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

阿尔茨海默病的特点是淀粉样原纤维沉积，其主要成分是β蛋白。β蛋白可能是由淀粉样蛋白前体（APP）的异常蛋白水解产生的。淀粉样蛋白形成的分子机制目前尚不清楚。我们利用培养细胞模型研究了淀粉样蛋白形成的分子机制。我们将APP互补DNA （cDNA）转染到畸胎瘤细胞（P19）中，建立过表达APP的细胞系。我们的研究结果如下：1)诱导P19细胞向神经细胞分化时，内源性APP 695 mRNA水平显著升高。APP 695 mRNA的变化模式与体内分化神经元相似。2)将APP cDNA转染到未分化的P19细胞中，建立了稳定的转染物。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）的细胞在溶酶体样细胞器中含有高水平的APP免疫反应性。因此，APP的细胞毒性片段很可能在溶酶体中产生。这些发现提示了一个新的概念，即神经元中APP的异常代谢会产生APP的细胞毒性片段，导致神经元死亡，并在细胞外空间形成淀粉样蛋白。转染APP基因的畸胎瘤细胞为淀粉样变性与神经元变性之间的分子解剖提供了有用的工具。

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.