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Altered Bad localization and interaction between Bad and Bcl-xL in the hippocampus after transient global ischemia
Brain Research 1009 (2004) 159 – 168 www.elsevier.com/locate/brainres
Research report
Altered Bad localization and interaction between Bad and Bcl-xL in the hippocampus after transient global ischemia Tsutomu Abe, Norio Takagi *, Midori Nakano, Mamiko Furuya, Satoshi Takeo Faculty of Pharmaceutical Sciences, Department of Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Accepted 2 March 2004
Abstract Accumulating evidence indicates that the mitochondrial cell-death pathway, which involves the release of cytochrome c from mitochondria, participates in neuronal cell death after transient cerebral ischemia. However, the upstream events, that induce cytochrome c release after transient global ischemia are not fully understood. Bad is a pro-apoptotic member of the bcl-2 gene family that promotes apoptosis by binding to and inhibiting functions of anti-apoptotic proteins Bcl-2 and Bcl-xL. We investigated the effects of transient (15 min) global ischemia on the intracellular localization of Bad and the interaction of Bad with calcineurin, Akt or Bcl-xL in the vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Immunoblotting analysis revealed that the amount of Bad in mitochondria significantly increased after ischemia. Co-immunoprecipitation studies showed decreased interactions of Bad with Akt and calcineurin in the cytosol and increased binding with Bcl-xL in the mitochondrial fraction of hippocampal CA1, but not in the CA3/dentate gyrus region. Further, we examined the effect of recombinant Bad on the cytochrome c release from isolated mitochondria. Treatment with both recombinant Bad and calcium, but not with recombinant Bad alone, induced cytochrome c release. These results suggest that changes in localization and complex formation by Bad are, at least in part, involved in the vulnerability of cells after transient global ischemia. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Bad; Bcl-xL; Mitochondria; Transient global ischemia
1. Introduction Morphological and biochemical features of apoptosis have been reproducibly detected in the ischemic brain, including cell membrane protrusion, chromatin condensation, formation of apoptotic bodies, and internucleosomal DNA degradation [20 – 22,24]. The results of several recent studies suggest that the cytochrome c-dependent apoptotic pathway contributes to the neuronal cell death after ischemia [10 – 12,26]. Cytochrome c release from mitochondria is also an important event in other central nervous system injuries, including brain trauma [6,30,41,49,50] and spinal cord injury [9,35]. However, the upstream events of cytochrome c release from mitochondria after transient global ischemia are not yet fully clear.
* Corresponding author. Tel./fax: +81-426-76-4584. E-mail address: [email protected] (N. Takagi). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.03.003
Anti-apoptotic members of the Bcl-2 family, including Bcl-2 and Bcl-xL, are associated with the mitochondrial outer membrane and can inhibit the release of cytochrome c [1,16,18,43]. In contrast, Bcl-2-associated death promoter (Bad), which is a pro-apoptotic member, binds to Bcl-2 and Bcl-xL, and inhibits their anti-apoptotic functions. Bad is phosphorylated by the serine– threonine kinase Akt, and phosphorylated Bad is normally maintained in the cytosol abundantly in an inactive form [7,8,14,17,33,47,48]. It was suggested that interaction of Bad with Bcl-2 or Bcl-xL on mitochondria depends on the dephosphorylation and translocation of Bad to mitochondria and that these interactions on mitochondria may initiate the opening of mitochondrial permeability transition pores and the consequent release of cytochrome c [46]. Therefore, phosphorylation states and intracellular localization of Bad may be important biochemical features in cell-death pathways. In this sense, it was shown that calcineurin, a Ca2 +-dependent protein phosphatase, contributes to apoptosis through dephosphorylation of
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Bad in cultured neurons [40]. In addition, recent study showed the translocation of Bad to the mitochondria in a mouse after transient focal cerebral ischemia [32]. However, the roles of Bad in the selective neuronal cell death in the hippocampal CA1 region after in vivo transient global ischemia are poorly understood. In the present study, we investigated the translocation of Bad to the mitochondrial fraction and changes in the interactions of Bad with calcineurin, Akt, and Bcl-xL after transient global ischemia in the vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. We also examined the direct effect of recombinant Bad on the release of cytochrome c from isolated mitochondria.
2. Materials and methods 2.1. Animal model of transient global ischemia Male Wistar rats (Charles River Japan, Atsugi, Japan), weighing 250 – 300 g, were conditioned at 23 F 1 jC with a constant humidity of 55 F 5% and a cycle of 12:12h light/darkness, given free access to food and water according to the National Institutes Guide for Care and Use of Laboratory Animals and the Guideline of Experimental Animal Care issued by the Prime Minister’s Office of Japan. The protocol of the present study was approved by the Committee of Animal Use and Welfare of Tokyo University of Pharmacy and Life Science. Transient (15 min) global ischemia was induced by a modification of the method of Sugio et al. [37], as described by Takagi et al. [38]. Sham operation was performed under the same surgical procedures except that the arteries were not occluded.
cytosol fraction. All procedures for protein fractionation were carried out at 4 jC. Determination of protein concentrations was conducted with a protein assay kit (Bio-Rad) according to the method of Bradford [3]. 2.3. Western blotting For immunoblotting, proteins were denatured in sodium dodecyl sulfate (SDS)-containing loading buffer (62.5 mM Tris – HCl [pH 6.8], 2% SDS, 10% h-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue) at 100 jC for 5 min and then separated on 10% SDS-polyacrylamide gels (40 Ag per lane) for total Akt, phospho-Akt (Ser-473) [pAkt (Ser473)], calcineurin, and a-tubulin, on 13% SDS-polyacrylamide gels for Bcl-xL or on 15% SDS-polyacrylamide gels for total Bad, phospho-Bad (Ser-112 and 136) [pBad (Ser112) and pBad (Ser-136)], cytochrome c, and cytochrome c oxidase subunit IV (COX IV). Immunoblotting was performed as described previously [36]. The working dilutions for antibodies against total Akt (Cell Signaling Technology), pAkt (Ser-473, Cell Signaling Technology), calcineurin (BD Transduction Laboratories), total Bad (Cell Signaling Technology), pBad (Ser-112, Cell Signaling Technology), pBad (Ser-136, Cell Signaling Technology), cytochrome c (Pharmingen), Bcl-xL (Santa Cruz Biotechnology), COX IV (Molecular Probes), and a-tubulin (Sigma) in the present
2.2. Mitochondrial and cytosolic protein isolation Animals were sacrificed by decapitation at 1, 12, and 24 h after the start of reperfusion after 15 min of ischemia. The hippocampi on ice were quickly removed in the cold room. Hippocampal slices (700 Am) were prepared with a McIlewain tissue chopper (Brinkmann, The Mickle Laboratory Engineering, Gomshall, Surrey, UK) and the CA1 and CA3/ dentate gyrus regions were dissected on ice in ice-cold isolation buffer containing 300 mM sucrose, 20 mM Tris – HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM glycerophosphate, 2.5 mM sodium diphosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 3.0 Ag/ml pepstatin A, 3.0 Ag/ml leupeptin, and 5.0 Ag/ml aprotinin. Tissues were homogenized in isolation buffer. Unbroken cells and nuclei were pelleted at 1200 g for 10 min. The supernatant was centrifuged at 10,000 g for 10 min to pellet the mitochondrial fraction. The pellet was washed and resuspended in isolation buffer. The resulting supernatant was then centrifuged at 100,000 g for 20 min to obtain the
Fig. 1. Effect of transient global ischemia on the levels of total Bad and phosphorylated Bad in total homogenates and cytosol fraction of the CA1 and CA3/dentate gyrus. (a) Total homogenates of the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with anti-Bad or anti-a-tubulin antibodies. Transient global ischemia had no effect on overall levels of Bad in either hippocampal region (n = 6 each). (b) Cytosol fractions in the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with anti-Bad, -pBad (Ser-112) or -pBad (Ser136) antibodies. Transient global ischemia had no effect on any form of Bad in either hippocampal region (n = 6 each).
