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Keywords:

  • AIF;
  • Bax;
  • Bcl-2;
  • hippocampus;
  • neuroprotection;
  • epilepsy

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2010) 115, 92–101.

Abstract

Prolonged seizures activate members of the Bcl-2 homology domain 3-only sub-group of the Bcl-2 protein family, which are essential for initiation of apoptosis signaling. Bid is a potent pro-apoptotic Bcl-2 homology domain 3-only protein, which upon proteolytic activation translocates to mitochondria to promote activation of the Bax/Bak sub-group of the pro-apoptotic Bcl-2 family and thereby contributes to release of apoptogenic molecules, such as cytochrome c and possibly apoptosis-inducing factor (AIF). Bid-deficient mice have been reported to show reduced lesion volumes after ischemia and trauma in vivo but a causal role for Bid in the setting of seizure-induced neuronal death has not been investigated. In this study, we studied Bid activation following status epilepticus in mice and compared hippocampal damage between wild-type and Bid-deficient animals. Full-length Bid was detected in normal mouse hippocampus and the cleaved (activated) p15 fragment of Bid was detected shortly after status epilepticus. Bid-deficient mice underwent equivalent electrographic seizure responses during status epilepticus as wild-type animals. Hippocampal counts of degenerating neurons and surviving neuron-specific nuclear protein-positive cells were not significantly different between wild-type and Bid-deficient mice. Additionally, nuclear translocation of AIF was not reduced in Bid-deficient compared with wild-type animals subjected to status epilepticus. The present study demonstrates that AIF is not dependent on Bid for mitochondrial release and nuclear import in this model and that while Bid is cleaved during seizure-induced neuronal death, it may be functionally redundant or even not essential.

Abbreviations used:
AIF

apoptosis-inducing factor

BH

Bcl-2 homology

Bid

BH3-interacting domain death agonist

CA

cornu ammonis

FJB

Fluoro-Jade B

KA

kainic acid

NeuN

neuron-specific nuclear protein

Bcl-2 family proteins are critical regulators of programmed cell death. The balance and interactions between pro- and anti-apoptotic members at the outer mitochondrial membrane play a critical role in determining the release of apoptogenic molecules, such as cytochrome c (Ow et al. 2008). The Bcl-2 homology domain 3-only (BH3) proteins constitute an upstream signal-dependent, pro-apoptotic subgroup that function by inhibiting anti-apoptotic Bcl-2 family proteins and/or directly activating pro-apoptotic multi-BH domain Bax/Bak (Youle and Strasser 2008). At least seven members of the BH3-only sub-group have been identified, which are activated via either transcriptional induction or post-translational modifications such as phosphorylation or proteolytic cleavage (Youle and Strasser 2008).

Bid (BH3-interacting domain death agonist) is a potently pro-apoptotic ∼22 kD BH3-only protein (Wang et al. 1996). Bid activation was originally demonstrated to occur via caspase 8-mediated cleavage downstream of death receptor stimulation (Li et al. 1998; Luo et al. 1998). The 15 kD truncated form (tBid) translocates to mitochondria where it activates Bax/Bak, either directly or indirectly, and thereby triggers cytochrome c release (Li et al. 1998; Luo et al. 1998; Gross et al. 1999). Bid has also been reported to be activated through cleavage by caspases 2 and 3 (Bossy-Wetzel and Green 1999; Upton et al. 2008), calpain (Chen et al. 2001; Mandic et al. 2002) as well as cathepsins (Stoka et al. 2001). Some (e.g. hepatocytes), but not all cell types (e.g. thymocytes) from Bid-deficient mice are resistant to Fas-induced apoptosis (Yin et al. 1999), but all undergo DNA damage- and replicative stress-induced apoptosis normally (Kaufmann et al. 2007).

