The toxins responsible for the clinical manifestations of botulism and tetanus are neurotoxins produced by strains of the bacterial genus Clostridium. These toxins block exocytosis of synaptic vesicles and thus inhibit neuronal signalling. Recent work has established that all clostridial neurotoxins function as metalloproteases. The toxins are composed of a heavy chain that targets and delivers them to neurons and a light chain that carries out their proteolytic activity. Individual toxins from each of several strains of Clostridium botulinum selectively proteolyze one of three synaptic proteins: synaptobrevin (VAMP), SNAP-25, or syntaxin. Synaptobrevin is a resident of synaptic vesicles, while SNAP- 25 and syntaxin are found on the synaptic plasma membrane. Several independent lines of evidence, including biochemical and genetic experiments, have recently converged to strongly implicate these three proteins as the core of a multiprotein complex which mediates exocytotic membrane fusion. Furthermore, the regulated assembly and disassembly of these proteins, in concert with cytoplasmic factors, are probably underlying vesicle docking and vesicle fusion. In this application, we plan to determine the molecular and physiological consequences of toxin poisoning in the nerve terminal with the goal of understanding how individual neurotoxins affect the protein-protein interactions which underlie neuronal exocytosis. This information should contribute to our understanding of the neuronal exocytotic, fission machine. We plan to use three approaches including in vitro studies with recombinant proteins, biochemical analysis of toxin-treated nerve terminals and experiments with cultured neurons. First, we will study the effects of toxin cleavage on protein-protein interactions between the components of the putative exocytotic fusion machine. These interactions include assembly and dissociation of the toxin substrates synaptobrevin, SNAP-25, and syntaxin, as well as their binding to regulatory proteins such as synaptophysin. The experiments will be carried out using purified proteins in solution as well as proteins reconstituted in proteoliposomes in order to imitate their native environment. Second, the interactions between the toxin substrate proteins will be studied in material isolated from nerve terminals which have been poisoned by the various toxins. Subfractionation of nerve terminals into defined membrane pools will allow us to determine if there is a specific point in the cycling of synaptic vesicles which is blocked by toxin action. Finally, we will use cultured neurons as a model system to expand on the information obtained in the in vitro experiments. These experiments will examine the effects of exogenous substrate proteins, protein fragments and toxin-resistant mutant proteins on synaptic transmission.We expect these studies to clarify how individual neurotoxins perturb neuronal exocytosis.
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