Cell Biology Of Neuropeptide And Catecholamine Biosynthe

肽能神经元需要“在线”转录、翻译、经由高尔基体包装成大的致密核心（分泌）囊泡（LDCV），以及LDCV的轴突运输至神经末梢以分泌神经肽。相比之下，儿茶酚胺能（CA）神经元产生和分泌后补充其囊泡CA存储在神经末梢的局部酶和膜运输机制。我们正在研究这些机制的肽能催产素（OT）和加压素（VP）的大细胞神经元（MCNs）的下丘脑神经垂体系统（HNS），和CA分泌神经元在中枢神经系统（CNS）。我们的主要目标是：（1）阐明OT和VP在下丘脑中的细胞特异性基因表达以及CNS中酪氨酸羟化酶的基因表达所涉及的机制;（2）在体内和体外将不同分子的基因表达靶向于这些特异性神经元，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，该假说指出小鼠OT和VP基因之间的3.6 kbp IGR包含MCNs中细胞特异性表达的关键增强子位点。我们最近的克隆和测序的人IGR及其与小鼠序列的比较，为这一假设提供了支持，并导致26个保守的序列作为推定的增强子位点的IGR的鉴定。此外，这合理化的几个OT和VP小鼠基因构建体的设计，现在正在完成和评估的细胞特异性表达在转基因小鼠和通过使用基因枪在下丘脑器官型培养。我们最近发现，在器官型培养基中加入10 ng/ml CNTF可使VP-MCNs的存活率提高6倍，使OT-MCNs的存活率提高3倍。CNTF处理后，SON中VP和OT-mRNA水平均发生相应变化。进一步的实验表明，CNTF处理并没有增加OT和VP基因的表达，并且在SON中观察到的OT和VP mRNA的增加是由于VP MCN数量的增加。CNTF对特别是更脆弱的VP-MCN表型的拯救，将首次允许对两种MCN表型进行长期的体外分子和生理研究。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LDCV，并使用荧光成像方法研究了转基因小鼠中来自单个垂体神经末梢的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中基因表达的分析。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。其中三个基因只在HNS中表达。其中一些似乎是转录激活因子，其他编码蛋白质参与其他细胞功能。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。四个转基因小鼠品系，表达EGFP在中枢神经系统中的儿茶酚胺能神经元中的细胞特异性的方式已经产生。对这些小鼠中EGFP表达的分析表明，弓状核中的多巴胺神经元和蓝斑中的去甲肾上腺素神经元特别强大。此外，我们开发了中枢神经系统CA神经元的器官型组织培养模型，并成功地产生了健康的切片外植体从前脑到后脑，其中包含经典的CA神经元系统。CA神经元在这些切片外植体成功地转染含有不同长度的酪氨酸羟化酶基因启动子的粒子介导的基因转移的结构，表明这些文化将是极好的模型，在中枢神经系统中的CA神经元的基因表达的生理调节的研究。

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.