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

1. 我们使用双位点ELISA和免疫细胞化学检测了许多尸检人脑中的淀粉样β蛋白（Abeta）沉积，发现在正常衰老过程中，Abeta在大脑中积累。大脑中β的水平与老年斑的丰度密切相关。此外，ELISA检测到的Abeta积累先于免疫细胞化学检测到的Abeta在脑内的沉积。本ELISA系统可捕获sds可解离的A13，但不能捕获sds稳定的β二聚体。对Abeta水平低于ELISA检测限的脑组织进行Western blot分析，发现许多样品含有sds稳定的Abeta二聚体，但不含sds可解离的Abeta单体。结果表明，sds稳定的β二聚体在大脑的初始阶段就积累了，这可能表明sds稳定二聚体在老年人脑的β -淀粉样蛋白形成和ad的神经变性中起重要作用。用CsCl等密度梯度离心对正常老年脑组织中沉积的Abeta进行分离，发现部分Abeta被分离成低密度膜组分。然而，从它们的电泳流动性和与各种β抗体的反应性分析来看，这些β分子不太可能与唐氏综合症患者大脑中发现的GM1神经节苷脂结合的β分子相同。我们已经证明，细胞内A13存在于人类神经母细胞瘤细胞的不可溶部分和可溶部分。不溶部分似乎也含有sds稳定的β二聚体。大约一半的不溶性β与小泡样低密度膜结构域有关。用同样的方法从正常衰老的大脑中分离匀浆，发现相当大比例的β定位在低密度的膜域。

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.