Bid is expressed in most (possibly all) cell types, including the developing and mature brain, particularly within the hippocampus (Krajewska et al. 2002). Although Bid-deficient mice do not display overt neuro-developmental abnormalities (Wang et al. 1996; Yin et al. 1999; Henshall et al. 2001; Krajewska et al. 2002). Bid has been proposed as a key effector of neuronal cell death (Culmsee and Plesnila 2006). Indeed, Bid was found to be cleaved following oxygen-glucose deprivation of neurons in vitro and after cerebral ischemia and traumatic brain injury in vivo (Plesnila et al. 2001; Franz et al. 2002; Yin et al. 2002; Bermpohl et al. 2006). Short interfering RNA targeting bid was reported to reduce the release of cytochrome c and apoptosis-inducing factor (AIF) after glutamate exposure or oxygen-glucose deprivation of neurons in vitro (Culmsee et al. 2005; Landshamer et al. 2008). Moreover, cytochrome c release was found to be reduced after cerebral ischemia in Bid-deficient mice in vivo (Plesnila et al. 2001; Yin et al. 2002) and histological signs of injury after ischemia or trauma in vivo, were also reportedly reduced in Bid-deficient mice (Plesnila et al. 2001; Yin et al. 2002; Bermpohl et al. 2006).

Seizure-induced neuronal death is associated with some features of apoptosis, including mitochondrial release of cytochrome c and AIF, proteolytic activation of effector caspases, DNA fragmentation, chromatin condensation and nuclear pyknosis (for review see Engel and Henshall 2009). BH3-only proteins may be critical upstream initiators since mice lacking bim or puma are protected against seizure-damage (Engel et al. 2010a; b; Murphy et al. 2010). Bid may also be important because it is rapidly cleaved following prolonged seizures (status epilepticus) in rats (Henshall et al. 2001, 2002; Shinoda et al. 2004b; Li et al. 2006). Cleaved Bid has also been detected in the mitochondrial fraction of hippocampus from patients with temporal lobe epilepsy (Henshall et al. 2004). However, the functional significance of Bid for seizure-induced neuronal death in vivo has not yet been examined. We studied the role of Bid in a mouse seizure model and assessed seizure-induced neuronal death in Bid-deficient mice.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Seizure model

All animal experiments were performed in accordance with the principles of the European Communities Council Directive (86/609/EEC) and National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Procedures were approved by the relevant Research Ethics Committees of the Royal College of Surgeons in Ireland and Legacy Clinical Research and Technology Center. Procedures were undertaken as previously described (Mouri et al. 2008; Murphy et al. 2010). Adult male C57BL/6 mice (20–25 g) were obtained from Harlan, UK. Mice deficient for bid were on a pure C57BL/6 background and were generated as described (Kaufmann et al. 2007). Mice were first anesthetized using isoflurane (3–5%) and maintained normothermic by means of a feedback-controlled heat blanket (Harvard Apparatus Ltd, Kent, UK). Mice were next placed in a stereotaxic frame and three partial craniectomies performed to affix cortical skull-mounted EEG electrodes (Bilaney Consultants Ltd, Sevenoaks, UK). EEG was recorded using a Grass Comet XL digital EEG (Medivent Ltd, Lucan, Ireland). A guide cannula was affixed (coordinates from Bregma: AP = −0.94; L = −2.85 mm) and the entire skull assembly fixed in place with dental cement. Anaesthesia was then discontinued and freely moving mice were placed inside a clear Perspex recording chamber. EEG recordings were commenced and after establishing baseline EEG for a few minutes, an injection cannula was lowered through the guide cannula for injection of kainic acid (KA) (Ocean Produce International, Nova Scotia, Canada) into the basolateral amygdala nucleus (0.3 μg in 0.2 μL phosphate-buffered saline). Non-seizure control mice underwent the same surgical procedure but received 0.2 μL intraamygdala vehicle. Forty minutes following injection of KA or vehicle, mice received intraperitoneal or intravenous lorazepam (6 mg/kg) to curtail status epilepticus, and the EEG was monitored for up to 1 h thereafter. Mice were killed 24 or 72 h later and perfused with saline to remove intravascular blood components. Brains were either flash-frozen whole in 2-methylbutane at −30°C and processed for histopathology, or dissected on ice to obtain hippocampus.

