Blockade of PKCδ proteolytic activation by loss of function mutants rescues mesencephalic dopaminergic neurons from methylcyclopentadienyl manganese tricarbonyl (MMT)-induced apoptotic cell death

The use of methytcyclopentadienyl manganese tricarbonyl (MMT) as a gasoline additive has raised health concerns and increased interest in understanding the neurotoxic effects of manganese. Chronic exposure to inorganic manganese causes Manganism, a neurological disorder somewhat similar to Parkinson's disease. However, the cellular mechanism by which MMT, an organic manganese compound, induces neurotoxicity in dopaminergic neuronal cells remains unclear. Therefore, we systematically investigated apoptotic cell-signaling events following exposure to 3-200 mu M MMT in mesencephalic dopaminergic neuronal (N27) cells. MMT treatment resulted in a time- and dose-dependent increase in reactive oxygen species generation and cell death in N27 cells. The cell death was preceded by sequential activation of mitochondrial-dependent proapoptotic events including cytochrome c release, caspase-3 activation, and DNA fragmentation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers MMT-induced apoptotic cell death. Importantly, MMT induced proteolytic cleavage of protein kinase C delta (PKC delta), resulting in persistently increased kinase activity. The proteolytic activation of PKC delta was suppressed by treatment with 100 mu M Z-VAD-FMK and 100 mu M Z-DEVD-FMK, suggesting that caspase-3 mediates the proteolytic activation of PKC delta. Pretreatment with 100 mu M Z-DEVD-FMK and 5 mu M rottlerin (a PKC delta inhibitor) also significantly attenuated MMT-induced DNA fragmentation. Furthermore, overexpression of either the kinase inactive dominant negative PKC delta(K376R) mutant or the caspase cleavage resistant PKC delta(D327A) mutant rescued N27 cells from MMT-induced DNA fragmentation. Collectively, these results demonstrate that the mitochondrial-dependent apoptotic cascade mediates apoptosis via proteolytic activation of PKC delta in MMT-induced dopaminergic degeneration and suggest that PKC delta may serve as an attractive therapeutic target in Parkinson-related neurological diseases.