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Caspase-3 dependent proteolytic activation of protein kinase Cδ mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration

Authors

  • Siddharth Kaul,

    1. Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, 2062 Veterinary Medicine Building, Iowa Sate University, Ames, IA 50011, USA
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  • Arthi Kanthasamy,

    1. Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, 2062 Veterinary Medicine Building, Iowa Sate University, Ames, IA 50011, USA
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  • Masashi Kitazawa,

    1. Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, 2062 Veterinary Medicine Building, Iowa Sate University, Ames, IA 50011, USA
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  • Vellareddy Anantharam,

    1. Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, 2062 Veterinary Medicine Building, Iowa Sate University, Ames, IA 50011, USA
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  • Anumantha G. Kanthasamy

    1. Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, 2062 Veterinary Medicine Building, Iowa Sate University, Ames, IA 50011, USA
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: Dr A.G. Kanthasamy, as above.
E-mail: akanthas@iastate.edu.

Abstract

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 Cδ (PKCδ) 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 PKCδ (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 PKCδ 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 PKCδ specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKCδ cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKCδ. Intracellular delivery of catalytically active recombinant PKCδ significantly increased caspase-3 activity, further indicating that PKCδ regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKCδK376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKCδ in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKCδ in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.

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