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study were 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:500, 1:1000, 1:1000, 1:1000, and 1:10 000, respectively. To control for equal sample loading, we also performed immunoblotting with antibody against a mitochondrial marker, COX IV, and with one against a cytosolic marker, a-tubulin. The bound antibody was detected by the enhanced chemiluminescence method (Amersham Biosciences, Piscataway, NJ, USA). Quantification was performed using computerized densitometry and an image analyzer (ATTO, Tokyo, Fig. 3. Effect of transient global ischemia on the levels of calcineurin and total Akt in the cytosol fraction, and on the Bcl-xL level in the mitochondrial fraction of the CA1 and CA3/dentate gyrus. Cytosol and mitochondrial fractions of the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with calcineurin, Akt, and Bcl-xL antibodies. Transient global ischemia had no effect on levels of calcineurin and Akt in the cytosol fraction or on the Bcl-xL level in the mitochondrial fraction of either hippocampal region (n = 4 each).
Japan). Care was taken to ensure that bands to be semiquantified were in the linear range of response. To remove bound antibodies, we heated the immunoblots for 30 min at 65 jC in 62.5 mM Tris –HCl buffer, pH 6.8, containing 2% SDS and 0.1 M h-mercaptoethanol. The efficacy of the stripping procedure was confirmed by reacting the stripped blot with secondary antibody alone to ensure that no bound antibodies could be detected. 2.4. Co-immunoprecipitation
Fig. 2. Effect of transient global ischemia on the levels of total Bad and phosphorylated Bad (pBad) in mitochondrial fractions of the CA1 and CA3/dentate gyrus. (a) Mitochondrial fractions in the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with antiBad, pBad (Ser-112) or pBad (Ser-136) antibodies. Phosphorylated Bad was not detected in mitochondrial fractions throughout the experiment. (b) Bands corresponding to total Bad in mitochondrial fractions were scanned. Results are the mean percentages of control F S.E. of six separate animals for each condition. *Significant difference from the naı¨ve controls ( p < 0.05). Additional immunoblotting analysis confirmed the presence of a-tubulin and cytochrome c oxidase subunit IV (COX IV) in the cytosol (c) and mitochondrial (d) fractions, respectively.
To examine protein – protein interactions in the mitochondrial or cytosol fraction, we performed co-immunoprecipitation experiments. Mitochondrial suspensions (100 Ag) were lysed in 10 mM HEPES (pH 7.4) buffer containing 142.5 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.5% Nonidet P-40, 0.5 mM PMSF, 10 Ag/ml aprotinin, and 1 Ag/ml each of leupeptin, antipain, and pepstatin A (lysis buffer) for 30 min at 4 jC. Cytosol fractions (400 Ag) were lysed in lysis buffer containing 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid instead of 0.5% Nonidet P-40. After solubilization, the samples were centrifuged at 100,000 g for 20 min. The resulting supernatant fluid of each sample was collected and pre-cleared with protein Gagarose beads. The supernatant was incubated overnight with anti-total Bad antibody (Cell Signaling Technology). Then, 20 Al protein G-agarose beads were added, and incubation was carried out for 2 h. The beads were collected by centrifugation and washed four times with lysis buffer. All procedures for co-immunoprecipitation were carried out at 4 jC. After a final wash, the beads were added to SDSloading buffer and boiled for 5 min. Bands of Akt, calcineurin and Bcl-xL, which were co-precipitated with Bad, were scanned and differences in the obtained optical densities between sham and ischemic group were evaluated statistically by the unpaired Student’s t-test. The results were expressed as the mean percentages relative to the corresponding sham-operated group.
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2.5. In vitro effect of bad on isolated mitochondria from brain To determine if Bad induces the release of cytochrome c from mitochondria, we examined the effect of Bad on isolated brain mitochondria. Mitochondria, purified from the forebrain of naı¨ve rat as previously described [34], were
suspended at 10 mg of protein/ml in 50 mM Tris –HCl buffer, pH 7.2, containing 400 mM mannitol, 5 mg/ml bovine serum albumin and 10 mM KH2PO4 and kept on ice [16]. For the assay of cytochrome c release, mitochondria (1 mg of protein/ml) were incubated with recombinant Bad (Upstate biotechnology), calcium or both in the Tris buffer for 1 h at 30 jC. Then, the mitochondria were
Fig. 4. Changes in Bad – Akt and Bad – calcineurin interactions in the cytosol fraction and in the Bad – Bcl-xL interaction in the mitochondrial fraction after transient global ischemia. (a) Lysates of cytosol fraction in the CA1 and CA3/dentate gyrus from sham-operated (S) and four-vessel-ligated animals at 24 h of reperfusion (I/R) underwent immunoprecipitation (IP) with anti-Bad antibody, and the immunoprecipitates were then analyzed by immunoblotting (IB) with anti-Akt antibody. Input indicates total homogenates. Bands corresponding to Akt were scanned, and the results were expressed as the mean percentages of sham-operated control F S.E. of four separate animals for each condition. *Significant difference from the sham-operated controls ( p < 0.05). (b) Lysates of the cytosol fraction from sham-operated (S) and four-vessel-ligated animals at 24 h of reperfusion (I/R) underwent immunoprecipitation (IP) with anti-Bad antibody, and immunoprecipitates were then analyzed by immunoblotting (IB) with anti-calcineurin antibody. Bands corresponding to calcineurin were scanned and the results were expressed as the mean percentages of sham-operated control F S.E. of four separate animals for each condition. *Significant difference from the sham-operated controls ( p < 0.05). (c) Lysates of mitochondrial fraction from sham-operated (S) and four-vessel-ligated animals at 24 h of reperfusion (I/R) underwent immunoprecipitation (IP) with anti-Bad antibody, and immunoprecipitates were subsequently analyzed by immunoblotting (IB) with anti-BclxL antibody. Bands corresponding to Bcl-xL were scanned, and the results were expressed as the mean percentages of sham-operated control F S.E. of four separate animals for each condition. *Significant difference from the sham-operated controls ( p < 0.05).
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pelleted by centrifugation, and pellet and supernatant were subjected to immunoblotting with antibodies against cytochrome c, COX IV, and total Bad. 2.6. Statistics The results were expressed as the means F S.E. Differences between two groups were evaluated statistically by the unpaired Student’s t-test. Statistical comparison among multiple groups was made by ANOVA followed by Dunnett’s post hoc test.
