- Email: [email protected]
Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation
Neurochemistry International 46 (2005) 73–81 www.elsevier.com/locate/neuint
Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation David Koubia, Hao Jianga,c, Lijie Zhanga, Wenxue Tangb, Jarret Kuoa, Alba I. Rodrigueza, Tangella Jackson Huntera, Michael D. Seidmanb, George B. Corcorand, Robert A. Levinea,c,d,* a
William T. Gossett Neurology Laboratories, Detroit, MI 48202, USA Otolaryngology Research, Henry Ford Health System, Detroit, MI 48202, USA c John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201, USA d Department of Pharmaceutical Sciences, Wayne State University School of Pharmacy, Detroit, MI 48201, USA b
Received 18 September 2003; received in revised form 11 May 2004; accepted 10 June 2004 Available online 29 September 2004
Abstract Apoptotic cell death has been observed in many in vivo and in vitro models of ischemia. However, the molecular pathways involved in ischemia-induced apoptosis remain unclear. We have examined the role of Bcl-2 family of proteins in mediating apoptosis of PC12 cells exposed to the conditions of oxygen and glucose deprivation (OGD) or OGD followed by restoration of oxygen and glucose (OGDrestoration, OGD-R). OGD decreased mitochondrial membrane potential and induced necrosis of PC12 cells, which were both prevented by the overexpression of Bcl-2 proteins. OGD-R caused apoptotic cell death, induced cytochrome C release from mitochondria and caspase-3 activation, decreased mitochondrial membrane potential, and increased levels of pro-apoptotic Bax translocated to the mitochondrial membrane, all of which were reversed by overexpression of Bcl-2. These results demonstrate that the cell death induced by OGD and OGD-R in PC12 cells is potentially mediated through the regulation of mitochondrial membrane potential by the Bcl-2 family of proteins. It also reveals the importance of developing therapeutic strategies for maintaining the mitochondrial membrane potential as a possible way of reducing necrotic and apoptotic cell death that occurs following an ischemic insult. # 2004 Published by Elsevier Ltd. Keywords: Ischemic stroke; Neuronal cell death; Homodimers
1. Introduction A significant amount of brain damage in ischemic stroke can be attributed to neuronal cell death, resulting from an insufficient supply of glucose and oxygen to brain tissue (Lipton, 1999). To understand the mechanisms of neuronal cell death after ischemic insult and to identify potential protective agents, in vitro cell culture model of ischemia using pheochromocytoma PC12 cells has been developed (Abu-Raya et al., 1993). The experimental paradigm Abbreviations: OGD, oxygen and glucose deprivation; DMEM, Dulbeccos modified Eagles medium; Rh123, 2-(6-Amino-3-imino-3H-xanthen9-yl) benzoic acid methyl ester * Corresponding author. Tel.: +1 313 874 3771; fax: +1 313 874 4570. E-mail address: [email protected] (R.A. Levine). 0197-0186/$ – see front matter # 2004 Published by Elsevier Ltd. doi:10.1016/j.neuint.2004.06.006
includes an initial short phase of oxygen and glucose deprivation (OGD) followed by a prolonged phase of restoration (adding back oxygen and glucose, OGD-R). The OGD phase mimics the lack of oxygen and glucose supply, such as a thrombus formation during stroke, while the OGDR phase reflects the restoration of oxygen and glucose supply to the injured brain. In addition to its extensive use as a model in studying the signaling mechanisms of neurotransmitter secretion, neuronal differentiation and various growth factors, PC12 cells have also been employed to study the mechanisms of neuronal survival under ischemic conditions (Abu-Raya et al., 1999; Abu-Raya et al., 2002; Tabakman et al., 2002). The involvement of apoptosis in ischemic cell death is strongly supported by the protective effects of specific
74
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
caspase inhibitors administered to animals exposed to ischemia (Love, 2003). In addition, the Bcl-2 family of proteins (e.g. anti-apoptotic Bcl-2 and Bcl-xL; pro-apoptotic Bcl-xS and Bax) has been suggested to play a role in ischemia-induced apoptosis. Bax can homodimerize with itself and heterodimerize with Bcl-2 or Bcl-xL. It appears that Bax homodimers activate apoptosis while heterodimers inhibit the process (Adams and Cory, 1998). Moreover, an elevated intracellular ratio of Bax to Bcl-2 occurs during increased apoptotic cell death (Zha and Reed, 1997). Overexpression of Bcl-2 reduces apoptosis during ischemia in vivo (Wang et al., 1999; Kitagawa et al., 1998). Recent studies show that Bcl-2 may control the release of cytochrome C from mitochondria into the cytosol, where it participates in caspase activation in response to various apoptotic stimuli (Kluck et al., 1997). Since proteins of the Bcl-2 family, such as Bax, regulate the activation of apoptosis when translocated to the mitochondrial membrane (Cao et al., 2001; Lindenboim et al., 2001), the function of this organelle appears to be critical in ischemia-induced apoptosis. The involvement of mitochondria in mediating apoptotic death is supported by recent studies showing a decrease in mitochondrial membrane potential (Dc) following ischemic insult (Bahar et al., 2000). We have examined the molecular mechanisms and role of the Bcl-2 family of proteins in mediating apoptosis under ischemia-like conditions using PC12 cells. We show that: (1) OGD results in PC12 cell death by necrosis rather than apoptosis; (2) overexpression of Bcl-2 proteins prevents necrosis induced by OGD through possible Dc regulation; (3) overexpression of Bcl-2 proteins prevents apoptosis induced by OGD followed by OGD-R through the regulation of Bax translocation to the mitochondria, cytochrome C release to cytosol, and caspase-3 activation.
2. Materials and methods
2.2. Cell culture PC12 cells were grown as monolayers in tissue culture flasks in DMEM supplemented with 7% FBS, 7% HS, streptomycin (100 mg/ml) and penicillin (100 units/ml) at 37 8C with 5% CO2 and 95% air (Jiang et al., 1997a). PC12 cells stably expressing human Bcl-2 (HB2-2) or empty vector (V1) were kindly provided by Dr. Eguchi (Shimizu et al., 1996). V1 and HB2-2 cells were cultured under the same conditions as wild type PC12 cells. 2.3. In vitro model of ischemia Cells were plated on dishes two days before each experiment. To initiate OGD, cell culture media was removed and cells were washed twice with glucose-free DMEM. Cells were then incubated in the glucose-free medium in an oxygen-free incubator (95% N2 and 5% CO2) for 4 h. Following OGD, glucose was added to normal levels (final concentration was 4.5 mg/ml) and cells were incubated under normal growth conditions for additional 20 h. 2.4. Flow cytometry analysis of cell death Apoptosis was determined using an apoptosis detection kit (Becton Dickinson, NJ). Briefly, cells were collected after treatment, washed twice in ice-cold PBS, and then resuspended in binding buffer (10 mM HEPES, pH 7.4, 0.14 M NaCl and 2.5 mM CaCl2) at a density of 1 106 cells/ml. Cells were incubated simultaneously with fluorescein-labeled annexin V and propidium iodide (PI) for 20 min and analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ). Annexin V-FITC generated signals were detected with an FITC signal detector (FL1, 525 nm). PI signals were monitored using a detector reserved for phycoerythrin emission (FL2, 575 nm). Data were analyzed using Cell Quest software from BD Biosciences.
2.1. Materials 2.5. DNA fragmentation Z-VAD-fmk (a general caspase inhibitor) and Z-DEVD-fmk (a selective caspase-3 inhibitor) were obtained from Calbiochem (San Diego, CA). 2-(6-amino3-imino-3H-xanthen-9-yl) benzoic acid methyl ester (Rhodamine123, Rh123) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were obtained from SigmaAldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), glucose-free DMEM, streptomycin, and penicillin were obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) and horse serum (HS) were purchased from Hyclone (Logan, UT). Antibodies against Bcl-2, Bax, and Bcl-xL/xS were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and antibody against actin was obtained from Oncogene Research Products (San Diego, CA).
PC12 cells (8 106 cells) were collected under various experimental conditions and subjected to DNA fragmentation assay as previously described (Anastasiadis et al., 2001). Briefly, cells were lysed with lysis buffer (0.5% Triton-X100, 5 mM Tris–HCl, pH 7.4, 20 mM EDTA) and lysates were collected by centrifugation. RNA was removed by incubation with RNase A (0.8 mg/ml) at 37 8C for 30 min. Cytosolic DNA was obtained after extraction with phenol–chloroform followed by precipitation with onetenth volume of 3 M sodium acetate, pH 5.2 and two volumes of 100% ethanol. DNA pellets were obtained by centrifugation, rinsed with 70% ethanol, dried and resuspended in 20 ml of 1 TE (10 mM Tris–HCl and 1 mM EDTA) and 4 ml of 6 loading buffer (40% sucrose in
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
H2O). The samples were separated on 2% agarose gels and then stained for 20 min with ethidium bromide (0.5 mg/l) in H2O. 2.6. Measurement of caspase-3 activity Caspase-3 activity under various experimental conditions was measured in PC12 cell extracts (2 106 cells/sample) using a colorimetric assay kit from BD Biosciences Clontech (Palo Alto, CA). The levels of chromophore p-nitroanilide (pNA), after cleavage from the labeled substrate of caspase-3, DEVD-pNA, were determined by using a spectrophotometry (l = 405 nm).
