The involvement of dityrosine crosslinking in α-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 alpha-synuclein (alpha-syn) amyloid fibrils. alpha-synuclein is an intrinsically disordered protein that has the capacity to assemble to form beta-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 postmortem 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 alpha-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 alpha-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of alpha-syn in PD.