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different from levels obtained from the naı¨ve control animal (Fig. 3, n = 4, each). Co-immunoprecipitation analysis using anti-Bad antibody showed that the interaction of Bad with either Akt or calcineurin in the cytosol fraction of the CA1 region, but not in that of the CA3/dentate gyrus region, after ischemia significantly decreased compared with the corresponding interaction for sham-operated animals (Fig. 4a and b, p < 0.05, n = 4 each). In contrast, the interaction of Bad with Bcl-xL in the CA1 region after ischemia significantly increased (Fig. 4c, p < 0.05, n = 4 each). 3.3. Changes in pAkt (Ser-473) after transient global ischemia
3. Results 3.1. Translocation of Bad to mitochondria after transient global ischemia In the initial set of experiments, changes in Bad protein levels in total homogenates and cytosol fraction at different time points after transient global ischemia were examined by immunoblotting analysis. The amount of Bad in total homogenates of the CA1 and CA3/dentate gyrus after ischemia was not different from the levels obtained from naı¨ve control animals (Fig. 1a, n = 6 each). Immunoblotting analysis of the cytosol fraction showed that the amounts of total Bad, pBad (Ser-112), and pBad (Ser-136) in each hippocampal region after ischemia were comparable to the levels found for the naı¨ve control animals (Fig. 1b, n = 6 each). We next examined the amount of Bad in the mitochondrial fraction after ischemia. It was noted that the amount of mitochondrial Bad in the CA1 region significantly increased at 1 h after ischemia and was maintained at significantly high levels for 24 h after ischemia (Fig. 2a and b, p < 0.05, n = 6, each). In contrast, there were no changes in the level of mitochondrial Bad in the CA3/dentate gyrus throughout the experiment (Fig. 2a and b). pBad (Ser-112 or Ser-136) in mitochondrial fraction was not detected in either hippocampal region throughout the experiment (Fig. 2a). Additional immunoblotting analysis showed that markers for the cytosol (a-tubulin) and mitochondrial (COX IV) fractions verified the separation of cytosol (Fig. 2c) and mitochondrial (Fig. 2d) fractions and the equal loading of protein samples in each lane. 3.2. Changes in levels Bad –Akt and Bad –calcineurin complexes in cytosol fraction and Bad –Bcl-xL complex in mitochondrial fraction at 24 h after transient global ischemia We next examined the interaction of Bad with calcineurin or Akt in the cytosol fraction and with Bcl-xL in the mitochondrial fraction at 24 h after transient global ischemia. The initial immunoblotting analysis showed that levels of calcineurin and total Akt in the cytosol fraction and BclxL in the mitochondrial fraction after ischemia were not
Next we examined the level of pAkt in total homogenates of the hippocampal region. In the CA1 and CA3/dentate gyrus regions, the level of pAkt significantly increased at 1 and 12 h after the start of reperfusion (Fig. 5, p < 0.05, n = 6 each), but there were no changes in total Akt levels throughout the experiment. 3.4. In vitro effect of Bad on release of cytochrome c using isolated mitochondria To determine the roles of Bad in mitochondria, we next examined the effect of Bad on cytochrome c release from
Fig. 5. Effect of transient global ischemia on the levels of phosphorylated Akt and total Akt in homogenates of the CA1 and CA3/dentate gyrus. (a) Total homogenates of the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with anti-phospho Akt [pAkt (Ser-473)] and total Akt antibodies. Transient global ischemia had no effect on overall levels of total Akt in either hippocampal region (n = 6 each). (b) Bands corresponding to pAkt (Ser-473) were scanned, and the results were expressed as the mean percentages of control F S.E. of six separate animals for each condition. *Significant difference from the naı¨ve controls ( p < 0.05).
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Fig. 6. Effects of Bad on cytochrome c release from isolated mitochondria. (a) Isolated mitochondria were incubated for 1 h at 30 jC with vehicle (C: C1 and C2 indicate samples of separate vehicle-controls) or the indicated concentrations of calcium. The mitochondria were then pelleted by centrifugation, and the resulting supernatants were analyzed by immunoblotting using anti-cytochrome c (Cyt. C) and COX IV antibodies. Bands corresponding to cytochrome c were scanned, and the results were expressed as the mean percentages of vehicle-treated control F S.E. of six separate animals for each condition. *Significant difference from the vehicle-treated controls ( p < 0.05). COX IV in the supernatant was not detected throught the experiment. (b) Mitochondrial pellets in ‘‘a’’ were analyzed by immunoblotting using anti-cytochrome c (Cyt. C) and COX IV antibodies. Bands corresponding to cytochrome c were scanned, and the results were expressed as the mean percentages of vehicle-treated control F S.E. of six separate animals for each condition. *Significant difference from the vehicle-treated controls ( p < 0.05). Mitochondrial pellet and input of equivalent amount were verified in every experiment by immunoblotting analysis using an antibody to the integral inner membrane protein COX IV. Isolated mitochondria were incubated with 0.1 or 0.3 AM recombinant Bad (reBad) alone (c) or reBad and 1 mM calcium (d). The mitochondria were then pelleted by centrifugation, and the resulting supernatants were analyzed by immunoblotting using anticytochrome c (Cyt. C) antibody. Bands corresponding to cytochrome c were scanned, and the results were expressed as the mean percentages of vehicle-treated control F S.E. of four separate animals for each condition. *Significant difference from the vehicle-treated controls ( p < 0.05). Mitochondrial pellets were analyzed using anti-Bad antibody. reBad was co-localized with the mitochondrial pellets in a concentration-dependent manner (c, d).
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isolated mitochondria. Incubation of mitochondria for 1 h in the presence of 2, 3, or 5 mM calcium resulted in a concentration-dependent release of cytochrome c into the supernatant, and COX IV was not detected in the supernatant (Fig. 6a, p < 0.05, n = 6 each), whereas the level of cytochrome c in the mitochondrial pellet was significantly decreased in the presence of 3 or 5 mM calcium (Fig. 6b, p < 0.05, n = 6 each). Additional immunoblotting showed that there were no changes in the level of COX IV in the mitochondrial pellet (Fig. 6b). Although incubation of mitochondria with recombinant Bad alone did not result in the release of cytochrome c into the supernatant (Fig. 6c, n = 4 each), co-incubation of 0.3 AM recombinant Bad with 1 mM calcium significantly increased the release of cytochrome c (Fig. 6d, p < 0.05, n = 4 each). In addition, immunoblotting analysis showed that the amount of Bad in the mitochondrial pellet increased in a concentration-dependent manner (Fig. 6c and d, n = 4 each).
