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Bax-inhibiting peptide protects glutamate-induced cerebellar granule cell death by blocking Bax translocation
Neuroscience Letters 451 (2009) 11–15
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Bax-inhibiting peptide protects glutamate-induced cerebellar granule cell death by blocking Bax translocation Takayuki Iriyama, Yoshimasa Kamei ∗ , Shiro Kozuma, Yuji Taketani Department of Obstetrics and Gynecology, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
a r t i c l e
i n f o
Article history: Received 22 October 2008 Received in revised form 7 December 2008 Accepted 12 December 2008 Keywords: Apoptosis Bax Bax-inhibiting peptide Caspase Excitotoxicity Glutamate
a b s t r a c t Glutamate-induced excitotoxicity has been implicated in the pathogenesis of various neurological damages and disorders. In the brain damage of immature animals such as neonatal hypoxic-ischemic brain injury, the excitotoxicity appears to be more intimately involved through apoptosis. Bax, a member of the Bcl-2 family proteins, plays a key role in the promotion of apoptosis by translocation from the cytosol to the mitochondria and the release of apoptogenic factors such as cytochrome c. Recently, Bax-inhibiting peptide (BIP), a novel membrane-permeable peptide which can bind Bax in the cytosol and inhibit its translocation to the mitochondria, was developed. To investigate the possibility of a new neuroprotection strategy targeting Bax translocation in glutamate-induced neuronal cell death, cerebellar granule neurons (CGNs) were exposed to glutamate with or without BIP. Pretreatment of CGNs with BIP elicited a dose-dependent reduction of glutamate-induced neuronal cell death as measured by MTT assay. BIP significantly suppressed both the number of TUNEL-positive cells and the increase in caspases 3 and 9 activities induced by glutamate. In addition, immunoblotting after subcellular fractionation revealed that BIP prevented the glutamate-induced Bax translocation to the mitochondria and the release of cytochrome c from the mitochondria. These results suggest that agents capable of inhibiting Bax activity such as BIP might lead to new drugs for glutamate-related diseases in the future. © 2008 Elsevier Ireland Ltd. All rights reserved.
Although glutamate is a major excitatory neurotransmitter in the central nervous system (CNS), its accumulation in CNS and excessive stimulation of glutamate receptors induce neurotoxic effects, which, specifically referred to as excitotoxicity, is involved in various acute and chronic neurological disorders such as stroke, Alzheimer’s disease, Parkinson’s disease, and neonatal hypoxicischemic brain injury [7,13]. Glutamate-induced excitotoxicity can be mediated through necrosis or apoptosis, depending on the population of neurons being examined and concentrations of agonist being used [4,8]. In neonatal hypoxic-ischemic brain injury, excitotoxicity appears to be even more intimately involved in the pathogenesis of cell destruction than in the adult brain [13]. In addition, comparison of immature and adult animal models of hypoxia-ischemia suggests that apoptosis is more prominent in the immature brain [12,14]. Bax is a member of the Bcl-2 family proteins that plays a key role in the promotion of apoptosis [28], and Bax-mediated cell death has been implicated as one of the major causes of neurological disorders described above [5,27]. Bax, normally residing in the cytosol in a quiescent state, translocates from the cytosol to the mitochondria in
∗ Corresponding author. Tel.: +81 3 5800 8657; fax: +81 3 3816 2017. E-mail address: [email protected] (Y. Kamei). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.12.021
response to various apoptotic stimuli [28]. Bax translocation causes the release of apoptogenic factors such as cytochrome c by permeabilization of the mitochondrial outer membrane [20]. Cytochrome c induces the oligomerization of APAF-1 to form the apoptosome, which recruits and dimerizes caspase 9. This association cleaves and activates the caspases 3 and 7, which in turn cause collapse of the cell what is known as the execution phase of apoptosis [26]. Because apoptotic neurons remain viable for a period of time and can be rescued, compounds that prevent apoptosis may have therapeutic significance [2]. Until recently, the development of caspase inhibitors that cross the blood–brain barrier has been the major focus of neuroprotective studies that target apoptosis [3]. However, because mitochondrial dysfunction often occurs even in the presence of caspase inhibitors, the inhibition of Bax function at the mitochondria could be potentially a more effective anti-apoptotic strategy [18]. So far, several pharmacologic agents which prevent Bax function have been identified [10,17,19]. The DNA repair protein Ku70 has been found to suppress Bax-mediated apoptosis by interacting with Bax in the cytosol and to prevent its translocation to the mitochondria [22]. A cell permeable five amino acid peptide, Bax-inhibiting peptide (BIP), designed from Bax-binding domain of Ku70, protected cells against Bax-dependent apoptotic stimuli in multiple cell types, demonstrating the possibility of inhibiting Bax-mediated cell death at the initial stage of cell death [21].