75
2.9. Preparation of mitochondrial and cytosolic fractions Mitochondrial and cytosolic fractions were prepared from cells collected at the end of OGD-R using an ApoAlert cell fractionation kit from BD Biosciences Clontech (Palo Alto, CA) according to the manufacturer’s instructions. 2.10. Statistical analyses A one-way analysis of variance followed by a Fisher’s protected LSD method (to address multiple comparisons issues) was used to determine statistical differences in cell death percentage, caspase-3 activity and mitochondrial membrane potential between the different groups. A P value of less than 0.05 was considered significant.
2.7. Measurement of mitochondrial membrane potential Following OGD-R, PC12 cells were collected, centrifuged at 1200 g for 5 min, and resuspended in DMEM. Cells were counted and 1 106 cells were used for flow cytometry analysis. Briefly, Rh123 was added to the cell samples at the final concentration of 1 mM. After 1 h of incubation at 37 8C, the Rh123 fluorescence (excitation = 488 nm; emission through a band-pass filter of 515–565 nm) was determined by flow cytometry (BD Biosciences, Franklin Lakes, NJ). All data acquisition and analysis was done using Cell Quest software from BD Biosciences. The specificity of Rh123 to translocate into the active mitochondria was verified by preincubating PC12 cells for 10 min with protonophore CCCP (50 mM), which depolarizes the mitochondrial membrane. Alterations in the fluorescence signal in the absence or presence of the protonophore were then observed under a fluorescent microscope. 2.8. Western blot analysis Cells were collected at the end of OGD and OGD-R and washed twice with ice-cold PBS, pH 7.4. Cells were then lysed for 30 min on ice in lysis buffer composed of 20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% deoxycholic acid, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 1 mg/ml aprotinin (Jiang et al., 1997b). Cell lysates were obtained by centrifugation at 12,000 g for 10 min at 4 8C, and the protein concentration in each sample was determined by the protein assay kit from Pierce (Rockford, IL). Equal amounts of protein were subjected to electrophoresis on 14% SDS–polyacrylamide gels. Separated proteins were then electro-transferred to Immobilon membranes (Millipore, Bedford, MA). After exposure to the desired antibodies, proteins were visualized using an enhanced chemiluminescence protein detection kit from Pierce (Rockford, IL) according to the manufacturer’s instructions.
3. Results 3.1. Overexpression of Bcl-2 inhibits cell death caused by both OGD and OGD-R To determine whether 4 h of OGD (OGD) or 4 h of OGD followed by 20 h of restored oxygen and glucose (OGD-R) induced necrosis and/or apoptosis, PC12/V1 cells were stained with both propidium iodide (PI) and FITC-labeled annexin V (AV-FITC), then analyzed by flow cytometry. Since the values obtained in PC12 and V1 cells over all experiments were not found to be statistically different, they were pooled as PC12/V1 group. Necrotic cells are demonstrated by AV /PI+ staining, since PI enters cells when membrane integrity is lost and binds nucleic acids. Apoptotic cells are demonstrated by AV+/PI staining, since annexin V binds to phosphatidylserine that translocates to the outer leaflet of the plasma membrane during apoptosis. AV+/PI+ stained cells are likely to be late apoptotic or necrotic and AV /PI cells represent viable cells. Fig. 1 shows representative dot plots of cells stained with PI and FITC-AV. Table 1 shows that OGD resulted in a significant increase in the percentage of necrotic cells (AV /PI+) from a control value of (7 2) to (53 7)%. No apoptotic cells (AV+/PI ) were detected. At the end of OGD-R, (1 2)% of the cell population was necrotic, whereas (41 4)% of the cells were apoptotic. Under control conditions, about (6 3)% of the cells were apoptotic. These results indicate that OGD caused necrosis, and delayed apoptosis occurred during the restoration period (OGD-R). To determine whether overexpressing Bcl-2 reversed either necrotic or apoptotic cell death, PC12 cells that stably expressed human Bcl-2 (HB22) or empty vector (V1) were exposed to OGD and OGD-R. Both necrosis and apoptosis were inhibited by Bcl-2 overexpression (Fig. 1; Table 1). To further confirm that overexpression of Bcl-2 inhibited death of PC12 cells exposed to OGD and OGD-R, DNA fragmentation was analyzed. Double-stranded breakdown of
76
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
Fig. 1. Representative dot plots of flow cytometry analysis of cell death in PC12 and HB2-2 cells. PC12/V1 and HB2-2 cells left untreated (Con) or were subjected to 4 h of OGD (OGD) followed by 20 h of restoration (OGD-R). Cells were stained with propidium iodide (PI) and FITC-Annexin V (AV). Samples were analyzed by flow cytometry.
DNA into nucleosomal segments is an indicator of apoptosis and is manifested as DNA laddering with fragments occurring in multiples of 180 bp. Fig. 2C shows that DNA breakdown was not observed at the end of OGD. Nevertheless, a DNA smear was visualized, suggesting the possible appearance of necrosis. DNA fragmentation was detected at the end of OGD-R, as indicated by the appearance of fragments in multiples of 180 bp. Both visualized smear and DNA fragmentation were strongly reduced when expressing Bcl-2 (Fig. 2C). Levels of expression of Bcl-2 in HB2-2, V1 and PC12 cells were examined (Fig. 2A and B). The absence of Bcl-2 protein expression in V1 and PC12 cells is consistent with a previously published study reporting that endogenous Bcl-2 expression is undetectable in PC12 cells (Maroto and PerezPolo, 1997).
3.2. Bcl-2 overexpression inhibits caspase-3 activation induced by OGD-R To investigate the involvement of caspases in apoptosis of PC12 cells exposed to OGD-R, cells were also treated with a general caspase inhibitor (Z-VAD-fmk, 100 mM) or a selective caspase-3 inhibitor (Z-DEVD-fmk, 100 mM) (Fig. 3A). DNA fragmentation induced by OGD-R was inhibited by both general and selective caspase-3 inhibitors. Activation of caspase-3 at the end of OGD-R was further demonstrated by assays of caspase-3 activity in PC12/V1 cells (Fig. 3B), which was increased to (262 12)% of the control levels, but caspase-3 activation induced by OGD-R did not occur in HB2-2 cells ((97 11)% of control).
3.3. Bcl-2 overexpression reduces Bax translocation to mitochondria during OGD-R Table 1 Flow cytometry analysis of the cell populations under various conditions Control (%)
OGD (%)
OGD-R (%)
PC12/V1 HB2-2 PC12/V1 HB2-2 PC12/V1 HB2-2 PI /FITC-AV 81 5 PI+/FITC-AV 72 PI /FITC-AV+ 6 3 PI+/FITC-AV+ 6 2
89 3 2 5
3 45 6a 2 53 7a 1 12 2 43
88 4 1 5
4 50 3a 2 32 2 41 4a 2 62
85 3 2 7
4 1 1 3
The percentages of cells stained with PI (AV /PI+) only, FITC-AV (AV+/ PI ) only. Neither PI nor FITC-AV (PI /AV ), or both (AV+/PI+) were calculated under control conditions at the end of OGD or OGD-R. Values obtained from four independent experiments are expressed as mean S.D. a P < 0.05, compared to control.
After characterizing the involvement of the Bcl-2 family of proteins in apoptosis induced by OGD-R, the expression of members of the Bcl-2 family (Bax, Bcl-xL, Bcl-xS) was examined by Western blotting (Fig. 4A). Bcl-xL protein level was increased by OGD and returned to control after 20 h of restoration. Bcl-xS was increased at the end of both OGD and OGD-R. Bax protein levels were increased only at the end of OGD-R. Translocation of Bax to mitochondria was detected in PC12 cells, and this translocation was reduced by approximately 50% after overexpression of Bcl-2 in HB2-2 cells (Fig. 4B). Reduction of Bax translocation to
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
77
Fig. 2. Effect of Bcl-2 overexpression during OGD and OGD-R on DNA fragmentation. (A) Western blot analysis of Bcl-2 expression in V1 and HB2-2 cells. (B) Western blot analysis of Bcl-2 expression in wild type PC12, C6 glioma and HB2-2 cells. (C) V1 and HB2-2 cells were subjected to 4 h of OGD (OGD) followed by 20 h of restoration (OGD-R). DNA fragmentation assay was performed. The appearance of fragments in multiples of 180 bp was indicated by arrows.
Fig. 3. Effect of Bcl-2 overexpression on DNA fragmentation and caspase-3 activity induced by OGD-R. (A) PC12 cells were subjected to OGD-R in the presence or absence of a general caspase inhibitor (Z-VAD-fmk, 100 mM) or a selective inhibitor of caspase-3 (Z-DEVD-fmk, 100 mM). Inhibitors were added immediately before the start of OGD. DNA fragmentation assay was performed. The appearance of fragments in multiples of 180 bp was indicated by arrows. (B) Caspase-3 activity was measured in V1 and HB2-2 cells under control conditions and after 4 h of OGD followed by 20 h of restoration (OGD-R). Caspase-3 activity was observed to be equivalent in both V1 and HB2-2 cells under control conditions (4.1 nmole DEVD-pNA cleaved/mg of protein/h). Results are the mean of five independent experiments and are expressed as percentage of the caspase-3 activity determined in control S.D. *P < 0.05, compared to control.
