Amyloid Beta Oligomer Formation Analysis by Photon Counting Histogram

Abstract

Amyloid beta (Ab) is 4 kDa peptide which is thought to form aggregates such as oligomers and fibrils, and to cause Alzheimer disease (AD). Recently, it has been suggested that soluble Ab oligomers are the causative agent of AD since such oligomers are more cytotoxic than fibrils. It was also suggested that Ab oligomers affect not only cell death but also early stage of cell dysfunction and cause memory loss.However, the mechanism how soluble oligomers are produced is still unknown. In this study, we analyzed formation of Ab oligomers in vitro at a single molecule level using photon counting histogram (PCH)(Chen, Y. et al. (1999) Biophys. J. 77, 553-67; Terada, N. et al. (2007) Biophys. J. 92, 2162-71.). Using PCH method, the number of protomers in oligomers and concentrations are obtained from histograms of photons from fluorescent molecules. Combination of a conforcal optics and a photon counting sensor enables us to catch the fluorescence from molecules diffusing through the conforcal volume at PCH. The concentration distribution of oligomers can be calculated from histograms.Fluorescent intensity of fluorescein-labeled Ab monomer (FL-Ab) was evaluated using PCH. After 30 min incubation of FL-Ab in buffer solution, dimer fraction was successfully observed with PCH, assuming that fluorescent intensity is in proportion to the number of protomers in oligomers. Detailed analysis of formation of Ab oligomers such as amyloid-beta derived diffusible ligands (ADDLs) using these techniques is in progress.