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.

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.

Oxidative stress and apoptosis are key mediators of numerous neurodegenerative processes in the nervous system, including Alzheimer (4, 5) and Huntington disease (6, 7), Friedrich ataxia (8, 9), and Parkinson disease (10 -12). Oxidative stress has been shown to trigger the apoptotic cell death process through activation of one or more signaling molecules (13-16). In dopaminergic neurons, oxidative stress-induced phosphorylation events involve mitogen-activated protein kinases including p38 mitogen-activated protein kinase (17) and stress-activated protein kinase c-Jun N-terminal kinase kinases (18). Recently, we showed that Parkinsonian toxin MPP ϩ1 -induced ROS generation promotes apoptotic cell death in dopaminergic neurons via caspase-3-mediated proteolytic cleavage of protein kinase C-␦ (PKC␦) (1).PKC␦ is a member of the PKC serine-threonine protein kinase family classified into three groups, namely the classical (␣, ␤, and ␥ activated by DAG and Ca 2ϩ ), the atypical ( and / DAG and Ca 2ϩ -independent), and the novel (␦, ⑀, , and activated by DAG but Ca 2ϩ -independent). PKC␦ activation requires either the phosphorylation of its activation loop residues, leading to enzyme translocation, or the proteolytic cleavage of the kinase to yield catalytically active fragments. In the cellular models of Parkinson disease, we observed a caspase-3 mediated activation of PKC␦ without any evidence of membrane translocation (1). PKC␦ is known to be phosphorylated at tyrosine residues Tyr-52, Tyr-155, Tyr-187, Tyr-311, Tyr-332, and Tyr-565 when activated in response to certain stimuli, particularly to the known oxidative stress-inducing agent hydrogen peroxide (H 2 O 2 ) (19 -21). Src kinase, a member of the nonreceptor protein-tyrosine kinase family, variably modulates PKC␦ activity by increasing tyrosine phosphorylation, depending on the cell type and the insult (22)(23)(24)(25). Other members of the Src family of kinases that influence PKC␦ activity via phosphorylative changes are Fyn and c-Abl kinase (26,27). Furthermore, recent studies have demonstrated that PKC␦, when phosphorylated on the tyrosine residue Tyr-311, exhibits an increased catalytic activity in H 2 O 2 -treated cells (28,29). However, the relationship between PKC␦ tyrosine phosphorylation and its proteolytic cleavage has never been explored, particularly whether PKC␦ tyrosine phosphorylation can regulate its proteolytic activation and proapoptotic function. Here we demonstrate that phosphorylation of the tyrosine residue Tyr-311 in PKC␦ is essential for proteolytic activation, and that inhibition of tyrosine phosphorylation can attenuate oxidative stress-induced apoptotic cell death in dopaminergic neuronal cells.

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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 cells express key NADPH oxidase subunits gp91 phox and p67 phox . We used structurally diverse NADPH oxidase inhibitors, aminoethyl-benzenesulfonylfluoride (AEBSF, 100-1000 μM), apocynin (100-1000 μM), and diphenylene iodonium (DPI, 3-30 μM), to inhibit intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300 μM MPP + -induced ROS production for over 45 min 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 24 hr 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.