CELL BIOLOGY OF NEUROPEPTIDE BIOSYNTHESIS AND SECRETION

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The peptidergic neuronal phenotype is distinct from conventional neurons in that the production and secretion of neuropeptides requires continual transcription, translation, packaging in the golgi, and axonal transport of the large dense core (secretory) vesicles (LDCVs) to nerve terminals before neurosecretion can occur. In the past year, we have primarily focused on the first step in the process, i.e., the cell-specific regulation of neuropeptide gene expression. We have specifically studied these issues in the magnocellular oxytocin (OT) and vasopressin (VP) neurons of the hypothalamo-neurohypophysial system (HNS), since these neuroendocrine (peptidergic) cells are particularly valuable models for such analyses. Our goals are twofold: The first is to elucidate the mechanisms that are involved in the cell-specific gene expression of OT and VP. The second is to use this information to target the gene expression of specific molecules to these neurons in vivo, in order to analyze their impact on neurosecretory processes. Given the absence of available homologous cell lines, we have used transgenic mice to evaluate the sites in the OT and VP genes that may be involved in the regulation of their cell-specific expression in the magnocellular neurons. Previous work has led us to propose the intergenic region( IGR.) hypothesis, which states that the 3.6-kb IGR between the OT and VP genes (located on Chromosome 2 in the mouse) contains the critical enhancer sites for cell-specific expression. Various OT and VP mouse gene constructs have been studied in transgenic mice and recent data in support of this hypothesis will be described. In addition, given the impracticality of using transgenic mice as an assay system for a systematic evaluation of further deletion constructs, we have also recently developed an in vitro model (hypothalamic organotypic cultures) in which the magnocellular OT and VP neurons can be successfully transfected with reporter containing constructs by using viral vectors and particle- mediated gene transfer (biolistics). This approach has provided a general alternative to transgenic mice for promoter/enhancer identification studies in CNS neurons. With respect to the determination of the possible transfactors and signal- transduction mechanisms that regulate OT and/or VP gene expression in magnocellular neurons, we have developed and employed subtractive and differential analyses of single-cell cDNA libraries (e.g., from OT versus VP cells), as well as analysis of cDNA libraries from microdissected supraoptic nuclei (SONs) obtained from hypo- versus hypernatremic rats (the latter differ in OT and VP gene expression by more than tenfold). Preliminary observations have revealed many specific molecular differences. These are currently being evaluated by in situ hybridization histochemistry for OT and VP cell relevance and specificity. Our future plans are: 1) to continue to test the IGR hypothesis, with a focus on the cis-regulatory elements and transcriptional activating factors that may be responsible for OT and VP cell-specific expression and 2) to target the fluorescent reporter, EGFP, to the LDCVs in HNS neurons, in order to study calcium-dependent secretion from dendrites and nerve terminals in these neurons.