Cell-surface Initial Aggregation Process of Amyloid-β Peptides Studied by Fluorescence Correlation Spectroscopy

The formation of neurotoxic aggregates by amyloid-beta peptide (A beta) is considered to be a key step in the onset of Alzheimer's disease. It is widely accepted that oligomers are more neurotoxic than amyloid fibrils in the aqueous-phase aggregation of A beta. Membrane-mediated amyloidogenesis is also relevant to the pathology, although the relationship between the aggregate size and cytotoxicity has remained elusive. Fluorescence correlation spectroscopy (FCS) is a sensitive method to monitor molecular aggregation processes by measuring diffusion of fluorophore-labeled molecules. Here, we monitored the initial aggregation process of A beta on cell membranes of SH-SY5Y neuroblastoma cells. For example, aggregation of A beta-(1.42) was evaluated by using Ab-(1.42) (5 mM) doped with fluorophore-A beta-(1.42) (10 nM). A membrane-bound Ab component was detected in FCS autocorrelation curve after 1-h incubation of the A beta s with the cells. Following incubation for similar to 10 h, A beta-(1.42) formed oligomers composed of similar to 10 A beta molecules. These Ab oligomers formed on membranes did not induce activation of caspase-3, an eSector caspase for apoptosis, therefore were not neurotoxic, in contrast to reported A beta oligomers prepared in aqueous phase. Formation of larger A beta flbrils on membranes was found to be critical for Ab neurotoxicity. We also report that trace amounts of pyroglutaminated A beta(3.42), a minor species of A beta, can enhance initial aggregation process of A beta-(1.42) on the cell membranes. These results indicate the usefulness of FCS detection of small A beta oligomers formed on cell surface, which can act as pathogenic seeds for amyloid fibrils responsible for neurotoxicity.