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Grupo de Neurofarmacología, Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Albacete, Spain (M.G.-L., M.F.G, R.M.M.-F.M., F.J.F.-G., J.J.); Department of Physiology and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland, Dublin, Ireland (C.G.C., J.H.M.P.); Cell Signaling and Apoptosis Group, Departament de Ciències Mèdiques Bàsiques, University of Lleida and Hospital Arnau de Vilanova, Lleida, Spain (M.F.S., J.X.C.); and Centro Regional de Investigaciones Biomedicas, Albacete, Spain (M.G.-L., M.F.G., R.M.M.-F.M., F.J.F.-G., J.J.)
Received September 8, 2006; accepted December 15, 2006
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Abstract |
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). Intrastriatal injection of the mitochondrial complex II inhibitor malonate induces striatal lesions similar to those described in cerebral ischemia and Huntington's disease (Brouillet et al., 1995). The mechanisms that account for these neurotoxic effects remain to be fully elucidated (Ferger et al., 1999). It has been proposed that malonate toxicity involves depletion of striatal ATP (Beal et al., 1993), resulting in neuronal depolarization and a secondary excitotoxic neuron loss (Beal et al., 1994). It has also been reported that malonate is capable of inducing a caspase-dependent apoptotic cell death (Schulz et al., 1998).
Mitochondria are being considered a main link between cellular stress signals activated during short- and long-term nerve cell injury and the execution of apoptotic and necrotic cell death (Jordan et al., 2003; Mattson and Kroemer, 2003). ATP depletion, pathophysiological increases in intracellular calcium (Ca2+), and enhanced reactive oxygen species (ROS) production frequently occur during necrotic cell death and can trigger an increase in the permeability of the inner mitochondrial membrane. This process is believed to involve the formation of a multiprotein channel referred to as mitochondrial permeability transitory pore (Bernardi, 1999), which triggers the release of solutes up to 1500 Da from the mitochondrial matrix into the cytoplasm. Apoptotic events in contrast can cause an increase in the permeability of the outer mitochondrial membrane (Green, 2006). This process triggers the release of intermembrane space proteins into the cytoplasm, including cytochrome c, Smac/DIABLO, and apoptosis-inducing factor. Cytochrome c and Smac/DIABLO are capable of activating a family of cytosolic cysteine proteases, the caspases, whereas apoptosis-inducing factor has been implicated in caspase-independent forms of apoptosis (Goldstein et al., 2002; Martinou et al., 2002).
The Bax protein has been identified as a key proapoptotic Bcl-2 family protein during neuronal apoptosis. Bax normally resides in the cytosol and translocates to mitochondria in response to a variety of apoptotic stimuli, including cerebral ischemia (Wolter et al., 1997; Putcha et al., 1999; Cao et al., 2001). The proapoptotic action of Bax is believed to be mediated by its insertion into the outer mitochondrial membrane, where it might directly form channels or regulate the activity of pre-existing channels (Goping et al., 1998; Sharpe et al., 2004). This step requires a conformational change in the Bax protein. The upstream events that induce this conformational change are still largely unknown. We have previously demonstrated that malonate causes apoptosis of human SH-SY5Y neuroblastoma cells, involving increased reactive oxygen species production, oxidative stress, and mitochondrial cytochrome c release (Fernandez-Gomez et al., 2005). In the present study, we were interested in studying the requirement of Bax for malonate-induced apoptosis and in elucidating the signaling pathways involved in malonate-induced Bax activation.
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Materials and Methods |
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) [in Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine, 20 units/ml penicillin, 5 µg/ml streptomycin, and 15% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA)]. Cells were grown in a humidified cell incubator at 37°C under a 5% CO2 atmosphere. For GFP-Bax translocation and viability experiments, cells were plated on glass coverslip at 2.9 x 105 cells/cm2 and allowed to attach overnight. Immediately before malonate addition, dilutions of malonate were made in phosphate-buffered saline (PBS) and added to fresh cell culture medium to achieve the required concentration.
Primary cultures of cerebellar granule neurons were obtained from dissociated cerebella of 7- to 8-day-old rats (Fernandez-Gomez et al., 2006). Dissection and dissociation were carried out in basal medium eagle (Invitrogen). Tissues were incubated with trypsin for 20 min at 37°C and dissociated by trituration in a medium containing DNase and trypsin. Cells were plated on 60-mm plastic Petri dishes precoated with poly-L-lysine (10 µg/ml) at a concentration of 8 x 106 cells /ml in basal medium eagle containing 25 mM KCl, 10% de-complemented fetal calf serum (Invitrogen), glutamine, and antibiotics. Cytosine-µ-D-arabino-furanoside (10 µM) was added at 3 days in vitro to prevent the growth of non-neuronal cells. All experiments were carried out after 7 days in culture.
