Video imaging of structural dynamics of amyloid β protofibrils.

Soluble aggregates including oligomers and protofibrils (PFs) of amyloid β (Aβ) have been reported to be more toxic than mature fibrils (MFs). Recently, clinical trials of antibodies targeting these aggregates are also in progress. Their structure is dynamic, which makes it difficult to extract and analyze them in a stable state. Therefore, the molecular process among the soluble Aβ aggregates has been unclear. Development of high-speed atomic force microscopy (HS-AFM) allows video-imaging of structural dynamics of individual amyloid aggregates in liquid. Here, we investigated structural dynamics and mechanical property of Aβ protofibrils and anti-Aβ antibody binding to PFs, using HS-AFM.Aβ1-42 PF and MF were prepared in vitro, which was deposited on the HS-AFM stage. Structural dynamics of the aggregates were observed by HS-AFM in phosphate buffered saline (PBS). Young's moduli of the individual aggregates were measured by force spectrometry mode of HS-AFM in PBS. The anti-Aβ antibody was added in the chamber, followed by HS-AFM observation of the antibody binding to PFs.Aβ1-42 PF showed diffusive 2D motion on bare mica and its typical structure was composed of several globular parts in a polygonal line. The angle between each segment of a PF was maintained during the diffusive motion. The length of PFs showed a single exponential distribution. Its mean length depended on PF concentration. The Young's modulus of PFs was higher than that of MFs in liquid. Lecanemab (BAN2401) bounded to PFs both most rapidly and abundantly, while 4G8 showed the weakest and least binding to PFs.Assembly and disassembly of the globular parts in PFs are dynamic, while their segment structure is stiffer than MFs and the bonds between the globular parts are rigid. These characteristics indicated the difference in internal structure between PFs and MFs, which supports that PFs may not be a direct precursor of MFs. Lecanemab binds to all various parts and length of PFs.