78
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
Fig. 4. Effect of OGD and OGD-R on Bax, Bcl-xL, and Bcl-xS protein levels. PC12 cells were subjected to 4 h of OGD (OGD) followed by 20 h of restoration (OGD-R). (A) Western blot analysis of Bax, Bcl-xL, Bcl-xS and actin protein expression. (B) Bax protein levels in mitochondrial fraction (mito) and cytochrome C levels in cytosolic fraction (cyto) were detected by Western blotting, respectively. Actin was used as an internal protein control, and band intensity was measured using Scion image-analysis software.
mitochondria was correlated to inhibition of cytochrome C release to the cytosol (Fig. 4B). 3.4. Bcl-2 overexpression and mitochondrial membrane potential during OGD-R
be caspase-3-dependent, since DNA fragmentation at the end of OGD-R was inhibited by a general caspace inhibitor or a selective caspase-3 inhibitor. This last result is consistent with reported in vivo data showing: (1) the appearance of activated caspase-3 at 24 h following 12 min of global ischemia (Xu et al., 1999); (2) elevated caspase-3-like activity
Mitochondrial membrane potential in PC12/V1 and HB2-2 cells exposed to OGD or OGD-R was assessed by the retention of rhodamine123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria. Rh123 fluorescence was decreased in PC12/V1 cells to (79 2)% of control levels after OGD and further decreased to (68 4)% of control levels after OGD-R (Fig. 5). Overexpression of Bcl-2 reversed the reduction of mitochondrial membrane potential that resulted from OGD and OGD-R.
4. Discussion Our novel results using an in vitro model of ischemic stroke demonstrate that PC12 cell death occurs by early necrosis upon exposure to OGD followed by delayed apoptosis during exposure to OGD-R. Both necrosis and apoptosis were precisely quantified using PI and annexin V dual staining. Also consistent with necrosis was the observed nucleic acid smear on agarose gels at the end of OGD, while the occurrence of apoptosis during OGD-R was indicated by DNA fragmentation and caspase-3 activation. Apoptosis was demonstrated to
Fig. 5. Effect of Bcl-2 overexpression during OGD and OGD-R on mitochondrial membrane potential. PC12/V1 and HB2-2 cells were subjected to 4 h of OGD (OGD) or OGD followed by 20 h of restoration (OGDR). Cells were collected and incubated with 1 mM Rhodamine123 (Rh123) to measure the mitochondrial membrane potential (Dc). Rh123 fluorescence was calculated in each cell population and the relative Dc was determined in each experimental condition as a percentage of control. Values obtained from five independent experiments in PC12 cells and four independent experiments in V1 and HB2-2 cells are expressed as means S.D. *P < 0.05, compared to control. DP < 0.05, compared to OGD.
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
(approximately 10-fold) in the hippocampus as early as 8 h after restoration and cleavage of caspase substrate, poly(ADP-ribose) polymerase (PARP), occurring 24 h later (Chen et al., 1998); and (3) significant reductions in cell death and DNA fragmentation in the CA1 layer of hippocampus up to 7 days after ischemia following ventricular infusion of the selective caspase-3 inhibitor, Z-DEVD-fmk (Chen et al., 1998). Knowing that PC12 cell death induced by OGD-R involved apoptosis, we focused on the role of the Bcl-2 family of proteins in the death process. AV and PI dual staining studies revealed that Bcl-2 overexpression inhibits both necrosis and apoptosis. We also showed that overexpression of Bcl-2 resulted in a reduction of DNA fragmentation along with a complete inhibition of cytochrome C release from mitochondria to cytosol and caspase3 activation following OGD-R. These results support the importance of the Bcl-2 family of proteins in mediating cell survival through regulating caspase activity, as suggested by a previous in vivo study of ischemia (Wang et al., 1999) and an in vitro study of resveratrol-induced apoptosis in U937 cells (Park et al., 2001). Bcl-2 has also been linked to mitochondrial function during inhibition of apoptosis. Many scenarios causing apoptosis have been reported to reduce mitochondrial membrane potential (Petit et al., 1996), suggesting that the opening of the mitochondrial permeability transition pore (a large conductance channel) occurs during apoptosis (Qian et al., 1997). Moreover, drugs that inhibit the opening of the permeability transition pore prevent apoptosis induced by various stimuli (Zamzami et al., 1996). Thus, loss of mitochondrial membrane potential through permeability transition pore opening appears to play a key role in mediating apoptosis. It has been shown that Bcl-2 may reduce apoptosis through regulating ion transport and preventing the decrease of mitochondrial membrane potential induced by pharmacological agents (Shimizu et al., 1996; Shimizu et al., 1998). Our results showing decreased mitochondrial membrane potential during OGD are consistent with previous data showing a loss of mitochondrial membrane potential in hippocampal neurons from CA1 layer when brain slice preparations were exposed to OGD (Bahar et al., 2000). The role of Bcl-2 in regulating mitochondrial membrane potential has been observed in isolated mitochondria following exposure to pharmacological agents (e.g. hydrogen peroxide) (Shimizu et al., 1998). We have shown for the first time that Bcl-2 overexpression in PC12 cells prevents the decrease in mitochondrial membrane potential caused by OGD and OGD-R. This indicates that Bcl-2 reduces necrosis through regulating mitochondrial membrane potential and apoptosis through regulating mitochondrial membrane potential and the release of cytochrome C. The regulation of caspase-3 activity by the Bcl-2 family of proteins has been suggested to be directly dependent on the elevation of Bax and its translocation to the mitochondrial membrane
79
(Murphy et al., 2000). When translocated to the mitochondrial membrane, Bax can homodimerize and trigger the activation of terminal caspases by increasing mitochondrial membrane permeability, which results in the release of apoptosis-promoting factors into the cytoplasm (Adams and Cory, 1998; Xu et al., 1999). Conversely, Bcl-2/Bax heterodimers will prevent such release (Adams and Cory, 1998). It was not possible to test the involvement of native Bcl-2 in PC12 cells, since it is not normally present (Maroto and Perez-Polo, 1997). However, we did examine the related pro-survival protein, Bcl-xL, and the pro-apoptotic proteins, Bax and Bcl-xS. Bax is capable of forming homodimers with itself and heterodimers with Bcl-xL or Bcl-2 (Lindenboim et al., 2001). However, the role of Bcl-xS in mediating apoptotic cell death remains unclear. We observed that BclxL was increased during OGD and returned to baseline at the end of OGD-R, whereas Bax protein level was maximal at the end of OGD-R. Bcl-xS protein level was increased at the end of OGD and decreased but remained higher than the control at the end of OGD-R. It is likely that the ratio of BclxS/Bcl-xL protein is increased at the end of OGD, whereas the Bax/Bcl-xL ratio is only increased at the end of OGD-R. The increase in Bcl-xL protein during restoration may be due to a stress response to increase the resistance of PC12 cells to OGD. The return of Bcl-xL protein to basal levels at the end of restoration is consistent with previous in vivo studies showing that Bcl-xL levels were not increased during increased apoptosis that followed an ischemic insult in CA1 layer of hippocampus (Ferrer et al., 1998; Antonawich et al., 1998). Our reports of Bcl-xS and Bax changes are consistent with previous studies showing that Bax is upregulated along with apoptosis in the CA1 layer of hippocampus after global ischemia and in the parietal cortex after focal ischemia (Isenmann et al., 1998; Krajewski et al., 1995), and Bcl-xS is upregulated in CA1 layer of hippocampus after global ischemia (Dixon et al., 1997). Our results indicate that alterations in the expression of Bcl-2 family members are responsible for the activation of caspase-3 in this model of ischemia. Indeed, the inhibition of caspase-3 activity by overexpression of Bcl-2 is likely to be mediated by the reduction of the pro-apoptotic Bax/Bax homodimers through the formation of Bcl-2/Bax heterodimers. The observation of increased Bax protein levels at 20 h after OGD-R when the caspase-3 activity was found to be greatly enhanced supports this hypothesis. More importantly, our results reveal that overexpression of Bcl2 markedly reduced the level of Bax in the mitochondrial fraction, indicating that Bcl-2 regulates Bax translocation to the mitochondria during OGD-R. This observation is further supported by recent studies showing that overexpression of Bcl-2 reduced apoptotic cell death induced by cytokine withdrawal, glucocorticoids, and cytotoxic drugs by preventing Bax translocation to the mitochondrial membrane (Murphy et al., 2000). Thus, Bcl-2 overexpression by reducing Bax translocation to the mitochondrial membrane may reduce the level of Bax/Bax homodimers and explain
80
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
the observed prevention of caspase-3 activation at the end of OGD-R. We suggest that Bcl-2 overexpression prevents the decrease of mitochondrial membrane potential induced by OGD through interaction with the pro-apoptotic Bcl-xS rather than Bax. Indeed, we noted a loss of mitochondrial membrane potential at the end of OGD when there was an upregulation of Bcl-xS but not Bax. These novel results are supported by an in vitro study showing that the general caspase inhibitor, Z-VAD-fmk, rescues serum-deprived brown adipocytes from apoptosis through the downregulation of Bcl-xS protein and prevention of the loss of mitochondrial membrane potential (Navarro et al., 1999). Moreover, the increase in Bcl-xS protein level at the end of OGD suggests a possible role of this protein in mediating necrosis. Since Bax levels were found to be elevated at the end of the restoration period, Bax may participate in the further decrease in mitochondrial membrane potential and contribute to mediating apoptosis. This concept is supported by studies showing that the loss of mitochondrial membrane potential occurring during apoptosis can be prevented by an adenoviral Bcl-2 homologue, E1B 19K, which interacts with Bax (Han et al., 1998). Another possibility is that caspase-3 activation following OGD (170% of control value, data not shown) may participate in the further decrease of mitochondrial membrane potential observed following restoration, which in turn induces further caspase activation through the release of cytochrome C and apoptosis-inducing factor. This is supported by previous studies in purified mitochondria showing that recombinant caspases and caspase inhibitors can affect Dc of isolated mitochondria (Marzo et al., 1998; Susin et al., 1997) and the fact that we observed a further increase in caspase-3 activity at the end of restoration (262% of control value). In summary, we have demonstrated that OGD followed by restoration of oxygen and glucose induces early necrosis followed by late apoptosis in PC12 cells, which is mediated by a decrease in the mitochondrial membrane potential and an increase in caspase-3 activity. Our novel results demonstrate the central role, played by the Bcl-2 family of proteins in mediating apoptotic death after OGD and OGD-R through a regulation of mitochondrial function. This work highlights the importance of developing therapeutic strategies to maintain mitochondrial membrane potential to reduce the deleterious effects of apoptotic cell death after an ischemic insult in vivo.
