Molecular biological study on the mechanism of neuronal degeneration using teratoma cells

利用畸胎瘤细胞研究神经元变性机制的分子生物学

• 批准号: 02455028
• 负责人: YOSHIKAWA Kazuaki
• 金额: $ 3.78万
• 依托单位: Tokyo Institute of Psychiatry
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02455028/
• 关键词: Alzheimer's disease; amyloid protein; Gene transfection; Teratoma cells; Neural differentiation; Neurons; Aging; Degeneration; アルツハイマ-病; テラト-マ細胞; ニュ-ロン; ニュ-ロン変性; アミロイド; 遺伝子発現

Alzheimer disease is characterized by the deposition of amyloid fibrils whose major constituent is beta protein. Beta-protein may be generated by abnormal proteolysis of the precursor, amyloid protein precursor (APP). Molecular mechanism of amyloid formation is as yet unclear at present. We have investigated the molecular mechanism of amyloidogenesis using cultured cell models. We have transfected APP complementary DNA (cDNA) to teratoma cells (P19) to establish the cell lines overexpressing APP. Our findings are as follows:1) The endogenous level of APP 695 mRNA was markedly increased when P19 cells were induced to differentiate into neural cells. The pat- tern of APP 695 mRNA changes resembles that in differentiating neurons in vivo.2) We transfected APP cDNA into undifferentiated P19 cells, and established stable transfectants. When the cells were treated to differentiate, almost all the differentiated neurons showed severe degeneration, which may be due to intracellular accumulations of potentially amyloidogenic fragments of APP.3) The remaining (surviving) cells contained a high level of APP immunoreactivity in lysosome-like organelles. Therefore, if is likely that the cytotoxic fragments of APP may be generated in lysosomes.These findings suggest a new concept that abnormal metabolism of APP in neurons generates cytotoxic fragments of APP, which cause neuronal death followed by amyloid formation in the extracellular space. The teratoma cells transfected with the APP gene is a useful tool for molecular dissection of the relationship between amyloidogenesis and neuronal degeneration.

阿尔茨海默病的特征是淀粉样纤维的沉积，其主要成分是β蛋白。β-蛋白可以通过前体淀粉样蛋白前体（APP）的异常蛋白水解产生。淀粉样蛋白形成的分子机制目前尚不清楚。我们使用培养的细胞模型研究了淀粉样蛋白生成的分子机制。我们将APP互补DNA（cDNA）转染到畸胎瘤细胞（P19）中，建立了过表达APP的细胞系，研究结果如下：1）诱导P19细胞分化为神经细胞时，内源性APP 695 mRNA水平明显增加。APP 695 mRNA的变化规律与体内分化神经元相似。2）将APP cDNA转染未分化的P19细胞，建立稳定的转染细胞。当细胞分化时，几乎所有分化的神经元都表现出严重的变性，这可能是由于APP潜在的淀粉样蛋白片段在细胞内积聚所致。3）剩余的（存活的）细胞在溶酶体样细胞器中含有高水平的APP免疫反应性。因此，如果APP的细胞毒性片段可能是在溶酶体中产生的，这些发现提出了一个新的概念，即APP在神经元中的异常代谢产生APP的细胞毒性片段，导致神经元死亡，然后在细胞外空间形成淀粉样蛋白。用APP基因转染的畸胎瘤细胞是一个有用的工具，分子解剖之间的关系淀粉样变性和神经元变性。

项目成果

丸山 敬: "Alzheimer病及タンパク質とその前駆体ーAmyloid protein precursor (APP)の分子生物学ー" 神経精神薬理. 13. 743-751 (1991)

Takashi Maruyama：“阿尔茨海默病蛋白及其前体-淀粉样蛋白前体（APP）的分子生物学”《神经精神药理学》13. 743-751（1991）。

吉川 和明: "アルツハイマ-病ーアミロイドβ/A4タンパク質からのアプロ-チー" 細胞工学. 10. 61-66 (1991)

Kazuaki Yoshikawa：“阿尔茨海默病 - 淀粉样蛋白 β/A4 蛋白的方法”《细胞工程》10. 61-66 (1991)。

Yoshikawa,Kazuaki: "Neural differentiation increases expression of Alzheimer amyloid protein precursor gene in murine embryonal carcinoma cells" Bioehemical and Biophysical Research Cammunications. 171. 204-209 (1990)

Yoshikawa，Kazuaki：“神经分化增加了小鼠胚胎癌细胞中阿尔茨海默淀粉样蛋白前体基因的表达”生物化学和生物物理研究通信。

Maruyawa,Kei: "Formation of amyloid-like fibrils in COS cells overexpressing part of the Alzheimer amyloid protein precursor" Nature. 347. 566-569 (1990)

Maruyawa, Kei：“在过度表达部分阿尔茨海默淀粉样蛋白前体的 COS 细胞中形成淀粉样蛋白样原纤维”，《Nature》。

Kazuaki Yoshikawa, Kei Maruyama, Kiyoshi Terakado, Mihoko Usami, and Takako Aizawa: "A DNA-transfected cell model for Alzheimer amyloidogenesis: Demonstration of primordial amyloid fibrils in COS cells" Frontiers of Alzheimer Research (Eds. Ishii, Allsop,

Kazuaki Yoshikawa、Kei Maruyama、Kiyoshi Terakado、Mihoko Usami 和 Takako Aizawa：“用于阿尔茨海默病淀粉样蛋白形成的 DNA 转染细胞模型：COS 细胞中原始淀粉样原纤维的演示”阿尔茨海默病研究前沿（Eds. Ishii，Allsop，