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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
Author(s) -
Kaul Siddharth,
Kanthasamy Arthi,
Kitazawa Masashi,
Anantharam Vellareddy,
Kanthasamy Anumantha G.
Publication year - 2003
Publication title -
european journal of neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.346
H-Index - 206
eISSN - 1460-9568
pISSN - 0953-816X
DOI - 10.1046/j.1460-9568.2003.02864.x
Subject(s) - rottlerin , protein kinase c , microbiology and biotechnology , caspase 3 , cytochrome c , caspase , dna fragmentation , apoptosis , chemistry , biology , programmed cell death , kinase , biochemistry
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 PKCδ K376R 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.