Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration.
1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.
D015655:1-Methyl-4-phenylpyridinium / Q000633:toxicity; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles / Q000378:metabolism; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D003374:Coumarins / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D004298:Dopamine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D016166:Free Radical Scavengers / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006540:Herbicides / Q000633:toxicity; D066298:In Vitro Techniques; D017895:Manganese Compounds / Q000494:pharmacology; D008636:Mesencephalon / Q000166:cytology / Q000378:metabolism; D008665:Metalloporphyrins / Q000494:pharmacology; D009410:Nerve Degeneration / Q000139:chemically induced / Q000503:physiopathology*; D009942:Organometallic Compounds; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods
D001562:Benzimidazoles; D061945:Caspase Inhibitors; D003374:Coumarins; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D006540:Herbicides; D017895:Manganese Compounds; D008665:Metalloporphyrins; D009942:Organometallic Compounds; D017382:Reactive Oxygen Species; C097284:manganese(III)-tetrakis(4-benzoic acid)porphyrin; C009907:2-methylcyclopentadienyl manganese tricarbonyl; D045304:Cytochromes c; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C028743:7-amino-4-methylcoumarin; C017807:bisbenzimide ethoxide trihydrochloride; D015655:1-Methyl-4-phenylpyridinium; D004298:Dopamine