Role of cytochrome c in α-synuclein radical formation: implications of α-synuclein in neuronal death in Maneb- and paraquat-induced model of Parkinson's disease.

Background: The pathological features of Parkinson's disease (PD) include an abnormal accumulation of α-synuclein in the surviving dopaminergic neurons. Though PD is multifactorial, several epidemiological reports show an increased incidence of PD with co-exposure to pesticides such as Maneb and paraquat (MP). In pesticide-related PD, mitochondrial dysfunction and α-synuclein oligomers have been strongly implicated, but the link between the two has not yet been understood. Similarly, the biological effects of α-synuclein or its radical chemistry in PD is largely unknown. Mitochondrial dysfunction during PD pathogenesis leads to release of cytochrome c in the cytosol. Once in the cytosol, cytochrome c has one of two fates: It either binds to apaf1 and initiates apoptosis or can act as a peroxidase. We hypothesized that as a peroxidase, cytochrome c leaked out from mitochondria can form radicals on α-synuclein and initiate its oligomerization.

Method: Samples from controls, and MP co-exposed wild-type and α-synuclein knockout mice were studied using immuno-spin trapping, confocal microscopy, immunohistochemistry, and microarray experiments.

Results: Experiments with MP co-exposed mice showed cytochrome c release in cytosol and its co-localization with α-synuclein. Subsequently, we used immuno-spin trapping method to detect the formation of α-synuclein radical in samples from an in vitro reaction mixture consisting of cytochrome c, α-synuclein, and hydrogen peroxide. These experiments indicated that cytochrome c plays a role in α-synuclein radical formation and oligomerization. Experiments with MP co-exposed α-synuclein knockout mice, in which cytochrome c-α synuclein co-localization and interaction cannot occur, mice showed diminished protein radical formation and neuronal death, compared to wild-type MP co-exposed mice. Microarray data from MP co-exposed wild-type and α-synuclein knockout mice further showed that the absence of α-synuclein per se or its co-localization with cytochrome c confers protection from MP co-exposure, as several important pathways were unaffected in α-synuclein knockout mice.

Conclusions: Altogether, these results show that peroxidase activity of cytochrome c contributes to α-synuclein radical formation and oligomerization, and that α-synuclein, through its co-localization with cytochrome c or on its own, affects several biological pathways which contribute to increased neuronal death in an MP-induced model of PD.