Western blot analysis

Western blotting was performed as previously described (Murphy et al. 2007, 2010). Whole hippocampus or amygdala tissue cores, prepared as before (Murphy et al. 2007), were homogenized in a lysis buffer containing a protease inhibitor cocktail. Protein concentration was determined and then 20 or 50 μg samples boiled in gel-loading buffer and separated by 12 or 15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride or nitrocellulose membranes and incubated with antibodies against the following: α-tubulin, AIF and Bim (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), acetyl-histone H3 and GluR6/7 (Millipore, Ireland B.V., Tullagreen, Ireland), Bid (R & D Systems, Minneapolis, MN, USA), Bad and Lamin A/C (Isis Ltd, Bray, Ireland), Porin (Merck KGaA, Darmstadt, Germany) and Puma (ProSci, Poway, CA, USA). Membranes were next incubated with horseradish peroxidise-conjugated secondary antibodies (Isis Ltd) and protein bands visualized using Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). Gel band image densities were captured using a Fuji-film LAS-3000 and analyzed using AlphaEaseFC4.0 software as described (Murphy et al. 2007, 2010).

Subcellular fractionation

Subcellular fractionation was performed to analyze AIF localization. Cytoplasm, mitochondria and nuclear fractions were prepared according to previously described techniques (Schindler et al. 2006). Briefly, samples were homogenized in a mannitol/sucrose buffer, mitochondria separated from the homogenate by centrifugation (10 000 g) and the crude cytosol fraction centrifuged (100 000 g) to obtain the cytosol and microsomal fraction (pellet). The crude nuclear fraction was further purified by centrifugation in sucrose buffer with Nonidet P-40 (10 mM Tris, pH 7.5, 300 mM sucrose, and 1 mM EDTA with 0.1% or 1% NP-40). All steps were performed at 4°C. Aliquots of fractionated samples were subject to western blotting to verify fraction purity, using antibodies against Bad for cytoplasm, Porin for mitochondria and Lamin A/C or acetyl-histone H3 for nuclear samples.

Histopathology and immunohistochemistry

Brains were sectioned at −20°C on a Leica cryostat and 12 μm sections collected at the level of dorsal and ventral hippocampus (−1.8 and −2.9 mm from Bregma according to a mouse stereotaxic atlas (Paxinos and Franklin 2001). Neurodegeneration was assessed using Fluoro-Jade® B (FJB), as previously described (Mouri et al. 2008). Briefly, sections were air-dried and post-fixed in formalin followed by hydrating through graded alcohols. Sections were then rinsed in distilled water and transferred to 0.006% potassium permanganate solution for 15 min. Sections were rinsed again and transferred to a 0.001% FJB solution according to manufacturer’s recommendations (Chemicon Europe Ltd, Chandlers Ford, UK). After staining, sections were rinsed again, dried, cleared and mounted in DPX (Sigma-Aldrich, St Louis, MO, USA). Hippocampal FJB-positive counts were the average of two adjacent sections for the cornu ammonis (CA)3 subfield under 40× lens magnification by an observer blinded to genotype. For neuron-specific nuclear protein (NeuN) immunohistochemistry, sections were fixed and permeabilized, blocked in goat serum and incubated overnight with anti-NeuN antibodies (Millipore) followed by incubation with goat anti-rabbit IgG antibodies coupled to AlexaFluor 568 (BioSciences Limited, Dun Laoghaire, Ireland). Sections were examined using a Nikon 2000s epifluorescence microscope under Ex/Em wavelengths of 472/520 nm (green) and 540 to 580/600 to 660 nm (red) and images captured using a Hamamatsu Orca 285 camera.

For investigation of brain architecture in wild-type and bid−/− mice, naïve brains were also prepared. Sections were prepared and immunostained for NeuN. Bi-lateral amygdala tissue cores were obtained from whole brains, as described (Murphy et al. 2007), for assessment of GluR6/7 expression.

For diaminobenzidine staining, mice were anesthetized with a xylazine/ketamine solution and transcardially perfused with 4% paraformaldehyde in Sorensen’s buffer for 10 min. Brains were post-fixed in 4% paraformaldehyde for 2 h at 4°C and cryoprotected in 30% sucrose solution. Thirty-micrometer sagittal sections were cut on a Leica cryostat and collected in 0.1% azide–phosphate-buffered saline solution. Next, brain sections were pre-treated for 1 h with 1% bovine serum albumin, 5% fetal bovine serum, and 0.2% Triton X-100, and then incubated with following primary antibodies against Bid (R & D Sysytems, Minneapolis, MN, USA) and Bid (pSer78) (antibodies-online GmbH, Germany). Finally, brain sections were incubated in avidin–biotin complex using the Elite Vectastain kit (Vector Laboratories, Burlingame, CA, USA). Chromogen reactions were performed with diaminobenzidine (Sigma) and 0.003% H2O2 for 10 min. Sections were coverslipped with Fluorosave.

