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

基本信息

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

项目摘要

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原纤维的沉积，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。目前淀粉样蛋白形成的分子机制尚不清楚。我们使用培养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) 转染至畸胎瘤细胞 (P19) 中，以建立过表达 APP 的细胞系。我们的研究结果如下：1）当P19细胞被诱导分化为神经细胞时，APP 695 mRNA的内源水平显着增加。 APP 695 mRNA的变化模式与体内分化神经元的变化模式相似。2）我们将APP cDNA转染到未分化的P19细胞中，并建立了稳定的转染子。当细胞进行分化处理时，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的潜在淀粉样蛋白片段在细胞内积累所致。3）剩余（存活）的细胞在溶酶体样中含有高水平的APP免疫反应性细胞器。因此，APP的细胞毒性片段很可能是在溶酶体中产生的。这些发现提出了一个新的概念，即神经元中APP代谢异常会产生APP的细胞毒性片段，导致神经元死亡，随后在细胞外空间形成淀粉样蛋白。转染 APP 基因的畸胎瘤细胞是分子解剖淀粉样蛋白生成和神经元变性之间关系的有用工具。