4. Discussion In the present study, we focused on the role of Bad after transient global ischemia and demonstrated the following points: (1) Bad was translocated to mitochondria in the hippocampal CA1 region after transient global ischemia. (2) Interaction of mitochondrial Bad with Bcl-xL increased after ischemia. (3) In the CA1 region, Bad dissociated from Akt and calcineurin in the cytosol fraction after ischemia, whereas in the CA3/DG region, their association did not change after ischemia. (4) Although recombinant Bad alone did not induce cytochrome c release from the isolated brain mitochondria, the release did occur when recombinant Bad was used in combination with calcium. Under physiological conditions, Bad is phosphorylated on Ser-136 by Akt, and is retained in the cytosol, where it is unable to induce apoptosis [47]. In contrast, dephosphorylated Bad interacts with and antagonizes the anti-apoptotic proteins Bcl-2 and Bcl-xL on the outer membrane of mitochondria [42]. Recently, it was suggested that apoptotic cell death was induced partially through calcineurin-dependent dephosphorylation of Bad [40,42]. Therefore, dephosphorylation and translocation of Bad to mitochondria are suggested to play an important role in apoptotic cascades and subsequent cell death. In accordance with this suggestion, it was earlier demonstrated that levels of pBad (Ser-112 and Ser-136) decreased in the CA1 region after transient global ischemia [40]. However, we could not detect any decrease in cytosolic pBad (Ser-112 and Ser-136) in the CA1 region. Although the differences between these findings are not clear, severities of ischemic conditions and/or periods of reperfusion might contribute to the phosphorylation states of Bad. Therefore, a large proportion of Bad might be phosphorylated and remain in the cytosol in the present study. To achieve further insight into the roles of Bad after transient global ischemia, we next focused on the
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level of Bad in the mitochondrial fraction. The results showed an increase in mitochondrial Bad only in the CA1 region after ischemia. Furthermore, Bad in the mitochondrial fraction was not phosphorylated, indicating that a part of Bad in the cytosol had been dephosphorylated and translocated to the mitochondria after transient global ischemia. As Bad, which associates with mitochondria, can inhibit the anti-apoptotic actions of other Bcl-2 family proteins, these results led us to further examine the changes in Bad complexes in the hippocampus after transient global ischemia. We next determined the interaction of Bad with Bcl-xL in the mitochondrial fraction. The results showed that in the CA1 region, the interaction of Bad with Bcl-xL in the mitochondrial fraction increased after transient global ischemia. In other studies, similar results were indicated after traumatic spinal cord injury [36] or after transient focal ischemia [32]. Bad – Bcl-xL complexes can induce the opening of mitochondrial permeability transition pores and subsequent release of cell death factors, such as cytochrome c [46]. Therefore, the results suggest that dephosphorylation of pBad and translocation of Bad to mitochondria may initiate the Bad – Bcl-xL interaction and that the increase in this interaction in mitochondria after ischemia contributes to cell vulnerability. In general, the level of protein phosphorylation depends on the balance between kinase and phosphatase activities. It has been shown that activation of Akt results in phosphorylation of Bad at Ser-136 [2,7,8] and that calcineurin dephosphorylates Bad at Ser-112 and Ser-136 [40]. To determine the possible mechanisms of Bad translocation, we next examined the level of pAkt and the interaction of Bad with Akt or calcineurin in the cytosol fraction. Changes in pAkt (Ser-473) levels were studied after transient global ischemia [27,29,44] and focal cerebral ischemia [15,45], and the results showed a temporal increase in pAkt (Ser-473), which was comparable to our results. Akt is also phosphorylated at threonine-308 (Thr-308) and activated. Uchino et al. [40] indicated that the pAkt (Thr-308) level decreased in the CA1 region after transient global ischemia. Therefore, although further studies will be required, phosphorylation at Thr-308 might have been decreased in the present study. Whereas the pAkt (Ser473) level in the CA1 region returned to the level of the naı¨ve control at 24 h after transient global ischemia, the interaction of Bad with Akt significantly decreased in our study. Our findings suggest that in addition to changes in Akt activities, altered association of Bad with Akt may also determine the phosphorylation and translocation of Bad. We further demonstrated that Bad associated with calcineurin, which finding is consistent with results of previous studies using cell lines [13,17,40,47] and that the association of Bad with calcineurin in the CA1 region decreased after ischemia. Although the pathophysiological consequences remain to be determined, the results suggest that dephosphorylated Bad might not require the interaction with calcineurin and that changes in interaction of Bad with calcineurin might contribute to phosphorylation and local-
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ization of Bad. However, the degree of changes in the levels of pAkt, Bad – Akt complex, and Bad – calcineurin complex were not completely comparable to phosphorylation states and localization of Bad after ischemia. It has been demonstrated that Ser-112 and Ser-155 residue on Bad can be phosphorylated by PKA and/or mitogen-activated protein kinase-activated protein kinase 1 (also called RSK) [23,39]. Furthermore, phosphorylation of Ser-155 residue on Bad has been shown to be a critical event for the translocation of Bad and dimerization with Bcl-xL [23,39]. In accordance with these findings, it has been shown that phosphorylation of Ser-155 residue on Bad decreased in the cytosol and that dephosphorylated Bad was translocated from the cytosol to the mitochondria after transient focal cerebral ischemia [32]. In addition, phosphorylation of Bad at Ser-155 residue causes the interaction with 14-3-3 protein to reside in the cytosol as an inactive form of Bad [23,32]. Therefore, we cannot fully exclude the possibility that other kinases and molecular chaperones contribute to the intracellular localization and the dimerization of Bad in the hippocampal CA1 region after transient global ischemia. It has been suggested that mitochondria are the crucial site for the regulation of apoptosis by Bcl-2 family proteins. For example, pro-apoptotic proteins such as Bax and Bak induce cytochrome c release from mitochondria. In this sense, it was shown that Bax was translocated from the cytosol to the mitochondria after transient focal ischemia and induced mitochondrial membrane permeabilization by interacting with proteins of the mitochondrial permeability transition pore complex such as voltage-dependent anion channel and adenine nucleotide translocator [4]. Thus, translocation of Bax is suggested to result in the release of apoptosis-promoting factors, including cytochrome c [5,16,19,28]. In the present study, we demonstrated the increased level of Bad and interaction of Bad with Bcl-xL in the mitochondria of the hippocampal CA1 region after transient global ischemia. To further determine the role of Bad in brain mitochondria, we examined the effect of recombinant Bad on the release of cytochrome c from mitochondria. The results showed that Bad alone had no effect on the release of cytochrome c from isolated mitochondria. This result is comparable to an earlier study that cleaved Bid, which belongs to Bcl-2 homology 3 (BH3)only protein, as well as Bad, had no effect on the release of cytochrome c [31]. We further showed that Bad increased the release of cytochrome c in the presence of calcium. This calcium concentration used with recombinant Bad did not induce cytochrome c release in the absence of recombinant Bad under our experimental conditions. Therefore, although the precise mechanism remains unclear, the translocation of Bad to mitochondria under pathological conditions such as ischemia may enhance calcium-induced cytochrome c release from mitochondria. In this sense, an accumulation of calcium in the hippocampal CA1 region, but not in the CA3 region, has been demonstrated in rats after transient global ischemia [25]. This finding suggests that the selective
neuronal cell death in the hippocampal CA1 region is, at least in part, mediated by an increase in calcium concentrations in the cytosol and a translocation of Bad to the mitochondria. Although cytochrome c is well known to be released from the mitochondria as a process of apoptosis, we cannot fully exclude the possibility that endoplasmic reticulum (ER) – Golgi stress may be involved in an increase in cytosolic cytochrome c after transient global ischemia. Moreover, as unfolded proteins are accumulated by ER – Golgi stress and are capable of inducing apoptosis, further studies are needed to clarify the possible links between the ER –Golgi stress and the mitochondrial stress in the neuronal cell death in the hippocampal CA1 region after transient global ischemia. In summary, our results showed the translocation of Bad to mitochondria in a region vulnerable to ischemia, hippocampal CA1, but not in one tolerant to it, CA3/dentate gyrus. Translocation of Bad to mitochondria and binding to mitochondrial Bcl-xL might be derived from dissociation of Bad from cytosolic Akt and calcineurin. Mitochondrial Bad induced release of cytochrome c and might be a trigger of the apoptotic cascade in the transient global ischemia.
Acknowledgements This work was supported by grants-in-aid from Ministry of Education, Science, Sports, and Culture of Japan.