Statistical analysis

Data are presented as mean ± SEM. Data were analyzed using Student’s t-test (StatView software; SAS Institute, Inc., Cary, NC, USA). Significance was accepted at < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Bid is rapidly cleaved following focal-onset status epilepticus in mice

Characteristic damage was observed in the hippocampus of mice subject to intra-amygdala KA-induced status epilepticus (Fig. 1). Typically, degenerating neurons were present within the ipsilateral CA3 subfield at 72 h (Fig. 1a). Damage extended through the full rostro-caudal extent of the hippocampal formation (Fig. 1b). Occasionally, degenerating hilar and CA1 neurons were also present in the dorsal and ventral hippocampus. Damage was not found within the contralateral hippocampus. Small numbers of FJB-positive cells were present outside the hippocampus in the neocortex and thalamus, as previously reported (Mouri et al. 2008).

image

Figure 1.  Bid cleavage following status epilepticus in mice. (a) Representative FJB-staining at the level of the dorsal hippocampus in a control mouse (Con) and a mouse 72 h after status epilepticus (SE). CA, cornu ammonis, H, hilus, a, b and c denote subdivisions of the CA3 subfield. Scale bar, 400 μm. (b) Representative FJB staining of seizure-damaged mouse brain sections at 72 h at the level of the ventral hippocampus. Arrows highlight regions of neurodegeneration. (c) Western blot showing Bid in control and seizure-damaged brain at various time-points. The p15-cleaved fragment of Bid is present in SE samples but not control. Each lane is a sample from a pool of = 2 ipsi-lateral hippocampi and the blot is representative of three independent experiments. α-Tubulin (Tub) is included as a guide to equal protein loading. (d) Immunoblot showing Bid and tBid in the mitochondrial (mito) fraction after SE (n = 3 per lane). Molecular weight markers are depicted to the left (in kD).

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To investigate whether Bid was activated in this model, we performed western blot analysis using whole cell lysates from the ipsilateral hippocampus of mice. As expected, full-length Bid was detected in whole cell lysates from control mouse hippocampus at its predicted size of ∼22–23 kD (Fig. 1c). Immunohistochemistry was performed to determine the cell type(s) expressing Bid. However, experiments with two different Bid antibodies detected staining in Bid-deficient hippocampus (see Figure S1).

Levels of full-length Bid remained largely unchanged in mice subjected to status epilepticus. However, a p15 fragment of Bid (tBid) was detected in the ipsi-lateral mouse hippocampus after status epilepticus, as early as 30 min following lorazepam administration, and continued to be detected until 72 h later (Fig. 1c).

To determine whether or not tBid migrated to mitochondria, we performed western blot analysis of the mitochondrial fraction. In control mouse brain, no tBid was detected in hippocampal mitochondrial fractions (data not shown). In contrast, tBid was present in the mitochondrial fraction 4 and 8 h after status epilepticus (Fig. 1d).

Phenotype of Bid-deficient mice

The generation of the Bid-deficient mice on a pure C57BL/6 background used for the present studies has been described previously (Kaufmann et al. 2007). Inspection of brains from these Bid-deficient mice revealed no apparent abnormalities, and hippocampal architecture appeared normal (Fig. 2a). To verify the absence of Bid in the relevant tissue, we performed western blot analysis on hippocampal extracts from naïve wild-type and Bid-deficient mice. Bid was present in all wild-type mice but was absent from the brains of Bid-deficient mice, confirming bid deletion (Fig. 2b). Levels of the KA receptor GluR6/7 was similar in the hippocampus of naïve wild-type and Bid-deficient mice (Fig. 2b). KA receptor levels in the amygdala of naïve wild-type and Bid-deficient mice were also comparable (Fig. 2c). Last, we used EEG to record electrographic status duration after intra-amygdala microinjection of KA in mice. The seizure durations elicited were similar between wild-type and Bid-deficient mice (Fig. 2d and e).

