Chronic low-dose oxidative stress induces caspase-3-dependent PKCdelta 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 H(2)O(2) (0-30 muM for 12-24 hr) exhibited dose- and time-dependent increases in cytotoxicity and ROS generation. In addition, the H(2)O(2)-induced neurotoxicity was accompanied by increased caspase-3 activity and PKCdelta cleavage. Notably, treatment with antioxidants Trolox and MnTBAP or PKCdelta 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 PKCdelta proteolytic activation may be important in the apoptotic process in dopaminergic neurons undergoing chronic oxidative insult.

Pharmacological inhibition of neuronal NADPH oxidase protects against 1-methyl-4-phenylpyridinium (MPP+)-induced oxidative stress and apoptosis in mesencephalic dopaminergic neuronal cells.

Oxidative stress is widely recognized as a key mediator of degenerative processes in Parkinson's disease (PD). Recently, we demonstrated that the dopaminergic toxin MPP+ initiates oxidative stress to cause caspase-3-dependent apoptotic cell death in mesencephalic dopaminergic neuronal (N27) cells. In this study, we determined the source of reactive oxygen species (ROS) produced during MPP+-induced apoptotic cell death. In addition to mitochondria, plasma membrane NADPH oxidase is considered a major producer of ROS inside the cell. Here, we show that N27 neuronal cells express key NADPH oxidase subunits gp91phox and p67phox. We used structurally diverse NADPH oxidase inhibitors, aminoethyl-benzenesulfonylfluoride (AEBSF, 100-1000microM), apocynin (100-1000microM), and diphenylene iodonium (DPI, 3-30microM), to inhibit intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300microM MPP+-induced ROS production for over 45min in N27 cells, in a dose-dependent manner. Further treatment with DPI, apocynin, and SOD also blocked MPP+-induced ROS production. In Sytox cell death assays, co-treatment with AEBSF, apocynin, or DPI for 24h significantly suppressed MPP+-induced cytotoxic cell death. Similarly, co-treatment with these inhibitors also significantly attenuated MPP+-induced increases in caspase-3 enzymatic activity. Furthermore, quantitative DNA fragmentation ELISA assays revealed that AEBSF, DPI, and apocynin rescue N27 cells from MPP+-induced apoptotic cell death. Together, these results indicate for the first time that intracellular ROS generated by NAPDH oxidase are present within the mesencephalic neuronal cells, and are a key determinant of MPP+-mediated dopaminergic degeneration in in vitro models of dopaminergic degeneration. This study supports a critical role of NADPH oxidase in the oxidative damage in PD; targeting this enzyme may lead to novel therapies for PD.

A novel peptide inhibitor targeted to caspase-3 cleavage site of a proapoptotic kinase protein kinase C delta (PKCdelta) protects against dopaminergic neuronal degeneration in Parkinson's disease models.

Oxidative stress and apoptosis are considered common mediators of many neurodegenerative disorders including Parkinson's disease (PD). Recently, we identified that PKCdelta, a member of the novel PKC isoform family, is proteolytically activated by caspase-3 to induce apoptosis in experimental models of PD [Eur. J. Neurosci. 18 (6):1387-1401, 2003; Antioxid. Redox Signal. 5 (5):609-620, 2003]. Since caspase-3 cleaves PKCdelta between proline and aspartate residues at the cleavage site 324DIPD327 to activate the kinase, we developed an irreversible and competitive peptide inhibitor, Z-Asp(OMe)-Ile-Pro-Asp(OMe)-FMK (z-DIPD-fmk), to mimic the caspase-3 cleavage site of PKCdelta and tested its efficacy against oxidative stress-induced cell death in PD models. Cotreatment of z-DIPD-fmk with the parkinsonian toxins MPP(+) and 6-OHDA dose dependently attenuated cytotoxicity, caspase-3 activation, and DNA fragmentation in a mesencephalic dopaminergic neuronal cell model (N27 cells). However, z-DIPD-fmk treatment did not block MPP(+)-induced increases in caspase-9 enzyme activity. The z-DIPD-fmk peptide was much more potent (IC50 6 microM) than the most widely used and commercially available caspase-3 inhibitor z-DEVD-fmk (IC50 18 microM). Additionally, z-DIPD-fmk more effectively blocked PKCdelta cleavage and proteolytic activation than the cleavage of another caspase-3 substrate, poly(ADP-ribose) polymerase (PARP). Importantly, the peptide inhibitor z-DIPD-fmk completely rescued TH(+) neurons from MPP(+)- and 6-OHDA-induced toxicity in mouse primary mesencephalic cultures. Collectively, these results demonstrate that the PKCdelta cleavage site is a novel target for development of a neuroprotective therapeutic strategy for PD.

Tyrosine phosphorylation regulates the proteolytic activation of protein kinase Cdelta in dopaminergic neuronal cells.