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Research report
Altered Bad localization and interaction between Bad and Bcl-xL in the hippocampus after transient global ischemia Tsutomu Abe, Norio Takagi *, Midori Nakano, Mamiko Furuya, Satoshi Takeo Faculty of Pharmaceutical Sciences, Department of Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Accepted 2 March 2004
Abstract Accumulating evidence indicates that the mitochondrial cell-death pathway, which involves the release of cytochrome c from mitochondria, participates in neuronal cell death after transient cerebral ischemia. However, the upstream events, that induce cytochrome c release after transient global ischemia are not fully understood. Bad is a pro-apoptotic member of the bcl-2 gene family that promotes apoptosis by binding to and inhibiting functions of anti-apoptotic proteins Bcl-2 and Bcl-xL. We investigated the effects of transient (15 min) global ischemia on the intracellular localization of Bad and the interaction of Bad with calcineurin, Akt or Bcl-xL in the vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Immunoblotting analysis revealed that the amount of Bad in mitochondria significantly increased after ischemia. Co-immunoprecipitation studies showed decreased interactions of Bad with Akt and calcineurin in the cytosol and increased binding with Bcl-xL in the mitochondrial fraction of hippocampal CA1, but not in the CA3/dentate gyrus region. Further, we examined the effect of recombinant Bad on the cytochrome c release from isolated mitochondria. Treatment with both recombinant Bad and calcium, but not with recombinant Bad alone, induced cytochrome c release. These results suggest that changes in localization and complex formation by Bad are, at least in part, involved in the vulnerability of cells after transient global ischemia. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Bad; Bcl-xL; Mitochondria; Transient global ischemia
1. Introduction Morphological and biochemical features of apoptosis have been reproducibly detected in the ischemic brain, including cell membrane protrusion, chromatin condensation, formation of apoptotic bodies, and internucleosomal DNA degradation [20 – 22,24]. The results of several recent studies suggest that the cytochrome c-dependent apoptotic pathway contributes to the neuronal cell death after ischemia [10 – 12,26]. Cytochrome c release from mitochondria is also an important event in other central nervous system injuries, including brain trauma [6,30,41,49,50] and spinal cord injury [9,35]. However, the upstream events of cytochrome c release from mitochondria after transient global ischemia are not yet fully clear.
* Corresponding author. Tel./fax: +81-426-76-4584. E-mail address: [email protected] (N. Takagi). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.03.003
Anti-apoptotic members of the Bcl-2 family, including Bcl-2 and Bcl-xL, are associated with the mitochondrial outer membrane and can inhibit the release of cytochrome c [1,16,18,43]. In contrast, Bcl-2-associated death promoter (Bad), which is a pro-apoptotic member, binds to Bcl-2 and Bcl-xL, and inhibits their anti-apoptotic functions. Bad is phosphorylated by the serine– threonine kinase Akt, and phosphorylated Bad is normally maintained in the cytosol abundantly in an inactive form [7,8,14,17,33,47,48]. It was suggested that interaction of Bad with Bcl-2 or Bcl-xL on mitochondria depends on the dephosphorylation and translocation of Bad to mitochondria and that these interactions on mitochondria may initiate the opening of mitochondrial permeability transition pores and the consequent release of cytochrome c [46]. Therefore, phosphorylation states and intracellular localization of Bad may be important biochemical features in cell-death pathways. In this sense, it was shown that calcineurin, a Ca2 +-dependent protein phosphatase, contributes to apoptosis through dephosphorylation of
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Bad in cultured neurons [40]. In addition, recent study showed the translocation of Bad to the mitochondria in a mouse after transient focal cerebral ischemia [32]. However, the roles of Bad in the selective neuronal cell death in the hippocampal CA1 region after in vivo transient global ischemia are poorly understood. In the present study, we investigated the translocation of Bad to the mitochondrial fraction and changes in the interactions of Bad with calcineurin, Akt, and Bcl-xL after transient global ischemia in the vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. We also examined the direct effect of recombinant Bad on the release of cytochrome c from isolated mitochondria.
2. Materials and methods 2.1. Animal model of transient global ischemia Male Wistar rats (Charles River Japan, Atsugi, Japan), weighing 250 – 300 g, were conditioned at 23 F 1 jC with a constant humidity of 55 F 5% and a cycle of 12:12h light/darkness, given free access to food and water according to the National Institutes Guide for Care and Use of Laboratory Animals and the Guideline of Experimental Animal Care issued by the Prime Minister’s Office of Japan. The protocol of the present study was approved by the Committee of Animal Use and Welfare of Tokyo University of Pharmacy and Life Science. Transient (15 min) global ischemia was induced by a modification of the method of Sugio et al. [37], as described by Takagi et al. [38]. Sham operation was performed under the same surgical procedures except that the arteries were not occluded.
cytosol fraction. All procedures for protein fractionation were carried out at 4 jC. Determination of protein concentrations was conducted with a protein assay kit (Bio-Rad) according to the method of Bradford [3]. 2.3. Western blotting For immunoblotting, proteins were denatured in sodium dodecyl sulfate (SDS)-containing loading buffer (62.5 mM Tris – HCl [pH 6.8], 2% SDS, 10% h-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue) at 100 jC for 5 min and then separated on 10% SDS-polyacrylamide gels (40 Ag per lane) for total Akt, phospho-Akt (Ser-473) [pAkt (Ser473)], calcineurin, and a-tubulin, on 13% SDS-polyacrylamide gels for Bcl-xL or on 15% SDS-polyacrylamide gels for total Bad, phospho-Bad (Ser-112 and 136) [pBad (Ser112) and pBad (Ser-136)], cytochrome c, and cytochrome c oxidase subunit IV (COX IV). Immunoblotting was performed as described previously [36]. The working dilutions for antibodies against total Akt (Cell Signaling Technology), pAkt (Ser-473, Cell Signaling Technology), calcineurin (BD Transduction Laboratories), total Bad (Cell Signaling Technology), pBad (Ser-112, Cell Signaling Technology), pBad (Ser-136, Cell Signaling Technology), cytochrome c (Pharmingen), Bcl-xL (Santa Cruz Biotechnology), COX IV (Molecular Probes), and a-tubulin (Sigma) in the present
2.2. Mitochondrial and cytosolic protein isolation Animals were sacrificed by decapitation at 1, 12, and 24 h after the start of reperfusion after 15 min of ischemia. The hippocampi on ice were quickly removed in the cold room. Hippocampal slices (700 Am) were prepared with a McIlewain tissue chopper (Brinkmann, The Mickle Laboratory Engineering, Gomshall, Surrey, UK) and the CA1 and CA3/ dentate gyrus regions were dissected on ice in ice-cold isolation buffer containing 300 mM sucrose, 20 mM Tris – HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM glycerophosphate, 2.5 mM sodium diphosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 3.0 Ag/ml pepstatin A, 3.0 Ag/ml leupeptin, and 5.0 Ag/ml aprotinin. Tissues were homogenized in isolation buffer. Unbroken cells and nuclei were pelleted at 1200 g for 10 min. The supernatant was centrifuged at 10,000 g for 10 min to pellet the mitochondrial fraction. The pellet was washed and resuspended in isolation buffer. The resulting supernatant was then centrifuged at 100,000 g for 20 min to obtain the
Fig. 1. Effect of transient global ischemia on the levels of total Bad and phosphorylated Bad in total homogenates and cytosol fraction of the CA1 and CA3/dentate gyrus. (a) Total homogenates of the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with anti-Bad or anti-a-tubulin antibodies. Transient global ischemia had no effect on overall levels of Bad in either hippocampal region (n = 6 each). (b) Cytosol fractions in the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with anti-Bad, -pBad (Ser-112) or -pBad (Ser136) antibodies. Transient global ischemia had no effect on any form of Bad in either hippocampal region (n = 6 each).