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Primary cerebellar granule neuron (CGN) culture has been the most commonly used cells in glutamate-induced excitotoxicityrelated studies. In this study, we hypothesized that Bax-mediated mitochondrial pathway has a critical role in neuronal apoptosis induced by long exposure of glutamate, and we examined the possibility of neuroprotection strategy targeting Bax translocation in glutamate-induced apoptosis using CGN culture. We demonstrated that inhibition of Bax translocation by BIP elicits a neuroprotective effect against glutamate-induced apoptosis in CGNs and suppresses the downstream changes of Bax. All media and supplements used for cell culture were purchased from Invitrogen (Carlsbad, CA). BIP and its negative control peptide (NCP) were obtained from Calbiochem (San Diego, CA), and glutamate was from Sigma (St. Louis, MO). Guidelines for the care and use of laboratory animals as adopted and promulgated by the University of Tokyo were followed. CGNs were prepared from 7-day-old Sprague Dawley rat male pups (Saitama Experimental Animals Supply Center, Japan) as described previously [11]. Briefly, neurons were seeded onto 5% poly-L lysine precoated plastic culture dishes at a density of 2.0 × 105 cells/cm2 in DMEM containing 10% fetal bovine serum and penicillin/streptomycin (100 g/ml). One hour later, this culture medium was replaced with NeuroBasal medium containing antioxidant-free B27 supplement, 20 mM KCl, 2 mM glutamine, and penicillin/streptomycin (100 g/ml) to limit the growth of nonneuronal cells. Half of the medium was replaced every 2 days. Under these protocols, >95% of the cultured cells were granule neurons. On day 7, CGNs were exposed to glutamate alone or glutamate with BIP or NCP for 24 h without replacing the medium in all the following experiments. MTT (3-[4,5-dimethylthiazole-2-yl]2,5-diphenyl tetrazolium bromide) assay was performed to determine the cell viability with the In Vitro Toxicology Assay Kit: MTT Based (Sigma) according to manufacturer’s instructions. The absorbance of the sample was measured at the reference wave length (570 and 690 nm). Unless otherwise indicated, the extent of MTT conversion in cells is expressed as a percentage of the control. Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 10 g/ml Hoechst 33342 fluorochrome, followed by examination on a fluorescence microscope. In some experiments, cells were double-stained with propidium iodide (PI, 5 g/ml) and Hoechst 33342 to distinguish apoptotic cells from necrotic cells as reported previously [25]. Intact blue nuclei, condensed/fragmented blue nuclei, condensed/fragmented pink nuclei, and intact pink nuclei were considered viable, early apoptotic, late apoptotic, and necrotic cells, respectively. At least three hundred nuclei were counted from randomly selected ten fields at ×400 by an investigator blinded to the experimental groups. To detect the terminal deoxyribonucleotidyl transferase (TdT)mediated biotin-16-dUTP nick-end labeling (TUNEL)-positive cells, we used the Apoptosis in situ Detection Kit (Wako Chemicals, Japan). All procedures were performed according to the specification of the manufacturer. Two different investigators blinded to the experimental groups examined each sample (n = 3 per group). At least three hundred nuclei were counted from randomly selected ten fields of each sample at ×400. Caspases 3 and 9 activity assays were performed using CaspaseGloTM Assays (Promega, Madison, WI) according to manufacturer’s instructions using a microplate luminometer LB96V (EG&G Berthold, Germany), and was expressed as fold increase/decrease compared with control group. Subcellular fractionation was performed using a Cytosol/Mitochondria Fractionation Kit (Calbiochem). Cells (3.6 × 107 ) were scraped into ice-cold PBS, collected by centrifugation and washed twice with PBS. The cells were resuspended in
350 l of 1× Cytosol Extraction Buffer, incubated for 10 min on ice, homogenized 15 strokes with a Potter-Elvehjem homogenizer at 4 ◦ C and centrifuged at 700 × g for 10 min at 4 ◦ C to remove debris. The supernatant was centrifuged again at 10,000 × g for 30 min at 4 ◦ C. The resulting supernatant was stored as the cytosolic fraction, and the pellet was resuspended in 100 l of 1× Mitochondria Extraction Buffer and stored as the mitochondrial fraction. Protein concentrations were determined with the Bradfold procedure. Ten micrograms of protein per sample were loaded on each lane, subjected to SDS-PAGE, and transferred to a PVDF membrane. The blots were probed with anti-Bax polyclonal antibody (1:1000, BD-Pharmingen, San Diego, CA) or anti-cytochrome c antibody (1:1000, 7H8.2C12, BD-Pharmingen), and incubated with ECL anti-rabbit or anti-mouse IgG (1:100,000, Amersham Biosciences, Aylesbury, UK) as secondary antibody. The blots were detected with ECL Advance detection reagent (Amersham Biosciences), and exposed to Medical X-ray Films (Konica Minolta, Japan). To confirm the subcellular fractionation, ␣-tubulin and F1 F0 -ATPase subunit␣ (F1␣ subunit) were also analyzed as markers of cytosolic and mitochondrial fractions, respectively, and anti-␣-tubulin antibody (DM1A, Sigma, 1:125,000) and anti-F1␣ subunit antibody (7H10, Molecular Probes, Eugene, OR, 1:5000) were used as primary antibodies. The data represented the mean and SEM from at least 3 independent experiments. p values were obtained using