image

Figure 2.  Phenotype of Bid-deficient mice. (a) Representative NeuN-stained sections at the level of dorsal hippocampus from wild-type (wt) and Bid-deficient (bid−/−) mice. Scale bar, 400 μm. (b) Western blots (= 1 per lane) confirming absence of Bid protein in brains of Bid-deficient mice but normal expression of the KA receptor GluR6/7 in the hippocampus. α-Tubulin (Tub) is shown below as a guide to protein loading. (c) Western blots (= 1 per lane) confirming that GluR6/7 levels in the amygdale are also comparable between wt and bid−/− mice. (d) Graph showing duration of high-amplitude high-frequency spiking recorded by EEG between the time of KA microinjection and lorazepam administration (= 5 each). (e) Representative traces of seizure activity recorded in a wt mouse and a bid−/− mouse. Seizure responses were not different between genotypes.

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Status epilepticus damage in the dorsal hippocampus is comparable between wild-type and Bid-deficient mice

We next examined hippocampal damage 72 h after status epilepticus in wild-type and Bid-deficient mice. Tissue sections at the level of dorsal hippocampus stained with FJB displayed a typical CA3 lesion in wild-type mice which mainly incorporated ipsi-lateral CA3a, whereas smaller numbers of degenerating neurons were found in CA3b,c (Fig. 3a). The location and extent of FJB staining was similar in Bid-deficient mice (Fig. 3a). That is, damage was also present in the ipsilateral CA3 subfield, extending into CA3b and CA3c sectors (Fig. 3a).

image

Figure 3.  Normal seizure damage in dorsal hippocampus in Bid-deficient mice. Representative photomicrographs of (a) FJB and (b) NeuN staining in two different regions of the CA3 subfield at the level of dorsal hippocampus 72 h after status epilepticus (SE) and NeuN staining in control (Con) wild-type (wt) mice (b). Note, similarity in FJB and NeuN staining between wt and Bid-deficient (bid−/−) mice. Scale bar, 80 μm. (c, d) Graphs showing hippocampal (c) FJB and (d) NeuN counts 72 h following status epilepticus in wt and bid−/− mice (= 9–10 per group). No significant differences were found between genotypes for either parameter.

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Tissue sections from wild-type and Bid-deficient mice stained for NeuN showed predictable loss of cellular staining within the same regions that contained FJB-positive neurons. That is, an overall reduction in NeuN immunoreactivity, particularly within the nucleus of neurons, within the CA3a, with lesser changes observed in CA3b and CA3c (Fig. 3b). No obvious differences were found between Bid-deficient and wild-type animals.

Semi-quantification of FJB and NeuN counts at the level of dorsal hippocampus are presented in Fig. 3c and d. No significant difference was found between wild-type and Bid-deficient mice for either parameter.

Seizure-induced damage in the ventral hippocampus is comparable between wild-type and Bid-deficient mice

To assure that differences in seizure-damage responses were not present in other parts of the hippocampus, we also analyzed FJB staining within the ventral hippocampus (Fig. 4). FJB staining in the ventral hippocampus 72 h after status epilepticus was similar between both genotypes (Fig. 4a). Damage was mainly restricted to the ipsilateral CA3 subfield and was of similar extent in wild-type and Bid-deficient mice (Fig. 4a). This similarity was confirmed by counting FJB-positive cells (Fig. 4b).

image

Figure 4.  Normal seizure damage in ventral hippocampus in Bid-deficient mice. (a) Representative photomicrographs of FJB staining in the ventral hippocampus 72 h after status epilepticus. Note similar FJB distribution and numbers of degenerating neurons (arrows). Scale bar, 450 μm. (b) Graph showing ventral hippocampal FJB counts 72 h following status epilepticus for both genotypes (= 8–9 per group). No significant differences were found between bid−/− and wt mice for either parameter.

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To exclude the possibility that differences in the extent of damage might be present at earlier time-points, we also investigated damage in separate groups of wild-type and Bid-deficient mice killed 24 h after status epilepticus. Assessment of damage 24 h after status epilepticus also revealed no differences in counts of FJB-stained hippocampal neurons between wild-type and Bid-deficient mice (data not shown).