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study were 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:500, 1:1000, 1:1000, 1:1000, and 1:10 000, respectively. To control for equal sample loading, we also performed immunoblotting with antibody against a mitochondrial marker, COX IV, and with one against a cytosolic marker, a-tubulin. The bound antibody was detected by the enhanced chemiluminescence method (Amersham Biosciences, Piscataway, NJ, USA). Quantification was performed using computerized densitometry and an image analyzer (ATTO, Tokyo, Fig. 3. Effect of transient global ischemia on the levels of calcineurin and total Akt in the cytosol fraction, and on the Bcl-xL level in the mitochondrial fraction of the CA1 and CA3/dentate gyrus. Cytosol and mitochondrial fractions of the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with calcineurin, Akt, and Bcl-xL antibodies. Transient global ischemia had no effect on levels of calcineurin and Akt in the cytosol fraction or on the Bcl-xL level in the mitochondrial fraction of either hippocampal region (n = 4 each).
Japan). Care was taken to ensure that bands to be semiquantified were in the linear range of response. To remove bound antibodies, we heated the immunoblots for 30 min at 65 jC in 62.5 mM Tris –HCl buffer, pH 6.8, containing 2% SDS and 0.1 M h-mercaptoethanol. The efficacy of the stripping procedure was confirmed by reacting the stripped blot with secondary antibody alone to ensure that no bound antibodies could be detected. 2.4. Co-immunoprecipitation
Fig. 2. Effect of transient global ischemia on the levels of total Bad and phosphorylated Bad (pBad) in mitochondrial fractions of the CA1 and CA3/dentate gyrus. (a) Mitochondrial fractions in the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with antiBad, pBad (Ser-112) or pBad (Ser-136) antibodies. Phosphorylated Bad was not detected in mitochondrial fractions throughout the experiment. (b) Bands corresponding to total Bad in mitochondrial fractions were scanned. Results are the mean percentages of control F S.E. of six separate animals for each condition. *Significant difference from the naı¨ve controls ( p < 0.05). Additional immunoblotting analysis confirmed the presence of a-tubulin and cytochrome c oxidase subunit IV (COX IV) in the cytosol (c) and mitochondrial (d) fractions, respectively.
To examine protein – protein interactions in the mitochondrial or cytosol fraction, we performed co-immunoprecipitation experiments. Mitochondrial suspensions (100 Ag) were lysed in 10 mM HEPES (pH 7.4) buffer containing 142.5 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.5% Nonidet P-40, 0.5 mM PMSF, 10 Ag/ml aprotinin, and 1 Ag/ml each of leupeptin, antipain, and pepstatin A (lysis buffer) for 30 min at 4 jC. Cytosol fractions (400 Ag) were lysed in lysis buffer containing 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid instead of 0.5% Nonidet P-40. After solubilization, the samples were centrifuged at 100,000 g for 20 min. The resulting supernatant fluid of each sample was collected and pre-cleared with protein Gagarose beads. The supernatant was incubated overnight with anti-total Bad antibody (Cell Signaling Technology). Then, 20 Al protein G-agarose beads were added, and incubation was carried out for 2 h. The beads were collected by centrifugation and washed four times with lysis buffer. All procedures for co-immunoprecipitation were carried out at 4 jC. After a final wash, the beads were added to SDSloading buffer and boiled for 5 min. Bands of Akt, calcineurin and Bcl-xL, which were co-precipitated with Bad, were scanned and differences in the obtained optical densities between sham and ischemic group were evaluated statistically by the unpaired Student’s t-test. The results were expressed as the mean percentages relative to the corresponding sham-operated group.
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2.5. In vitro effect of bad on isolated mitochondria from brain To determine if Bad induces the release of cytochrome c from mitochondria, we examined the effect of Bad on isolated brain mitochondria. Mitochondria, purified from the forebrain of naı¨ve rat as previously described [34], were
suspended at 10 mg of protein/ml in 50 mM Tris –HCl buffer, pH 7.2, containing 400 mM mannitol, 5 mg/ml bovine serum albumin and 10 mM KH2PO4 and kept on ice [16]. For the assay of cytochrome c release, mitochondria (1 mg of protein/ml) were incubated with recombinant Bad (Upstate biotechnology), calcium or both in the Tris buffer for 1 h at 30 jC. Then, the mitochondria were
Fig. 4. Changes in Bad – Akt and Bad – calcineurin interactions in the cytosol fraction and in the Bad – Bcl-xL interaction in the mitochondrial fraction after transient global ischemia. (a) Lysates of cytosol fraction in the CA1 and CA3/dentate gyrus from sham-operated (S) and four-vessel-ligated animals at 24 h of reperfusion (I/R) underwent immunoprecipitation (IP) with anti-Bad antibody, and the immunoprecipitates were then analyzed by immunoblotting (IB) with anti-Akt antibody. Input indicates total homogenates. Bands corresponding to Akt were scanned, and the results were expressed as the mean percentages of sham-operated control F S.E. of four separate animals for each condition. *Significant difference from the sham-operated controls ( p < 0.05). (b) Lysates of the cytosol fraction from sham-operated (S) and four-vessel-ligated animals at 24 h of reperfusion (I/R) underwent immunoprecipitation (IP) with anti-Bad antibody, and immunoprecipitates were then analyzed by immunoblotting (IB) with anti-calcineurin antibody. Bands corresponding to calcineurin were scanned and the results were expressed as the mean percentages of sham-operated control F S.E. of four separate animals for each condition. *Significant difference from the sham-operated controls ( p < 0.05). (c) Lysates of mitochondrial fraction from sham-operated (S) and four-vessel-ligated animals at 24 h of reperfusion (I/R) underwent immunoprecipitation (IP) with anti-Bad antibody, and immunoprecipitates were subsequently analyzed by immunoblotting (IB) with anti-BclxL antibody. Bands corresponding to Bcl-xL were scanned, and the results were expressed as the mean percentages of sham-operated control F S.E. of four separate animals for each condition. *Significant difference from the sham-operated controls ( p < 0.05).
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pelleted by centrifugation, and pellet and supernatant were subjected to immunoblotting with antibodies against cytochrome c, COX IV, and total Bad. 2.6. Statistics The results were expressed as the means F S.E. Differences between two groups were evaluated statistically by the unpaired Student’s t-test. Statistical comparison among multiple groups was made by ANOVA followed by Dunnett’s post hoc test.
