Induction of alpha-synuclein aggregation by intracellular nitrative insult

Brain lesions containing filamentous and aggregated alpha -synuclein are ha llmarks of neurodegenerative synucleinopathies. Oxidative stress has been i mplicated in the formation of these lesions. Using HEK 293 cells stably tra nsfected with wild-type and mutant alpha -synuclein, we demonstrated that i ntracellular generation of nitrating agents results in the formation of alp ha -synuclein aggregates. Cells were exposed simultaneously to nitric oxide - and superoxide-generating compounds, and the intracellular formation of p eroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodami ne 123 and the nitration of alpha -synuclein. Light microscopy using antibo dies against alpha -synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was gene rated but not when cells were exposed to nitric oxide- or superoxide-genera ting 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 ma jority of the untransfected cells and cells expressing alpha -synuclein. Ho wever, alpha -synuclein did not colocalize with these aggregates, indicatin g that inhibition of the proteasome does not promote alpha -synuclein aggre gation. In addition, proteasome inhibition did not alter the steady-state l evels of alpha -synuclein, but addition of the lysosomotropic agent ammoniu m chloride significantly increased the amount of alpha -synuclein, indicati ng that lysosomes are involved in degradation of alpha -synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha -synuclein aggregates.