Bim and Puma expression following seizures in wild-type and Bid-deficient mice

We have recently established causal roles for the BH3-only proteins Bim and Puma in seizure-induced neuronal death in the present model (Engel et al. 2010a; b; Murphy et al. 2010). We were therefore interested in whether or not Bim or Puma compensated for the absence of Bid in the Bid-deficient animals. Western blot analysis showed that hippocampal Bim expression was significantly higher in Bid-deficient mice than wild-type mice after status epilepticus (see Figure S2). A similar trend was observed for Puma. These data suggest some compensatory responses of other BH3-only proteins may occur in the absence of Bid.

Nuclear accumulation of AIF after status epilepticus is not prevented in Bid-deficient mice

Although Bid was originally shown to mediate cytochrome c release from mitochondria, studies have also suggested that Bid can mediate AIF release from neurons (Konig et al. 2007; Landshamer et al. 2008). AIF translocates from mitochondria to the nucleus during seizure-induced neuronal death (Zhao et al. 2010) and AIF hypomorphic mice were reportedly resistant to seizure-induced neuronal death (Cheung et al. 2005). Accordingly, we were interested in whether AIF was activated in the present model and, critically, whether nuclear translocation was impacted by the absence of Bid.

Hippocampal levels of AIF were similar between wild-type and Bid-deficient mice (Fig. 5a), indicating that loss of Bid does not influence expression of AIF. Next, we prepared nuclear fractions from wild-type mice at various time points following status epilepticus (Fig. 5c). Western blot analysis of nuclear fractions demonstrated that AIF levels began to increase ∼4 to 8 h following status epilepticus, peaking at 24 h. Finally, we compared AIF translocation from mitochondria to the nucleus between wild-type and Bid-deficient mice subject to status epilepticus. No significant difference was found between wild-type and Bid-deficient mice in nuclear AIF levels after status epilepticus (Fig. 5d and e).

image

Figure 5.  Normal AIF translocation following status epilepticus in Bid-deficient mice. (a) Representative western blot showing total AIF levels in hippocampal whole cell lysates 8 h after status epilepticus in wild-type (wt) and Bid-deficient (bid−/−) mice. α-Tubulin (Tub) is shown below as a guide to protein loading. (b) Panel of western blots from fractionated bid−/− hippocampi confirming enrichment of appropriate compartment markers Bad (cytoplasm, Cyto), lamin A/C [LamA/C; nucleus (Nuc)] and Porin (Mitochondria, Mito), and absence of significant contamination between fractions (each lane is a pool of = 3 hippocampi). (c) Representative western blot showing nuclear AIF accumulation over time in wt mice following status epilepticus. Lamin A/C is shown below as a guide to protein loading. (d) Representative western blot showing AIF in nuclear fractions from wt and bid−/− mice 8 h following status epilepticus. Probing for acetyl-histone 3 (AcH3) is included as a control for protein loading. (e) Graph showing semi-quantification of nuclear AIF levels (arbitrary units) in hippocampus 8 h after status epilepticus for both genotypes. Levels were not significantly different between the two genotypes (= 3 per group). All lanes are from pools of = 3 hippocampi.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The BH3-only proteins are an emerging focus of interest as potential initiators of seizure-induced neuronal death. We show here that intra-amygdala KA-induced status epilepticus results in early Bid cleavage in the hippocampus. However, Bid-deficient mice are not protected against seizure-induced neuronal death in vivo. We also show that nuclear translocation of AIF does not appear to be Bid-dependent in this model. These studies demonstrate that Bid plays no role or a redundant role in seizure-induced neuronal death in vivo.

