CELL BIOLOGY OF NEUROPEPTIDE AND CATECHOLAMINE BIOSYNTHESIS AND SECRETION

肽能神经元表型与常规神经元的表型不同，因为神经肽的产生和分泌需要持续的转录、翻译、高尔基体中的包装以及大的致密核心（分泌）囊泡（LDCV）在神经分泌可以发生之前向神经末梢的轴突运输。与此相反，其他神经递质分泌神经元（包括儿茶酚胺神经元）分泌后能够通过局部转运机制补充其在神经末梢中的囊泡神经递质储存。我们通过研究下丘脑-神经垂体系统的大细胞催产素（OT）和加压素（VP）神经元以及多巴胺（DA）神经元来研究潜在的细胞生物学机制。中脑分泌神经元作为这两种表型的代表。我们的目标是：（1）阐明参与下丘脑OT和VP的细胞特异性基因表达以及中脑酪氨酸羟化酶基因表达的机制;（2）在体内和体外靶向这些神经元特异性分子的基因表达，以干扰和可视化两种表型的神经分泌过程。我们以前的工作使我们提出了基因间区（IGR）假说，该假说指出，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。支持这一假设已经来自我们最近的克隆和测序的人IGR，其与小鼠序列的比较，这合理化的各种OT和VP小鼠基因构建体的设计，现在正在评估细胞特异性表达的转基因小鼠和下丘脑器官型培养。此外，通过使用这些信息，我们已经能够将绿色荧光蛋白（GFP）靶向LDCV，并使用荧光成像方法研究了转基因小鼠和PC-12细胞中单个垂体神经末梢的OT-GFP和VP-GFP融合蛋白的钙依赖性分泌。我们还完成了我们的差异分析OT和VP神经元的基因表达采用单细胞（RT-PCR衍生）催产素和加压素神经元的cDNA文库。这些差异表达的基因中有几个已被克隆，并代表已知酶的同种型。其他的是父系表达的不同功能的基因（例如，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。在一个新的倡议，我们已经研究了细胞特异性表达的酪氨酸羟化酶在中枢神经系统中使用的9 kbp的上游区域的基因，再加上一个绿色荧光蛋白报告。目前正在生产含有该构建体的转基因小鼠，并且细胞特异性表达的分析将很快进行。如果成功，这种转基因模型将对研究儿茶酚胺系统（例如，黑质和蓝斑）参与帕金森病。本实验室目前正在建立两种组织培养模型，分别在下丘脑和中脑的分离神经元和器官型培养物中研究了各种生长因子对OT、VP和多巴胺神经元的存活和生长的影响，发现GDNF和BDNF作为营养因子是非常无效的，但在中脑中需要cAMP预处理。我们未来的计划是继续分析OT，VP和酪氨酸羟化酶基因启动子在这些模型中，以确定细胞特异性增强剂和研究钙依赖性分泌树突和神经末梢在下丘脑VP和OT神经元和多巴胺神经元在黑质。

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.