Cell Biology Of Neuropeptide And Catecholamine Biosynthe

中枢神经系统神经元中细胞特异性基因表达的研究一直受到缺乏同源神经元细胞系的限制，这些细胞系充分代表了特定中枢神经元的表型。因此，对于大多数这些神经元表型的相关信息主要是通过研究转基因小鼠中的特定神经元获得的。转基因小鼠研究催产素（OT）和血管加压素（VP）基因表达的大细胞神经元（MNCs）的下丘脑-神经垂体系统（HNS）在我们自己和其他实验室导致我们提出的基因间区（IGR）假说，其中指出，3？VP基因的侧翼区（IGR）分隔OT-和VP-基因，含有这些神经肽基因的细胞特异性表达的关键增强子位点。我们测试了这一假设，使用转基因小鼠含有小鼠基因组DNA衍生的结构连接到氯霉素乙酰转移酶（CAT）报告。将小鼠VP基因的5'侧翼区3.8kbp和全部外显子、内含子融合在CAT报告基因的第3外显子末端，再融合一个2.1kb的IGR片段，构建了VP基因表达载体。使用催产素CAT转基因的类似构建体，其含有全长小鼠IGR（3.6kbp）。两种转基因构建体在适当的OT或VP大细胞神经元中产生CAT报告基因的细胞特异性表达。电子显微镜免疫细胞化学显示，CAT融合蛋白生产的OT-和VP-转基因被有效地贩运通过调节分泌途径在各自的大细胞神经元，包装成大致密的核心囊泡（LDCV）和运输到神经末梢在垂体后叶。IGR假说的进一步测试，现在正在使用一种新的高通量的方法，最近在我们的实验室，使用生物枪转染的器官型下丘脑文化中确定的神经元。我们最近证明，下丘脑大细胞神经元（MCNs）的广泛凋亡，发生在器官型文化由于轴突切断可以救出CNTF。最近，我们证明了抗凋亡剂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。CNTF的使用，以挽救MCN的第一次使我们能够进行长期的分子和生理研究的MCN表型在体外。使用含有EGFP报告基因的质粒构建体进行下丘脑器官型切片外植体的生物射弹转染，以研究HNS中VP基因的表达。使用这种高通量的策略，我们发现，减少5？小鼠VP基因构建体中UTR从3.5 kbp到288 bp和IGR从3.6 kbp到2.1 kbp没有改变其在下丘脑切片中表达的功效。所有随后的构建体都是基于这288 bp的5？仅对IGR区域进行改变的UTR VP基因构建体。我们的第一个用于测试IGR假设的构建体完全消除了IGR，并且在转染测定中给出了阴性结果，与假设一致。根据对人、大鼠和小鼠IGR保守序列的生物信息学分析，将2.1kb的IGR分为176、374、298、446和834 bp 5个片段。使用含有上述IGR片段的5个构建体的实验正在进行中。除了上述对MCNs的研究外，我们还研究了内源性VP基因在下丘脑小细胞神经元中的表达。VP在视交叉上核（SCN）中以昼夜节律模式合成和分泌。我们研究了AVP基因的转录在大鼠SCN器官型培养用内含子原位杂交。培养SCN中AVP基因的转录维持日节律，白天达到高峰。抑制自发活动的钠通道阻断剂，河豚毒素（TTX），显着降低AVP异源RNA水平和抑制节律性。此外，MAP激酶通路抑制剂PD 98059，深刻降低VP转录和取消其每日节律。因此，神经活性和功能性MAP激酶信号通路似乎是SCN中AVP基因表达的关键。在另一项研究中，VP基因转录在大鼠室旁核（PVN）的器官型培养使用内含子原位杂交，我们可以证明，cAMP直接激活AVP转录的小细胞神经元的PVN。最后，细胞特异性基因表达数据使我们能够将特定分子的基因表达靶向体内HNS中MCN中的LDCV，并直接可视化MCN神经末梢中的神经分泌过程。我们表明，AI-03转基因，其中含有增强的绿色荧光蛋白（EGFP）融合到神经垂体素的末端在OT前激素原的C-末端，选择性地表达在OT-大细胞神经元，并被贩运到转基因小鼠的分泌颗粒。含EGFP的分泌颗粒然后被运输到神经垂体中的OT-神经分泌末端，在那里可以看到EGFP荧光经历去极化诱导的钙依赖性分泌。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，在体外和体内。三个转基因小鼠品系，表达EGFP的细胞特异性的方式在中枢神经系统中的儿茶酚胺能神经元已经产生，并正在回交。对这些小鼠中的EGFP表达的分析显示，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显示出非常稳健的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.