A novel molecular mechanism for nitrated {alpha}-synuclein-induced cell death.

Although previous studies have demonstrated the involvement of nitrated α-synuclein in neurodegenerative disorders (synucleinopathies), the effects of nitrated α-synuclein and the molecular mechanisms underlying its toxicity are still unclear. In the present study, nitrated α-synuclein with four 3-nitrotyrosines (Tyr(39), Tyr(125), Tyr(133), and Tyr(136)) was obtained non-enzymatically by incubation with nitrite. The nitrated protein existed as a mixture of monomers, dimers, and polymers in solution. The nitrated α-synuclein could induce cell death in a time- and concentration-dependent manner when SH-SY5Y cells (a human neuroblastoma cell line) were incubated with the dimers and polymers. Treatment with anti-integrin α5β1 antibody partially rescued the SH-SY5Y cells from the cell death. Dot blotting and immunoprecipitation revealed that the nitrated protein bound to integrin on the cell membranes. Level of nitric oxide (NO) and calcium-independent inducible NO synthase (iNOS) activity increased during the initial stages of the treatment. The expression of phosphorylated focal adhesion kinase (FAK) decreased in the cells. Subsequently, an increase in caspase 3 activity was observed in SH-SY5Y cells. Our results demonstrate that activation of iNOS and inhibition of FAK may both be responsible for the cell death induced by nitrated α-synuclein. These data suggest that the cytotoxicity of nitrated α-synuclein is mediated via an integrin-iNOS/-FAK signaling pathway, and that the nitration of α-synuclein plays a role in neuronal degeneration.