OLIGOMERIZATION OF ENDOGENOUS AND SYNTHETIC AMYLOID BETA-PROTEIN AT NANOMOLAR LEVELS IN CELL-CULTURE AND STABILIZATION OF MONOMER BY CONGO RED

Amyloid beta-proteins (A beta) are proteolytic fragments of the beta-a myloid precursor protein (beta APP) that are secreted by mammalian cel ls throughout life but also accumulate progressively as insoluble cere bral 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 aggreg ation have been conducted using synthetic peptides under generally non physiological conditions and concentrations. We recently described the oligomerization of A beta peptides secreted by beta APP-expressing ce lls 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 congruent to 3.4 mu M). Addition of radio iodinated synthetic A beta(1-40) to the cultures or to their condition ed media at physiological concentrations (0.25-2.5 nM) reveals that it undergoes progressive aggregation into SDS-stable oligomers of 6-25 k Da during brief (similar to 4 h) incubation at 37 degrees C, and this is inhibitable by Congo red. The level of A beta oligomers can be quan titated in the Chinese hamster ovary (CHO) conditioned medium by size- exclusion chromatography as well as by SDS-polyacrylamide gel electrop horesis (PAGE), and comparison of these two methods suggests that aggr egation of A beta into higher molecular weight polymers that are not d etectable by SDS-PAGE occurs in the cultures. We conclude that both en dogenous and synthetic A beta can assemble into stable oligomers at ph ysiological concentrations in cell culture, providing a manipulable sy stem for studying the mechanism of early A beta aggregation and identi fying inhibitors thereof under biologically relevant conditions.