Cell Biology Of Neuropeptide And Catecholamine Biosynthe

由于缺乏能够充分代表特定中枢神经元表型的同源神经细胞系，对中枢神经系统神经元细胞特异性基因表达的研究受到了限制。因此，大多数神经元表型的相关信息主要是通过研究转基因小鼠的特定神经元来获得的。在我们自己和其他实验室对下丘脑-神经垂体系统（HNS）大细胞神经元（MNCs）中催产素（OT）和抗利尿激素（VP）基因表达的转基因小鼠研究使我们提出了基因间区（IGR）假说，该假说认为3？VP基因的侧翼区域（IGR）分隔OT-和VP-基因，包含这些神经肽基因细胞特异性表达的关键增强子位点。我们使用含有与氯霉素乙酰转移酶（CAT）报告基因相关的小鼠基因组dna衍生构建体的转基因小鼠验证了这一假设。利用含有3.8 kbp的5'侧翼区和小鼠VP基因的所有外显子和内含子的构建物研究VP基因的表达，该基因在外显子3的末端与CAT报告子融合，随后是2.1kb的IGR片段。使用了一个类似的催产素CAT转基因结构，其中包含全长小鼠IGR（3.6 kbp）。两种转基因构建体在适当的OT或VP大细胞神经元中都产生了cat报告基因的细胞特异性表达。电镜免疫细胞化学显示，由OT-和vp -转基因产生的CAT融合蛋白通过各自大细胞神经元的调节分泌途径被有效运输，包装成大而致密的核心囊泡（ldcv），并运输到垂体后叶的神经末梢。IGR假说的进一步测试现在正在使用我们实验室最近开发的一种新的高通量方法进行，该方法使用生物转染在器官型下丘脑培养中鉴定的神经元。我们最近证明，在器官型培养中，由于轴切引起的下丘脑大细胞神经元（mcn）的广泛凋亡可以通过CNTF来挽救。最近，我们证明抗凋亡药物Bcl-XL和z-VAD也可以在体外拯救VP和OT MCN，这与VP和OT MCN细胞死亡是通过凋亡机制发生的观点一致。使用CNTF来拯救MCN首次使我们能够在体外对两种MCN表型进行长期的分子和生理研究。利用含有EGFP报告基因的质粒对下丘脑器官型切片外植体进行生物转染，研究VP基因在HNS中的表达。使用这种高吞吐量策略，我们发现减少5？小鼠VP基因结构的UTR从3.5kbp增加到288bp， IGR从3.6 kbp增加到2.1 kbp，并没有改变其在下丘脑切片中的表达效果。所有后续构建均基于288 bp 5？仅对IGR区域进行改变的UTR VP基因构建。我们用于测试IGR假设的第一个结构完全消除了IGR，并且在转染试验中给出了阴性结果，与假设一致。基于对人、大鼠和小鼠IGR中保守序列的生物信息学分析，将2.1 kb的IGR划分为5个片段，分别为176、374,298、446和834 bp。使用包含上述IGR片段的5种结构的实验正在进行中。除了上述对mcn的研究外，我们还研究了内源性VP基因在下丘脑细小细胞神经元中的表达。VP是由视交叉上核（SCN）合成和分泌的。采用内含子原位杂交技术研究了AVP基因在大鼠SCN器官型培养中的转录。AVP基因转录在培养的SCN中保持每日节律，在白天达到峰值。钠通道阻滞剂河豚毒素（TTX）抑制自发性活动，显著降低AVP异核RNA水平，抑制节律性。此外，MAP激酶途径抑制剂PD98059可显著降低VP转录并取消其日常节律。因此，神经活动和功能性MAP激酶信号通路似乎对AVP基因在SCN中的表达至关重要。在另一项利用内含子原位杂交技术对大鼠室旁核（PVN）中VP基因转录的研究中，我们可以证明cAMP直接激活PVN旁细胞神经元中AVP的转录。最后，细胞特异性基因表达数据使我们能够在体内将特定分子的基因表达靶向HNS中MCN中的LDCVs，并直接观察MCN神经末梢的神经分泌过程。我们发现AI-03转基因基因含有融合到OT前激素c端神经physin末端的增强型绿色荧光蛋白（EGFP），在OT大细胞神经元中选择性表达，并被转运到转基因小鼠的分泌颗粒中。然后，含有EGFP的分泌颗粒被运输到神经垂体的ot神经分泌终端，在那里可以看到EGFP荧光进行去极化诱导的钙依赖性分泌。我们还在体外和体内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。在中枢神经系统的儿茶酚胺能神经元中以细胞特异性方式表达EGFP的三种转基因小鼠系已经产生并正在进行回交。对这些小鼠EGFP的表达分析表明，弓形核的多巴胺神经元和蓝斑的去甲肾上腺素神经元表现出非常强的GFP表达。

Studies of cell-specific gene expression in central nervous system neurons have been limited by the paucity of homologous neuronal cell lines that adequately represent the phenotypes of specific central neurons. Hence, for most of these neuronal phenotypes relevant information has been mainly obtained by studying the specific neurons in transgenic mice. Transgenic mouse studies on oxytocin (OT) and vasopressin (VP) gene expression in magnocellular neurons (MNCs) of the hypothalamo-neurohypophysial system (HNS) in our own and other laboratories have led us to propose the intergenic region (IGR) hypothesis, which states that the 3?flanking region of the VP gene (the IGR) separating the OT- and VP-genes, contains critical enhancer sites for cell-specific expression of these neuropeptide genes. We tested this hypothesis using transgenic mice containing mouse genomic DNA-derived constructs linked to chloramphenicol acetyltransferase (CAT) reporters. VP gene' expression was studied using constructs containing 3.8 kbp of the 5' flanking region and all the exons and introns in the mouse VP gene which was fused at the end of exon 3 to a CAT reporter followed by a 2.1kb IGR fragment. A similar construct for the oxytocin CAT transgene was used which contained the full-length mouse IGR(3.6 kbp). Both transgenic constructs produced cell-specific expression of the CAT-reporter in the appropriate OT or VP magnocellular neurons. Electron microscopic immunocytochemistry showed that the CAT fusion proteins produced from the OT- and VP-transgenes were efficiently trafficked through the regulated secretory pathways in their respective magnocellular neurons, packaged into large dense core vesicles (LDCVs) and transported to nerve terminals in the posterior pituitary. Further tests of the IGR hypothesis are now being done using a novel high throughput method recently developed in our laboratory which uses biolistic transfections of identified neurons in