The involvement of dityrosine crosslinking in ?-synuclein assembly and deposition in Lewy Bodies in Parkinson's disease

Parkinson's disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable ?-synuclein (?-syn) amyloid fibrils. ?-synuclein is an intrinsically disordered protein that has the capacity to assemble to form ?-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD patients. In vitro, we show that dityrosine cross-links in ?-syn are formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress by fluorescence and confirmed using mass-spectrometry. A covalently cross-linked dimer isolated by SDS-PAGE and mass analysis showed that dityrosine dimer was formed via the coupling of Y39-Y39 to give a homo dimer peptide that may play a key role in formation of oligomeric and seeds for fibril formation. Atomic force microscopy analysis reveals that the covalent dityrosine contributes to the stabilization of ?-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of ?-syn in PD.