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

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。将APP互补DNA（cDNA）转染畸胎瘤细胞（P19），建立APP过表达细胞系，结果发现：（1）P19细胞经诱导分化为神经细胞后，内源性APP 695 mRNA水平明显升高; 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。

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.