Transfection. Cells were plated 24 h before transfection at a density of 5.3 x 104 cells/cm2, on poly(D-lysine)-coated glass slides. Transfection was achieved using Lipofectamine reagent (Invitrogen) according to the manufacturer's protocol. Cells were transfected with the following plasmids encoding caspase-9 dominant-negative mutant caspase-9 (C287A; casp 9DN, a gift from Ding HF, Medical College of Ohio, Toledo, OH), Bcl-2, Bcl-xL, and p35 (Dr. J Merino, Universidad de Santander, Santander, Spain), GFP (pGFP-C1; Clontech, Mountain View, CA), GFP-Bax (Poppe et al., 2002). After 4-h incubation, the transfection mixture was removed and replaced with fresh complete medium.
Confocal Microscopy. For time-lapse analysis, cells were grown in 24-mm poly(D-lysine)-coated glass slides and mounted in a chamber for confocal microscopy with Krebs-HEPES buffer with the following ionic composition: 140 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 15 mM HEPES, 10 mM glucose, and 2.5 mM CaCl2, pH 7.4. Images were captured with a Leica microscope using a 63x objective with a 1.4 numerical aperture. The excitation wavelengths for GFP and tetramethylrhodamine ethyl ester (TMRE) were 488 and 543 nm, respectively. Images were taken for an hour every 5 min for control cells, and treated cells were injured with malonate after the first photograph.
Mitochondrial Potential. The cationic, lipophilic dye TMRE (Invitrogen) enters the cell in the form of an ester that is subsequently hydrolyzed, and the product, tetramethylrhodamine, is accumulated in mitochondria because of a high membrane potential. Cell cultures were washed in Krebs-HEPES buffer, and incubated at 37°C for 30 min in with TMRE (0.1 µM). Cells were then washed with Krebs-HEPES buffer, and resuspended in Krebs-HEPES buffer. Cell fluorescence was analyzed by confocal microscopy as it has been described above.
Detection of Peroxides/Reactive Oxygen Species. We used the oxidation-sensitive fluorescent dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate to measure the production of ROS, mainly hydrogen peroxide and hydroxyl radicals. 2',7'-Dichlorfluorescein-diacetate is deacetylated by esterases to dichlorofluorescein. This nonfluorescent product is then converted by reactive species into DCF, which can easily be visualized by fluorescence at 530 nm when excited at 485 nm. SH-SY5Y cells seeded in 96-well culture plates were incubated with dichlorodihydrofluorescein diacetate (10 µg/ml) for 5 min, and fluorescence intensity was measured in a Spectra Max Gemini XS (Molecular Devices, Sunnyvale, CA). Average ROS production (relative to level of vehicle-treated controls) was calculated from four individual wells in at least three independent platings.
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Western Blot. SH-SY5Y cell cultures were washed with ice-cold PBS twice and then collected by mechanical scraping with 1 ml of PBS in each tissue culture dish. The suspension was centrifuged at 12,000–14,000 rpm for 5 min. The supernatant was discarded, and the pellet was brought up in 150 µl of sample buffer. The protein from each condition was quantified spectrophotometrically (Micro BCA Protein Reagent Kit; Pierce, Rockford, IL), and an equal amount of protein (30 µg) was loaded onto 10% SDS-PAGE gels. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Immobilon; Millipore Corporation, Billerica, MA). Nonspecific protein binding was blocked with Blotto [4% w/v nonfat dried milk, 4% bovine serum albumin (Sigma), and 0.1% Tween 20 Sigma)] in PBS for 1 h. The membranes were incubated with anti-p53 [1:1000 dilution of anti-mouse monoclonal (Pab240) (Santa Cruz Biotechnology, Santa Cruz, CA)], anti-pan p38, and anti-phospho-p38 (1:1000 dilution of polyclonal) overnight at 4°C. After washing with Blotto, the membranes were incubated with a secondary antibody (1:5000 dilution of peroxidase-labeled anti-mouse; Promega, Madison, WI) in Blotto. The signal was detected using an enhanced chemiluminescence detection kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Immunoblots were developed by exposure to X-ray film (Eastman Kodak, Rochester, NY).
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Statistics. The results were expressed at the mean ± S.D. of at least three independent experiments. Student's two-tailed, unpaired t test was used, and values of P < 0.05 were considered significant. When comparing more than two conditions, statistically significant differences between groups were determined by analysis of variance followed by a Newman–Keuls post hoc analysis. The level of statistical significance was set at P < 0.05.
