Aggregation of secreted amyloid β-protein into sodium dodecyl sulfate- stable oligomers in cell culture

Abstract

Filamentous aggregates of the 40-42-residue amyloid β-protein (Aβ) 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 Aβ is relatively inert, whereas aggregated Aβ reproducibly exerts a variety of neurotoxic effects. The processes that mediate the conversion of monomeric Aβ 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 Aβ peptides under nonbiological conditions. We report bore the detection of small amounts (<10-9 M) of SDS-stable Aβ oligomers in the culture media of Chinese hamster ovary cells expressing endogenous or transfected amyloid β- protein precursor genes. The identity of these oligomers (primarily dimers and trimers) was established by immunoprecipitation with a panel of Aβ antibodies, by electrophoretic comigration with synthetic Aβ oligomers, and by amino acid sequencing. The oligomeric Aβ species comprised ~10-20% of the total immunoprecipitable Aβ in these cultures. A truncated Aβ species beginning at Arg 5 was enriched in the oligomers, suggesting that amino- terminal heterogeneity can influence Aβ oligomerization in this system. Addition of Congo red (10 μM) during metabolic labeling of the cells led to increased monomeric and decreased oligomeric Aβ. The ability to detect and quantitate oligomers of secreted Aβ peptides in cell culture should facilitate dynamic studies of the critical process of initial Aβ aggregation under physiological conditions.