Cell Biology Of Neuropeptide And Catecholamine Biosynthe

肽能神经元需要“在线”转录、翻译、通过高尔基体包装成大而致密的核心（分泌）囊泡（ldcv），并将ldcv轴突运输到神经末梢，以分泌神经肽。相比之下，儿茶酚胺能（catecholaminergic， CA）神经元分泌后通过局部酶和膜运输机制在神经末梢补充其囊状CA储存。我们正在下丘脑-神经垂体系统（HNS）的多肽性催产素（OT）和加压素（VP）大细胞神经元（MCNs）和中枢神经系统（CNS）的ca分泌神经元中研究这些机制。我们的主要目标是：(1)阐明下丘脑中OT和VP的细胞特异性基因表达以及中枢神经系统中酪氨酸羟化酶的基因表达的机制；(2)在体内和体外将不同分子的基因表达靶向到这些特定神经元，以干扰和可视化它们的神经分泌过程。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mcn中细胞特异性表达的关键增强子位点。我们最近对人类IGR的克隆和测序及其与小鼠序列的比较为这一假设提供了支持，并鉴定出26个保守序列作为IGR的假定增强子位点。此外，这使得一些OT和VP小鼠基因构建的设计合理化，这些构建目前正在完成，并通过在下丘脑器官型培养中使用生物学来评估转基因小鼠的细胞特异性表达。我们最近发现，在有机型培养基中添加10ng/ml CNTF可使VP-MCNs的存活率提高6倍，OT-MCNs的存活率提高3倍。在CNTF处理的结果中，SON中的vp - mrna和OT-mRNA水平都发生了相应的变化。进一步的实验表明，CNTF处理并没有增加OT-和VP-基因的表达，在SON中观察到的OT和VP mRNA的增加是由于VP MCN数量的增加。CNTF对更脆弱的VP-MCN表型的拯救，将首次允许在体外对两种MCN表型进行长期的分子和生理研究。作为这些研究的副产品，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转基因小鼠个体垂体神经末梢中OT-GFP融合蛋白的钙依赖性分泌。我们通过差异筛选单细胞cDNA文库完成了OT-和VP-MCNs基因表达的分析。大多数差异表达基因的功能未知，原位杂交研究证实了它们的细胞特异性表达。其中3个基因仅在HNS中表达。其中一些似乎是转录激活因子，另一些则编码涉及其他细胞功能的蛋白质。这些差异表达基因的作用目前正在积极研究中。我们还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。四种转基因小鼠系在中枢神经系统的儿茶酚胺能神经元中以细胞特异性的方式表达EGFP。EGFP在这些小鼠中的表达分析表明，弓形核中的多巴胺神经元和蓝斑的去甲肾上腺素神经元特别旺盛。此外，我们建立了中枢神经系统CA神经元的器官型组织培养模型，并成功地从前脑到后脑制备了含有经典CA神经元系统的健康切片外植体。这些薄片外植体中的CA神经元通过颗粒介导的基因转移成功转染了含有不同长度酪氨酸羟化酶基因启动子的构建物，这表明这些培养物将成为研究中枢神经系统CA神经元基因表达生理调控的极好模型。

Peptidergic neurons require "on-line" transcription, translation, packaging into large dense core (secretory) vesicles (LDCVs) via the golgi, and axonal transport of the LDCVs to nerve terminals in order to secrete neuropeptides. In contrast, catecholaminergic (CA) neurons produce and after secretion replenish their vesicular CA stores in the nerve terminal by local enzymatic and membrane transport mechanisms. We are studying these mechanisms in peptidergic oxytocin (OT) and vasopressin (VP) magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system (HNS), and CA-secreting neurons in the central nervous system (CNS). Our principal goals are: (1) to elucidate the mechanisms that are involved in the cell-specific gene expression of OT and VP in the hypothalamus, and the gene expression of tyrosine hydroxylase in the CNS and (2) to target the gene expression of diverse molecules to these specific neurons both in vivo and in vitro, in order to perturb and visualize their neurosecretory processes. We have proposed 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 in the MCNs. Our recent cloning and sequencing of the human IGR and its comparison with the mouse sequence provided support for this hypothesis and has lead to the identification of 26 conserved sequences as putative enhancer sites in the IGR. In addition, this rationalized the design of several OT and VP mouse gene constructs which are now being completed and evaluated for cell-specific expression in transgenic mice and by the use of biolistics in hypothalamic organotypic cultures. We recently found that 10ng/ml CNTF added to the organotypic culture medium increased the survival of the VP-MCNs by 6-fold and the OT-MCNs by 3-fold. Corresponding changes were found in both VP-and OT-mRNA levels in the SON as a result of the CNTF treatment. Further experiments indicated that the CNTF treatment did not increase OT- and VP-gene expression, and that the increase of OT and VP mRNA observed in the SON was due to the increase in VP MCN number. This rescue of especially the more vulnerable VP-MCN phenotype by CNTF, will for the first time allow for long-term molecular and physiological studies of both MCN phenotypes in vitro. As a by-product of these studies, we have been able to target green fluorescent protein (GFP) to LDCVs and have studied the calcium-dependent secretion of OT-GFP fusion proteins from individual pituitary nerve terminals in transgenic mice using a fluorescence imaging approach. We completed an analysis of gene expression in OT- and VP-MCNs by differentially screening single-cell cDNA libraries. Most of the differentially expressed genes were of unknown function, and in situ hybrization studies confirmed their cell-specific expression. Three of these genes were expressed exclusively in the HNS. Some of these appear to be transcriptional-activating factors, and others encode proteins involved in other cell functions. The roles of these differentially expressed genes are presently under active study. We also studied the cell-specific expression of tyrosine hydroxylase in the CNS using a 9kbp upstream region of the gene, coupled to an EGFP reporter. Four transgenic mouse lines which express EGFP in a cell-specific manner in catecholaminergic neurons in the CNS have been produced. Analysis of EGFP expression in these mice shows that dopamine neurons in the arcuate nucleus and norepinephrine neurons in the locus coeruleus are particularly robust. In addition, we developed organotypic tissue culture models of CA neurons in the CNS, and have successfully produced healthy slice-explants from the forebrain to the hindbrain which contained classical CA neuronal systems. CA neurons in these slice explants were successfully transfected with constructs containing various length tyrosine hydroxlase gene promoters by particle mediated gene transfer, indicating that these cultures will be superb models for the study of the physiological regulation of gene expression in CA neurons in the CNS.