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different from levels obtained from the naı¨ve control animal (Fig. 3, n = 4, each). Co-immunoprecipitation analysis using anti-Bad antibody showed that the interaction of Bad with either Akt or calcineurin in the cytosol fraction of the CA1 region, but not in that of the CA3/dentate gyrus region, after ischemia significantly decreased compared with the corresponding interaction for sham-operated animals (Fig. 4a and b, p < 0.05, n = 4 each). In contrast, the interaction of Bad with Bcl-xL in the CA1 region after ischemia significantly increased (Fig. 4c, p < 0.05, n = 4 each). 3.3. Changes in pAkt (Ser-473) after transient global ischemia
3. Results 3.1. Translocation of Bad to mitochondria after transient global ischemia In the initial set of experiments, changes in Bad protein levels in total homogenates and cytosol fraction at different time points after transient global ischemia were examined by immunoblotting analysis. The amount of Bad in total homogenates of the CA1 and CA3/dentate gyrus after ischemia was not different from the levels obtained from naı¨ve control animals (Fig. 1a, n = 6 each). Immunoblotting analysis of the cytosol fraction showed that the amounts of total Bad, pBad (Ser-112), and pBad (Ser-136) in each hippocampal region after ischemia were comparable to the levels found for the naı¨ve control animals (Fig. 1b, n = 6 each). We next examined the amount of Bad in the mitochondrial fraction after ischemia. It was noted that the amount of mitochondrial Bad in the CA1 region significantly increased at 1 h after ischemia and was maintained at significantly high levels for 24 h after ischemia (Fig. 2a and b, p < 0.05, n = 6, each). In contrast, there were no changes in the level of mitochondrial Bad in the CA3/dentate gyrus throughout the experiment (Fig. 2a and b). pBad (Ser-112 or Ser-136) in mitochondrial fraction was not detected in either hippocampal region throughout the experiment (Fig. 2a). Additional immunoblotting analysis showed that markers for the cytosol (a-tubulin) and mitochondrial (COX IV) fractions verified the separation of cytosol (Fig. 2c) and mitochondrial (Fig. 2d) fractions and the equal loading of protein samples in each lane. 3.2. Changes in levels Bad –Akt and Bad –calcineurin complexes in cytosol fraction and Bad –Bcl-xL complex in mitochondrial fraction at 24 h after transient global ischemia We next examined the interaction of Bad with calcineurin or Akt in the cytosol fraction and with Bcl-xL in the mitochondrial fraction at 24 h after transient global ischemia. The initial immunoblotting analysis showed that levels of calcineurin and total Akt in the cytosol fraction and BclxL in the mitochondrial fraction after ischemia were not
Next we examined the level of pAkt in total homogenates of the hippocampal region. In the CA1 and CA3/dentate gyrus regions, the level of pAkt significantly increased at 1 and 12 h after the start of reperfusion (Fig. 5, p < 0.05, n = 6 each), but there were no changes in total Akt levels throughout the experiment. 3.4. In vitro effect of Bad on release of cytochrome c using isolated mitochondria To determine the roles of Bad in mitochondria, we next examined the effect of Bad on cytochrome c release from
Fig. 5. Effect of transient global ischemia on the levels of phosphorylated Akt and total Akt in homogenates of the CA1 and CA3/dentate gyrus. (a) Total homogenates of the CA1 and CA3/dentate gyrus from naı¨ve control animals (C) and four-vessel-ligated animals at 1, 12, and 24 h of reperfusion were analyzed by immunoblotting with anti-phospho Akt [pAkt (Ser-473)] and total Akt antibodies. Transient global ischemia had no effect on overall levels of total Akt in either hippocampal region (n = 6 each). (b) Bands corresponding to pAkt (Ser-473) were scanned, and the results were expressed as the mean percentages of control F S.E. of six separate animals for each condition. *Significant difference from the naı¨ve controls ( p < 0.05).
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Fig. 6. Effects of Bad on cytochrome c release from isolated mitochondria. (a) Isolated mitochondria were incubated for 1 h at 30 jC with vehicle (C: C1 and C2 indicate samples of separate vehicle-controls) or the indicated concentrations of calcium. The mitochondria were then pelleted by centrifugation, and the resulting supernatants were analyzed by immunoblotting using anti-cytochrome c (Cyt. C) and COX IV antibodies. Bands corresponding to cytochrome c were scanned, and the results were expressed as the mean percentages of vehicle-treated control F S.E. of six separate animals for each condition. *Significant difference from the vehicle-treated controls ( p < 0.05). COX IV in the supernatant was not detected throught the experiment. (b) Mitochondrial pellets in ‘‘a’’ were analyzed by immunoblotting using anti-cytochrome c (Cyt. C) and COX IV antibodies. Bands corresponding to cytochrome c were scanned, and the results were expressed as the mean percentages of vehicle-treated control F S.E. of six separate animals for each condition. *Significant difference from the vehicle-treated controls ( p < 0.05). Mitochondrial pellet and input of equivalent amount were verified in every experiment by immunoblotting analysis using an antibody to the integral inner membrane protein COX IV. Isolated mitochondria were incubated with 0.1 or 0.3 AM recombinant Bad (reBad) alone (c) or reBad and 1 mM calcium (d). The mitochondria were then pelleted by centrifugation, and the resulting supernatants were analyzed by immunoblotting using anticytochrome c (Cyt. C) antibody. Bands corresponding to cytochrome c were scanned, and the results were expressed as the mean percentages of vehicle-treated control F S.E. of four separate animals for each condition. *Significant difference from the vehicle-treated controls ( p < 0.05). Mitochondrial pellets were analyzed using anti-Bad antibody. reBad was co-localized with the mitochondrial pellets in a concentration-dependent manner (c, d).
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isolated mitochondria. Incubation of mitochondria for 1 h in the presence of 2, 3, or 5 mM calcium resulted in a concentration-dependent release of cytochrome c into the supernatant, and COX IV was not detected in the supernatant (Fig. 6a, p < 0.05, n = 6 each), whereas the level of cytochrome c in the mitochondrial pellet was significantly decreased in the presence of 3 or 5 mM calcium (Fig. 6b, p < 0.05, n = 6 each). Additional immunoblotting showed that there were no changes in the level of COX IV in the mitochondrial pellet (Fig. 6b). Although incubation of mitochondria with recombinant Bad alone did not result in the release of cytochrome c into the supernatant (Fig. 6c, n = 4 each), co-incubation of 0.3 AM recombinant Bad with 1 mM calcium significantly increased the release of cytochrome c (Fig. 6d, p < 0.05, n = 4 each). In addition, immunoblotting analysis showed that the amount of Bad in the mitochondrial pellet increased in a concentration-dependent manner (Fig. 6c and d, n = 4 each).
