Podlisny M B, Walsh D M, Amarante P, Ostaszewski B L, Stimson E R, Maggio J E, Teplow D B, Selkoe D J
Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 1998 Mar 17;37(11):3602-11. doi: 10.1021/bi972029u.
Amyloid beta-proteins (A beta) are proteolytic fragments of the beta-amyloid precursor protein (beta APP) that are secreted by mammalian cells throughout life but also accumulate progressively as insoluble cerebral aggregates in Alzheimer's disease (AD). Because mounting evidence indicates that A beta aggregation and deposition are early, critical features of AD leading to neurotoxicity, many studies of A beta aggregation have been conducted using synthetic peptides under generally nonphysiological conditions and concentrations. We recently described the oligomerization of A beta peptides secreted by beta APP-expressing cells at low nanomolar (20-30 ng/mL) levels into sodium dodecyl sulfate- (SDS-) stable oligomers of 6-16 kDa. Here, we extensively characterize this in vitro system and show that the amyloid binding dye, Congo red, acts to markedly decrease oligomer/monomer ratios by stabilizing the 4 kDa A beta monomers (ID50 approximately equal to 3.4 microM). Addition of radioiodinated synthetic A beta 1-40 to the cultures or to their conditioned media at physiological concentrations (0.25-2.5 nM) reveals that it undergoes progressive aggregation into SDS-stable oligomers of 6-25 kDa during brief (approximately 4 h) incubation at 37 degrees C, and this is inhibitable by Congo red. The level of A beta oligomers can be quantitated in the Chinese hamster ovary (CHO) conditioned medium by size-exclusion chromatography as well as by SDS-polyacrylamide gel electrophoresis (PAGE), and comparison of these two methods suggests that aggregation of A beta into higher molecular weight polymers that are not detectable by SDS-PAGE occurs in the cultures. We conclude that both endogenous and synthetic A beta can assemble into stable oligomers at physiological concentrations in cell culture, providing a manipulable system for studying the mechanism of early A beta aggregation and identifying inhibitors thereof under biologically relevant conditions.
淀粉样β蛋白（Aβ）是β淀粉样前体蛋白（βAPP）的蛋白水解片段，哺乳动物细胞在整个生命过程中都会分泌该片段，但在阿尔茨海默病（AD）中，它也会逐渐累积形成不溶性脑聚集体。由于越来越多的证据表明Aβ聚集和沉积是AD导致神经毒性的早期关键特征，许多关于Aβ聚集的研究都是在一般非生理条件和浓度下使用合成肽进行的。我们最近描述了表达βAPP的细胞分泌的Aβ肽在低纳摩尔（20 - 30 ng/mL）水平下寡聚化形成6 - 16 kDa的十二烷基硫酸钠（SDS）稳定寡聚物。在此，我们对该体外系统进行了广泛表征，并表明淀粉样结合染料刚果红通过稳定4 kDa的Aβ单体（半数抑制浓度约等于3.4 μM），显著降低寡聚物/单体比例。在生理浓度（0.25 - 2.5 nM）下向培养物或其条件培养基中添加放射性碘化合成Aβ1 - 40，结果显示在37℃短暂孵育（约4小时）期间，它会逐渐聚集成6 - 25 kDa的SDS稳定寡聚物，并且这一过程可被刚果红抑制。可以通过尺寸排阻色谱以及SDS - 聚丙烯酰胺凝胶电泳（PAGE）对中国仓鼠卵巢（CHO）条件培养基中的Aβ寡聚物水平进行定量，这两种方法的比较表明，培养物中会发生Aβ聚集成SDS - PAGE无法检测到的更高分子量聚合物的情况。我们得出结论，内源性和合成的Aβ在细胞培养中都能在生理浓度下组装成稳定的寡聚物，为研究早期Aβ聚集机制以及在生物学相关条件下鉴定其抑制剂提供了一个可操控的系统。
