CELL BIOLOGY OF NEUROPEPTIDE BIOSYNTHESIS AND SECRETION

在过去的二十年中，神经肽作为外周和中枢神经系统的细胞间信使，作为神经激素、神经调节剂、神经营养因子和/或神经递质而日益突出。多肽能神经元的表型与传统神经元不同，因为神经肽的产生和分泌需要持续的转录、翻译、高尔基体包装，以及在神经分泌发生之前将大致密核心（分泌）囊泡（ldcv）轴突运输到神经末梢。我们在下丘脑-神经垂体系统（HNS）的大细胞催产素（OT）和抗利尿激素（VP）神经元中专门研究了这些问题。我们的目标是：(1)阐明OT和VP细胞特异性基因表达的机制；(2)在体内针对这些神经元特异性分子的基因表达，以分析其对神经分泌过程的影响。我们之前的工作使我们提出了基因间区（IGR）假说，该假说认为小鼠OT和VP基因之间的3.6 kb IGR包含细胞特异性表达的关键增强子位点。我们最近在转基因小鼠和下丘脑器官型培养中对大细胞OT和VP神经元进行颗粒介导的基因转移（生物学）转染的各种OT和VP小鼠基因构建的研究支持了这一假设。此外，我们目前正在使用一种新的成像方法分析转基因小鼠单个神经末梢中OT-GFP和VP- GFP融合蛋白的钙依赖性分泌。我们还研究了各种生长因子对出生后下丘脑和中脑分离神经元和器官型培养中OT、VP和多巴胺神经元存活和过程生长的影响，发现GDNF和BDNF都非常有效，而CNTF和LIF则非常有害。我们完成了HNS基因在高钠血症和低钠血症大鼠中表达的差异分析，发现除了低钠血症中基因表达的整体增加外，还有一个新的基因在低钠血症中表达增加。后者基因是用cDNA微阵列（芯片）发现的，目前正在克隆中。利用我们的单细胞（rt - pcr衍生）催产素和加压素神经元cDNA文库发现了许多在这两种表型中差异表达的基因。其中一些新颖的、差异表达的基因正在被克隆。我们已经成功地将AVP- GFP融合蛋白在体内HNS神经元和体外PC-12细胞中分选到分泌颗粒，并有效地利用这些模型进行分泌研究。我们未来的计划是：1)继续测试IGR假说，2)研究这些神经元的树突和神经末梢的钙依赖性分泌。-加压素，催产素，下丘脑，垂体，基因表达，基因转移，器官型培养，神经肽，分泌，钙

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