DIABLO in the rat hippocampus following transient forebrain ischemia

Neurochemistry International 46 (2005) 41–51 www.elsevier.com/locate/neuint

Regulation of XIAP and Smac/DIABLO in the rat hippocampus following transient forebrain ischemia M.D. Siegelin, L.S. Kossatz, J. Winckler, A. Rami* Institute of Anatomy III, Dr. Senckenbergische Anatomie, Clinic of the JWG-University, Theodor-Stern-Kai 7, Frankfurt/Main 60590, Germany Received 15 April 2004; received in revised form 7 July 2004; accepted 9 July 2004 Available online 3 October 2004 This paper is dedicated to the memory of Professor Ju¨rgen Winckler, who died on May 15, 2004.

Abstract We investigated the expression of XIAP (X chromosome-linked inhibitor of apoptosis protein) and Smac/DIABLO, a newly identified mitochondrila apoptogenig molecule in the hippocampus following transient global ischemia. Transient global ischemia produced by twovessel occlusion triggers the delayed neuronal death of CA1 neurons in the hippocampus. We demonstrate that CA1 neuronal loss induced by ischemia (10 min) is preceded by a selective and marked elevation of catalytically active caspase-3 in these neurons, indicative of apoptosis. XIAP (X chromosome-linked inhibitor of apoptosis protein) is a member of the inhibitor of apoptosis (IAP) gene family that, in addition to suppressing cell death by inhibition of caspases, is involved in an increasing number of signalling cascades. The present study shows alterations in the levels of XIAP and of Smac/DIABLO (second mitochondrial activator of caspase) after cerebral ischemia. The protein levels of XIAP and the number of XIAP-positive cells were regulated by cerebral ischemia in a strictly time and region dependent manner. The largest change in XIAP-IR was observed in the CA1 sub field, which is the most vulnerable area of hippocampus. The mitochondrial expression level of Smac/DIABLO increased during reperfusion. Smac/DIABLO expression was associated with alteration of the XIAP levels and the appearance of activated form of caspase-3 within the hippocampus during reperfusion in spatial and temporal manners. # 2004 Elsevier Ltd. All rights reserved. Keywords: XIAP; Smac/DIABLO; Hippocampus; Ischemia; Apoptosis

1. Introduction A number of recent studies suggest that cell death after ischemia involves apoptosis, an active and genetically controlled cell suicide process. Histological and biochemical characteristics of apoptosis are present in dying neurones after ischemia (Rami et al., 2003a,b; Rami, 2003). One basic challenge is to define the temporal and regional pattern of apoptotic cell changes after transient forebrain ischemia, and to correlate this localisation with principal effectors of the apoptotic pathways. It is evident that apoptosis is controlled by the caspase family (Deveraux et al., 1998, 1999; Roy et al., 1997; Takahashi et al., 1998). It has been suggested that release of cytochrome c from * Corresponding author. Tel.: +49 69 63016929; fax: +49 69 63016920. E-mail address: [email protected] (A. Rami). 0197-0186/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2004.07.009

mitochondria is sufficient to induce the nucleation of apoptosome, multiprotein complex that contain the zymogen forms of caspase-9 and-3, Apaf-1, and perhaps other factors (Srinivasan et al., 1998). When recruited to the apoptosome, caspase-9 is activated by autolytic cleavage and in turn cleaves and activates caspase-3. Consistent with this mechanism, direct injection of cytochrome c into the cell cytosol is reported to induce apoptosis in several cell types. The activation of initiator caspases is thought to irreversibly trigger the caspase cascade, necessitating that caspase activation be tightly regulated by layered control mechanisms. To avoid disease and inappropriate cell death, apoptotic mechanisms must be tightly regulated. Recent experimental evidence has revealed that apoptosis can be abolished and regulated at several distinct check points in the apoptoptic pathway. Among the growing number of cellular proteins