Acknowledgements This work was supported by grants to R.A. Levine from NIH and the Veterans Administration. We thank the Flow Cytometry Core Facility at the Wayne State University for assistance in the analysis of the extent of cell death and changes in mitochondrial membrane potential.
References Abu-Raya, S., Trembovler, V., Shohami, E., Lazarovici, P., 1993. A tissue culture ischemic device to study eicosanoid release by pheochromocytoma PC12 cultures. J. Neurosci. Methods 50, 197–203. Abu-Raya, S., Blaugrund, E., Trembovler, V., Shilderman-Bloch, E., Shohami, E., Lazarovici, P., 1999. Rasagiline, a monoamine oxidase-B inhibitor, protects NGF-differentiated PC12 cells against oxygen–glucose deprivation. J. Neurosci. Res. 58, 456–463. Abu-Raya, S., Tabakman, R., Blaugrund, E., Trembovler, V., Lazarovici, P., 2002. Neuroprotective and neurotoxic effects of monoamine oxidase-B inhibitors and derived metabolites under ischemia in PC12 cells. Eur. J. Pharmacol. 434, 109–116. Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326. Anastasiadis, P.Z., Jiang, H., Bezin, L., Kuhn, D.M., Levine, R.A., 2001. Tetrahydrobiopterin enhances apoptotic PC12 cell death following withdrawal of trophic support. J. Biol. Chem. 276, 9050– 9058. Antonawich, F.J., Krajewski, S., Reed, J.C., Davis, J.N., 1998. Bcl-xL Bax interaction after transient global ischemia. J. Cereb. Blood Flow Metab. 18, 882–886. Bahar, S., Fayuk, D., Somjen, G.G., Aitken, P.G., Turner, D.A., 2000. Mitochondrial and intrinsic optical signals imaged during hypoxia and spreading depression in rat hippocampal slices. J. Neurophysiol. 84, 311–324. Cao, G., Minami, M., Pei, W., Yan, C., Chen, D., O’Horo, C., Graham, S.H., Chen, J., 2001. Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death. J. Cereb. Blood Flow Metab. 21, 321–333. Chen, J., Nagayama, T., Jin, K., Stetler, R.A., Zhu, R.L., Graham, S.H., Simon, R.P., 1998. Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J. Neurosci. 18, 4914–4928. Dixon, E.P., Stephenson, D.T., Clemens, J.A., Little, S.P., 1997. Bcl-x short is elevated following severe global ischemia in rat brains.. Brain Res. 776, 222–229. Ferrer, I., Lopez, E., Blanco, R., Rivera, R., Ballabriga, J., Pozas, E., Marti, E., 1998. Bcl-2, Bax, and Bcl-x expression in the CA1 area of the hippocampus following transient forebrain ischemia in the adult gerbil. Exp. Brain Res. 121, 167–173. Han, J., Modha, D., White, E., 1998. Interaction of E1B 19K with Bax is required to block Bax-induced loss of mitochondrial membrane potential and apoptosis. Oncogene 17, 2993–3005. Isenmann, S., Stoll, G., Schroeter, M., Krajewski, S., Reed, J.C., Bahr, M., 1998. Differential regulation of Bax, Bcl-2, and Bcl-x proteins in focal cortical ischemia in the rat. Brain Pathol. 8, 49–62. Jiang, H., St. Ulme, D., Dickens, G., Chabuk, A., Lavarreda, M., Lazarovici, P., Guroff, G., 1997a. Both p140trk and p75NGFR nerve growth factor receptors mediate nerve growth factor-stimulated calcium uptake. J. Biol. Chem. 272, 6835–6837. Jiang, H., Movsesyan, V., Fink, D.W., Fasler, M., Whalin, M., Katagiri, Y., Monshipouri, M., Dickens, G., Lelkes, P.I., Guroff, G., Lazarovici, P., 1997b. Expression of human trkA receptors in trkA-deficient PC12/endothelial cells results in nerve growth factorinduced signal transduction and DNA synthesis. J. Cell. Biochem. 66, 229–244. Kitagawa, K., Matsumoto, M., Tsujimoto, Y., Ohtsuki, T., Kuwabara, K., Matsushita, K., Yang, G., Tanabe, H., Martinou, J.C., Hori, M., Yanagihara, T., 1998. Amelioration of hippocampal neuronal damage after global ischemia by neuronal overexpression of Bcl-2 in transgenic mice. Stroke 29, 2616–2621. Kluck, R.M., Bossy-Wetzel, E., Green, D.R., Newmeyer, D.D., 1997. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132–1136.
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81 Krajewski, S., Mai, J.K., Krajewska, M., Sikorska, M., Mossakowski, M.J., Reed, J.C., 1995. Upregulation of bax protein levels in neurons following cerebral ischemia. J. Neurosci. 15, 6364– 6376. Lindenboim, L., Borner, C., Stein, R., 2001. Bcl-xS can form homodimers and heterodimers and its apoptotic activity requires localization of BclxS to the mitochondria and its BH3 and loop domains. Cell Death Differ. 8, 933–942. Lipton, P., 1999. Ischemic cell death in brain neurons. Physiological Rev. 79, 1431–1568. Love, S., 2003. Apoptosis and brain ischaemia. Prog. Neuropsychopharmacol. Biol. Psychiatry 27, 267–282. Maroto, R., Perez-Polo, J.R., 1997. Bcl-2-related protein expression in apoptosis: oxidative stress versus serum deprivation in PC12 cells. J. Neurochem. 69, 514–523. Marzo, I., Susin, S.A., Petit, P.X., Ravagnan, L., Brenner, C., Larochette, N., Zamzami, N., Kroemer, G., 1998. Caspases disrupt mitochondrial membrane barrier function. FEBS Lett. 427, 198–202. Murphy, K.M., Ranganathan, V., Farnsworth, M.L., Kavallaris, M., Lock, R.B., 2000. Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells. Cell Death Differ. 7, 102–111. Navarro, P., Valverde, A.M., Conejo, R., Benito, M., Lorenzo, M., 1999. Inhibition of caspases rescues brown adipocytes from apoptosis downregulating Bcl-xS and upregulating Bcl-2 gene expression. Exp. Cell Res. 246, 301–307. Park, J.W., Choi, Y.J., Suh, S.I., Baek, W.K., Suh, M.H., Jin, I.N., Min, D.S., Woo, J.H., Chang, J.S., Passaniti, A., Lee, Y.H., Kwon, T.K., 2001. Bcl-2 overexpression attenuates resveratrol-induced apoptosis in U937 cells by inhibition of caspase-3 activity. Carcinogenesis 22, 1633–1639. Petit, P.X., Susin, S.A., Zamzami, N., Mignotte, B., Kroemer, G., 1996. Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition. FEBS Lett. 396, 7–13.
81
Qian, T., Nieminen, A.L., Herman, B., Lemasters, J.J., 1997. Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am. J. Physiol. 273, C1783–C1792. Shimizu, S., Eguchi, Y., Kamiike, W., Waguri, S., Uchiyama, Y., Matsuda, H., Tsujimoto, Y., 1996. Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors. Oncogene 13, 21–29. Shimizu, S., Eguchi, Y., Kamiike, W., Funahashi, Y., Mignon, A., Lacronique, V., Matsuda, H., Tsujimoto, Y., 1998. Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux. Proc. Natl. Acad. Sci. USA 95, 1455–1459. Susin, S.A., Zamzami, N., Castedo, M., Daugas, E., Wang, H.G., Geley, S., Fassy, F., Reed, J.C., Kroemer, G., 1997. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J. Exp. Med. 186, 25–37. Tabakman, R., Lazarovici, P., Kohen, R., 2002. Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia. J. Neurosci. Res. 68, 463–469. Wang, H.D., Fukuda, T., Suzuki, T., Hashimoto, K., Liou, S.Y., Momoi, T., Kosaka, T., Yamamoto, K., Nakanishi, H., 1999. Differential effects of Bcl-2 overexpression on hippocampal CA1 neurons and dentate granule cells following hypoxic ischemia in adult mice. J. Neurosci. Res. 57, 1–12. Xu, D., Bureau, Y., McIntyre, D.C., Nicholson, D.W., Liston, P., Zhu, Y., Fong, W.G., Crocker, S.J., Korneluk, R.G., Robertson, G.S., 1999. Attenuation of ischemia-induced cellular and behavioral deficits by X chromosome-linked inhibitor of apoptosis protein overexpression in the rat hippocampus. J. Neurosci. 19, 5026–5033. Zamzami, N., Marchetti, P., Castedo, M., Hirsch, T., Susin, S.A., Masse, B., Kroemer, G., 1996. Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett. 384, 53–57. Zha, H., Reed, J.C., 1997. Heterodimerization-independent functions of cell death regulatory proteins Bax and Bcl-2 in yeast and mammalian cells. J. Biol. Chem. 272, 31482–31488.
Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation David Koubia, Hao Jianga,c, Lijie Zhanga, Wenxue Tangb, Jarret Kuoa, Alba I. Rodrigueza, Tangella Jackson Huntera, Michael D. Seidmanb, George B. Corcorand, Robert A. Levinea,c,d,* a
William T. Gossett Neurology Laboratories, Detroit, MI 48202, USA Otolaryngology Research, Henry Ford Health System, Detroit, MI 48202, USA c John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201, USA d Department of Pharmaceutical Sciences, Wayne State University School of Pharmacy, Detroit, MI 48201, USA b
Received 18 September 2003; received in revised form 11 May 2004; accepted 10 June 2004 Available online 29 September 2004
Abstract Apoptotic cell death has been observed in many in vivo and in vitro models of ischemia. However, the molecular pathways involved in ischemia-induced apoptosis remain unclear. We have examined the role of Bcl-2 family of proteins in mediating apoptosis of PC12 cells exposed to the conditions of oxygen and glucose deprivation (OGD) or OGD followed by restoration of oxygen and glucose (OGDrestoration, OGD-R). OGD decreased mitochondrial membrane potential and induced necrosis of PC12 cells, which were both prevented by the overexpression of Bcl-2 proteins. OGD-R caused apoptotic cell death, induced cytochrome C release from mitochondria and caspase-3 activation, decreased mitochondrial membrane potential, and increased levels of pro-apoptotic Bax translocated to the mitochondrial membrane, all of which were reversed by overexpression of Bcl-2. These results demonstrate that the cell death induced by OGD and OGD-R in PC12 cells is potentially mediated through the regulation of mitochondrial membrane potential by the Bcl-2 family of proteins. It also reveals the importance of developing therapeutic strategies for maintaining the mitochondrial membrane potential as a possible way of reducing necrotic and apoptotic cell death that occurs following an ischemic insult. # 2004 Published by Elsevier Ltd. Keywords: Ischemic stroke; Neuronal cell death; Homodimers
1. Introduction A significant amount of brain damage in ischemic stroke can be attributed to neuronal cell death, resulting from an insufficient supply of glucose and oxygen to brain tissue (Lipton, 1999). To understand the mechanisms of neuronal cell death after ischemic insult and to identify potential protective agents, in vitro cell culture model of ischemia using pheochromocytoma PC12 cells has been developed (Abu-Raya et al., 1993). The experimental paradigm Abbreviations: OGD, oxygen and glucose deprivation; DMEM, Dulbeccos modified Eagles medium; Rh123, 2-(6-Amino-3-imino-3H-xanthen9-yl) benzoic acid methyl ester * Corresponding author. Tel.: +1 313 874 3771; fax: +1 313 874 4570. E-mail address: [email protected] (R.A. Levine). 0197-0186/$ – see front matter # 2004 Published by Elsevier Ltd. doi:10.1016/j.neuint.2004.06.006
includes an initial short phase of oxygen and glucose deprivation (OGD) followed by a prolonged phase of restoration (adding back oxygen and glucose, OGD-R). The OGD phase mimics the lack of oxygen and glucose supply, such as a thrombus formation during stroke, while the OGDR phase reflects the restoration of oxygen and glucose supply to the injured brain. In addition to its extensive use as a model in studying the signaling mechanisms of neurotransmitter secretion, neuronal differentiation and various growth factors, PC12 cells have also been employed to study the mechanisms of neuronal survival under ischemic conditions (Abu-Raya et al., 1999; Abu-Raya et al., 2002; Tabakman et al., 2002). The involvement of apoptosis in ischemic cell death is strongly supported by the protective effects of specific
74
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
caspase inhibitors administered to animals exposed to ischemia (Love, 2003). In addition, the Bcl-2 family of proteins (e.g. anti-apoptotic Bcl-2 and Bcl-xL; pro-apoptotic Bcl-xS and Bax) has been suggested to play a role in ischemia-induced apoptosis. Bax can homodimerize with itself and heterodimerize with Bcl-2 or Bcl-xL. It appears that Bax homodimers activate apoptosis while heterodimers inhibit the process (Adams and Cory, 1998). Moreover, an elevated intracellular ratio of Bax to Bcl-2 occurs during increased apoptotic cell death (Zha and Reed, 1997). Overexpression of Bcl-2 reduces apoptosis during ischemia in vivo (Wang et al., 1999; Kitagawa et al., 1998). Recent studies show that Bcl-2 may control the release of cytochrome C from mitochondria into the cytosol, where it participates in caspase activation in response to various apoptotic stimuli (Kluck et al., 1997). Since proteins of the Bcl-2 family, such as Bax, regulate the activation of apoptosis when translocated to the mitochondrial membrane (Cao et al., 2001; Lindenboim et al., 2001), the function of this organelle appears to be critical in ischemia-induced apoptosis. The involvement of mitochondria in mediating apoptotic death is supported by recent studies showing a decrease in mitochondrial membrane potential (Dc) following ischemic insult (Bahar et al., 2000). We have examined the molecular mechanisms and role of the Bcl-2 family of proteins in mediating apoptosis under ischemia-like conditions using PC12 cells. We show that: (1) OGD results in PC12 cell death by necrosis rather than apoptosis; (2) overexpression of Bcl-2 proteins prevents necrosis induced by OGD through possible Dc regulation; (3) overexpression of Bcl-2 proteins prevents apoptosis induced by OGD followed by OGD-R through the regulation of Bax translocation to the mitochondria, cytochrome C release to cytosol, and caspase-3 activation.
2. Materials and methods
2.2. Cell culture PC12 cells were grown as monolayers in tissue culture flasks in DMEM supplemented with 7% FBS, 7% HS, streptomycin (100 mg/ml) and penicillin (100 units/ml) at 37 8C with 5% CO2 and 95% air (Jiang et al., 1997a). PC12 cells stably expressing human Bcl-2 (HB2-2) or empty vector (V1) were kindly provided by Dr. Eguchi (Shimizu et al., 1996). V1 and HB2-2 cells were cultured under the same conditions as wild type PC12 cells. 2.3. In vitro model of ischemia Cells were plated on dishes two days before each experiment. To initiate OGD, cell culture media was removed and cells were washed twice with glucose-free DMEM. Cells were then incubated in the glucose-free medium in an oxygen-free incubator (95% N2 and 5% CO2) for 4 h. Following OGD, glucose was added to normal levels (final concentration was 4.5 mg/ml) and cells were incubated under normal growth conditions for additional 20 h. 2.4. Flow cytometry analysis of cell death Apoptosis was determined using an apoptosis detection kit (Becton Dickinson, NJ). Briefly, cells were collected after treatment, washed twice in ice-cold PBS, and then resuspended in binding buffer (10 mM HEPES, pH 7.4, 0.14 M NaCl and 2.5 mM CaCl2) at a density of 1 106 cells/ml. Cells were incubated simultaneously with fluorescein-labeled annexin V and propidium iodide (PI) for 20 min and analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ). Annexin V-FITC generated signals were detected with an FITC signal detector (FL1, 525 nm). PI signals were monitored using a detector reserved for phycoerythrin emission (FL2, 575 nm). Data were analyzed using Cell Quest software from BD Biosciences.
2.1. Materials 2.5. DNA fragmentation Z-VAD-fmk (a general caspase inhibitor) and Z-DEVD-fmk (a selective caspase-3 inhibitor) were obtained from Calbiochem (San Diego, CA). 2-(6-amino3-imino-3H-xanthen-9-yl) benzoic acid methyl ester (Rhodamine123, Rh123) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were obtained from SigmaAldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), glucose-free DMEM, streptomycin, and penicillin were obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) and horse serum (HS) were purchased from Hyclone (Logan, UT). Antibodies against Bcl-2, Bax, and Bcl-xL/xS were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and antibody against actin was obtained from Oncogene Research Products (San Diego, CA).
PC12 cells (8 106 cells) were collected under various experimental conditions and subjected to DNA fragmentation assay as previously described (Anastasiadis et al., 2001). Briefly, cells were lysed with lysis buffer (0.5% Triton-X100, 5 mM Tris–HCl, pH 7.4, 20 mM EDTA) and lysates were collected by centrifugation. RNA was removed by incubation with RNase A (0.8 mg/ml) at 37 8C for 30 min. Cytosolic DNA was obtained after extraction with phenol–chloroform followed by precipitation with onetenth volume of 3 M sodium acetate, pH 5.2 and two volumes of 100% ethanol. DNA pellets were obtained by centrifugation, rinsed with 70% ethanol, dried and resuspended in 20 ml of 1 TE (10 mM Tris–HCl and 1 mM EDTA) and 4 ml of 6 loading buffer (40% sucrose in
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
H2O). The samples were separated on 2% agarose gels and then stained for 20 min with ethidium bromide (0.5 mg/l) in H2O. 2.6. Measurement of caspase-3 activity Caspase-3 activity under various experimental conditions was measured in PC12 cell extracts (2 106 cells/sample) using a colorimetric assay kit from BD Biosciences Clontech (Palo Alto, CA). The levels of chromophore p-nitroanilide (pNA), after cleavage from the labeled substrate of caspase-3, DEVD-pNA, were determined by using a spectrophotometry (l = 405 nm).
75
2.9. Preparation of mitochondrial and cytosolic fractions Mitochondrial and cytosolic fractions were prepared from cells collected at the end of OGD-R using an ApoAlert cell fractionation kit from BD Biosciences Clontech (Palo Alto, CA) according to the manufacturer’s instructions. 2.10. Statistical analyses A one-way analysis of variance followed by a Fisher’s protected LSD method (to address multiple comparisons issues) was used to determine statistical differences in cell death percentage, caspase-3 activity and mitochondrial membrane potential between the different groups. A P value of less than 0.05 was considered significant.