Biochemical hallmarks of apoptosis-associated signaling pathways are present in hippocampal and extra-hippocampal brain regions after experimental status epilepticus and in temporal lobe material from patients with long-standing epilepsy (Engel and Henshall 2009). However, the apical initiators of neuronal killing in this setting remain incompletely understood. Evidence already points to involvement of anti-apoptotic Bcl-2 family proteins as inhibitors of cell death after seizures. Indeed, loss of bcl-w or over-expression of Bcl-xL modulates hippocampal damage in seizure models (Murphy et al. 2007; Ju et al. 2008). BH3-only proteins may be critical for the initiation of cell death after seizures. Several have been shown to be induced or post-translationally modified within a few hours of seizures including Bim and Puma, and the pro-apoptotic multi-BH domain protein Bax accumulates at mitochondria (Henshall et al. 2001, 2002; Shinoda et al. 2004b; Li et al. 2005; Noh et al. 2006; Engel et al. 2010b; Murphy et al. 2010). Here we studied Bid, which is particularly interesting because it can be activated downstream of the death receptor pathway, which is induced after seizures (Henshall et al. 2001; Shinoda et al. 2003). Bid activation has previously been reported in multiple in vivo models of neurologic insults, including ischemia, trauma and status epilepticus (Henshall et al. 2001; Plesnila et al. 2001; Franz et al. 2002; Yin et al. 2002; Bermpohl et al. 2006). Our studies show that full-length Bid is expressed in adult mouse hippocampus, in agreement with other mouse as well as rat and human data (Wang et al. 1996; Henshall et al. 2001; Franz et al. 2002; Krajewska et al. 2002; Yin et al. 2002; Shinoda et al. 2004b). Cleavage of Bid greatly enhances its cytochrome c releasing activity (Li et al. 1998; Luo et al. 1998) and our data show that Bid is cleaved into the p15 form after status epilepticus in mice. Thus, as in rats, Bid cleavage is a feature of seizure-induced neuronal death in vivo in mice. This contrasts with certain in vitro data reporting glutamate- and N-methyl-d-aspartate treatment of neurons does not result in Bid cleavage (Ward et al. 2006). Indeed, in vitro studies suggested that a translocation of full-length Bid to mitochondria may also occur during excitotoxic cell death, and that over-expression of a caspase 8 cleavage resistant mutant of Bid is sufficient to trigger release of cytochrome c and AIF in neurons (Ward et al. 2006; Konig et al. 2007). These differences likely reflect elements of the in vivo setting not being reproduced in vitro, such as caspase 8 involvement, and use of a variety of cell types, which may display differential BH3-only protein responses, as we have observed in vivo (Murphy et al. 2010). However, in the original studies on Bid, full-length Bid at high concentrations was also capable of releasing cytochrome c (Li et al. 1998; Luo et al. 1998). The amount of tBid we detected is certainly equivalent and in some cases greater than is induced after ischemia or trauma (Plesnila et al. 2001; Franz et al. 2002; Bermpohl et al. 2006), suggesting status epilepticus is particularly effective at generating tBid.

Our analysis of the temporal profile over which Bid cleavage occurred in mice parallels the time course observed in rats after status epilepticus (Henshall et al. 2001; Li et al. 2006). Bid cleavage is an early event in the present model, preceding up-regulation of both Bim and Puma, two other potent BH3-only proteins (Engel et al. 2010b; Murphy et al. 2010). The immediacy of tBid formation after status epilepticus creates a problem in causally linking Bid to mitochondrial release of cytochrome c or AIF because these only emerge at ∼4 h (Murphy et al. 2007 and present data). In contrast, Bim and Puma induction coincide with the release of apoptogenic factors from mitochondria in this model (Engel et al. 2010b; Murphy et al. 2010).

Caspase 8 is a potential mediator of Bid cleavage in the present model. Bid has previously been shown to be efficiently cleaved to tBid by caspase 8 in mouse brain (Plesnila et al. 2001), and caspase 8 is cleaved after seizures in our model (Shinoda et al. 2004a). Moreover, tBid formation after seizures in rats is reduced by treatment with a caspase 8 inhibitor (Henshall et al. 2001; Li et al. 2006). Caspase 3 is unlikely to be the cause, at least initially, because it is not activated until ∼4 h after seizures in the model (Engel et al. 2010b). Although the tBid fragment was 15 kD, which argues for caspase involvement, there are reports that calpain can produce a similar-sized fragment of Bid (Mandic et al. 2002). Nevertheless, the requirement for Bid to be cleaved to effect neuronal death in response to excitotoxic insults remains unproven (Konig et al. 2007).

