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-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.
Podlisny
M B
MB
Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walsh
D M
DM
Amarante
P
P
Ostaszewski
B L
BL
Stimson
E R
ER
Maggio
J E
JE
Teplow
D B
DB
Selkoe
D J
DJ
D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002850:Chromatography, Gel; D004396:Coloring Agents / Q000378:metabolism / Q000494:pharmacology*; D003224:Congo Red / Q000378:metabolism / Q000494:pharmacology*; D006224:Cricetinae; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism*; D011233:Precipitin Tests
D016229:Amyloid beta-Peptides; D004396:Coloring Agents; D010446:Peptide Fragments; D011108:Polymers; C078592:amyloid beta-protein (1-40); D003224:Congo Red