title: Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKCδ in cell culture and animal models of Parkinson's disease.
abstract: 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 μM) for 24h 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 μM) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKCδ) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 μM). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKCδ(D327A) and kinase dead PKCδ(K376R) or siRNA-mediated knockdown of PKCδ protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKCδ promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKCδ expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKCδ cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKCδ(D327A) protein protected against 6-OHDA-induced PKCδ activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKCδ is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD.
authors.affiliation: Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011-1250, USA.
authors.forename: Calivarathan
authors.initials: C
authors.lastname: Latchoumycandane
authors.forename: Vellareddy
authors.initials: V
authors.lastname: Anantharam
authors.forename: Huajun
authors.initials: H
authors.lastname: Jin
authors.forename: Anumantha
authors.initials: A
authors.lastname: Kanthasamy
authors.forename: Arthi
authors.initials: A
authors.lastname: Kanthasamy
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D059290:Dopaminergic Neurons / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D016627:Oxidopamine / Q000633:toxicity*; D010302:Parkinson Disease, Secondary / Q000139:chemically induced*; D051745:Protein Kinase C-delta / Q000378:metabolism*; D059748:Proteolysis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D016627:Oxidopamine; D045304:Cytochromes c; D051745:Protein Kinase C-delta; D053148:Caspase 3; D053453:Caspase 9