Dityrosine (DiY), via the cross-linking of tyrosine residues, is a marker of protein oxidation, which increases with aging. Amyloid-b (Ab) forms DiY in vitro and DiYcross-linked Ab is found in the brains of patients with Alzheimer disease. Metal-or UV-catalyzed oxidation of Ab42 results in an increase in DiY cross-links. Using DiY as a marker of oxidation, we compare the self-assembly propensity and DiY crosslink formation for a non-assembly competent variant of Ab42 (vAb) with wildtype Ab42. Oxidation results in the formation of trapped wild-type Ab assemblies with increased DiY cross-links that are unable to elongate further. Assemblyincompetent vAb and trapped Ab assemblies are non-toxic to neuroblastoma cells at all stages of self-assembly, in contrast to oligomeric, non-cross-linked Ab. These findings point to a mechanism of toxicity that necessitates dynamic self-assembly whereby trapped Ab assemblies and assembly-incompetent variant Ab are unable to result in cell death.

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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 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 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 H202, 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.

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A characteristic hallmark of Alzheimers Disease (AD) is the pathological aggregation and deposition of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs). Oxidative stress is an early event during AD pathogenesis and is associated with tau-mediated AD pathology. Oxidative environments can result in the formation of covalent dityrosine crosslinks that can increase protein stability and insolubility. Dityrosine cross-linking has been shown to occur in vivo in Abeta; plaques and alphasynuclein aggregates in Lewy bodies, and this modification may increase the insolubility of these aggregates and their resistance to degradation. Using the PHF-core tau fragment (residues 297-391) as a model, we have previously demonstrated that dityrosine formation traps tau assemblies to reduce further elongation. However, it is unknown whether dityrosine crosslinks are found in tau deposits in vivo in AD and its relevance to disease mechanism is unclear. Here, using transmission electron microscope (TEM) double immunogold-labelling, we reveal that neurofibrillary NFTs in AD are heavily decorated with dityrosine crosslinks alongside tau. Single immunogold-labelling TEM and fluorescence spectroscopy revealed the presence of dityrosine on AD brain-derived tau oligomers and fibrils. Using the tau (297-391) PHF-core fragment as a model, we further showed that prefibrillar tau species are more amenable to dityrosine crosslinking than tau fibrils. Dityrosine formation results in heat and SDS stability of oxidised prefibrillar and fibrillar tau assemblies. This finding has implications for understanding the mechanism governing the insolubility and toxicity of tau assemblies in vivo.