| Parkinson's disease (PD) is characterized by the loss of pigmented neurons of the substantia nigra. These neurons are particularly susceptible to oxidative stress, perhaps exacerbated by dopamine contained within. It is believed that the pigment in these neurons is formed as a result of oxidation of dopamine to dopaminochrome (DAC). We believe that DAC may be responsible, in part, for the loss of these neurons in the pathogenesis of PD. Using the murine mesencephalic cell line MN9D, we show that DAC is cytotoxic to MN9D cells in a concentration-dependent manner, and that this toxicity is caused by a rapid increase in oxidative stress within the cell. This increase in oxidative stress is also accompanied by a rapid decrease in the intracellular antioxidant, reduced glutathione, suggesting that the toxicity caused by DAC is directly dependent upon oxidative stress.;Furthermore, we investigate the mode of cell death induced by DAC and show that DAC is cytotoxic despite the presence of the pan-caspase inhibitor, Z-VAD-fmk, suggesting that the caspase system is not involved in DAC-induced cytotoxicity in MN9D cells. While caspases are not activated, other indicators of apoptosis are present, including membrane blebbing, DNA oxidation and fragmentation, and the presentation of phosphatidylserine on the cell membrane surface.;Finally, the toxicity induced by DAC is not affected by blockade of common dopaminergic transport systems, such as the monoamine trasporter or the organic cation transporter. DAC also does not signal through the dopamine receptor, nor other receptors dependent upon the function of lipid rafts. However, blockade of intracellular monoamine oxidase, an enzyme key to dopamine metabolism, is effective at improving cell viability in the presence of DAC, while blockade of xanthine oxidase and NADPH oxidase, both producers of superoxide, is not.;These results demonstrate that DAC causes MN9D cell death in a manner that is dependent upon oxidative stress, yet independent of caspases, with evidence suggesting the involvement of apoptosis inducing factor. This study provides new insights into the pathogenesis of neurodegeneration observed in PD. |
