Single Molecule Studies of

[摘要] The formation of beta-amyloid fibrils in patient’s brain tissues has been the hallmark of Alzheimer’s disease. However, recent evidence suggests the early oligomers of beta-amyloid peptides are the origin of neurotoxicity. While the importance of identifying the toxic oligomeric species is widely recognized, its realization has been challenging because these oligomers are metastable, occur at low concentration, and are characterized by a high degree of heterogeneity.This doctoral thesis focuses on the study of beta-amyloid(1-40) oligomer and its interaction with lipid membrane through a novel single molecule approach. In the first part of the thesis (Chapters II & III), a single molecule methodology based on photobleaching is developed to identify the beta-amyloid(1-40) oligomeric species. By directly counting the photobleaching steps in the fluorescence, we can determine the number of subunits in individual beta-amyloid(1-40) oligomers. The results are further analyzed by comparison with stochastic simulations to show that the variability seen in the size of photobleaching steps can be explained by assuming random dipole orientations for the fluorophores in a given oligomer. In addition, by accounting for biasing the oligomer size distribution due to thresholding, the results can be made more quantitative, and show good agreement with the oligomer size distribution determined using HPLC gel filtration.In the second part of this thesis (Chapter IV), the interaction of beta-amyloid(1-40) peptide with supported planar lipid membrane is investigated in detail through single molecule imaging techniques. The evolution of beta-amyloid species on lipid membranes is monitored for up to a few days. The results indicate a tight, uniform binding of beta-amyloid(1-40) peptides onto lipid membranes, followed by oligomer formation. The size of the beta-amyloid(1-40) oligomers and the rate of their formation are highly dependent on the peptide concentration. Our results suggest there are two different pathways of oligomer formation, which lead to drastically different oligomeric species formed in the membrane.

[发布机构] University of Michigan

[效力级别] Beta-amyloid

[关键词] Single Molecule;Beta-amyloid;Alzheimer"s Disease;Photobleaching;Oligomer;Membrane;Physics;Science;Biophysics