2.7. Measurement of mitochondrial membrane potential Following OGD-R, PC12 cells were collected, centrifuged at 1200 g for 5 min, and resuspended in DMEM. Cells were counted and 1 106 cells were used for flow cytometry analysis. Briefly, Rh123 was added to the cell samples at the final concentration of 1 mM. After 1 h of incubation at 37 8C, the Rh123 fluorescence (excitation = 488 nm; emission through a band-pass filter of 515–565 nm) was determined by flow cytometry (BD Biosciences, Franklin Lakes, NJ). All data acquisition and analysis was done using Cell Quest software from BD Biosciences. The specificity of Rh123 to translocate into the active mitochondria was verified by preincubating PC12 cells for 10 min with protonophore CCCP (50 mM), which depolarizes the mitochondrial membrane. Alterations in the fluorescence signal in the absence or presence of the protonophore were then observed under a fluorescent microscope. 2.8. Western blot analysis Cells were collected at the end of OGD and OGD-R and washed twice with ice-cold PBS, pH 7.4. Cells were then lysed for 30 min on ice in lysis buffer composed of 20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% deoxycholic acid, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 1 mg/ml aprotinin (Jiang et al., 1997b). Cell lysates were obtained by centrifugation at 12,000 g for 10 min at 4 8C, and the protein concentration in each sample was determined by the protein assay kit from Pierce (Rockford, IL). Equal amounts of protein were subjected to electrophoresis on 14% SDS–polyacrylamide gels. Separated proteins were then electro-transferred to Immobilon membranes (Millipore, Bedford, MA). After exposure to the desired antibodies, proteins were visualized using an enhanced chemiluminescence protein detection kit from Pierce (Rockford, IL) according to the manufacturer’s instructions.
3. Results 3.1. Overexpression of Bcl-2 inhibits cell death caused by both OGD and OGD-R To determine whether 4 h of OGD (OGD) or 4 h of OGD followed by 20 h of restored oxygen and glucose (OGD-R) induced necrosis and/or apoptosis, PC12/V1 cells were stained with both propidium iodide (PI) and FITC-labeled annexin V (AV-FITC), then analyzed by flow cytometry. Since the values obtained in PC12 and V1 cells over all experiments were not found to be statistically different, they were pooled as PC12/V1 group. Necrotic cells are demonstrated by AV /PI+ staining, since PI enters cells when membrane integrity is lost and binds nucleic acids. Apoptotic cells are demonstrated by AV+/PI staining, since annexin V binds to phosphatidylserine that translocates to the outer leaflet of the plasma membrane during apoptosis. AV+/PI+ stained cells are likely to be late apoptotic or necrotic and AV /PI cells represent viable cells. Fig. 1 shows representative dot plots of cells stained with PI and FITC-AV. Table 1 shows that OGD resulted in a significant increase in the percentage of necrotic cells (AV /PI+) from a control value of (7 2) to (53 7)%. No apoptotic cells (AV+/PI ) were detected. At the end of OGD-R, (1 2)% of the cell population was necrotic, whereas (41 4)% of the cells were apoptotic. Under control conditions, about (6 3)% of the cells were apoptotic. These results indicate that OGD caused necrosis, and delayed apoptosis occurred during the restoration period (OGD-R). To determine whether overexpressing Bcl-2 reversed either necrotic or apoptotic cell death, PC12 cells that stably expressed human Bcl-2 (HB22) or empty vector (V1) were exposed to OGD and OGD-R. Both necrosis and apoptosis were inhibited by Bcl-2 overexpression (Fig. 1; Table 1). To further confirm that overexpression of Bcl-2 inhibited death of PC12 cells exposed to OGD and OGD-R, DNA fragmentation was analyzed. Double-stranded breakdown of
76
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
Fig. 1. Representative dot plots of flow cytometry analysis of cell death in PC12 and HB2-2 cells. PC12/V1 and HB2-2 cells left untreated (Con) or were subjected to 4 h of OGD (OGD) followed by 20 h of restoration (OGD-R). Cells were stained with propidium iodide (PI) and FITC-Annexin V (AV). Samples were analyzed by flow cytometry.
DNA into nucleosomal segments is an indicator of apoptosis and is manifested as DNA laddering with fragments occurring in multiples of 180 bp. Fig. 2C shows that DNA breakdown was not observed at the end of OGD. Nevertheless, a DNA smear was visualized, suggesting the possible appearance of necrosis. DNA fragmentation was detected at the end of OGD-R, as indicated by the appearance of fragments in multiples of 180 bp. Both visualized smear and DNA fragmentation were strongly reduced when expressing Bcl-2 (Fig. 2C). Levels of expression of Bcl-2 in HB2-2, V1 and PC12 cells were examined (Fig. 2A and B). The absence of Bcl-2 protein expression in V1 and PC12 cells is consistent with a previously published study reporting that endogenous Bcl-2 expression is undetectable in PC12 cells (Maroto and PerezPolo, 1997).
3.2. Bcl-2 overexpression inhibits caspase-3 activation induced by OGD-R To investigate the involvement of caspases in apoptosis of PC12 cells exposed to OGD-R, cells were also treated with a general caspase inhibitor (Z-VAD-fmk, 100 mM) or a selective caspase-3 inhibitor (Z-DEVD-fmk, 100 mM) (Fig. 3A). DNA fragmentation induced by OGD-R was inhibited by both general and selective caspase-3 inhibitors. Activation of caspase-3 at the end of OGD-R was further demonstrated by assays of caspase-3 activity in PC12/V1 cells (Fig. 3B), which was increased to (262 12)% of the control levels, but caspase-3 activation induced by OGD-R did not occur in HB2-2 cells ((97 11)% of control).
3.3. Bcl-2 overexpression reduces Bax translocation to mitochondria during OGD-R Table 1 Flow cytometry analysis of the cell populations under various conditions Control (%)
OGD (%)
OGD-R (%)
PC12/V1 HB2-2 PC12/V1 HB2-2 PC12/V1 HB2-2 PI /FITC-AV 81 5 PI+/FITC-AV 72 PI /FITC-AV+ 6 3 PI+/FITC-AV+ 6 2
89 3 2 5
3 45 6a 2 53 7a 1 12 2 43
88 4 1 5
4 50 3a 2 32 2 41 4a 2 62
85 3 2 7
4 1 1 3
The percentages of cells stained with PI (AV /PI+) only, FITC-AV (AV+/ PI ) only. Neither PI nor FITC-AV (PI /AV ), or both (AV+/PI+) were calculated under control conditions at the end of OGD or OGD-R. Values obtained from four independent experiments are expressed as mean S.D. a P < 0.05, compared to control.
After characterizing the involvement of the Bcl-2 family of proteins in apoptosis induced by OGD-R, the expression of members of the Bcl-2 family (Bax, Bcl-xL, Bcl-xS) was examined by Western blotting (Fig. 4A). Bcl-xL protein level was increased by OGD and returned to control after 20 h of restoration. Bcl-xS was increased at the end of both OGD and OGD-R. Bax protein levels were increased only at the end of OGD-R. Translocation of Bax to mitochondria was detected in PC12 cells, and this translocation was reduced by approximately 50% after overexpression of Bcl-2 in HB2-2 cells (Fig. 4B). Reduction of Bax translocation to
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
77
Fig. 2. Effect of Bcl-2 overexpression during OGD and OGD-R on DNA fragmentation. (A) Western blot analysis of Bcl-2 expression in V1 and HB2-2 cells. (B) Western blot analysis of Bcl-2 expression in wild type PC12, C6 glioma and HB2-2 cells. (C) V1 and HB2-2 cells were subjected to 4 h of OGD (OGD) followed by 20 h of restoration (OGD-R). DNA fragmentation assay was performed. The appearance of fragments in multiples of 180 bp was indicated by arrows.
Fig. 3. Effect of Bcl-2 overexpression on DNA fragmentation and caspase-3 activity induced by OGD-R. (A) PC12 cells were subjected to OGD-R in the presence or absence of a general caspase inhibitor (Z-VAD-fmk, 100 mM) or a selective inhibitor of caspase-3 (Z-DEVD-fmk, 100 mM). Inhibitors were added immediately before the start of OGD. DNA fragmentation assay was performed. The appearance of fragments in multiples of 180 bp was indicated by arrows. (B) Caspase-3 activity was measured in V1 and HB2-2 cells under control conditions and after 4 h of OGD followed by 20 h of restoration (OGD-R). Caspase-3 activity was observed to be equivalent in both V1 and HB2-2 cells under control conditions (4.1 nmole DEVD-pNA cleaved/mg of protein/h). Results are the mean of five independent experiments and are expressed as percentage of the caspase-3 activity determined in control S.D. *P < 0.05, compared to control.
