Nanoprobing of the effect of Cu²⁺ cations on misfolding, interaction and aggregation of amyloid β peptide

Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer's disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu²⁺. Cu²⁺ cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu²⁺ regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu²⁺ cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu²⁺ cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu²⁺ cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu²⁺ cations induce Aβ42 peptide structural changes resulting in N-termini interactions within the dimers. Cu²⁺ cations also stabilize the dimers. No effects of Cu²⁺ cations on Aβ-Aβ interactions were observed at pH 4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu²⁺ cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu²⁺ cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu²⁺ cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu²⁺ cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu²⁺.