Amyloid Beta Oligomer Studied by Newly Developed Single Molecule Analysis Method

Abstract

Amyloid beta (Ab) is 4 kDa peptide which forms aggregates such as oligomers and fibrils. They have been considered to cause Alzheimer's diseases (AD). Recent results have 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 formation mechanism of these soluble oligomers is still unknown. In this study, we developed new single molecule analysis method that can analyze Ab oligomer distribution. For this purpose, we combined total internal reflection fluorescence microscopy (TIRFM) with photon counting histogram (PCH) (Terada, N. et al. (2007) Biophys. J. 92, 2162). Using TIRFM, fluorescent intensity of monomer is obtained from discrete photobleaching. Using PCH method, the number of protomers in oligomers and concentrations are obtained from histograms of photons from fluorescent molecules diffusing through the confocal volume.A model Ab oligomer was prepared by sonication treatment of Ab fibrils made from FITC labeled Ab monomer. The number of Ab monomer in a single Ab oligomer was estimated by TIRFM, which agrees well with the results of PCH analysis. Thus we concluded that PCH can be applied to analyze Ab oligomer formation. Next, Ab oligomers were formed at physiological Ab concentration (LeVineIII, H. (2004) Anal. Biochem. 335, 81). Oligomer formation was observed with TIRFM analysis. PCH analysis is now in pregress.