Key Nucleation Stages and Associated Molecular Determinants and Processes in pH-Induced Formation of Amyloid Beta Oligomers as Revealed by High-Speed AFM

Non-fibrillar oligomers formed via nucleation from amyloid beta (AB) peptides are currently implicated in the neurotoxicity of Alzheimers disease. Thus, shedding light on the molecular mechanisms underlying their formation is important for identifying targets for drug therapies. This is however an enduring challenge due to the inability to detect AB nucleation processes in the lag phase from bulk kinetic assays, while time-course analyses using a series of peptide solutions involve the discontinuous observation of dynamic nucleation processes and pathways. In this study, by adjusting the pH of AB42 peptide samples while simultaneously imaging with high-speed atomic force microscopy (HS-AFM), we show the in-situ, continuous visualization of dynamic AB42 nucleation at the molecular level. The process reveals a pH-induced saturation regime, enabling a critical monomer substrate concentration to initiate the birth of nucleation. A number of key nucleation phases are identified, including pre-nucleation, saturation regime (mass surface adsorption), nucleation and post-nucleation growth, eventually leading to the formation of predominately oligomer species. HS-AFM observations further reveal the distinct, molecular processes associated with each nucleation phase that constitute the path-dependent formation of different AB species, namely an intial monomer "diffuse-like" surface layer, followed by the emergence of nuclei and then subsequent formation and growth of new complexes and oligomers. In particular, the ability of individual nuclei to undergo surface diffusion and establish new complexes via binding interactions with other species encountered in the system was found to be a significant mechanism influencing the growth of oligomers. Herein, the study contributes to current AB nucleation theories by ascribing new molecular mechanisms. More generally, the knowledge gained from single molecule techniques can greatly assist in our current understanding of various biological processes of AB peptide such as nucleation, growth, aggregation and their related kinetic pathways.