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 intracellul ar targets that mediate the mechanism of action of botulinum toxin at the m ammalian neuromuscular junction are the same as those identified in nontarg et tissues. Previous studies of this nature have been limited to nontarget tissues because of the perceived low abundance of neural proteins in a neur omuscular preparation. In the current study we have used differential centr ifugation to concentrate neural proteins in a synaptosomal-enriched fractio n from the mouse phrenic nerve-hemidiaphragm preparation. Immunoblot detect ion 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 botuli num toxin substrates in neuromuscular tissue was determined by measuring to xin-induced proteolysis. Exposure of the intact hemidiaphragm preparation t o botulinum serotypes A, C, and D (10(-8) M, 5-6-h exposure) resulted in si gnificant 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 d irect 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 ex tended to a recognized mammalian target tissue preparation.