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that have been shown to regulate caspase activation are the IAPs (Inhibitor of apoptosis proteins), including c-IAP1, cIAP2, XIAP, and Survivin (LaCasse et al., 1998; Rami, 2003). These proteins have been reported to block mitochondrially-mediated apoptotic pathways by directly inhibiting initiator and effector caspases. Several of the human IAP family proteins have been reported to directly bind and inhibit specific members of the caspase family. For instance, XIAP, c-IAP1, c-IAP2, and Survivin directly bind and inhibit caspases-3, -7, and -9 but not caspase 1, 6, 8, or 10 (Deveraux et al., 1997, 1998; Holcik et al., 2001). Moreover, IAPs themselves are regulated by cellular proteins (Silke et al., 2000) such as Smac/DIABLO, a mitochondrial protein released together with cytochrome c from the mitochondria into the cytosol in response to apoptotic stimuli, and found to promote caspase activation by binding and eliminating IAP function. Smac/DIABLO binds to most known human IAP family members and relieves their inhibition of caspase activity. Of all the IAP family members known to date, the X-linked inhibitor of apoptosis protein (XIAP) is recognized as the most potent caspase inhibitor (Deveraux et al., 1997, 1999; Liston et al., 1996). In contrast to other cell types, the expression and functions of IAPs in the nervous system have been less studied. However, preliminary data indicate that IAP family proteins, can compromise cell demise in various neuropathological situations. For instance, adenovirus mediated overexpression of XIAP inhibited cell death in the substantia nigra induced by MPTP (Eberhardt et al., 2000). In addition, apoptosis in cerebellar granule cells (Simons et al., 1999) and glioma (Wagenknecht et al., 2000) was attenuated by XIAP. Furthermore, increased levels of XIAP have been reported in the spinal cord following moderate traumatic injury (Keane et al., 2001b) and after cardiac arrest (Katz et al., 2001). However, little is known about the regulation of XIAP and Smac/DIABLO in the hippocampal formation following transient forebrain ischemia in rats. In this study we demonstrated the subcellular redistribution of XIAP and Smac/DIABLO by Western blot and immunyohistochemistry in the rat hippocampus following transient global cerebral ischemia by using the 2-vessel occlusion model of Smith et al. (1984).

2. Materials and methods 2.1. Cerebral ischemia Transient forebrain ischemia was performed in male Wistar rats (250–300 g) according to Smith et al. (1984). Cerebral ischemia was induced by clamping both common carotid arteries and lowering the mean arterial blood pressure to 40 mmHg. After 10 min ischemia the blood pressure was restored by the infusion of blood and the removal of the clamps. Arterial pH, pCO2, pO2, arterial

blood pressure and plasma glucose concentration were determined 15 min before and 15 min after the induction of ischemia. Thirty rats (controls sham operated and ischemic rats taken at 6, 12, 24, 48, and 72 h post-ischemia, n = 5) were used for histology, immunocytochemistry (n = 2) and immunoblotting (n = 3). Rats have been perfused 6, 12, 24, 48 and 72 h post-ischemia. For Western blotting, animals were decapitated and brains were processed at the same post-ischemic times used for histology and immunohistochemistry. For histological evaluation of degenerating neurons, brain coronal sections (5 mM) were stained with 1% celestine blue and 1% acid fuchsin. In the staining procedure used, necrotic cells appeared bright red on examination under the light microscope, showed extensive cytorrhexis and/or karyorrhexis, and were readily distinguished from surviving neurons. 2.2. Immunohistochemistry Rats were anaesthetized with 3.5% halothane and perfused transcardially with phosphate buffer (pH 7.4) containing 4% paraformaldehyde. The sections were deparaffinized in xylene and rehydrated through graded ethanol. The sections were washed in several changes of PBS, containing 0.3% Triton X100 at pH 7.4, and treated with 1% hydrogen peroxide in PBS to eliminate endogenous peroxidase activity. Polyclonal antibodies raised against caspase-9, caspase-3, fodrin, Smac/DIABLO and XIAP were used. Activated caspase-3 (work dilution 1:200 in PBS, 18 h at 4 8C) was used (Cell Signaling, Germany). Activated caspase-3 antibodies recognise the p17 active subunit of caspase-3 at optimal concentrations such as 1:200, but do not detect or poorly detect the precursor form. The antibodies may cross-react with the inactive form of caspase-3 at higher concentrations. AntiSmac/DIABLO (R&D, Germany) was used at 1/200. AntiXIAP (Biocarta, Germany) was used at 1/500. The sections were washed in PBS three times for 20 min each and then incubated with the secondary antibody (Chemicon, Germany), biotinylated goat-anti-rabbit (1:200). The sections were then rinsed in PBS three times for 20 min each, and subsequently incubated in streptavidin– biotin–peroxidase complex for 40 min. Following this, the sections were again rinsed in PBS and allowed to react with a solution containing 3,30 -diaminobenzidine tetrahydrochloride, hydrogen peroxide (0.006%) and Tris–HCl-buffer (pH 7.6) for 10 min. To ensure comparable immunostaining, sections were processed together at the same time under the same conditions. For the assessment of non-specific immunostaining, alternating sections from each experimental group were incubated without the primary antibody. 2.3. Western blotting A rat hippocampal homogenate standard was prepared by homogenising frozen hippocampus in 10 volumes of

