CELL BIOLOGY OF NEUROPEPTIDE BIOSYNTHESIS AND SECRETION

肽能神经元表型不同于 传统的神经元， 神经肽需要持续的转录，翻译， 包装在高尔基体，和轴突运输的大致密核心 （分泌）囊泡（LDCV）到神经末梢之前 神经分泌可以发生。在过去的一年里，我们主要 专注于该过程的第一步，即，小区特定 调节神经肽基因的表达。我们特别 在大细胞催产素（OT）中研究了这些问题， 下丘脑-神经垂体的血管加压素（VP）神经元 神经内分泌系统（HNS），因为这些神经内分泌（肽能）细胞 特别是有价值的模型进行这种分析。我们的目标是 双重：第一是阐明所涉及的机制 OT和VP的细胞特异性基因表达。二是 利用这些信息来靶向特定的基因表达， 分子到这些神经元，以分析它们的影响， 神经分泌过程由于缺乏可用的 同源细胞系，我们使用转基因小鼠来评估 OT和VP基因中可能参与调节的位点 它们在大细胞神经元中的细胞特异性表达。 以前的工作使我们提出了基因间区（IGR）。 假设，该假设指出OT和 VP基因（位于小鼠的2号染色体上）含有 细胞特异性表达的关键增强子位点。各种OT和 已经在转基因小鼠中研究了VP小鼠基因构建体， 将描述支持这一假设的最新数据。在 此外，考虑到使用转基因小鼠作为 用于系统评价进一步缺失的测定系统 构建，我们最近还开发了一种体外模型， （下丘脑器官型培养），其中大细胞OT VP神经元可以成功转染报告基因 通过使用病毒载体和颗粒介导的 基因转移（基因枪）。这种方法提供了一个通用的 用于启动子/增强子鉴定的转基因小鼠的替代物 CNS神经元的研究。关于确定 可能的转因子和信号转导机制， 调节大细胞神经元中OT和/或VP基因的表达， 我们开发并使用了减法和微分 单细胞cDNA文库的分析（例如，从OT到VP 细胞），以及分析来自显微切割的 视上核（SONs），从hypo-对比 高钠血症大鼠（后者在OT和VP基因表达上存在差异 超过10倍）。初步观察显示， 特定的分子差异。目前正在对这些措施进行评估 通过原位杂交组织化学检测OT和VP细胞相关性 和特异性。我们未来的计划是：1）继续测试IGR 假设，重点是顺式调节元件， 可能负责OT的转录激活因子 和VP细胞特异性表达，以及2）靶向荧光蛋白， 报告基因EGFP与HNS神经元中的LDCV的结合，以研究 树突和神经末梢的钙依赖性分泌 这些神经元。

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. In the past year, we have primarily focused on the first step in the process, i.e., the cell-specific regulation of neuropeptide gene expression. We have specifically studied these issues in the magnocellular oxytocin (OT) and vasopressin (VP) neurons of the hypothalamo-neurohypophysial system (HNS), since these neuroendocrine (peptidergic) cells are particularly valuable models for such analyses. Our goals are twofold: The first is to elucidate the mechanisms that are involved in the cell-specific gene expression of OT and VP. The second is to use this information to target the gene expression of specific molecules to these neurons in vivo, in order to analyze their impact on neurosecretory processes. Given the absence of available homologous cell lines, we have used transgenic mice to evaluate the sites in the OT and VP genes that may be involved in the regulation of their cell-specific expression in the magnocellular neurons. Previous work has led us to propose the intergenic region( IGR.) hypothesis, which states that the 3.6-kb IGR between the OT and VP genes (located on Chromosome 2 in the mouse) contains the critical enhancer sites for cell-specific expression. Various OT and VP mouse gene constructs have been studied in transgenic mice and recent data in support of this hypothesis will be described. In addition, given the impracticality of using transgenic mice as an assay system for a systematic evaluation of further deletion constructs, we have also recently developed an in vitro model (hypothalamic organotypic cultures) in which the magnocellular OT and VP neurons can be successfully transfected with reporter containing constructs by using viral vectors and particle- mediated gene transfer (biolistics). This approach has provided a general alternative to transgenic mice for promoter/enhancer identification studies in CNS neurons. With respect to the determination of the possible transfactors and signal- transduction mechanisms that regulate OT and/or VP gene expression in magnocellular neurons, we have developed and employed subtractive and differential analyses of single-cell cDNA libraries (e.g., from OT versus VP cells), as well as analysis of cDNA libraries from microdissected supraoptic nuclei (SONs) obtained from hypo- versus hypernatremic rats (the latter differ in OT and VP gene expression by more than tenfold). Preliminary observations have revealed many specific molecular differences. These are currently being evaluated by in situ hybridization histochemistry for OT and VP cell relevance and specificity. Our future plans are: 1) to continue to test the IGR hypothesis, with a focus on the cis-regulatory elements and transcriptional activating factors that may be responsible for OT and VP cell-specific expression and 2) to target the fluorescent reporter, EGFP, to the LDCVs in HNS neurons, in order to study calcium-dependent secretion from dendrites and nerve terminals in these neurons.