78
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
Fig. 4. Effect of OGD and OGD-R on Bax, Bcl-xL, and Bcl-xS protein levels. PC12 cells were subjected to 4 h of OGD (OGD) followed by 20 h of restoration (OGD-R). (A) Western blot analysis of Bax, Bcl-xL, Bcl-xS and actin protein expression. (B) Bax protein levels in mitochondrial fraction (mito) and cytochrome C levels in cytosolic fraction (cyto) were detected by Western blotting, respectively. Actin was used as an internal protein control, and band intensity was measured using Scion image-analysis software.
mitochondria was correlated to inhibition of cytochrome C release to the cytosol (Fig. 4B). 3.4. Bcl-2 overexpression and mitochondrial membrane potential during OGD-R
be caspase-3-dependent, since DNA fragmentation at the end of OGD-R was inhibited by a general caspace inhibitor or a selective caspase-3 inhibitor. This last result is consistent with reported in vivo data showing: (1) the appearance of activated caspase-3 at 24 h following 12 min of global ischemia (Xu et al., 1999); (2) elevated caspase-3-like activity
Mitochondrial membrane potential in PC12/V1 and HB2-2 cells exposed to OGD or OGD-R was assessed by the retention of rhodamine123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria. Rh123 fluorescence was decreased in PC12/V1 cells to (79 2)% of control levels after OGD and further decreased to (68 4)% of control levels after OGD-R (Fig. 5). Overexpression of Bcl-2 reversed the reduction of mitochondrial membrane potential that resulted from OGD and OGD-R.
4. Discussion Our novel results using an in vitro model of ischemic stroke demonstrate that PC12 cell death occurs by early necrosis upon exposure to OGD followed by delayed apoptosis during exposure to OGD-R. Both necrosis and apoptosis were precisely quantified using PI and annexin V dual staining. Also consistent with necrosis was the observed nucleic acid smear on agarose gels at the end of OGD, while the occurrence of apoptosis during OGD-R was indicated by DNA fragmentation and caspase-3 activation. Apoptosis was demonstrated to
Fig. 5. Effect of Bcl-2 overexpression during OGD and OGD-R on mitochondrial membrane potential. PC12/V1 and HB2-2 cells were subjected to 4 h of OGD (OGD) or OGD followed by 20 h of restoration (OGDR). Cells were collected and incubated with 1 mM Rhodamine123 (Rh123) to measure the mitochondrial membrane potential (Dc). Rh123 fluorescence was calculated in each cell population and the relative Dc was determined in each experimental condition as a percentage of control. Values obtained from five independent experiments in PC12 cells and four independent experiments in V1 and HB2-2 cells are expressed as means S.D. *P < 0.05, compared to control. DP < 0.05, compared to OGD.
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
(approximately 10-fold) in the hippocampus as early as 8 h after restoration and cleavage of caspase substrate, poly(ADP-ribose) polymerase (PARP), occurring 24 h later (Chen et al., 1998); and (3) significant reductions in cell death and DNA fragmentation in the CA1 layer of hippocampus up to 7 days after ischemia following ventricular infusion of the selective caspase-3 inhibitor, Z-DEVD-fmk (Chen et al., 1998). Knowing that PC12 cell death induced by OGD-R involved apoptosis, we focused on the role of the Bcl-2 family of proteins in the death process. AV and PI dual staining studies revealed that Bcl-2 overexpression inhibits both necrosis and apoptosis. We also showed that overexpression of Bcl-2 resulted in a reduction of DNA fragmentation along with a complete inhibition of cytochrome C release from mitochondria to cytosol and caspase3 activation following OGD-R. These results support the importance of the Bcl-2 family of proteins in mediating cell survival through regulating caspase activity, as suggested by a previous in vivo study of ischemia (Wang et al., 1999) and an in vitro study of resveratrol-induced apoptosis in U937 cells (Park et al., 2001). Bcl-2 has also been linked to mitochondrial function during inhibition of apoptosis. Many scenarios causing apoptosis have been reported to reduce mitochondrial membrane potential (Petit et al., 1996), suggesting that the opening of the mitochondrial permeability transition pore (a large conductance channel) occurs during apoptosis (Qian et al., 1997). Moreover, drugs that inhibit the opening of the permeability transition pore prevent apoptosis induced by various stimuli (Zamzami et al., 1996). Thus, loss of mitochondrial membrane potential through permeability transition pore opening appears to play a key role in mediating apoptosis. It has been shown that Bcl-2 may reduce apoptosis through regulating ion transport and preventing the decrease of mitochondrial membrane potential induced by pharmacological agents (Shimizu et al., 1996; Shimizu et al., 1998). Our results showing decreased mitochondrial membrane potential during OGD are consistent with previous data showing a loss of mitochondrial membrane potential in hippocampal neurons from CA1 layer when brain slice preparations were exposed to OGD (Bahar et al., 2000). The role of Bcl-2 in regulating mitochondrial membrane potential has been observed in isolated mitochondria following exposure to pharmacological agents (e.g. hydrogen peroxide) (Shimizu et al., 1998). We have shown for the first time that Bcl-2 overexpression in PC12 cells prevents the decrease in mitochondrial membrane potential caused by OGD and OGD-R. This indicates that Bcl-2 reduces necrosis through regulating mitochondrial membrane potential and apoptosis through regulating mitochondrial membrane potential and the release of cytochrome C. The regulation of caspase-3 activity by the Bcl-2 family of proteins has been suggested to be directly dependent on the elevation of Bax and its translocation to the mitochondrial membrane
79
(Murphy et al., 2000). When translocated to the mitochondrial membrane, Bax can homodimerize and trigger the activation of terminal caspases by increasing mitochondrial membrane permeability, which results in the release of apoptosis-promoting factors into the cytoplasm (Adams and Cory, 1998; Xu et al., 1999). Conversely, Bcl-2/Bax heterodimers will prevent such release (Adams and Cory, 1998). It was not possible to test the involvement of native Bcl-2 in PC12 cells, since it is not normally present (Maroto and Perez-Polo, 1997). However, we did examine the related pro-survival protein, Bcl-xL, and the pro-apoptotic proteins, Bax and Bcl-xS. Bax is capable of forming homodimers with itself and heterodimers with Bcl-xL or Bcl-2 (Lindenboim et al., 2001). However, the role of Bcl-xS in mediating apoptotic cell death remains unclear. We observed that BclxL was increased during OGD and returned to baseline at the end of OGD-R, whereas Bax protein level was maximal at the end of OGD-R. Bcl-xS protein level was increased at the end of OGD and decreased but remained higher than the control at the end of OGD-R. It is likely that the ratio of BclxS/Bcl-xL protein is increased at the end of OGD, whereas the Bax/Bcl-xL ratio is only increased at the end of OGD-R. The increase in Bcl-xL protein during restoration may be due to a stress response to increase the resistance of PC12 cells to OGD. The return of Bcl-xL protein to basal levels at the end of restoration is consistent with previous in vivo studies showing that Bcl-xL levels were not increased during increased apoptosis that followed an ischemic insult in CA1 layer of hippocampus (Ferrer et al., 1998; Antonawich et al., 1998). Our reports of Bcl-xS and Bax changes are consistent with previous studies showing that Bax is upregulated along with apoptosis in the CA1 layer of hippocampus after global ischemia and in the parietal cortex after focal ischemia (Isenmann et al., 1998; Krajewski et al., 1995), and Bcl-xS is upregulated in CA1 layer of hippocampus after global ischemia (Dixon et al., 1997). Our results indicate that alterations in the expression of Bcl-2 family members are responsible for the activation of caspase-3 in this model of ischemia. Indeed, the inhibition of caspase-3 activity by overexpression of Bcl-2 is likely to be mediated by the reduction of the pro-apoptotic Bax/Bax homodimers through the formation of Bcl-2/Bax heterodimers. The observation of increased Bax protein levels at 20 h after OGD-R when the caspase-3 activity was found to be greatly enhanced supports this hypothesis. More importantly, our results reveal that overexpression of Bcl2 markedly reduced the level of Bax in the mitochondrial fraction, indicating that Bcl-2 regulates Bax translocation to the mitochondria during OGD-R. This observation is further supported by recent studies showing that overexpression of Bcl-2 reduced apoptotic cell death induced by cytokine withdrawal, glucocorticoids, and cytotoxic drugs by preventing Bax translocation to the mitochondrial membrane (Murphy et al., 2000). Thus, Bcl-2 overexpression by reducing Bax translocation to the mitochondrial membrane may reduce the level of Bax/Bax homodimers and explain
80
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81
the observed prevention of caspase-3 activation at the end of OGD-R. We suggest that Bcl-2 overexpression prevents the decrease of mitochondrial membrane potential induced by OGD through interaction with the pro-apoptotic Bcl-xS rather than Bax. Indeed, we noted a loss of mitochondrial membrane potential at the end of OGD when there was an upregulation of Bcl-xS but not Bax. These novel results are supported by an in vitro study showing that the general caspase inhibitor, Z-VAD-fmk, rescues serum-deprived brown adipocytes from apoptosis through the downregulation of Bcl-xS protein and prevention of the loss of mitochondrial membrane potential (Navarro et al., 1999). Moreover, the increase in Bcl-xS protein level at the end of OGD suggests a possible role of this protein in mediating necrosis. Since Bax levels were found to be elevated at the end of the restoration period, Bax may participate in the further decrease in mitochondrial membrane potential and contribute to mediating apoptosis. This concept is supported by studies showing that the loss of mitochondrial membrane potential occurring during apoptosis can be prevented by an adenoviral Bcl-2 homologue, E1B 19K, which interacts with Bax (Han et al., 1998). Another possibility is that caspase-3 activation following OGD (170% of control value, data not shown) may participate in the further decrease of mitochondrial membrane potential observed following restoration, which in turn induces further caspase activation through the release of cytochrome C and apoptosis-inducing factor. This is supported by previous studies in purified mitochondria showing that recombinant caspases and caspase inhibitors can affect Dc of isolated mitochondria (Marzo et al., 1998; Susin et al., 1997) and the fact that we observed a further increase in caspase-3 activity at the end of restoration (262% of control value). In summary, we have demonstrated that OGD followed by restoration of oxygen and glucose induces early necrosis followed by late apoptosis in PC12 cells, which is mediated by a decrease in the mitochondrial membrane potential and an increase in caspase-3 activity. Our novel results demonstrate the central role, played by the Bcl-2 family of proteins in mediating apoptotic death after OGD and OGD-R through a regulation of mitochondrial function. This work highlights the importance of developing therapeutic strategies to maintain mitochondrial membrane potential to reduce the deleterious effects of apoptotic cell death after an ischemic insult in vivo.