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homogenisation buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 0,5% sodium deoxycholate, 0,1% SDS, 1% Nonidet P-40, for 10 s with polytron). Aliquots were stored at 70 8C and 30 mg of total proteins was used per lane.sub cellular fractionation was performed as follows. The homogenates were spun at 500  g for 5 min. The 500  g supernatant was spun at 11000  g for 10 min. This procedure was repeated and the pellet was designated as the mitochondrial fraction. The protein concentration of each sample was determined using the Bradford assay (Bio-Rad KIT, Hercules CA). Samples were resuspended to contain 30 mg of total protein in loading buffer, heated for 5 min at 95 8C and separated using a MINI-PROTEAN III electrophoresis system from Bio-Rad on 5% Tris–Glycine gel (Cleaved Fodrin), 10% Tris–glycine gel (XIAP) and 15% Tris–glycine gel (Smac/DIABLO, cleaved Caspase-9, Caspase-3) with 4% stacking gel. Gels were run at 180 V for 1 h. The matched gel was soaked for 15 min in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol) then transferred to a PVDF membrane for 30 min at 100 mA constant current by using the Bio-Rad Semi–Dry–Blotting apparatus. The blot was blocked with 10% rehydrated nonfat dry milk for 1 h at room temperature. The primary antibodies were 1:500 dilution of rabbit polyclonal antibody against Smac/DIABO (R&D), 1:1000 dilution of rabbit polyclonal antibody against XIAP (Biocarta, Germany),

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1:500 dilution of rabbit polyclonal antibody against cleaved caspase-3 p17 subunit (Cell Signaling, Germany), 1:250 dilution of rabbit polyclonal antibody against cleaved caspase-9 and 1:4000 dilution of mouse monoclonal antibody against tubulin (Sigma, Germany). Western blots were performed with horseradish peroxidase conjugated anti-rabbit IgG (Chemicon, Germany) using enhanced chemiluminescence Western blotting detection reagents (Amersham International). The detection reagents were placed on the blot for 1 min, then Hyperfilm-ECL was exposed to the blot for two through 30 min and developed. The film was scanned and the results were quantified using ‘‘Quantity one basic (Bio-Rad)’’. Blots were stripped for 40 min and reprobed with antibodies for b-tubulin (1/1000 from Sigma, Germany) or for cytochrome c oxidase (1/1000, from Abcam). 2.4. Data analysis The levels of protein expression were quantified by using the NIH gel analysis software (http://rsb.info.nih.gov/ nih-image/download.html). Data are reported as means  S.D. of n experiments. Significant differences between means were statistically assessed by ANOVA. Significant differences between treated and control animals at each time point were assessed by paired Student’s-t-test. A level of *p < 0.05 was considered statistically significant **p < 0.01.

