Amyloid fibrils comprising primarily the peptides Aβ40 and Aβ42 are a defining feature of the Alzheimer's disease (AD) brain, and convergent evidence suggests that the process of their formation plays a central role in the AD pathogenic pathway. Elucidation of fibril assembly is critical for the discovery of potential AD diagnostics and therapeutics, since the pathogenic entity is not necessarily the product fibril, but could be a precursor species whose formation is linked to fibrillogenesis in vivo. Atomic force microscopy allowed the identification of an unanticipated intermediate in in vitro fibril formation, the Aβ amyloid protofibril. This manuscript describes studies of the structure of the Aβ40 protofibril and its in vitro assembly and disassembly using atomic force microscopy (AFM). The Aβ40 protofibril has a height of ca. 4.3 ± 0.5 nm and a periodicity of ca. 20 ± 4.7 nm. The rate of its elongation depends on the total concentration of Aβ40, the temperature, and ionic strength of the medium. Aβ42 and Aβ40 protofibrils elongate at a comparable rate. Statistical analysis of AFM data reveals a decrease in the number of protofibrils with time, indicating that coalescence of smaller protofibrils contributes to protofibril elongation. Similar analysis reveals that protofibrils shorten while the number of protofibrils also decrease following dilution, indicating that protofibril disassembly does not proceed by a reverse of the assembly process. These investigations provide systematic data defining factors affecting Aβ fibrillization and, thus, should be valuable in the design of high-throughput assays to identify agents which alter Aβ protofibril assembly.
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