STUDY OF CNS NEUROPEPTIDES WITH PEPTIDE-TOXIN COMPLEXES

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The goal of this project is to utilize hybrid molecules between neuropeptides and fragments of diptheria toxin or lectins as neurotoxins or pharmacological antagonists to investigate neuropeptide receptor-containing cells in the central nervous system (CNS) with emphasis on the basal ganglia. Thyrotropin releasing hormone (TRH), substance P and somatostatin will be studied. The rationale for the use of these complexes is to target an otherwise nontoxic fragment of diptheria toxin or a lectin to specific neuropeptide receptor-bearing cells by complexing a toxin to a neuropeptide. This project seeks to determine the conditions under which such hybrids are toxic to CNS neurons in the basal ganglia bearing specific neuropeptide receptors in vivo and in vitro. Using an antibody directed against the diptheria toxin fragment, this project will also attempt to visualize neuropeptide receptors on neurons by immunohistochemistry. Previous work demonstrated receptor-specific toxicity of the TRH-diptheria toxin hybrid in vitro against rat pituitary tumor cells. This project will establish conditions for in vivo toxicity of the TRH-toxin hybrid in spinal cord and pituitary, regions where TRH fibers and receptors are present, by examining morphological changes and receptor visualization after systemic or intrathecal delivery of the complex. Having established the feasibility of this methodology for CNS neurons, the neuropeptides substance P and somatostatin, complexed to toxins, will be evaluated for receptor binding and toxicity in vitro in primary cultures of mesencephalic, striatal or cortical neurons. In vivo analysis of substance P and somatostatin-toxin complexes will be performed in the nigrostriatal system by local intracerebral injections and subsequent changes in nigrostriatal neurotransmitter metabolism and behaviors dependent on the integrity of the nigrostriatal system. This tool may provide a means to a better understanding of the processes responsible for the apparent selective vulnerability of some CNS neurons to premature death in degenerative diseases of the nervous system, especially in the basal ganglia (e.g.: Parkinson's, Huntington's, Alzheimer's), by permitting (1) functional and pharmacological analysis of neuropeptide neurons, (2) characterization of distribution of neuropeptide receptors in postmortem tissue, (3) analysis of the factors which regulate the degenerative response to peptide-toxin hybrids, and (4) models of selective neuron loss.