Fig. 1. The Time course of cell fragmentation in the CA1 subfield of hippocamus. (A) controls; (B) and (C) 6 and 12 h post-ischemia; (D) 3 days post-ischemia. Sections were stained with 1% celestine blue and 1% acid fuchsin. Some cells showed an ischemic change, such as triangle-shaped shrunken nuclei or a condensed nucleus with visible nucleoli (magnification: 600).

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3. Results 3.1. Physiologic parameters Physiologic data showed no significant differences in body temperature, MABP, and arterial blood gas analysis between groups. The preischemic physiologic parameters were as follows: body temperature 37, 3  0.4 8C; MABP, 130  7.6 mmHg; pH, 7.2  0.2; PaO2, 137.9  21 mmHg; PaCO2, 35  7.5 mmHg (n = 5). There was no deviation from these values over the period of measurement. 3.2. Histological analysis of hippocampal neuronal damage Histological changes in the CA1 pyramidal cell layer first appeared 2–3 days post-ischemia. Some cells showed morphological changes, such as triangle-shaped shrunken nuclei or a condensed nucleus with visible nucleoli (Fig. 1). Five and 7 days post-ischemia, the majority of the pyramidal cells in the CA1 subfield had undergone ischemic damage. These histological changes were compatible with our previous reports (Rami et al., 2000). 3.3. XIAP-expression after cerebral ischemia To examine the regulation of XIAP protein expression in ischemic brains, the temporal pattern of changes in XIAP-

immunostaining was assessed. A specific anti-XIAP antibody raised in rabbits recognised a band of 57 kDa in hippocampus. Western blot analysis showed that XIAP immuno-reactivity was evident as a single band with molecular mass of 57 kDa in the homogenates from isolated hippocampi. A slight increase of XIAP levels at 12 and 24 h post-ischemia and normalisation at 48 h postischemia (Fig. 2(a)). A consistent amount of tubulin was observed in control and ischemic samples, suggesting that the amount of the loaded protein was consistent. The immunocytochemical analysis of XIAP-immunoreactivity (XIAP-IR) showed also changes in hippocampus following cerebral ischemia: Fig. 2(b) shows that the number of XIAP-positive cells significantly increased in the CA1 region at 6, 12 and 24 h post-ischemia (Fig. 2b). The intensity of XIAP-IR was localised predominantly in cytosol and in the perinuclear region of cytoplasm. At 6, 12 and 24 h post-reperfusion. XIAP-IR exhibited a diffuse pattern within the cell. This diffuse pattern of expression was observed in cytoplasmic compartments. Compared with the CA1 region, the alterations in XIAP by ischemia were modest in the CA3 subfield (not shown). This suggests that cerebral ischemia induced a time and region specific alteration in XIAP expression in hippocampus. Moreover XIAP was quite absent in cells undergoing cell death in the CA1 region at 3 days postischemia, suggested that the protein may be degraded in degenerating neurons.

Fig. 2. (a) Western blot analysis XIAP from the brain homogenate samples in the non-ischemic control hippocampi (control lane) and ischemic hippocampi (lanes 12, 24 and 72 h). XIAP is evident as band with molecular mass of 57 kDa. XIAP was expressed constitutively in the control brain and showed an increase by 24 h post-ischemia. *p < 0.5 and **p < 0.01 different from controls. (b) Regulation of XIAP by the ischemic insult in the selectively vulnerable hippocampal CA1 subfield. XIAP immunoreactivity in controls (A), 6 h (B), 12 h (C), 24 h (D), 48 h (E) and 72 h (F), 3 days (H), and 5 days (G) after transient global ischemia. The XIAP-IR was localised predominantly in the perinuclear region of cytoplasm. At 2, 6, 12 and 24 h post-reperfusion XIAP-IR exhibited a diffuse pattern within the cell. This diffuse pattern of expression was observed in cytoplasmic compartments (magnification: 600).

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Fig. 2. (Continued ).