two-way ANOVA and Tukey’s tests. p value <0.05 was considered significant. CGNs were exposed to various concentrations of glutamate for 24 h at day 7, and the cell viability was measured by MTT assay. Glutamate evoked a dose-dependent neurotoxic effect starting from a concentration of 10 M and peaking at 200 M (Fig. 1A). The mode of neuronal cell death could be apoptosis or necrosis depending on the concentration of glutamate [25]. These two mechanisms of cell death can be distinguished from each other by the difference in Hoechst/PI double staining [1]. We carried out dose–response studies and found that the mode of cell death switched from apoptosis to necrosis between 20 and 50 M glutamate (Fig. 1B). Exposure to 20 M glutamate for 24 h predominantly induced apoptosis; apoptotic cells accounted for 80% of all dead cells. However, at 50 M, necrotic cells accounted for 90% of all dead cells, and necrosis was the dominant mode of cell death when cells were exposed to more than 50 M. To investigate the effect of BIP on apoptosis, we chose 20 M glutamate for all further experiments. Preincubation of CGNs was started with BIP at various concentrations or with MK-801 (5 M), an NMDA receptor antagonist, at 1 h before glutamate exposure and was maintained for 24 h. BIP significantly protected CGNs from glutamate-induced cell death in a dose-dependent manner (Fig. 1C). As expected, MK-801 also blocked the cell death, confirming that glutamate toxicity largely depends on NMDA receptors. However, cell death induced by 50 M glutamate was not prevented by BIP (data not shown). To further examine the effect of BIP on neuronal apoptosis, we performed Hoechst 33342 staining for morphological assessment. Apoptotic nuclei were confirmed with chromatin condensation and/or fragmentation. Twenty micromolar glutamate caused a significant increase in the number of apoptotic nucleated-cells, which was prevented by BIP pretreatment (Fig. 2A). In addition, we conducted TUNEL staining to detect glutamate-induced apoptosis in CGNs. Pretreatment of neurons with BIP successfully blocked glutamate-induced apoptosis when compared with NCP pretreatment (Fig. 2B). To further confirm the apoptosis of CGNs induced by glutamate, we next assayed the activities of caspases 3 and 9 after glutamate exposure. Several lines of investigations have demonstrated that in CGNs, the activation of caspase 3 plays an important role in glutamate-induced apoptosis [2]. In addition, caspase 9 has been shown to be activated only via the mitochondrial pathway after cytochrome c release from the mitochondria in
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Fig. 1. Inhibition of glutamate-induced apoptotic cell death by BIP. (A) Quantitative analysis of cell viability measured by MTT assay. CGNs were exposed to 10–200 M glutamate (Glu) for 24 h without replacing the culture medium. Data are shown as the fold decrease of the absorbance in the control (con) (n = 3). (B) The mode of cell death switched from apoptosis to necrosis between 20 and 50 M Glu as judged by Hoechst 33342/PI double-staining. Cells with blue intact nuclei (green arrows) were determined as viable cells, whereas those with blue condensed/fragmented nuclei (yellow arrows) were as early apoptotic cells. Cells with pink intact nuclei (white arrows) were considered necrotic cells, whereas cells with pink condensed/fragmented nuclei (red arrows) were late apoptotic cells. Scale bar, 20 m. (C) The neuroprotective effect of BIP was dose-dependent. CGNs were preincubated with different concentration of BIP (+BIP), 200 M NCP (+NCP), or 5 M MK-801 for 1 h, and then exposed together with 20 M Glu for 24 h. Cell viability was measured by MTT assay. Data are expressed as percentage of the control of three independent experiments (n = 8 for each group). * p < 0.05 and ** p < 0.01 versus control in (A), versus NCP in (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
neuronal apoptosis [18]. In this study, the activities of caspases 3 and 9 were significantly increased by glutamate, and were suppressed by BIP pretreatment (Fig. 2C and D). These results strongly suggest that Bax is closely involved in glutamate-induced apoptosis via the mitochondrial pathway. To evaluate the temporal profile of the neuroprotective activity of BIP, CGNs were treated with BIP at various time points before and
after glutamate exposure. BIP maintained significant neuroprotective effect not only at 1 h before but also at 15 min after glutamate exposure. However, BIP was no longer effective when added 30 min after glutamate exposure (Fig. 3). We further investigated the changes in the subcellular distribution of Bax and cytochrome c (Fig. 4A and B). In the control, basal Bax immunoreactivity was detectable in the cytosol and to
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Fig. 2. Protective effect of BIP against glutamate-induced apoptosis. (A) Quantitative analysis of the apoptotic rate measured by Hoechst 33342 staining after Glu exposure. (B) Quantitative analysis of the rate of TUNEL-positive cells 24 h after Glu exposure. (C and D) BIP prevents the increase of activities of caspase 3 (C) and caspase 9 (D) induced by glutamate. These activities were measured at 24 h after addition of 20 M Glu in the presence of 200 M BIP or NCP. Data are obtained from 3 independent experiments (n = 3 per experiment). * p < 0.05, ** p < 0.01 versus control, # p < 0.01 versus NCP.