4. Discussion In the present study, we focused on the role of Bad after transient global ischemia and demonstrated the following points: (1) Bad was translocated to mitochondria in the hippocampal CA1 region after transient global ischemia. (2) Interaction of mitochondrial Bad with Bcl-xL increased after ischemia. (3) In the CA1 region, Bad dissociated from Akt and calcineurin in the cytosol fraction after ischemia, whereas in the CA3/DG region, their association did not change after ischemia. (4) Although recombinant Bad alone did not induce cytochrome c release from the isolated brain mitochondria, the release did occur when recombinant Bad was used in combination with calcium. Under physiological conditions, Bad is phosphorylated on Ser-136 by Akt, and is retained in the cytosol, where it is unable to induce apoptosis [47]. In contrast, dephosphorylated Bad interacts with and antagonizes the anti-apoptotic proteins Bcl-2 and Bcl-xL on the outer membrane of mitochondria [42]. Recently, it was suggested that apoptotic cell death was induced partially through calcineurin-dependent dephosphorylation of Bad [40,42]. Therefore, dephosphorylation and translocation of Bad to mitochondria are suggested to play an important role in apoptotic cascades and subsequent cell death. In accordance with this suggestion, it was earlier demonstrated that levels of pBad (Ser-112 and Ser-136) decreased in the CA1 region after transient global ischemia [40]. However, we could not detect any decrease in cytosolic pBad (Ser-112 and Ser-136) in the CA1 region. Although the differences between these findings are not clear, severities of ischemic conditions and/or periods of reperfusion might contribute to the phosphorylation states of Bad. Therefore, a large proportion of Bad might be phosphorylated and remain in the cytosol in the present study. To achieve further insight into the roles of Bad after transient global ischemia, we next focused on the
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level of Bad in the mitochondrial fraction. The results showed an increase in mitochondrial Bad only in the CA1 region after ischemia. Furthermore, Bad in the mitochondrial fraction was not phosphorylated, indicating that a part of Bad in the cytosol had been dephosphorylated and translocated to the mitochondria after transient global ischemia. As Bad, which associates with mitochondria, can inhibit the anti-apoptotic actions of other Bcl-2 family proteins, these results led us to further examine the changes in Bad complexes in the hippocampus after transient global ischemia. We next determined the interaction of Bad with Bcl-xL in the mitochondrial fraction. The results showed that in the CA1 region, the interaction of Bad with Bcl-xL in the mitochondrial fraction increased after transient global ischemia. In other studies, similar results were indicated after traumatic spinal cord injury [36] or after transient focal ischemia [32]. Bad – Bcl-xL complexes can induce the opening of mitochondrial permeability transition pores and subsequent release of cell death factors, such as cytochrome c [46]. Therefore, the results suggest that dephosphorylation of pBad and translocation of Bad to mitochondria may initiate the Bad – Bcl-xL interaction and that the increase in this interaction in mitochondria after ischemia contributes to cell vulnerability. In general, the level of protein phosphorylation depends on the balance between kinase and phosphatase activities. It has been shown that activation of Akt results in phosphorylation of Bad at Ser-136 [2,7,8] and that calcineurin dephosphorylates Bad at Ser-112 and Ser-136 [40]. To determine the possible mechanisms of Bad translocation, we next examined the level of pAkt and the interaction of Bad with Akt or calcineurin in the cytosol fraction. Changes in pAkt (Ser-473) levels were studied after transient global ischemia [27,29,44] and focal cerebral ischemia [15,45], and the results showed a temporal increase in pAkt (Ser-473), which was comparable to our results. Akt is also phosphorylated at threonine-308 (Thr-308) and activated. Uchino et al. [40] indicated that the pAkt (Thr-308) level decreased in the CA1 region after transient global ischemia. Therefore, although further studies will be required, phosphorylation at Thr-308 might have been decreased in the present study. Whereas the pAkt (Ser473) level in the CA1 region returned to the level of the naı¨ve control at 24 h after transient global ischemia, the interaction of Bad with Akt significantly decreased in our study. Our findings suggest that in addition to changes in Akt activities, altered association of Bad with Akt may also determine the phosphorylation and translocation of Bad. We further demonstrated that Bad associated with calcineurin, which finding is consistent with results of previous studies using cell lines [13,17,40,47] and that the association of Bad with calcineurin in the CA1 region decreased after ischemia. Although the pathophysiological consequences remain to be determined, the results suggest that dephosphorylated Bad might not require the interaction with calcineurin and that changes in interaction of Bad with calcineurin might contribute to phosphorylation and local-
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ization of Bad. However, the degree of changes in the levels of pAkt, Bad – Akt complex, and Bad – calcineurin complex were not completely comparable to phosphorylation states and localization of Bad after ischemia. It has been demonstrated that Ser-112 and Ser-155 residue on Bad can be phosphorylated by PKA and/or mitogen-activated protein kinase-activated protein kinase 1 (also called RSK) [23,39]. Furthermore, phosphorylation of Ser-155 residue on Bad has been shown to be a critical event for the translocation of Bad and dimerization with Bcl-xL [23,39]. In accordance with these findings, it has been shown that phosphorylation of Ser-155 residue on Bad decreased in the cytosol and that dephosphorylated Bad was translocated from the cytosol to the mitochondria after transient focal cerebral ischemia [32]. In addition, phosphorylation of Bad at Ser-155 residue causes the interaction with 14-3-3 protein to reside in the cytosol as an inactive form of Bad [23,32]. Therefore, we cannot fully exclude the possibility that other kinases and molecular chaperones contribute to the intracellular localization and the dimerization of Bad in the hippocampal CA1 region after transient global ischemia. It has been suggested that mitochondria are the crucial site for the regulation of apoptosis by Bcl-2 family proteins. For example, pro-apoptotic proteins such as Bax and Bak induce cytochrome c release from mitochondria. In this sense, it was shown that Bax was translocated from the cytosol to the mitochondria after transient focal ischemia and induced mitochondrial membrane permeabilization by interacting with proteins of the mitochondrial permeability transition pore complex such as voltage-dependent anion channel and adenine nucleotide translocator [4]. Thus, translocation of Bax is suggested to result in the release of apoptosis-promoting factors, including cytochrome c [5,16,19,28]. In the present study, we demonstrated the increased level of Bad and interaction of Bad with Bcl-xL in the mitochondria of the hippocampal CA1 region after transient global ischemia. To further determine the role of Bad in brain mitochondria, we examined the effect of recombinant Bad on the release of cytochrome c from mitochondria. The results showed that Bad alone had no effect on the release of cytochrome c from isolated mitochondria. This result is comparable to an earlier study that cleaved Bid, which belongs to Bcl-2 homology 3 (BH3)only protein, as well as Bad, had no effect on the release of cytochrome c [31]. We further showed that Bad increased the release of cytochrome c in the presence of calcium. This calcium concentration used with recombinant Bad did not induce cytochrome c release in the absence of recombinant Bad under our experimental conditions. Therefore, although the precise mechanism remains unclear, the translocation of Bad to mitochondria under pathological conditions such as ischemia may enhance calcium-induced cytochrome c release from mitochondria. In this sense, an accumulation of calcium in the hippocampal CA1 region, but not in the CA3 region, has been demonstrated in rats after transient global ischemia [25]. This finding suggests that the selective
neuronal cell death in the hippocampal CA1 region is, at least in part, mediated by an increase in calcium concentrations in the cytosol and a translocation of Bad to the mitochondria. Although cytochrome c is well known to be released from the mitochondria as a process of apoptosis, we cannot fully exclude the possibility that endoplasmic reticulum (ER) – Golgi stress may be involved in an increase in cytosolic cytochrome c after transient global ischemia. Moreover, as unfolded proteins are accumulated by ER – Golgi stress and are capable of inducing apoptosis, further studies are needed to clarify the possible links between the ER –Golgi stress and the mitochondrial stress in the neuronal cell death in the hippocampal CA1 region after transient global ischemia. In summary, our results showed the translocation of Bad to mitochondria in a region vulnerable to ischemia, hippocampal CA1, but not in one tolerant to it, CA3/dentate gyrus. Translocation of Bad to mitochondria and binding to mitochondrial Bcl-xL might be derived from dissociation of Bad from cytosolic Akt and calcineurin. Mitochondrial Bad induced release of cytochrome c and might be a trigger of the apoptotic cascade in the transient global ischemia.
Acknowledgements This work was supported by grants-in-aid from Ministry of Education, Science, Sports, and Culture of Japan.
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