Dityrosine cross-link trapping of amyloid-β intermediates reveals that self-assembly is required for Aβ-induced cytotoxicity

Abstract

Multiple chemical reactions, such as the production of reactive oxygen species (ROS) can lead to dityrosine (DiY) formation via the cross-linking of closely spaced tyrosine residues and this can serve as a marker for aging. Amyloid-{beta} (A{beta}) has been found to be DiY cross-linked in the brains of AD patients. In vitro, A{beta} forms DiY cross-links via metal-catalysed oxidation (Cu2+ and H202) (MCO) leading to the formation of fibrils that are resistant to formic acid denaturation. However, copper is well known to influence and enhance self-assembly. Here, to investigate the interplay between self-assembly and DiY cross-linking we have utilised a non-assembly competent variant of A{beta} (vA{beta}). MCO and UV oxidation experiments using vA{beta} and wild-type A{beta}, revealed that DiY cross-linking stabilises, but does not induce or promote A{beta} assembly. Cu2+ alone, without H202, facilitates the formation and DiY cross-linking of wild-type A{beta} into long-lived oligomers. Our work reveals DiY formation halts further A{beta} self-assembly. DiY cross-linked A{beta} is non-toxic to neuroblastoma cells at all stages of self-assembly in contrast to oligomeric non-cross-linked A{beta}. These findings point to a mechanism of toxicity that necessitates continuing self-assembly of the A{beta} peptide, whereby trapped DiY A{beta} assemblies and assembly incompetent variant A{beta} are unable to result in cell death.