a lesser extent in the mitochondria, and glutamate exposure dramatically translocated Bax to the mitochondria at 24 h. In addition, cytochrome c immunoreactivity was exclusively detected in the mitochondrial fraction in the control, but upon glutamate exposure for 24 h, cytochrome c was detected mainly in the cytosolic fraction. These changes were observed even at 2 h after glutamate exposure (data not shown). BIP pretreatment elicited the inhibitory effect against the translocation of both Bax and cytochrome c. In our experiments, contrary to some previous reports suggesting that Bax
Fig. 3. BIP elicits time-dependent neuroprotective effects against glutamateinduced cell death. CGNs were exposed to 200 M BIP or NCP at various time points before and after 20 M Glu administration (−60, −30, 0, +15, +30, and +60 min). Cell viability was measured by MTT assay at 24 h after glutamate. Three independent experiments were performed (n = 8 per experiment). ** p < 0.01 versus NCP.
protein expression induced by glutamate is increased [6,23], total amount of Bax did not change (data not shown). Glutamate has been well known as an important factor in various acute and chronic neurological disorders [7,13]. In the present study using CGN culture, we demonstrated that there is a threshold between apoptosis and necrosis in neuronal cell death induced by glutamate. In addition, we showed that, at lower glutamate concentrations, the neuronal cell death is apoptosis-dominant, which was determined by TUNEL staining, Hoechst staining, and caspases 3 and 9 assays. These results are in agreement with the previous studies demonstrating that mild or low concentrations of excitotoxic agents promote predominantly apoptosis, while intense or high concentrations cause necrotic neuronal cell death [4]. Considering the pathophysiological conditions in stroke, trauma, and some neurodegenerative diseases, the state of excessive extracellular glutamate accumulation persists and neuronal damage evolves over hours to days [16]. A better way to reproduce these phenomena in vitro would be slowly triggered excitotoxicity induced by long glutamate exposure. Therefore, in this study, we used lower concentrations of glutamate to further analyze the intracellular mechanism of excitotoxic neuronal apoptosis. It has been well established that Bax plays a key role in apoptosis induced by various stressors [28], although the insights about the potential involvement of Bax in glutamate-induced neuronal apoptosis remain elusive. While the pathophysiological implications of glutamate control on the expression of Bax have been reported recently [6,23,24], there have been no studies examining the translocation of Bax itself in primary neurons. In this study, where glutamate-induced neuronal cell death was apoptosis-dominant, Bax was translocated from the cytosol to the mitochondria. We
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Fig. 4. BIP prevented glutamate-induced translocation of Bax and the release of cytochrome c. CGNs were treated with 20 M glutamate for 24 h in the presence of 200 M BIP or NCP. After the subcellular fractionation, cell lysates were subjected to immunoblotting. ␣-tubulin and F1 F0 ATP synthase subunit ␣ (F1␣) were used as markers for cytosolic and mitochondrial fractions, respectively. Note that BIP pretreatment significantly prevented the translocation of both Bax (A) and cytochrome c (B).
