Copper Mediates Dityrosine Cross-Linking of Alzheimer's Amyloid-β

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Abstract
We have previously reported that amyloid Aβ, the major component of senile plaques in Alzheimer's disease (AD), binds Cu with high affinity via histidine and tyrosine residues [Atwood, C. S., et al. (1998) J. Biol. Chem. 273, 12817−12826; Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219−1233] and produces H2O2 by catalyzing the reduction of Cu(II) or Fe(III) [Huang, X., et al. (1999) Biochemistry 38, 7609−7616; Huang, X., et al. (1999) J. Biol. Chem. 274, 37111−37116]. Incubation with Cu induces the SDS-resistant oligomerization of Aβ [Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219−1233], a feature characteristic of neurotoxic soluble Aβ extracted from the AD brain. Since residues coordinating Cu are most vulnerable to oxidation, we investigated whether modifications of these residues were responsible for Aβ cross-linking. SDS-resistant oligomerization of Aβ caused by incubation with Cu was found to induce a fluorescence signal characteristic of tyrosine cross-linking. Using ESI-MS and a dityrosine specific antibody, we confirmed that Cu(II) (at concentrations lower than that associated with amyloid plaques) induces the generation of dityrosine-cross-linked, SDS-resistant oligomers of human, but not rat, Aβ peptides. The addition of H2O2 strongly promoted Cu-induced dityrosine cross-linking of Aβ1−28, Aβ1−40, and Aβ1−42, suggesting that the oxidative coupling is initiated by interaction of H2O2 with a Cu(II) tyrosinate. The dityrosine modification is significant since it is highly resistant to proteolysis and is known to play a role in increasing structural strength. Given the elevated concentration of Cu in senile plaques, our results suggest that Cu interactions with Aβ could be responsible for causing the covalent cross-linking of Aβ in these structures.