Siddharth Kaul

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

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.

Suppression of caspase-3-dependent proteolytic activation of protein kinase Cδ by small interfering RNA prevents MPP+-induced dopaminergic degeneration

The cellular mechanisms underlying the neurodegenerative process in Parkinsons disease are not well understood. Using RNA interference (RNAi), we demonstrate that caspase-3-dependent proteolytic activation of protein kinase Cdelta (PKCdelta) contributes to the degenerative process in dopaminergic neurons. The Parkinsonian toxin MPP(+) activated caspase-3 and proteolytically cleaved PKCdelta into catalytic and regulatory subunits, resulting in persistent kinase activation in mesencephalic dopaminergic neuronal cells. The caspase-3 inhibitor Z-DEVD-FMK and the caspase-9 inhibitor Z-LEHD-FMK effectively blocked MPP(+)-induced PKCdelta proteolytic activation. To characterize the functional role of PKCdelta activation in MPP(+)-induced dopaminergic cell death, RNAi-mediated gene knockdown was performed. Among four siRNAs designed against PKCdelta, two specifically suppressed PKCdelta expression. The application of siRNA abolished the MPP(+)-induced PKCdelta activation, DNA fragmentation, and tyrosine hydroxylase (TH)-positive neuronal loss. Together, these results suggest that proteolytic activation of PKCdelta may be a critical downstream event in the degenerative process of Parkinsons disease.

Chronic low dose oxidative stress induces caspase-3 dependent PKCδ proteolytic activation and apoptosis in a cell culture model of dopaminergic neurodegeneration

Oxidative stress has been implicated as a key event in the degenerative process of dopaminergic neurons; however, the cellular mechanisms underlying chronic oxidative stress–induced neurodegeneration remain to be established. In this study, N27 cells, a dopaminergic neuronal cell line derived from rat mesencephalon, exposed to low doses of H2O2 (0–30 μM for 12–24 hr) exhibited dose‐ and time‐dependent increases in cytotoxicity and ROS generation. In addition, the H2O2‐induced neurotoxicity was accompanied by increased caspase‐3 activity and PKCδ cleavage. Notably, treatment with antioxidants Trolox and MnTBAP or PKCδ cleavage inhibitor z‐DIPD‐fmk significantly protected against oxidative stress–induced apoptotic cell death. These results demonstrate that the N27 cell line is a useful model for the study of the chronic low‐dose oxidative stress–induced apoptotic cell death cascade and that caspase‐3‐dependent PKCδ proteolytic activation may be important in the apoptotic process in dopaminergic neurons undergoing chronic oxidative insult.