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

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-β (Aβ) has been found to be DiY cross-linked in the brains of AD patients. In vitro, Aβ forms DiY cross-links via metal-catalysed oxidation (Cu 2+ and H 2 0 2 ) (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 Aβ (vAβ). MCO and UV oxidation experiments using vAβ and wild-type Aβ, revealed that DiY cross-linking stabilises, but does not induce or promote Aβ assembly. Cu 2+ alone, without H 2 0 2 , facilitates the formation and DiY cross-linking of wild-type Aβ into long-lived oligomers. Our work reveals DiY formation halts further Aβ self-assembly. DiY cross-linked Aβ is non-toxic to neuroblastoma cells at all stages of self-assembly in contrast to oligomeric non-cross-linked Aβ. These findings point to a mechanism of toxicity that necessitates continuing self-assembly of the Aβ peptide, whereby trapped DiY Aβ assemblies and assembly incompetent variant Aβ are unable to result in cell death.