, exerts a secondary effect on SNAP-25.

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K+-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K+ depolarization, in the presence of Ca2+, triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A–blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca2+ is required for synaptic vesicle membrane retrieval.

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