further demonstrated that BIP was capable of protecting CGNs against glutamate-induced apoptosis by preventing Bax translocation. These findings suggest that the classical Bax-dependent pathway is critically involved in glutamate-induced neuronal apoptosis. Several studies with Bax-null mutant mice have suggested that Bax is dispensable for glutamate-induced neuronal cell death [9,15]. However, it has been well established that both the mechanism and the type of cell death that occurs both in vivo and in vitro depends on both the concentration and the exposure time of glutamate or glutamate agonist [8]. Slowly triggered excitotoxicity, as used in our study, and rapidly triggered excitotoxicity, as in the Bax-null mutant mice experiments, might be mediated via physiologically different pathways or stimulated by different sets of ionotropic glutamate receptors. Clinically, in many cases such as the neonatal hypoxic-ischemic brain damage in which glutamate-induced apoptosis plays a pivotal role [12,13], neuroprotective therapy has no choice but to be initiated after the insult. In our experiments, most importantly, BIP was shown to be still effective even after the glutamate exposure. Therefore, in terms of pharmacological therapy against glutamaterelated diseases, anti-Bax agents might be promising therapies in the future. Acknowledgments We are grateful to C. Luckey for the critical reading of the manuscript. This work was supported in part by a grant from Japan Society for the Promotion of Science. References [1] M. Ankarcrona, J.M. Dypbukt, E. Bonfoco, B. Zhivotovsky, S. Orrenius, S.A. Lipton, P. Nicotera, Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function, Neuron 15 (1995) 961–973. [2] A. Bachis, A.M. Colangelo, S. Vicini, P.P. Doe, M.A. De Bernardi, G. Brooker, I. Mocchetti, Interleukin-10 prevents glutamate-mediated cerebellar granule cell death by blocking caspase-3-like activity, J. Neurosci. 21 (2001) 3104–3112. [3] J. Bilsland, S. Harper, Caspases and neuroprotection, Curr. Opin. Investig. Drugs 3 (2002) 1745–1752. [4] E. Bonfoco, D. Krainc, M. Ankarcrona, P. Nicotera, S.A. Lipton, Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-d-aspartate or nitric oxide/superoxide in cortical cell cultures, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 7162–7166. [5] G. Cao, M. Minami, W. Pei, C. Yan, D. Chen, C. O’Horo, S.H. Graham, J. Chen, Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death, J. Cereb. Blood Flow Metab. 21 (2001) 321–333. [6] R.W. Chen, D.M. Chuang, Long term lithium treatment suppresses p53 and Bax expression but increases Bcl-2 expression. A prominent role in neuroprotection against excitotoxicity, J. Biol. Chem. 274 (1999) 6039–6042.
[7] D.W. Choi, Glutamate neurotoxicity and diseases of the nervous system, Neuron 1 (1988) 623–634. [8] D.W. Choi, Excitotoxic cell death, J. Neurobiol. 23 (1992) 1261–1276. [9] R. Dargusch, D. Piasecki, S. Tan, Y. Liu, D. Schubert, The role of Bax in glutamateinduced nerve cell death, J. Neurochem. 76 (2001) 295–301. [10] B. Guo, D. Zhai, E. Cabezas, K. Welsh, S. Nouraini, A.C. Satterthwait, J.C. Reed, Humanin peptide suppresses apoptosis by interfering with Bax activation, Nature 423 (2003) 456–461. [11] C.A. Harris, E.M. Johnson Jr., BH3-only Bcl-2 family members are coordinately regulated by the JNK pathway and require Bax to induce apoptosis in neurons, J. Biol. Chem. 276 (2001) 37754–37760. [12] B.R. Hu, C.L. Liu, Y. Ouyang, K. Blomgren, B.K. Siesjo, Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation, J. Cereb. Blood Flow Metab. 20 (2000) 1294–1300. [13] M.V. Johnston, W.H. Trescher, A. Ishida, W. Nakajima, Neurobiology of hypoxicischemic injury in the developing brain, Pediatr. Res. 49 (2001) 735–741. [14] M. Koike, M. Shibata, M. Tadakoshi, K. Gotoh, M. Komatsu, S. Waguri, N. Kawahara, K. Kuida, S. Nagata, E. Kominami, K. Tanaka, Y. Uchiyama, Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxicischemic injury, Am. J. Pathol. 172 (2008) 454–469. [15] T. Lindsten, J.A. Golden, W.X. Zong, J. Minarcik, M.H. Harris, C.B. Thompson, The proapoptotic activities of Bax and Bak limit the size of the neural stem cell pool, J. Neurosci. 