Figure 2.

Metal-Catalyzed Oxidation Results in the Formation of Stabilized Aβ Assemblies

(A–E) Th-T fluorescence was monitored for the freshly prepared Aβ and vAβ (50 μM) incubated at 37°C, without CuCl₂, with CuCl₂ (400 μM), or CuCl₂ in combination with H₂O₂ (A). Dot blotting using NU-1 antibody identified Aβ oligomers in the Aβ samples, but not in vAβ reactions (B). Quantification of dot blotting signal over time reveals that the oligomers in the CuCl₂-oxidized Aβ remain stable for 5 days, unlike in the other reactions in which very low NU-1 affinity signal was observed (C). CD at 5 days showed a high β-sheet content in the unoxidized Aβ sample, with reduced signal for CuCl₂ Aβ, and CuCl₂/H₂O₂Aβ. All vAβ samples showed spectra consistent with random coil conformation (D). TEM imaging at 5 days revealed a network of fibers in the unoxidized Aβ, whereas the CuCl₂ Aβ showed clumped assemblies with very little fiber density and the CuCl₂/H₂O₂ sample revealed amorphous-like assemblies. Unoxidized vAβ showed no assemblies, whereas both oxidized vAβ reactions showed amorphous-like aggregates (E). A minimum of three independent experiments was repeated to ensure the reproducibility of the findings. Scale bars, 500 nm. Error bars are expressed as ±SEM.