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.