Aggregation of secreted amyloid beta-protein into sodium dodecyl sulfate-stable oligomers in cell culture.
Filamentous aggregates of the 40-42-residue amyloid beta-protein (A beta) accumulate progressively in the limbic and cerebral cortex in Alzheimer's disease, where they are intimately associated with neuronal and glial cytopathology. Attempts to model this cytotoxicity in vitro using synthetic peptides have shown that monomeric A beta is relatively inert, whereas aggregated A beta reproducibly exerts a variety of neurotoxic effects. The processes that mediate the conversion of monomeric A beta into a toxic aggregated state are thus of great interest. Previous studies of this conversion have employed high concentrations (10(-5)-10(-3) M) of synthetic A beta peptides under nonbiological conditions. We report here the detection of small amounts (< 10(-9) M) of SDS-stable A beta oligomers in the culture media of Chinese hamster ovary cells expressing endogenous or transfected amyloid beta-protein precursor genes. The identity of these oligomers (primarily dimers and trimers) was established by immunoprecipitation with a panel of A beta antibodies, by electrophoretic comigration with synthetic A beta oligomers, and by amino acid sequencing. The oligomeric A beta species comprised approximately 10-20% of the total immunoprecipitable A beta in these cultures. A truncated A beta species beginning at Arg 5 was enriched in the oligomers, suggesting that amino-terminal heterogeneity can influence A beta oligomerization in this system. Addition of Congo red (10 microM) during metabolic labeling of the cells led to increased monomeric and decreased oligomeric A beta. The ability to detect and quantitate oligomers of secreted A beta peptides in cell culture should facilitate dynamic studies of the critical process of initial A beta aggregation under physiological conditions.
Podlisny
M B
MB
Department of Neurology, Harvard Medical School, Boston, Massachusetts, USA.
Ostaszewski
B L
BL
Squazzo
S L
SL
Koo
E H
EH
Rydell
R E
RE
Teplow
D B
DB
Selkoe
D J
DJ
D016229:Amyloid beta-Peptides / Q000378:metabolism* / Q000633:toxicity; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D011108:Polymers / Q000378:metabolism; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*
D016229:Amyloid beta-Peptides; D011108:Polymers; D012967:Sodium Dodecyl Sulfate