Oligomerization of Endogenous and Synthetic Amyloid β-Protein at Nanomolar Levels in Cell Culture and Stabilization of Monomer by Congo Red

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Abstract
Amyloid β-proteins (Aβ) are proteolytic fragments of the β-amyloid precursor protein (βAPP) 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 Aβ aggregation and deposition are early, critical features of AD leading to neurotoxicity, many studies of Aβ aggregation have been conducted using synthetic peptides under generally nonphysiological conditions and concentrations. We recently described the oligomerization of Aβ peptides secreted by βAPP-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 Aβ monomers (ID50 ≅ 3.4 μM). Addition of radioiodinated synthetic Aβ1-40 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 (∼4 h) incubation at 37 °C, and this is inhibitable by Congo red. The level of Aβ 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 Aβ into higher molecular weight polymers that are not detectable by SDS−PAGE occurs in the cultures. We conclude that both endogenous and synthetic Aβ can assemble into stable oligomers at physiological concentrations in cell culture, providing a manipulable system for studying the mechanism of early Aβ aggregation and identifying inhibitors thereof under biologically relevant conditions.