Induction of α-synuclein aggregation by intracellular nitrative insult

Brain lesions containing filamentous and aggregated alpha -synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha -synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha -synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha -synuclein. Light microscopy using antibodies against alpha -synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha -Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha -synuclein and in 5% of cells expressing A30P mutant alpha -synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta -synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha -synuclein. However, alpha -synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha -synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha -synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha -synuclein, indicating that lysosomes are involved in degradation of alpha -synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha -synuclein aggregates.