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Results |
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m) has been demonstrated to occur downstream of mitochondrial cytochrome c release (Waterhouse et al., 2001). By analyzing tetramethylrhodamine ethyl ester fluorescence intensities, we studied m changes in cells exhibiting either a clustered or diffuse GFP-Bax distribution. By 12 h after malonate treatment, we found that clustered punctate GFP-Bax cells showed an approximately 75% decrease in TMRE fluorescence compared with those cells with cytoplasmic diffuse GFP-Bax distribution (n = 114 cells). In malonate-treated cells where Bax translocation had not yet occurred, mitochondrial potential remained at the level observed in untreated cells (Fig. 2D). Epifluorescence observation suggested that cells with a clustered GFP-Bax fluorescence also exhibited a nuclear apoptotic morphology, and analyzed by chromatin using Hoechst 33342 (Fig. 2E).
Bax Was Required for Malonate-Induced Apoptosis. To determine the Bax involvement in malonate-induced apoptosis, we blocked Bax translocation using the chloride channel inhibitor furosemide (Karpinich et al., 2002). As shown in Fig. 3A, a 3-h pretreatment with 10 µM furosemide significantly reduced Bax translocation and afforded significant protection to the SH-SY5Y cells treated for 12 h with 50 mM malonate (Fig. 3B).
In the next set of experiments, we addressed the question of whether Bax was required for malonate-induced apoptosis. Western blotting analysis of lysates prepared from mouse embryonic fibroblasts (MEF) cultures revealed that malonate failed to activate caspase-3 in cells derived from Bax-deficient mice (Fig. 3). Indeed, the lack of Bax protein conferred resistance against malonate toxicity, as Bax–/– MEFs were protected against malonate-induced apoptosis (Fig. 3D).
Inhibition of Ros Production Inhibited Malonate-Induced Bax Translocation and Apoptosis. We have previously shown that ROS production is increased in SH-SY5Y cell cultures challenged with malonate (Fernandez-Gomez et al., 2005). To investigate whether this increased ROS production is functionally linked to malonate-induced Bax translocation, we treated SH-SY5Y neuroblastoma cells with the ROS scavenger vitamin E. One hour of pretreatment with 50 µM vitamin E significantly inhibited the formation of peroxides detected with the fluorescent indicator 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (Fig. 4A). Pretreatment with vitamin E also reduced malonate-induced GFP-Bax translocation compared with vehicle-treated control cultures (Fig. 4B). We next tested whether vitamin E treatment also conferred protection against malonate-induced apoptosis. Consistent with the effect on Bax-translocation, a pretreatment for 1 h with 50 µM vitamin E afforded significant protection against malonate-induced apoptosis (Fig. 4C). A similar protection was observed in vitamin E-pretreated cultured cerebellar granule neurons exposed to malonate (Fig. 4D).
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ROS are an important source of toxin-, ischemia-, and age-related DNA damage (Lombard et al., 2005). Indeed, there is growing evidence for a pivotal function of p53 in neuronal death (Gomez-Lazaro et al., 2004). To elucidate the participation of p53 in malonate-induced cell death, we tested whether malonate induced an increase in the total p53 protein levels in SH-SY5Y cell cultures. Western blotting analysis revealed that malonate did not increase p53 protein levels at any time point investigated (Fig. 5A). We found that SH-SY5Y neuroblastoma cells treated with 6-hydoxydopamine on the contrary showed a significant increase in the cellular p53 levels (Biswas et al., 2005). To confirm a lack of p53 in the signaling pathways leading to malonate-induced cell death, we also performed a set of experiments in p53-deficient MEFs (p53 –/–MEF). The lack of p53 did not afford a significant protection against malonate-induced apoptosis (Fig. 5B). These experiments suggested that the effects of ROS on Bax activation and cell death were independent of p53.
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). To analyze whether p38 MAPK was activated by malonate, SH-SY5Y cells were challenged with malonate and cytoplasmic extracts were assayed by Western blotting using a phosphospecific antibody recognizing active p38 MAP kinase (Sanchez-Prieto et al., 2002). Protein extracts from SH-SY5Y cells challenged with 50 mM malonate showed a marked increase in the phosphorylation status of p38 MAPK (Fig. 6A). Maximal increases in p38 MAPK phosphorylation occurred after 1 h of treatment. Phosphorylation levels slowly declined subsequently, returning to basal levels after 12 h. Next, we employed the p38 MAP kinase inhibitor SKF86002 to study the relevance of p38 MAP kinase activation in malonate-induced Bax translocation. At 10 µM, SKF86002 has been shown to inhibit all p38 MAPK isoforms, 
, 
, 
, and 
. SH-SY5Y cell cultures were pretreated for 12 h with 10 µM SKF86002. As shown in Fig. 6, B and C, SKF86002 potently prevented the appearance of cells showing a clustered GFP-Bax fluorescence. It is noteworthy that SKF86002 pretreatment also prevented apoptosis of SH-SY5Y cells treated with malonate (Fig. 6D). A similar, potent protection was observed in SKF86002-pretreated cultured cerebellar granule neurons exposed to malonate (Fig. 4D).
