Protein chemical analysis of amyloid beta-protein in the brain of early stages of beta-amyloidogenesis

1.我们用双点酶联免疫吸附试验和免疫细胞化学方法检测了许多尸检人脑组织中淀粉样β蛋白(Abeta)的沉积，发现在正常老化过程中Abeta在脑内积聚。大脑中的Abeta水平与老年斑块的丰富程度密切相关。此外，在免疫细胞化学检测到的脑内可检测到的Abeta沉积之前，可通过ELISA法检测到的Abeta积聚。目前的酶联免疫吸附试验系统捕获了SDS可解离的A13，但不能捕获SDS稳定的Aβ二聚体。对Abeta水平低于ELISA检测下限的大脑进行的蛋白质印迹分析表明，许多这类标本含有稳定的Abeta二聚体，但不含有可分解的Abeta单体。结果表明，稳定的十二烷基硫酸酯二聚体在脑内的初始阶段就积累了，这增加了十二烷基硫酸钠稳定的二聚体在老年脑淀粉样蛋白的形成和AD的神经退行性变中发挥重要作用的可能性。用氯化铯等密度梯度离心法对正常老年人脑内沉积的Abeta组分进行分级，发现部分Abeta组分形成低密度膜组分。然而，从它们的电泳迁移率和与各种Abeta抗体的反应性分析来看，这些Abeta分子不太可能与唐氏综合症患者脑中发现的GM1神经节苷脂结合的Abeta分子相同。我们已经证明，细胞内A13存在于人神经母细胞瘤细胞的不溶部分和可溶部分中。不溶部分似乎也含有十二烷基硫酸钠稳定的Aβ二聚体。大约一半的不溶性Abeta与凹陷样的低密度膜结构域有关。用同样的程序对正常老化大脑的匀浆进行分级，发现相当大比例的Abeta定位于低密度膜结构域。

1. We have examined amyloid beta-protein (Abeta) deposition in many autopsied human brains using two-site ELISA and immunocytochemistry, and found that Abeta accumulates in the brain during normal aging. The level of Abeta in the brain correlated well with the abundance of senile plaque. In addition, Abeta accumulation which could be detected by ELISA preceded immunocytochemically detectable Abeta deposition in the brain.2. The present ELISA system captured SDS-dissociable A13 but not SDS-stable Abeta dimer. Western blot analysis of the brains in which the level of Abeta was below the detection limit of ELISA showed that many of such specimens contained SDS-stable Abeta dimer, but not SDS-dissociable Abeta monomer. The results indicate that SDS-stable Abeta dimer accumulates in the brain at the very initial stages, raising a possibility that SDS-stable dimer plays important roles in beta-amyloidogenesis in the aged human brain and the neurodegeneration of AD.3. Fractionation of the deposited Abeta species in normal aged brains by CsCl isopycnic density gradient centrifugation showed that a part of the Abeta was fractionated into the low-density membrane fraction. From analyses of their electrophoretic mobilities and reactivities with various Abeta antibodies, however, these Abeta molecules were not likely to be the same as GM1 ganglioside-bound Abeta found in the Down's syndrome brains.4. We have shown that intracellular A13 is present in the insoluble fractions as well as in the soluble fractions of human neuroblastoma cells. The insoluble fraction appeared to contain also SDS-stable Abeta dimer. Approximately half an amount of the insoluble Abeta was associated with caveolae-like low-density membrane domains. Fractionation of the homogenates from normal aged brains by the same procedure revealed that a substantial proportion of Abeta is localized in the low-density membrane domains.