23 (2003) 11112–11119. [16] B. Pettmann, C.E. Henderson, Neuronal cell death, Neuron 20 (1998) 633–647. [17] B.M. Polster, G. Basanez, M. Young, M. Suzuki, G. Fiskum, Inhibition of Baxinduced cytochrome c release from neural cell and brain mitochondria by dibucaine and propranolol, J. Neurosci. 23 (2003) 2735–2743. [18] B.M. Polster, G. Fiskum, Mitochondrial mechanisms of neural cell apoptosis, J. Neurochem. 90 (2004) 1281–1289. [19] C.M. Rodrigues, S. Sola, J.C. Sharpe, J.J. Moura, C.J. Steer, Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria, Biochemistry 42 (2003) 3070–3080. [20] M. Saito, S.J. Korsmeyer, P.H. Schlesinger, BAX-dependent transport of cytochrome c reconstituted in pure liposomes, Nat. Cell Biol. 2 (2000) 553–555. [21] M. Sawada, P. Hayes, S. Matsuyama, Cytoprotective membrane-permeable peptides designed from the Bax-binding domain of Ku70, Nat. Cell Biol. 5 (2003) 352–357. [22] M. Sawada, W. Sun, P. Hayes, K. Leskov, D.A. Boothman, S. Matsuyama, Ku70 suppresses the apoptotic translocation of Bax to mitochondria, Nat. Cell Biol. 5 (2003) 320–329. [23] W.R. Schelman, R.D. Andres, K.J. Sipe, E. Kang, J.A. Weyhenmeyer, Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line, Brain Res. Mol. Brain Res. 128 (2004) 160–169. [24] E.A. Sribnick, S.K. Ray, M.W. Nowak, L. Li, N.L. Banik, 17beta-estradiol attenuates glutamate-induced apoptosis and preserves electrophysiologic function in primary cortical neurons, J. Neurosci. Res. 76 (2004) 688–696. [25] K. Suk, I. Chang, Y.H. Kim, S. Kim, J.Y. Kim, H. Kim, M.S. Lee, Interferon gamma (IFNgamma) and tumor necrosis factor alpha synergism in ME-180 cervical cancer cell apoptosis and necrosis. IFNgamma inhibits cytoprotective NF-kappa B through STAT1/IRF-1 pathways, J. Biol. Chem. 276 (2001) 13153–13159. [26] R.C. Taylor, S.P. Cullen, S.J. Martin, Apoptosis: controlled demolition at the cellular level, Nat. Rev. Mol. Cell Biol. 9 (2008) 231–241. [27] M. Vila, V. Jackson-Lewis, S. Vukosavic, R. Djaldetti, G. Liberatore, D. Offen, S.J. Korsmeyer, S. Przedborski, Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 2837–2842. [28] R.J. Youle, A. Strasser, The BCL-2 protein family: opposing activities that mediate cell death, Nat. Rev. Mol. Cell Biol. 9 (2008) 47–59.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Bax-inhibiting peptide protects glutamate-induced cerebellar granule cell death by blocking Bax translocation Takayuki Iriyama, Yoshimasa Kamei ∗ , Shiro Kozuma, Yuji Taketani Department of Obstetrics and Gynecology, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
a r t i c l e
i n f o
Article history: Received 22 October 2008 Received in revised form 7 December 2008 Accepted 12 December 2008 Keywords: Apoptosis Bax Bax-inhibiting peptide Caspase Excitotoxicity Glutamate
a b s t r a c t Glutamate-induced excitotoxicity has been implicated in the pathogenesis of various neurological damages and disorders. In the brain damage of immature animals such as neonatal hypoxic-ischemic brain injury, the excitotoxicity appears to be more intimately involved through apoptosis. Bax, a member of the Bcl-2 family proteins, plays a key role in the promotion of apoptosis by translocation from the cytosol to the mitochondria and the release of apoptogenic factors such as cytochrome c. Recently, Bax-inhibiting peptide (BIP), a novel membrane-permeable peptide which can bind Bax in the cytosol and inhibit its translocation to the mitochondria, was developed. To investigate the possibility of a new neuroprotection strategy targeting Bax translocation in glutamate-induced neuronal cell death, cerebellar granule neurons (CGNs) were exposed to glutamate with or without BIP. Pretreatment of CGNs with BIP elicited a dose-dependent reduction of glutamate-induced neuronal cell death as measured by MTT assay. BIP significantly suppressed both the number of TUNEL-positive cells and the increase in caspases 3 and 9 activities induced by glutamate. In addition, immunoblotting after subcellular fractionation revealed that BIP prevented the glutamate-induced Bax translocation to the mitochondria and the release of cytochrome c from the mitochondria. These results suggest that agents capable of inhibiting Bax activity such as BIP might lead to new drugs for glutamate-related diseases in the future. © 2008 Elsevier Ireland Ltd. All rights reserved.
