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Anantharam V, Kitazawa M, Wagner J, Kaul S, Kanthasamy AGCaspase-3-dependent proteolytic cleavage of protein kinase Cdelta is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl. J Neurosci 22:1738-1751

Parkinson Disorders Research Program, Department of Biomedical Sciences, Iowa Sate University, Ames, Iowa 50011, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.75). 04/2002; 22(5):1738-51.
Source: PubMed

ABSTRACT 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.

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    • "In addition, mhtt-induced cell death was partially blocked by the over-expression of a dominant negative form of PKCδ or by knockingdown endogenous PKCδ by using a siRNA thus supporting the idea that PKCδ plays a role in the regulation of striatal neuron death induced by mhtt. Similarly, a dominant negative form of PKCδ blocks for example phorbol ester-induced apoptosis in prostate cancer cells (Fujii et al., 2000) oxidative stress-mediated dopaminergic neuronal death (Anantharam et al., 2002) fatty acid-induced apoptosis of insulin secreting cells (Eitel et al., 2003) and acinus-induced apoptosis in PC12 cells (Hu et al., 2007) whereas down-regulation of PKCδ has been shown to be protective in different cell death paradigms (Zhong et al., 2002; Humphries et al., 2006 Shin et al., 2011). "
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    ABSTRACT: A balance between cell survival and apoptosis is crucial to avoid neurodegeneration. Here, we analyzed whether the pro-apoptotic protein PKCδ, and the pro-survival PKCα and βII, were dysregulated in the brain of R6/1 mouse model of Huntington's disease (HD). Protein levels of the three PKCs examined were reduced in all the brain regions analyzed being PKCδ the most affected isoform. Interestingly, PKCδ protein levels were also decreased in the striatum and cortex of R6/2 and Hdh(Q111/Q111) mice, and in the putamen of HD patients. Nuclear PKCδ induces apoptosis, but we detected reduced PKCδ in both cytoplasmic and nuclear enriched fractions from R6/1 mouse striatum, cortex and hippocampus. In addition, we show that phosphorylation and ubiquitination of PKCδ are increased in 30-week-old R6/1 mouse brain. All together these results suggest a pro-survival role of reduced PKCδ levels in response to mutant huntingtin-induced toxicity. In fact, we show that over-expression of PKCδ increases mutant huntingtin-induced cell death in vitro, whereas over-expression of a PKCδ dominant negative form or silencing of endogenous PKCδ partially blocks mutant huntingtin-induced cell death. Finally, we show that the analysis of lamin B protein levels could be a good marker of PKCδ activity, but it is not involved in PKCδ-mediated cell death in mutant huntingtin-expressing cells. In conclusion, our results suggest that neurons increase the degradation of PKCδ as a compensatory pro-survival mechanism in response to mutant huntingtin-induced toxicity that can help to understand why cell death appears late in the disease.
    Neuromolecular medicine 07/2013; 16(1). DOI:10.1007/s12017-013-8248-8 · 3.89 Impact Factor
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    • "This has been confirmed both in vivo and in vitro (Ghayur et al. 1996), and thus the C-terminal catalytic fragment is released from inhibition of the N-terminal regulatory domain (Emoto et al. 1995, 1996). PKCd is activated by a variety of apoptosis stimuli such as DNA damaging agents, ultraviolet (UV) radiation , phorbol 12-myristate12-acetate (PMA) and reactive oxygen species (ROS) (Anantharam et al. 2002; Chen et al. 1999; Konishi et al. 2001; Majumder et al. 2001; Reyland et al. 1999; Song et al. 2005). In PKCd null mice, the mitochondrial functions are compromised, which caused more ROS generation and a shift from glucose to lipid metabolism in murine hearts (Mayr et al. 2004a, b), which indicated the role of PKCd in mitochondria-related apoptosis . "
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    ABSTRACT: Protein kinase C-delta (PKCδ), a member of the lipid-regulated serine/threonine PKC family, has been implicated in a wide range of important cellular processes. In the past decade, the critical role of PKCδ in the regulation of both intrinsic and extrinsic apoptosis pathways has been widely explored. In most cases, over-expression or activation of PKCδ results in the induction of apoptosis. The phosphorylations and multiple cell organelle translocations of PKCδ initiate apoptosis by targeting multiple downstream effectors. During apoptosis, PKCδ is proteolytically cleaved by caspase-3 to generate a constitutively activated catalytic fragment, which amplifies apoptosis cascades in nucleus and mitochondria. However, PKCδ also exerts its anti-apoptotic and pro-survival roles in some cases. Therefore, the complicated role of PKCδ in apoptosis appears to be stimulus and cell type dependent. This review is mainly focused on how PKCδ gets activated in diverse ways in response to apoptotic signals and how PKCδ targets different downstream regulators to sponsor or restrain apoptosis induction.
    Archivum Immunologiae et Therapiae Experimentalis 08/2012; 60(5):361-72. DOI:10.1007/s00005-012-0188-8 · 2.82 Impact Factor
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    • "Our previous study showed that Mn(II) exposure increases phosphorylation of PKCd and the interaction of this isoform with the Glu transporter, ASCT2 (Sidoryk-Wegrzynowicz et al. 2011). Based on this finding, as well as evidence on the important role of PKCd in Mn(II) neurotoxicity, we hypothesized that this isoform may be specifically involved in Mn(II) – dependent disruption of GGC (Anantharam et al. 2002; Kitazawa et al. 2005; Latchoumycandane et al. 2005). Previous studies showed that both PKC activation and treatment with Mn(II) led to protein polyubiquitination (Sidoryk-Wegrzynowicz et al. 2011). "
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    ABSTRACT: J. Neurochem. (2012) 122, 856–867. Manganese (Mn) has been implicated in the impairment of the glutamate–glutamine cycling (GGC) by deregulation of Glu and glutamine (Gln) turnover in astrocytes. Here, we have examined possible mechanisms involved in the Mn(II)-mediated disruption of Glu turnover, including those related to protein degradation, such as the proteasomal and lysosomal machinery. Our study revealed that lysosome but not proteasomal inhibition is responsible for down-regulation of the Glu transporter after Mn(II) treatment. Because protein kinase C (PKC) activation leads to the down-regulation of Glu carriers, and Mn(II) increases PKC activity, we hypothesized that the PKC signaling contributes to the Mn(II)-mediated disruption of Glu turnover. Our results show that PKC activation causes a decrease in Glu uptake and that inhibition of PKC reverses Mn(II)-dependent down-regulation of Glu influx as well as glutamate transporter 1 (GLT1) and glutamate–aspartate transporter (GLAST) protein level. Co-immunoprecipitation studies show association of GLT1 with the PKCδ and PKCα isoforms and Mn(II)-induced specific increase in PKCδ-GLT1 interaction. In addition, astrocytes transfected with shRNA against PKCδ show decreased sensitivity to Mn(II) compared with those transfected with control shRNA or shRNA targeted against PKCα. Taken together, these findings demonstrate that PKCδ signaling is involved in the Mn(II)-induced deregulation of Glu turnover in astrocytes.
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