Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKCδ in cell culture and animal models of Parkinson's disease

The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 mu M) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 mu M) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKC delta) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 mu M). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKC delta(D327A) and kinase dead PKC delta(K376R) or siRNA-mediated knockdown of PKC delta protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKC delta promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKC delta expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKC delta cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of pKC delta(D327A) protein protected against 6-OHDA-induced PKC delta activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKC delta is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD. Published by Elsevier Inc.