CELL BIOLOGY OF NEUROPEPTIDE BIOSYNTHESIS AND SECRETION

点击翻译按钮获取中文摘要

Over the past twenty years, neuropeptides have become increasingly prominent as intercellular messengers in the peripheral and central nervous system, acting as neurohormones, neuromodulators, neurotrophic factors, and/or neurotransmitters. 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. We have specifically studied these issues in the magnocellular oxytocin (OT) and vasopressin (VP) neurons of the hypothalamo-neurohypophysial system (HNS. Our goals are:(1)to elucidate the mechanisms that are involved in the cell-specific gene expression of OT and VP, and (2) to target the gene expression of specific molecules to these neurons in vivo, in order to analyze their impact on neurosecretory processes. Our previous work led us to propose the intergenic region( IGR.) hypothesis,which states that the 3.6-kb IGR between the mouse OT and VP genes contains the critical enhancer sites for cell-specific expression.Support for this hypothesis has come from our recent studies of various OT and VP mouse gene constructs in transgenic mice and in hypothalamic organotypic cultures in which the magnocellular OT and VP neurons are transfected by particle- mediated gene transfer (biolistics). In addition, we are presently analysing the calcium -dependent secretion of OT-GFP & VP- GFP fusion proteins from individual nerve terminals in transgenic mice using a novel imaging approach . We also studied the effects of various growth factors on the survival & process outgrowth of OT, VP and dopamine neurons in our postnatal dissociated neuronal and organotypic cultures of hypothalamus and mesencephalon, respectively, and found that both GDNF and BDNF were very effacacious whereas CNTF & LIF were very deleterious. We completed our differential analysis of HNS gene expression in hyper- vs hyponatremic rats and found in addition to a global increase in gene expression in hyponatremia, that there was a novel gene increased in expression during hyponatremia. The latter gene was discovered using a a cDNA microarray(chip) & is now being cloned . Use of our single-cell (RT-PCR-derived) oxytocin and vasopressin neuronal cDNA libraries uncovered many genes that are differentially expressed in these two phenotypes. Several of these novel, differentially expressed genes are now being cloned. We have succeeded in sorting the AVP- GFP fusion proteins to secretory granules in HNS neurons in vivo as well as PC-12 cells in vitro and have effectively used these models for secretion studies. Our future plans are: 1) to continue to test the IGR hypothesis and 2) to study calcium-dependent secretion from dendrites and nerve terminals in these neurons. - vasopressin, oxytocin, hypothalamus, pituitary, gene expression, gene transfer, organotypic culture,neuropeptides,secretion, calcium