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Finally, we determined whether ROS participated in the activation of p38 MAP kinase in response to malonate. It is noteworthy that a pretreatment with the anti-oxidant vitamin E reduced the early accumulation of active p38 MAP kinase (Fig. 7A) after 0.5 and 3 h of malonate treatment, suggesting that ROS are required for malonate-induced p38 MAP kinase activation.
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; Düßmann et al., 2003). In agreement with these findings, the levels of MDA, a marker for oxidative stress (Esterbauer et al., 1991), were reduced at a later stage (12 h after addition of malonate) in MEF cells treated with the p38 kinase inhibitor SKF86002 compared with vehicle-treated control cells (Fig. 7B). The levels of MDA in MEFs challenged with 50 mM malonate were also significantly reduced in cells derived from Bax-deficient mice (Fig. 7B). Finally, to confirm the existence of a functionally separated primary and secondary increase in ROS production in more detail, we investigated the effects of vitamin E and SKF86002 pretreatment on ROS production in SH-SY5Y neuroblastoma cells at 1 h (early increase) and at 12 h (late increase). As expected, pretreatment with the p38 kinase inhibitor significantly reduced the late increase in ROS production but had no effect on the early increase (Fig. 7C). In contrast, pretreatment with vitamin E reduced ROS formation at both time points.
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Discussion |
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Cytochrome c release during apoptosis results from the permeabilization of the mitochondrial outer membrane, a process mediated via activation of the proapoptotic, multidomain Bcl-2 family members Bax and Bak. In our study, malonate-induced apoptosis was highly dependent on the expression of Bax; apoptosis was very significantly impaired in Bax-deficient cells. Furthermore, overexpression of the Bax antagonists Bcl-2 and Bcl-xL, as well as a dominant-negative caspase-9, was sufficient to suppress malonate-induced apoptosis. This requirement for Bax expression is surprising given that Bax and Bak have functionally redundant roles in a variety of cell death models (Kuwana et al., 2005). However, evidence suggests that neurons express a tissue-specific truncated isoform of Bak, N-Bak, which may lack the proapoptotic function of full-length Bax (Uo et al., 2005). Translocation and clustering of Bax at mitochondria was clearly evident after malonate treatment. Indeed, Bax translocation seemed to be a prerequisite step in malonate-induced apoptosis as a pretreatment of SH-SY5Y cells with furosemide, which has been shown to inhibit Bax translocation (Lin et al., 2005), also reduced the extent of malonate-induced apoptosis. However, it should also be mentioned that furosemide did not result in a complete inhibition of cell death. Indeed, it has previously been suggested that translocation of roughly 20% of the cellular Bax to the mitochondria may be sufficient to induce apoptosis (Annis et al., 2001).
Several mechanisms have been proposed to account for Bax conformational change responsible for its redistribution. Transcriptional or post-translational activation of BH3-only proteins, such as Bid, Bim, and PUMA, can activate Bax directly or indirectly by binding to and neutralizing the function of Bcl-xL and Bcl-2. Bax activation may be triggered by modifications in intracellular pH (Khaled et al., 1999) or by phosphorylation of critical amino acid residues by c-Jun NH2-terminal kinase and pK38 MAP kinases (De Chiara et al., 2006). Malonate produced a marked increase in the phosphorylation of p38 MAP kinase in SH-SY5Y cells. Furthermore, use of the p38 MAP kinase inhibitor SKF86002 potently inhibited Bax translocation and offered significant protection against malonate-induced apoptosis. Indeed, a significant body of evidence suggests that p38 MAPK activation plays an important role during excitotoxic and neurodegenerative processes (Cao et al., 2004). In addition to the p38 MAPK inhibitor, treatment of SH-SY5Y cells with the anti-oxidant vitamin E also inhibited Bax translocation resulting in decreased levels of apoptosis. It is noteworthy that the inhibition of ROS levels ameliorated the effect of malonate on p38 MAP kinase phosphorylation, suggesting that malonate activates ROS, which subsequently activates p38 kinase (Ghatan et al., 2000; Choi et al., 2004).
In summary, we demonstrate that malonate is able to induce mitochondrial cytochrome c releases through the redistribution of Bax, and that this event is mediated by ROS and the p38 MAP kinase pathway. Interfering with signaling pathways activated either by modulating the accumulation of ROS or by pharmacological inhibition of p38 MAP kinase may afford protection against malonate toxicity. These findings may have important therapeutic implications for the treatment of disorders such as HD and ischemic stroke.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: m, mitochondrial transmembrane potential; GFP, green fluorescent protein; MDA, malondialdehyde; MEF, mouse embryonic fibroblasts; ROS, reactive oxygen species; TMRE, tetramethylrhodamine ethyl ester.
Address correspondence to: Joaquin Jordán, Grupo de Neurofarmacología, Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, 02006, Albacete, Spain. E-mail: joaquin.jordan{at}uclm.es
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