Oxidative stress is a key apoptotic stimulus in neuronal cell death and has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson disease (PD). Recently, we demonstrated that protein kinase C-delta (PKCdelta) is an oxidative stress-sensitive kinase that can be activated by caspase-3-dependent proteolytic cleavage to induce apoptotic cell death in cell culture models of Parkinson disease (Kaul, S., Kanthasamy, A., Kitazawa, M., Anantharam, V., and Kanthasamy, A. G. (2003) Eur. J. Neurosci. 18, 1387-1401 and Kanthasamy, A. G., Kitazawa, M., Kanthasamy, A., and Anantharam, V. (2003) Antioxid. Redox. Signal. 5, 609-620). Here we showed that the phosphorylation of a tyrosine residue in PKCdelta can regulate the proteolytic activation of the kinase during oxidative stress, which consequently influences the apoptotic cell death in dopaminergic neuronal cells. Exposure of a mesencephalic dopaminergic neuronal cell line (N27 cells) to H(2)O(2)(0-300 microm) induced a dose-dependent increase in cytotoxicity, caspase-3 activation and PKCdelta cleavage. H(2)O(2)-induced proteolytic activation of PKC was delta mediated by the activation of caspase-3. Most interestingly, both the general Src tyrosine kinase inhibitor genistein (25 microm) and the p60(Src) tyrosine-specific kinase inhibitor (TSKI; 5 microm) dramatically inhibited H(2)O(2) and the Parkinsonian toxin 1-methyl-4-phenylpyridinium-induced PKCdelta cleavage, kinase activation, and apoptotic cell death. H(2)O(2) treatment also increased phosphorylation of PKCdelta at tyrosine site 311, which was effectively blocked by co-treatment with TSKI. Furthermore, N27 cells overexpressing a PKCdelta(Y311F) mutant protein exhibited resistance to H(2)O(2)-induced PKCdelta cleavage, caspase activation, and apoptosis. To our knowledge, these data demonstrate for the first time that phosphorylation of Tyr-311 on PKCdelta can regulate the proteolytic activation and proapoptotic function of the kinase in dopaminergic neuronal cells.

Wild-type alpha-synuclein interacts with pro-apoptotic proteins PKCdelta and BAD to protect dopaminergic neuronal cells against MPP+-induced apoptotic cell death.

Alpha-synuclein is a pre-synaptic protein of unknown function that has been implicated in the pathogenesis of Parkinson's disease (PD). Recently, we demonstrated that 1-methyl-4-phenylpyridinium (MPP+) induces caspase-3-dependent proteolytic activation of PKCdelta, which subsequently contributes to neuronal apoptotic cell death in mesencephalic dopaminergic neuronal cells. In the present study, we examined whether PKCdelta interacts with alpha-synuclein to modulate MPP+-induced dopaminergic degeneration. Over-expression of wild-type human alpha-synuclein in mesencephalic dopaminergic neuronal cells (N27 cells) attenuated MPP+-induced (300 microM) cytotoxicity, release of mitochondrial cytochrome c, and subsequent caspase-3 activation, without affecting reactive oxygen species (ROS) generation. Wild-type alpha-synuclein over-expression also dramatically reduced MPP+-induced caspase-3-mediated proteolytic cleavage of PKCdelta, whereas over-expression of the mutant human alpha-synucleinA53T did not alter the PKCdelta cleavage under similar conditions. Immunoprecipitation-kinase assay revealed reduced PKCdelta kinase activity in wild-type alpha-synuclein over-expressing cells in response to MPP+ treatment. Wild-type alpha-synuclein over-expression also rescued mesencephalic dopaminergic neuronal cells from MPP+-induced apoptotic cell death, while alpha-synucleinA53T exacerbated the MPP+-induced DNA fragmentation. Furthermore, co-immunoprecipitation studies revealed that alpha-synuclein interacts with the pro-apoptotic proteins PKCdelta and BAD, but not with the anti-apoptotic protein Bcl-2 following MPP+ treatment. We also observed that the interaction between PKCdelta and alpha-synuclein does not involve direct phosphorylation. Together, our results demonstrate that wild-type alpha-synuclein interacts with the pro-apoptotic molecules BAD and PKCdelta to protect dopaminergic neuronal cells against neurotoxic insults.

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

The cellular mechanisms underlying the neurodegenerative process in Parkinson's 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 Parkinson's disease.

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.

Caspase-3-dependent proteolytic cleavage of protein kinase Cdelta is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl.

In the present study, we characterized oxidative stress-dependent cellular events in dopaminergic cells after exposure to an organic form of manganese compound, methylcyclopentadienyl manganese tricarbonyl (MMT). In pheochromocytoma cells, MMT exposure resulted in rapid increase in generation of reactive oxygen species (ROS) within 5--15 min, followed by release of mitochondrial cytochrome C into cytoplasm and subsequent activation of cysteine proteases, caspase-9 (twofold to threefold) and caspase-3 (15- to 25-fold), but not caspase-8, in a time- and dose-dependent manner. Interestingly, we also found that MMT exposure induces a time- and dose-dependent proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield 41 kDa catalytically active and 38 kDa regulatory fragments. Pretreatment with caspase inhibitors (Z-DEVD-FMK or Z-VAD-FMK) blocked MMT-induced proteolytic cleavage of PKCdelta, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCdelta activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCdelta cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCdelta. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCdelta fragment. Attenuation of ROS generation, caspase-3 activation, and PKCdelta activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCdelta(K376R) (dominant-negative mutant) prevented MMT-induced apoptosis in immortalized mesencephalic dopaminergic cells. For the first time, these data demonstrate that caspase-3-dependent proteolytic activation of PKCdelta plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.