Previous studies in Bid-deficient mice demonstrated significantly smaller infarcts compared with wild-type mice following ischemia and, at least transiently, reduced lesion size after trauma (Plesnila et al. 2001; Yin et al. 2002; Bermpohl et al. 2006). In the present study, we found that neuronal death after status epilepticus was not different in Bid-deficient compared with wild-type mice. Thus, Bid is not required for seizure-induced neuronal death in vivo in this model. The difference could rest with the functional significance of inflammation, which for ischemia and trauma is a critical contributor to damage via disruption of the blood–brain barrier and invasion of inflammatory cells, such as macrophages and cytotoxic lymphocytes (Waterhouse et al. 2005; Wang et al. 2007). Regardless, the lack of importance of Bid for seizure-induced neuronal death was surprising given its role in other neurologic injury models. Taken together, our data indicate that there is specificity for BH3-only proteins between neurologic insults with Puma, and possibly Bim being more important for seizure-induced neuronal death in vivo, and Bid being more important for ischemia and trauma.

An interesting finding was that AIF translocation was not altered in Bid-deficient mice after status epilepticus. Bid has been shown to regulate AIF release during neuronal death in some models (Konig et al. 2007; Landshamer et al. 2008), so our data appear to conflict with such a role. The trigger for AIF release in the present studies is unknown. However, Bid-independent mediators of AIF release have been demonstrated, including calpain (Chen et al. 2001; Polster et al. 2005; Cao et al. 2007), and calpains contribute to seizure-induced neuronal death in vivo (Takano et al. 2005; Wang et al. 2008).

Taken together, our data show cleavage of Bid is a conserved feature of seizure-induced neuronal death in vivo but it does not appear to be functionally important. This is not surprising because the pathogenic mechanisms driving cell death after seizures versus ischemia or trauma have overlap in some areas (e.g. excessive glutamate release) but also differences (e.g. cerebral blood flow, inflammation, the extent of tissue oxygen and glucose depletion, oedema and oxidative stress, among others) (Nedergaard 1988; Meldrum 1994; Liou et al. 2003). At least two predications can be made. First, pathophysiological features of the specific neurologic insult impose cell death stimuli with unique characteristics which drive to a greater or lesser extent the importance of one or more BH3-only protein, but that these may later converge on common post-mitochondrial downstream effectors (e.g. caspases or AIF). Second, one or more other members of the more potent BH3-only proteins, such as Puma or Bim, may be critical for seizure-induced neuronal death while being functionally redundant for ischemia- or trauma-induced neuronal death. As noted, seizure-damage in Puma- and Bim-deficient mice is reduced compared with wild-type animals (Engel et al. 2010a; b; Murphy et al. 2010). This suggests that tailoring of treatment to differences in the pathophysiology of the insult continues to be a likely requirement of any therapeutic strategy for acute neurologic insults.

In conclusion, the present study demonstrates that prolonged seizures in vivo activate the BH3-only protein Bid. However, cell death and AIF translocation in this model was not significantly influenced by the absence of Bid. These data further resolve the functional influence of cell death pathways during seizure-induced neuronal death which may be important for future approaches to neuroprotection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported by Health Research Board Ireland (RP/2005/24, RP/2007/37), Science Foundation Ireland (08/IN1/B1875 and 08/RFP/NSC1745), post-doctoral fellowships from the Irish Research Council for Science Engineering and Technology and Health Research Board (to T.E.) and the NHMRC (AS).

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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. Bid immunohistochemistry in bid−/− mouse hippocampus. Representative photomicrographs showing Bid-deficient (bid−/−) mouse hippocampus stained with two different Bid antibodies (left panel, R & D systems’ anti-Bid; right panel, anti-phosphoBid from www.antibodies-online.com). Note there is staining of many hippocampal cells with either antibody in Bid-deficient mice, in particular the CA3 pyramidal neurons and cells in the hilus (arrows). Inset shows a western blot confirming the absence of Bid in Biddeficient mouse hippocarnpus.

Figure S2. Bim and Puma expression following seizures in wild- type and Bid-deficient mice (a) Representative western blots (n = 1 per lane) showing expression of Bid, Bim and Puma 8 h following status epilepticus. Note, Bim and Puma staining are somewhat elevated in Bid- deficient (bid’) mice compared to controls. (b) Graphs semi-quantifying Bim and Puma levels in wild-type and Bid-deficient mice (n = 3 each). *p < 0.05 compared to wild-type (wt). ns, non-significant.

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