Cleavage of intracellular substrates of botulinum toxins A, C, and D in a mammalian target tissue.

The objective of the current study was to determine whether the intracellular targets that mediate the mechanism of action of botulinum toxin at the mammalian neuromuscular junction are the same as those identified in nontarget tissues. Previous studies of this nature have been limited to nontarget tissues because of the perceived low abundance of neural proteins in a neuromuscular preparation. In the current study we have used differential centrifugation to concentrate neural proteins in a synaptosomal-enriched fraction from the mouse phrenic nerve-hemidiaphragm preparation. Immunoblot detection revealed the presence of discrete immunoreactive bands corresponding to SNAP-25, synaptobrevin II, and syntaxin I in the innervated region of the neuromuscular preparation. The ability of these proteins to serve as botulinum toxin substrates in neuromuscular tissue was determined by measuring toxin-induced proteolysis. Exposure of the intact hemidiaphragm preparation to botulinum serotypes A, C, and D (10(-8) M, 5-6-h exposure) resulted in significant reductions in SNAP-25 (67%), syntaxin I (56%), and synaptobrevin II (72%) immunoreactivity, respectively. The toxin-induced proteolysis was specific for each serotype examined. Collectively, these findings provide direct confirmation that botulinum toxin targets integral components of the molecular machinery mediating neurotransmitter release at the neuromuscular junction. To the best of our knowledge this is the first time that studies of this nature on the intracellular action of botulinum toxin have been extended to a recognized mammalian target tissue preparation.