This proposal sets forth the design and creation of two highly specific diphtheria-based neurotoxins paralleling previous work with IL-2. Each fusion toxin will consist of an abbreviated diphtheria toxin bound to the neuropeptides substance P or neurotensin. Thus when the extended peptide bind to its receptor, the diphtheria toxin will be incorporated into the neuron and destroy it. The ability to lesion specific neurons confers the means of directly observing which neurons bind the peptide while also observing the functional consequences in vivo of eliminating these neurons. Initial steps in the production of the toxin have entailed modifying a plasmid encoding an abbreviated form of the diphtheria toxin. Oligonucleotides coding for the substance P precursor peptide, substance P-glycine have been inserted. The enlarged plasmid was transformed into E. coli bacteria and these bacteria were induced to express the protein which will be reacted with an enzyme to cleave the glycine from the end of substance P and thereby make it recognizable to cells bearing substance P receptors. The neurotensin toxin will be produced similarly. The toxins will be extensively characterized in vitro and in vivo, with the ultimate goal of using them experimentally to ascertain neural function. The ability to selectively lesion neurons expressing receptors for these peptides should lead to better understanding of the function of these peptides as neurotransmitters, and may help to elucidate their role in mental disorders. The substance P fusion toxin may also have clinical applications in the treatment of chronic pain and inflammation.
