Molecular Genetic Studies on the Structure, Function and Pathology of Catecholamine Neurons

儿茶酚胺神经元结构、功能和病理学的分子遗传学研究

• 批准号: 03102004
• 负责人: NAGATSU Toshiharu
• 金额: $ 131.2万
• 依托单位: Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Specially Promoted Research
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03102004/
• 关键词: Catecholamine Neurons; Tyrosine Hydroxylase; Aromatic L-Amino Acid Decarboxylase; Dopamine beta-Hydroxylase; Phenylethanolamine N-Methyltransferase; Gene Expression; Gene Targeting; Transgenic Mouse; ドーパミンbeta-水酸化酵素; ドーパミンβー水酸化酵素; 分子生物学; カテコ-ルア

(1)Regulatiory Mechanism of Tyrosine Hydroxylase--- We investigated the existence of the TH heterogeneity by RT-PCR analysis of the TH mRNAs from several species and analysis of the TH genes from some monkeys, to explore evolution of the TH gene. Also, we examined changes in the expression pattern of the human TH mRNA types in several disorders including Parkinson's disease and schizophrenia. Four kinds of the human TH isoform were expressed in E.coli, and purified to characterize their enzymatic properties. (2)Molecular Mechanism of the Neuron-Specific Gene Expression for Catecholamine- Synthesizing Enzymes---Analysis of the human TH gene promoter in transgenic mice has shown that tissue-specific expression of the TH gene requires the cis-element(s) located in the exon-intron region or 3'-downstream region of the gene in addition to its 5'-upstream promoter. Several transgenic analyzes have demonstrated that the 4-kb 5'-flanking region of the human DBH gene is sufficient for tissue-specific expression of the gene in both norepinephrine- and epinephrine-producing cells, and that the 2-kb 5'-flanking region of the human PNMT gene drives the transgene expression in adrenal gland chromaffin cells. Moreover, we have investigated activities of the alternative promoters which regulate tissue-specific expression of the human AADC gene. In transgenic mice carrying the 7.0-kb upstream region including the two alternative promoters of the AADC gene, the reporter gene expression was detected in the peripheral tissues including liver and kidney. (3) Functional Analysis of Catecholamine Neurons Using Transgenic Mice---We have succeeded in genetic conversion of catecholamine specificity from norepinephrine phenotype to epinephrine phenotype, by expressing PNMT under the control of the human DBH gene promoter in transgenic mice (DPN). In DPN mice, epinephrine release from the sympathetic neurons induced down-regulation of the adrenergic receptor subtype. A novel transgenic techni

(1)酪氨酸羟化酶的调控机制——通过RT-PCR分析多个物种的TH mRNA以及部分猴子的TH基因分析，探讨TH异质性的存在，探讨TH基因的进化。此外，我们还研究了帕金森病和精神分裂症等多种疾病中人类 TH mRNA 类型表达模式的变化。四种人类 TH 同种型在大肠杆菌中表达，并纯化以表征其酶性质。 (2)儿茶酚胺合成酶神经元特异性基因表达的分子机制——对转基因小鼠中人TH基因启动子的分析表明，TH基因的组织特异性表达需要顺式元件位于除了其 5'-上游启动子之外，还位于基因的外显子-内含子区域或 3'-下游区域。一些转基因分析表明，人 DBH 基因的 4-kb 5'-侧翼区域足以在去甲肾上腺素和肾上腺素产生细胞中实现该基因的组织特异性表达，并且 2-kb 5'-侧翼区域足以实现该基因在去甲肾上腺素和肾上腺素产生细胞中的组织特异性表达。人类 PNMT 基因区域驱动肾上腺嗜铬细胞中的转基因表达。此外，我们还研究了调节人 AADC 基因组织特异性表达的替代启动子的活性。在携带7.0-kb上游区域（包括AADC基因的两个替代启动子）的转基因小鼠中，在包括肝脏和肾脏在内的外周组织中检测到报告基因表达。 (3)利用转基因小鼠进行儿茶酚胺神经元的功能分析——我们通过在转基因小鼠(DPN)中在人DBH基因启动子的控制下表达PNMT，成功地将儿茶酚胺特异性从去甲肾上腺素表型遗传转化为肾上腺素表型。在 DPN 小鼠中，交感神经元释放肾上腺素诱导肾上腺素能受体亚型下调。一种新颖的转基因技术

项目成果

D.Nakahara: "Normalization of throsine hydroxylase activity in vivo in the striatum of transgenic mice carrying human tyrosine hydroxylase gene:a microdialysis study" Neuroscience Letters. 158. 44-46 (1993)

D.Nakahara：“携带人酪氨酸羟化酶基因的转基因小鼠纹状体体内苏氨酸羟化酶活性的正常化：一项微透析研究”《神经科学快报》。

A.Ota: "Nicotine-induced regulation of tyrosinchydroxylase acitivity in adrenal gland of transgenic mouse carrying human tyrosine hydroxylase gene" Neuroscience Letters. 166. 55-58 (1994)

A.Ota：“尼古丁诱导的携带人酪氨酸羟化酶基因的转基因小鼠肾上腺中酪氨酸羟化酶活性的调节”《神经科学快报》。

Nagatsu, T.: Tyrosine Hydroxylase : Historical reflections.Tyrosine Hydroxylase : From Discovery to Cloning, VSP, Utrecht, 311(3-6) (1993)

Nagatsu, T.：酪氨酸羟化酶：历史反思。酪氨酸羟化酶：从发现到克隆，VSP，乌得勒支，311(3-6) (1993)

Nagatsu, I., Yamada, K., Karasawa, N., Kaneda, N., Sasaoka, T., Kobayashi, K., Fujita, K., Nagatsu, T.: Noncatecholaminergic neuronal expression in the brain of transgenic mice with special reference to aromatic L-amino acid decarboxylase.Tyrosine Hydroxy

Nagatsu, I., Yamada, K., Karasawa, N., Kaneda, N., Sasaoka, T., Kobayashi, K., Fujita, K., Nagatsu, T.：转基因小鼠大脑中的非儿茶酚胺能神经元表达

永津 俊治: "ビオプテリンの分子生物学" ビタミン. 66. 661-666 (1992)

Shunji Nagatsu：“生物蝶呤的分子生物学”维生素 66. 661-666 (1992)