3.4. Smac/DIABLO expression after cerebral ischemia Western blot analysis revealed that Smac/DIABLO immunoreactivity was evident as a single band of molecular

mass of 25 kDa (Fig. 3(a)). Analysis of the amount of Smac/ DIABLO expression in the homogenates of hippocampus subjected to ischemia/reperfusion showed a tendency to increase, but did not change significantly at any time points

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examined (Fig. 3(a)). By examining the mitochondrial fraction, we found a significant decrease of Smac/DIABLOIR at 24 h post-ischemia (Fig. 3(b)). We also examined the activated form of caspase-9 in order detect the beginning of the caspase cascade reaction. The cleaved p38 subunit

expression is considered to be an initiator of the caspase cascade. The cleaved p38 subunit was detected as a single band of 38 kDa (Fig. 3(c)). The cleaved caspase-9 was observed by 12 h after reperfusion, and was increased drastically by 72 h after the ischemic insult (Fig. 3(c)). A

Fig. 3. (a) Western blot analysis of Smac/DIABLO from the brain homogenate samples in the non-ischemic control hippocampi (control lane) and ischemic hippocampi (lanes 12, 24 and 48 h). Smac/DIABLO is evident as band with molecular mass of 25 kDa. The immunoblot of Smac/DIABLO fragment did not reveal any drastic increase of immunoreactivity post-ischemia. No significant changes between controls and post-ischemic specimens. (b) Western blot analysis of Smac/DIABLO from the mitochondrial fraction samples in the non-ischemic control hippocampi (control lane) and ischemic hippocampi (lanes 6, 12 and 24 h). COX; cytochrome oxidase c was used as control marker for mitochondrial fraction. Smac/DIABLO is evident as band with molecular mass of 25 kDa. The immunoblot of Smac/DIABLO fragment revealed a drastic decrease of immunoreactivity by 24 h post-ischemia. *p < 0.5 and **p < 0.01 different from controls. (c) Western blot analysis of the cleaved caspase-9 from the brain homogenate samples in the non-ischemic control hippocampi (control lane) and ischemic hippocampi (lanes 6, 12 and 24 h). Caspase cleaved subunit is evident as band with molecular mass of 38 kDa. The immunoblot of cleaved Caspase-9 (38 kDa) fragment revealed an increase of immunoreactivity, peaking at 24 h post-ischemia. *p < 0.5 and **p < 0.01 different from controls. (d) Distribution of Smac/DIABLO immunoreactivity in the CA1 region in controls (A), 6 h (B), 12 h (C), 24 h (D), 48 h (E) and 72 h (F). Note the gradual global increase of Smac/ DIABLO-immunoreactivity during the reperfusion phase (6–24 h) in the vulnerable CA1 sub field of the hippocampus (magnification: 600).

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Fig. 3. (Continued ).

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consistent amount of tubulin was observed in the control and ischemic samples. Western blot analysis of Smac/DIABLO from the mitochondrial fraction samples ischemic hippocampi (lanes 12, 24 and 72 h in Fig. 3(b)) showed that Smac/DIABLO is evident as band with molecular mass of 25 kDa. The

immunoblot of Smac/DIABLO fragment revealed a drastic decrease of immunoreactivity post-ischemia. The immunocytochemical analysis of Smac/DIABLO showed an ubiquituous distribution in all hippocampal areas. There was only a negligible amount of Smac/DIABLO in the cytosol in the normal state. We observed a gradual global

Fig. 4. (a) Western blot analysis of the cleaved caspase-3 from the brain homogenate samples in the non-ischemic control hippocampi (control lane) and ischemic hippocampi (lanes 12, 24 and 72 h). Caspase cleaved subunit is evident as band with molecular mass of 17 kDa. The immunoblot of cleaved Caspase-3 (17 kDa) fragment revealed an increase of immunoreactivity from 12 to 72 h, peaking at 72 h post-ischemia. *p < 0.5 and **p < 0.01 different from controls. (b) Western blot analysis of the cleaved fodrin from the brain homogenate samples in the non-ischemic control hippocampi (control lane) and ischemic hippocampi (lanes 12, 24 and 72 h). Caspase cleaved subunit is evident as band with molecular mass of 150 kDa. The immunoblot of cleaved fodrin fragment revealed an increase of immunoreactivity, peaking at 72 h post-ischemia. *p < 0.5 and **p < 0.01 different from controls. (c) Immunoreactivity corresponding to the 17 kDa cleavage product of caspase-3 appeared quite evidently within 6 h in specific regions such as the CA1 subregion (B). At 6 and 12 h (B and C, respectively) postischemia the 17 kDa cleavage product of caspase-3 was detectable in all vulnerable hippocampal areas. At 72 h post-ischemia the p17-IR was seen mostly in nuclei (D). A shows lack of immunoreactivity in control sham-operated rats. (magnification: 600).