Acknowledgements This work was supported by grants to R.A. Levine from NIH and the Veterans Administration. We thank the Flow Cytometry Core Facility at the Wayne State University for assistance in the analysis of the extent of cell death and changes in mitochondrial membrane potential.
References Abu-Raya, S., Trembovler, V., Shohami, E., Lazarovici, P., 1993. A tissue culture ischemic device to study eicosanoid release by pheochromocytoma PC12 cultures. J. Neurosci. Methods 50, 197–203. Abu-Raya, S., Blaugrund, E., Trembovler, V., Shilderman-Bloch, E., Shohami, E., Lazarovici, P., 1999. Rasagiline, a monoamine oxidase-B inhibitor, protects NGF-differentiated PC12 cells against oxygen–glucose deprivation. J. Neurosci. Res. 58, 456–463. Abu-Raya, S., Tabakman, R., Blaugrund, E., Trembovler, V., Lazarovici, P., 2002. Neuroprotective and neurotoxic effects of monoamine oxidase-B inhibitors and derived metabolites under ischemia in PC12 cells. Eur. J. Pharmacol. 434, 109–116. Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326. Anastasiadis, P.Z., Jiang, H., Bezin, L., Kuhn, D.M., Levine, R.A., 2001. Tetrahydrobiopterin enhances apoptotic PC12 cell death following withdrawal of trophic support. J. Biol. Chem. 276, 9050– 9058. Antonawich, F.J., Krajewski, S., Reed, J.C., Davis, J.N., 1998. Bcl-xL Bax interaction after transient global ischemia. J. Cereb. Blood Flow Metab. 18, 882–886. Bahar, S., Fayuk, D., Somjen, G.G., Aitken, P.G., Turner, D.A., 2000. Mitochondrial and intrinsic optical signals imaged during hypoxia and spreading depression in rat hippocampal slices. J. Neurophysiol. 84, 311–324. Cao, G., Minami, M., Pei, W., Yan, C., Chen, D., O’Horo, C., Graham, S.H., Chen, J., 2001. Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death. J. Cereb. Blood Flow Metab. 21, 321–333. Chen, J., Nagayama, T., Jin, K., Stetler, R.A., Zhu, R.L., Graham, S.H., Simon, R.P., 1998. Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J. Neurosci. 18, 4914–4928. Dixon, E.P., Stephenson, D.T., Clemens, J.A., Little, S.P., 1997. Bcl-x short is elevated following severe global ischemia in rat brains.. Brain Res. 776, 222–229. Ferrer, I., Lopez, E., Blanco, R., Rivera, R., Ballabriga, J., Pozas, E., Marti, E., 1998. Bcl-2, Bax, and Bcl-x expression in the CA1 area of the hippocampus following transient forebrain ischemia in the adult gerbil. Exp. Brain Res. 121, 167–173. Han, J., Modha, D., White, E., 1998. Interaction of E1B 19K with Bax is required to block Bax-induced loss of mitochondrial membrane potential and apoptosis. Oncogene 17, 2993–3005. Isenmann, S., Stoll, G., Schroeter, M., Krajewski, S., Reed, J.C., Bahr, M., 1998. Differential regulation of Bax, Bcl-2, and Bcl-x proteins in focal cortical ischemia in the rat. Brain Pathol. 8, 49–62. Jiang, H., St. Ulme, D., Dickens, G., Chabuk, A., Lavarreda, M., Lazarovici, P., Guroff, G., 1997a. Both p140trk and p75NGFR nerve growth factor receptors mediate nerve growth factor-stimulated calcium uptake. J. Biol. Chem. 272, 6835–6837. Jiang, H., Movsesyan, V., Fink, D.W., Fasler, M., Whalin, M., Katagiri, Y., Monshipouri, M., Dickens, G., Lelkes, P.I., Guroff, G., Lazarovici, P., 1997b. Expression of human trkA receptors in trkA-deficient PC12/endothelial cells results in nerve growth factorinduced signal transduction and DNA synthesis. J. Cell. Biochem. 66, 229–244. Kitagawa, K., Matsumoto, M., Tsujimoto, Y., Ohtsuki, T., Kuwabara, K., Matsushita, K., Yang, G., Tanabe, H., Martinou, J.C., Hori, M., Yanagihara, T., 1998. Amelioration of hippocampal neuronal damage after global ischemia by neuronal overexpression of Bcl-2 in transgenic mice. Stroke 29, 2616–2621. Kluck, R.M., Bossy-Wetzel, E., Green, D.R., Newmeyer, D.D., 1997. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132–1136.
D. Koubi et al. / Neurochemistry International 46 (2005) 73–81 Krajewski, S., Mai, J.K., Krajewska, M., Sikorska, M., Mossakowski, M.J., Reed, J.C., 1995. Upregulation of bax protein levels in neurons following cerebral ischemia. J. Neurosci. 15, 6364– 6376. Lindenboim, L., Borner, C., Stein, R., 2001. Bcl-xS can form homodimers and heterodimers and its apoptotic activity requires localization of BclxS to the mitochondria and its BH3 and loop domains. Cell Death Differ. 8, 933–942. Lipton, P., 1999. Ischemic cell death in brain neurons. Physiological Rev. 79, 1431–1568. Love, S., 2003. Apoptosis and brain ischaemia. Prog. Neuropsychopharmacol. Biol. Psychiatry 27, 267–282. Maroto, R., Perez-Polo, J.R., 1997. Bcl-2-related protein expression in apoptosis: oxidative stress versus serum deprivation in PC12 cells. J. Neurochem. 69, 514–523. Marzo, I., Susin, S.A., Petit, P.X., Ravagnan, L., Brenner, C., Larochette, N., Zamzami, N., Kroemer, G., 1998. Caspases disrupt mitochondrial membrane barrier function. FEBS Lett. 427, 198–202. Murphy, K.M., Ranganathan, V., Farnsworth, M.L., Kavallaris, M., Lock, R.B., 2000. Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells. Cell Death Differ. 7, 102–111. Navarro, P., Valverde, A.M., Conejo, R., Benito, M., Lorenzo, M., 1999. Inhibition of caspases rescues brown adipocytes from apoptosis downregulating Bcl-xS and upregulating Bcl-2 gene expression. Exp. Cell Res. 246, 301–307. Park, J.W., Choi, Y.J., Suh, S.I., Baek, W.K., Suh, M.H., Jin, I.N., Min, D.S., Woo, J.H., Chang, J.S., Passaniti, A., Lee, Y.H., Kwon, T.K., 2001. Bcl-2 overexpression attenuates resveratrol-induced apoptosis in U937 cells by inhibition of caspase-3 activity. Carcinogenesis 22, 1633–1639. Petit, P.X., Susin, S.A., Zamzami, N., Mignotte, B., Kroemer, G., 1996. Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition. FEBS Lett. 396, 7–13.
81
Qian, T., Nieminen, A.L., Herman, B., Lemasters, J.J., 1997. Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am. J. Physiol. 273, C1783–C1792. Shimizu, S., Eguchi, Y., Kamiike, W., Waguri, S., Uchiyama, Y., Matsuda, H., Tsujimoto, Y., 1996. Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors. Oncogene 13, 21–29. Shimizu, S., Eguchi, Y., Kamiike, W., Funahashi, Y., Mignon, A., Lacronique, V., Matsuda, H., Tsujimoto, Y., 1998. Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux. Proc. Natl. Acad. Sci. USA 95, 1455–1459. Susin, S.A., Zamzami, N., Castedo, M., Daugas, E., Wang, H.G., Geley, S., Fassy, F., Reed, J.C., Kroemer, G., 1997. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J. Exp. Med. 186, 25–37. Tabakman, R., Lazarovici, P., Kohen, R., 2002. Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia. J. Neurosci. Res. 68, 463–469. Wang, H.D., Fukuda, T., Suzuki, T., Hashimoto, K., Liou, S.Y., Momoi, T., Kosaka, T., Yamamoto, K., Nakanishi, H., 1999. Differential effects of Bcl-2 overexpression on hippocampal CA1 neurons and dentate granule cells following hypoxic ischemia in adult mice. J. Neurosci. Res. 57, 1–12. Xu, D., Bureau, Y., McIntyre, D.C., Nicholson, D.W., Liston, P., Zhu, Y., Fong, W.G., Crocker, S.J., Korneluk, R.G., Robertson, G.S., 1999. Attenuation of ischemia-induced cellular and behavioral deficits by X chromosome-linked inhibitor of apoptosis protein overexpression in the rat hippocampus. J. Neurosci. 19, 5026–5033. Zamzami, N., Marchetti, P., Castedo, M., Hirsch, T., Susin, S.A., Masse, B., Kroemer, G., 1996. Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett. 384, 53–57. Zha, H., Reed, J.C., 1997. Heterodimerization-independent functions of cell death regulatory proteins Bax and Bcl-2 in yeast and mammalian cells. J. Biol. Chem. 272, 31482–31488.