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TOXICOLOGY

Protein Kinase C Is a Key Downstream Mediator of Manganese-Induced Apoptosis in Dopaminergic Neuronal Cells

Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, Iowa State University, Ames, Iowa

Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn induces dopaminergic neuronal cell death remains unclear. In the present study, we sought to investigate the key downstream apoptotic cell signaling events that contribute to Mn-induced cell death in mesencephalic dopaminergic neuronal (N27) cells. Mn exposure induced a dose-dependent increase in neuronal cell death in N27 cells. The cell death was accompanied by sequential activation of mitochondrial-dependent proapoptotic events, including cytochrome c release, caspase-3 activation, and DNA fragmentation, but not caspase-8 activation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers Mn-induced apoptosis. Notably, Mn treatment proteolytically activated protein kinase C (PKC), a member of a novel class of protein kinase C. The caspase-3 specific inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK) significantly blocked PKC cleavage and its kinase activity, indicating that caspase-3 mediates the proteolytic activation. Cotreatment with the PKC inhibitor rottlerin or the caspase-3 inhibitor Z-DEVD-FMK almost completely blocked Mn-induced DNA fragmentation. Additionally, N27 cells expressing a catalytically inactive PKC^K376R protein (PKC dominant negative mutant) or a caspase cleavage resistant PKC^D327A protein (PKC cleavage resistant mutant) were found to be resistant to Mn-induced apoptosis. To further establish the proapoptotic role of PKC, RNA interference-mediated gene knockdown was performed. Small interfering RNA suppression of PKC expression protected N27 cells from Mn-induced apoptotic cell death. Collectively, these results suggest that caspase-3-dependent proteolytic activation of PKC plays a key role in Mn-induced apoptotic cell death.

Address correspondence to: Dr. A. G. Kanthasamy, Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, 2008 Veterinary Medicine Bldg., Iowa State University, Ames, IA 50011-1250. E-mail: akanthas{at}iastate.edu

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