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Fig. 4. (Continued ).

increase of Smac/DIABLO-immunoreactivity during the reperfusion phase (6–24 h) in the vulnerable CA1 sub field of the hippocampus (Fig. 3(d)). Collectively these datas implied that there was a slight up regulation of Smac/ DIABLO expression on immunoreactivity levels initiating directly after reperfusion. 3.5. Caspase-3 activation following cerebral ischemia Western blot analysis revealed that cleaved caspase-3immunoreactivity was evident as a single band of molecular mass of 17 kDa (Fig. 4(a)). Western blot analysis of the amount of p17 subunit expression in the hippocampus subjected to ischemia/reperfusion showed a tendency to increase beginning at 12 h post-ischemia. We examined the cleaved form fodrin, the cleaved p150 subunit expression as a substrate of caspase-3, to detect the beginning of the caspase cascade reaction. The cleaved subunit of fodrin was detected as a single band of 150 kDa (Fig. 4(b)). The cleaved fodrin was observed by 12 h after reperfusion, and was increased by 24 and 72 h after the ischemic insult. A consistent amount of tubulin was observed in the control and ischemic samples. The immunocytochemical analysis of immunoreactivity corresponding to the 17 kDa cleavage product of caspase-3 appeared quite evidently within specific regions such as the CA1 sub region. At 12 and 24 h post-ischemia the 17 kDa cleavage product of caspase-3 was detectable in all vulnerable hippocampal areas. At 2 and 3 days postischemia the p17-IR was seen mostly in nuclei of cells undergoing cell death (Fig. 4(c)).

4. Discussion Transient global cerebral ischemia in rats activated caspsae-3, redistributed XIAP and Smac/DIABLO in selectively vulnerable subfields of the hippocampal formation. Among the IAP family, XIAP is the most potent apoptotic inhibitor (Deveraux et al., 1999); however, little is known about its regulation in the CNS following cerebral ischemia. The present study provided evidence that XIAP levels are altered after cerebral ischemia, and that the protein levels are regulated by the ischemic insult. These changes were found to be temporally and spatially specific, with a large enhancement in the CA1 subfield at 6, 12 and 24 h postischemia. Moreover, XIAP increased concurrently with the peak expression of activated caspase-9. Recent evidence has showed that XIAP is also involved in the caspase-9 activation machinery (Holcik and Korneluk, 2001). The IAPs are defined by the existence of a zinc-finger motif referred to as the baculoviral IAP repeat (BIR) in their Nterminal portion. There are three BIR domains in XIAP, cIAP1, cIAP2 and NAIP (Holcik and Korneluk, 2001). The IAPs inhibits the activity of cleaved caspase-9 through the interaction of their BIR3 domain with the IAP binding motif of caspase-9. At later time points such as 48 and 72 h post-ischemia, a decrease of XIAP levels took place. This could reflect a subsequent down-regulation in the synthesis of the protein or a breakdown of XIAP. Although the expression level of XIAP exhibited no drastic changes in our Western blot data, immunohistochemical analysis revealed that XIAP-IR