organotypic hypothalamic cultures. We recently demonstrated that the extensive apoptosis of magnocellular neurons (MCNs) in the hypothalamus that occurs in organotypic cultures due to axotomy could be rescued by CNTF. More recently, we demonstrated that the anti-apoptotic agents, Bcl-XL and z-VAD, could also rescue VP and OT MCNs in vitro, consistent with the view that the VP and OT MCN cell death is occurring by apoptotic mechanisms. The use of CNTF to rescue the MCNs has for the first time allowed us to perform long-term molecular and physiological studies of both MCN phenotypes in vitro. Biolistic transfections of hypothalamic organotypic slice explants were done using plasmid constructs containing an EGFP reporter to study VP gene expression in the HNS. Using this high throughput strategy we found that reducing the 5?UTR from 3.5kbp to 288bp and the IGR from 3.6 to 2.1 kbp in the mouse VP gene construct did not alter the efficacy of its expression in the hypothalamic slices. All subsequent constructs were based on this 288 bp 5? UTR VP gene construct with changes made only to the IGR region. Our first construct used to test the IGR hypothesis eliminated the IGR completely, and gave negative results in transfection assays, consistent with the hypothesis. The 2.1 kb IGR was then divided into 5 segments of 176, 374, 298, 446, and 834 bp based on our bioinformatic anayses of conserved sequences found in the human, rat and mouse IGRs. Experiments using the 5 constructs containing the above IGR segments are now in progress. In addition to the above studies on the MCNs, we have also studied the expression of the endogenous VP gene in parvocellular neurons in the hypothalamus. VP is synthesized in and secreted by the suprachiasmatic nucleus (SCN) in a circadian pattern. We studied transcription of the AVP gene in organotypic cultures of rat SCN by using intronic in situ hybridization. AVP gene transcription in the cultured SCN maintained a daily rhythm with a peak in the daytime. Inhibition of spontaneous activity by the sodium channel blocker, tetrodotoxin (TTX), dramatically decreased AVP heteronuclear RNA levels and suppressed rhythmicity. In addition, the MAP kinase pathway inhibitor, PD98059, profoundly decreased VP transcription and abolished its daily rhythm. Hence, neural activity and a functional MAP kinase signaling pathway appears to be critical for AVP gene expression in the SCN. In another study on VP gene transcription in the rat paraventricular nucleus (PVN) in organotypic cultures using intronic in situ hybridization we could demonstrated that cAMP directly activates AVP transcription in parvocellular neurons of the PVN. Finally, the cell-specific gene expression data has allowed us to target the gene expression of specific molecules to LDCVs in the MCNs in the HNS in vivo and to directly visualize neurosecretory processes in the MCN nerve endings. We showed that the AI-03 transgene which contains enhanced green fluorescent protein (EGFP) fused to the end of the neurophysin at the C-terminus of the OT pre-prohormone, is expressed selectively in OT-magnocellular neurons,and is trafficked to secretory granules in transgenic mice. The EGFP-containing secretory granules are then transported to OT-neurosecretory terminals in the neurohypophysis, where the EGFP fluorescence could be seen to undergo depolarization-induced calcium-dependent secretion. 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, both in vitro and in vivo. Three transgenic mouse lines which express EGFP in a cell-specific manner in catecholaminergic neurons in the CNS have been produced and are being backcrossed. Analysis of EGFP expression in these mice shows that dopamine neurons in the arcuate nucleus and norepinephrine neurons in the locus coeruleus show very robust expression of the GFP.