CELL BIOLOGY OF NEUROPEPTIDE AND CATECHOLAMINE BIOSYNTHESIS AND SECRETION

多肽能神经元的表型与传统神经元的表型不同，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。相比之下，其他神经递质分泌神经元（包括儿茶酚胺神经元）在分泌后能够通过局部运输机制补充其在神经末梢的囊状神经递质储存。我们正在通过研究下丘脑-神经垂体系统的大细胞催产素（OT）和加压素（VP）神经元以及中脑分泌多巴胺的神经元作为这两种表型的代表来研究潜在的细胞生物学机制。我们的目标是：(1)阐明下丘脑中OT和VP的细胞特异性基因表达以及中脑中酪氨酸羟化酶的基因表达的机制；(2)在体内和体外针对这些神经元的特定分子的基因表达，以干扰和可视化这两种表型的神经分泌过程。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 kbp IGR包含细胞特异性表达的关键增强子位点。我们最近对人类IGR的克隆和测序，以及与小鼠序列的比较，支持了这一假设，使各种OT和VP小鼠基因结构的设计合理化，这些基因结构目前正在转基因小鼠和下丘脑器官型培养中进行细胞特异性表达的评估。此外，利用这些信息，我们已经能够将绿色荧光蛋白（GFP）靶向LDCVs，并使用荧光成像方法研究了OT-GFP和VP-GFP融合蛋白在转基因小鼠和PC-12细胞的个体垂体神经末梢的钙依赖性分泌。我们还利用单细胞（rt - pcr衍生）催产素和加压素神经元cDNA文库完成了OT和VP神经元基因表达的差异分析。这些差异表达的基因中有几个已被克隆，并代表了已知酶的同种异构体。其他是不同功能的父系表达基因（如peg-3）。这种差异基因表达的生物学意义目前正在积极研究中。在一项新的研究中，我们研究了酪氨酸羟化酶在中枢神经系统中的细胞特异性表达，使用该基因的上游9kbp区域，与EGFP报告基因偶联。含有这种结构的转基因小鼠目前正在生产中，细胞特异性表达的分析将很快进行。如果获得成功，这种转基因模型将对儿茶酚胺系统的研究具有巨大的价值。如黑质和蓝斑)与帕金森病有关。我们实验室目前正在开发两种组织培养模型，在我们出生后下丘脑和中脑的分离神经元和器官型培养中，我们分别研究了各种生长因子对OT、VP和多巴胺神经元存活和生长过程的影响，发现GDNF和BDNF作为营养因子都是非常有效的，但在中脑中需要cAMP预处理。我们未来的计划是继续分析这些模型中的OT、VP和酪氨酸羟化酶基因启动子，以确定细胞特异性增强子，并研究下丘脑VP和OT神经元的树突和神经末梢以及黑质多巴胺神经元的钙依赖性分泌。

The peptidergic neuronal phenotype is distinct from that of 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 contrast, other neurotransmitter secreting neurons (including catecholamine neurons) are able to replenish their vesicular neurotransmitter stores in the nerve terminal by local transport mechanisms after secretion.We are examining the underlying cell biological mechanisms by studying magnocellular oxytocin (OT) and vasopressin (VP) neurons of the hypothalamo-neurohypophysial system and dopamine-secreting neurons in the mesencephalon as representatives of these two phenotypes. Our goals are:(1)to elucidate the mechanisms that are involved in the cell-specific gene expression of OT and VP in the hypothalamus, and gene expression of tyrosine hydroxylase in the mesencephalon and (2) to target the gene expression of specific molecules to these neurons both in vivo and in vitro, in order to perturb and visualize the neurosecretory processes in both phenotypes. Our previous work led us to propose the intergenic region( IGR) hypothesis, which states that the 3.6-kbp 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 cloning and sequencing of the human IGR, and its comparison with the mouse sequence, which has rationalized the design of various OT and VP mouse gene constructs which are now being evaluated for cell-specific expression in transgenic mice and in hypothalamic organotypic cultures. In addition, by using this information we have been able to target green fluorescent protein (GFP) to LDCVs and have studied the calcium-dependent secretion of OT-GFP & VP-GFP fusion proteins from individual pituitary nerve terminals in transgenic mice and PC-12 cells using an fluorescence imaging approach. We also completed our differential analysis of gene expression in OT and VP neurons by employing single-cell (RT-PCR-derived) oxytocin and vasopressin neuronal cDNA libraries. Several of these differentially expressed genes have been cloned and represent isoforms of known enzymes. Others are paternally expressed genes of varied function (e.g., peg-3). The biological significance of this differential gene expression is presently under active study. In a new initiative, we have studied the cell-specific expression of tyrosine hydroxylase in the CNS using a 9kbp upstream region of the gene, coupled to an EGFP reporter. Transgenic mice containing this construct are presently under production and analysis of cell-specific expression will soon be underway. Given success, this transgenic model will be of immense value for the study of catecholamine systems (e,g., substantia nigra and locus coeruleus) involved in Parkinsons Disease. Two tissue culture models are currently under development in our lab, and we have 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 as trophic factors but in the mesencephalon require cAMP pretreatment. Our future plans are to continue to analyse the OT , VP and tyrosine hydroxlase gene promoters in these models in order to identify the cell-specfic enhancers and to study calcium-dependent secretion from dendrites and nerve terminals in the hypothalamic VP and OT neurons and from dopamine neurons in the substantia nigra.