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became more extensive in neurons during reperfusion. This may refelct the binding of XIAP with cleaved caspases that is accumulated in response to ischemia/reperfusion within the cells. Similar data have been reported by Korhonen et al., 2001 in a model of kainic acid-induced cell death in the rat hippocampus. Moreover, it has been recently reported that the XIAP expression in the hippocampus is up regulated following traumatic brain injury (Keane et al., 2001a). Keane et al., (2001b) have shown that death of spinal cord cells after moderate spinal cord injury is associated with cleavage of XIAP. These observations suggest that XIAP may act as a suicide inactivator of caspases and undergo cleavage to allow the suicide program to ensue when sufficient levels of caspases activation are reached. Whether XIAP functions in a similar fashion in the hippocampus after cerebral ischemia is unclear, but the present study suggest that dysregulation of normal control mechanisms of XIAP expression contributes partly to apoptosis induced by cerebral ischemia. On the other hand, recently Hu et al. (2001) showed that ubiquitinated protein aggregates are produced within the cytoplasm after focal cerebral ischemia. Therefore, XIAP would potentially have a dual means of inhibiting caspase function, through direct inhibition and through ubiquitination leading to degradation. However, because there are several binding partners for XIAP and other IAP proteins can bind caspases, predictions based on analysis of limited set of proteins should be interpreted with caution. To sum up, it seems that the relative level of XIAP in neurons may be important for the outcome and the survival of neurons after brain ischemia. Moreover, the elevation of XIAP by ischemia indicates that this protein may be part of a survival pathway by which neurons counteract degeneration after cerebral ischemia. As matter of fact, Xu et al. (1997, 1999) and recently Trapp et al. (2003) have demonstrated in very elegant studies that transgenic mice overexpressing XIAP in neurons exhibit better outcome after cerebral ischemia and stroke in mice, respectively. However, it is known that the apoptosis promoter Smac/ DIABLO is encoded by a nuclear gene and is subsequently imported into mitochondria (Silke et al., 2000). Smac/ DIABLO is localised within the mitochondrial intermembranous space under the normal circumstances (Holcik and Korneluk, 2001). Upon the induction of any cell deathrelated stimuli, Smac/DIABLO is released into the cytosol, and facilitates cell death by abrogating the caspase inhibiting actions of the IAPs (Srinivasula et al., 2000). Therefore, the presence of the IAPs bound to cleaved caspases is required for the cell death inducing actions of Smac/DIABLO. We found in our study that Smac/DIABLO levels are enhanced shortly after ischemia in the selectively vulnerable area of hippocampus and this correlates well temporally and spatially with the activation of caspase-3. Hence, it seems reasonable to assume that Smac/DIABLO could contribute to the apoptotic cell death in our model of global cerebral ischemia. For that reason, we have examined the Samc/

DIABLO redistribution not only in homogenates of hippocampus, but also in a mitochondrial subcellular fraction. We found a drastic decrease of Smac/DIABLOIR in the mitochondrial fraction during reperfusion. This may lead to an enhanced translocation from mitochondria to cytosol. During apoptosis, Smac/DIABLO is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. However, the increase in Smac/DIABLO-IR was concurrent with the appearance of activated casapse-9. Activated caspase-9 regulates the initiation of mitochondrial caspase cascade way. This result suggests that in analogy to cytochrome c activation, Smac/DIABLO may also regulate caspase initiation upstream of the caspase cascade. Our observation is supported by the findings of Saito et al. (2003, 2004), who reported on postischemic translocation of Smac/ DIABLO from mitochondria to cytosol in mice after transient focal ischemia by using the model of transitory occlusion of the middle cerebral artery. Taken together, our results clearly demonstrate the coexistence of cleaved caspase-9, cleaved caspase-3, cleaved fodrin, Smac/DIABLO and XIAP in the hippocampus during reperfusion. Results presented in this communication further (1) emphasize the role of mitochondria in the regulation of apoptosis (2) provide evidence that Smac/ DIABLO and XIAP are redistributed in the hippocampus after cerebral ischemia in rats and that (3) this redistribution is well correlated to the activation of the initiator of the apoptotic program, caspase-9 and of the executioner of the apoptotic program, caspase-3. Our results imply that Smac/ DIABLO and XIAP are implicated in the pathophysiological mechanisms of reperfusion injury.

Acknowledgements Support of this work by the ‘‘Dr. Paul und Cilli WeillStiftung (Grant 8598924-2003 to Dr. A. Rami)’’ is gratefully acknowledged.

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