Although glutamate is a major excitatory neurotransmitter in the central nervous system (CNS), its accumulation in CNS and excessive stimulation of glutamate receptors induce neurotoxic effects, which, specifically referred to as excitotoxicity, is involved in various acute and chronic neurological disorders such as stroke, Alzheimer’s disease, Parkinson’s disease, and neonatal hypoxicischemic brain injury [7,13]. Glutamate-induced excitotoxicity can be mediated through necrosis or apoptosis, depending on the population of neurons being examined and concentrations of agonist being used [4,8]. In neonatal hypoxic-ischemic brain injury, excitotoxicity appears to be even more intimately involved in the pathogenesis of cell destruction than in the adult brain [13]. In addition, comparison of immature and adult animal models of hypoxia-ischemia suggests that apoptosis is more prominent in the immature brain [12,14]. Bax is a member of the Bcl-2 family proteins that plays a key role in the promotion of apoptosis [28], and Bax-mediated cell death has been implicated as one of the major causes of neurological disorders described above [5,27]. Bax, normally residing in the cytosol in a quiescent state, translocates from the cytosol to the mitochondria in
∗ Corresponding author. Tel.: +81 3 5800 8657; fax: +81 3 3816 2017. E-mail address: [email protected] (Y. Kamei). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.12.021
response to various apoptotic stimuli [28]. Bax translocation causes the release of apoptogenic factors such as cytochrome c by permeabilization of the mitochondrial outer membrane [20]. Cytochrome c induces the oligomerization of APAF-1 to form the apoptosome, which recruits and dimerizes caspase 9. This association cleaves and activates the caspases 3 and 7, which in turn cause collapse of the cell what is known as the execution phase of apoptosis [26]. Because apoptotic neurons remain viable for a period of time and can be rescued, compounds that prevent apoptosis may have therapeutic significance [2]. Until recently, the development of caspase inhibitors that cross the blood–brain barrier has been the major focus of neuroprotective studies that target apoptosis [3]. However, because mitochondrial dysfunction often occurs even in the presence of caspase inhibitors, the inhibition of Bax function at the mitochondria could be potentially a more effective anti-apoptotic strategy [18]. So far, several pharmacologic agents which prevent Bax function have been identified [10,17,19]. The DNA repair protein Ku70 has been found to suppress Bax-mediated apoptosis by interacting with Bax in the cytosol and to prevent its translocation to the mitochondria [22]. A cell permeable five amino acid peptide, Bax-inhibiting peptide (BIP), designed from Bax-binding domain of Ku70, protected cells against Bax-dependent apoptotic stimuli in multiple cell types, demonstrating the possibility of inhibiting Bax-mediated cell death at the initial stage of cell death [21].
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Primary cerebellar granule neuron (CGN) culture has been the most commonly used cells in glutamate-induced excitotoxicityrelated studies. In this study, we hypothesized that Bax-mediated mitochondrial pathway has a critical role in neuronal apoptosis induced by long exposure of glutamate, and we examined the possibility of neuroprotection strategy targeting Bax translocation in glutamate-induced apoptosis using CGN culture. We demonstrated that inhibition of Bax translocation by BIP elicits a neuroprotective effect against glutamate-induced apoptosis in CGNs and suppresses the downstream changes of Bax. All media and supplements used for cell culture were purchased from Invitrogen (Carlsbad, CA). BIP and its negative control peptide (NCP) were obtained from Calbiochem (San Diego, CA), and glutamate was from Sigma (St. Louis, MO). Guidelines for the care and use of laboratory animals as adopted and promulgated by the University of Tokyo were followed. CGNs were prepared from 7-day-old Sprague Dawley rat male pups (Saitama Experimental Animals Supply Center, Japan) as described previously [11]. Briefly, neurons were seeded onto 5% poly-L lysine precoated plastic culture dishes at a density of 2.0 × 105 cells/cm2 in DMEM containing 10% fetal bovine serum and penicillin/streptomycin (100 g/ml). One hour later, this culture medium was replaced with NeuroBasal medium containing antioxidant-free B27 supplement, 20 mM KCl, 2 mM glutamine, and penicillin/streptomycin (100 g/ml) to limit the growth of nonneuronal cells. Half of the medium was replaced every 2 days. Under these protocols, >95% of the cultured cells were granule neurons. On day 7, CGNs were exposed to glutamate alone or glutamate with BIP or NCP for 24 h without replacing the medium in all the following experiments. MTT (3-[4,5-dimethylthiazole-2-yl]2,5-diphenyl tetrazolium bromide) assay was performed to determine the cell viability with the In Vitro Toxicology Assay Kit: MTT Based (Sigma) according to manufacturer’s instructions. The absorbance of the sample was measured at the reference wave length (570 and 690 nm). Unless otherwise indicated, the extent of MTT conversion in cells is expressed as a percentage of the control. Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 10 g/ml Hoechst 33342 fluorochrome, followed by examination on a fluorescence microscope. In some experiments, cells were double-stained with propidium iodide (PI, 5 g/ml) and Hoechst 33342 to distinguish apoptotic cells from necrotic cells as reported previously [25]. Intact blue nuclei, condensed/fragmented blue nuclei, condensed/fragmented pink nuclei, and intact pink nuclei were considered viable, early apoptotic, late apoptotic, and necrotic cells, respectively. At least three hundred nuclei were counted from randomly selected ten fields at ×400 by an investigator blinded to the experimental groups. To detect the terminal deoxyribonucleotidyl transferase (TdT)mediated biotin-16-dUTP nick-end labeling (TUNEL)-positive cells, we used the Apoptosis in situ Detection Kit (Wako Chemicals, Japan). All procedures were performed according to the specification of the manufacturer. Two different investigators blinded to the experimental groups examined each sample (n = 3 per group). At least three hundred nuclei were counted from randomly selected ten fields of each sample at ×400. Caspases 3 and 9 activity assays were performed using CaspaseGloTM Assays (Promega, Madison, WI) according to manufacturer’s instructions using a microplate luminometer LB96V (EG&G Berthold, Germany), and was expressed as fold increase/decrease compared with control group. Subcellular fractionation was performed using a Cytosol/Mitochondria Fractionation Kit (Calbiochem). Cells (3.6 × 107 ) were scraped into ice-cold PBS, collected by centrifugation and washed twice with PBS. The cells were resuspended in
350 l of 1× Cytosol Extraction Buffer, incubated for 10 min on ice, homogenized 15 strokes with a Potter-Elvehjem homogenizer at 4 ◦ C and centrifuged at 700 × g for 10 min at 4 ◦ C to remove debris. The supernatant was centrifuged again at 10,000 × g for 30 min at 4 ◦ C. The resulting supernatant was stored as the cytosolic fraction, and the pellet was resuspended in 100 l of 1× Mitochondria Extraction Buffer and stored as the mitochondrial fraction. Protein concentrations were determined with the Bradfold procedure. Ten micrograms of protein per sample were loaded on each lane, subjected to SDS-PAGE, and transferred to a PVDF membrane. The blots were probed with anti-Bax polyclonal antibody (1:1000, BD-Pharmingen, San Diego, CA) or anti-cytochrome c antibody (1:1000, 7H8.2C12, BD-Pharmingen), and incubated with ECL anti-rabbit or anti-mouse IgG (1:100,000, Amersham Biosciences, Aylesbury, UK) as secondary antibody. The blots were detected with ECL Advance detection reagent (Amersham Biosciences), and exposed to Medical X-ray Films (Konica Minolta, Japan). To confirm the subcellular fractionation, ␣-tubulin and F1 F0 -ATPase subunit␣ (F1␣ subunit) were also analyzed as markers of cytosolic and mitochondrial fractions, respectively, and anti-␣-tubulin antibody (DM1A, Sigma, 1:125,000) and anti-F1␣ subunit antibody (7H10, Molecular Probes, Eugene, OR, 1:5000) were used as primary antibodies. The data represented the mean and SEM from at least 3 independent experiments. p values were obtained using two-way ANOVA and Tukey’s tests. p value <0.05 was considered significant. CGNs were exposed to various concentrations of glutamate for 24 h at day 7, and the cell viability was measured by MTT assay. Glutamate evoked a dose-dependent neurotoxic effect starting from a concentration of 10 M and peaking at 200 M (Fig. 1A). The mode of neuronal cell death could be apoptosis or necrosis depending on the concentration of glutamate [25]. These two mechanisms of cell death can be distinguished from each other by the difference in Hoechst/PI double staining [1]. We carried out dose–response studies and found that the mode of cell death switched from apoptosis to necrosis between 20 and 50 M glutamate (Fig. 1B). Exposure to 20 M glutamate for 24 h predominantly induced apoptosis; apoptotic cells accounted for 80% of all dead cells. However, at 50 M, necrotic cells accounted for 90% of all dead cells, and necrosis was the dominant mode of cell death when cells were exposed to more than 50 M. To investigate the effect of BIP on apoptosis, we chose 20 M glutamate for all further experiments. Preincubation of CGNs was started with BIP at various concentrations or with MK-801 (5 M), an NMDA receptor antagonist, at 1 h before glutamate exposure and was maintained for 24 h. BIP significantly protected CGNs from glutamate-induced cell death in a dose-dependent manner (Fig. 1C). As expected, MK-801 also blocked the cell death, confirming that glutamate toxicity largely depends on NMDA receptors. However, cell death induced by 50 M glutamate was not prevented by BIP (data not shown). To further examine the effect of BIP on neuronal apoptosis, we performed Hoechst 33342 staining for morphological assessment. Apoptotic nuclei were confirmed with chromatin condensation and/or fragmentation. Twenty micromolar glutamate caused a significant increase in the number of apoptotic nucleated-cells, which was prevented by BIP pretreatment (Fig. 2A). In addition, we conducted TUNEL staining to detect glutamate-induced apoptosis in CGNs. Pretreatment of neurons with BIP successfully blocked glutamate-induced apoptosis when compared with NCP pretreatment (Fig. 2B). To further confirm the apoptosis of CGNs induced by glutamate, we next assayed the activities of caspases 3 and 9 after glutamate exposure. Several lines of investigations have demonstrated that in CGNs, the activation of caspase 3 plays an important role in glutamate-induced apoptosis [2]. In addition, caspase 9 has been shown to be activated only via the mitochondrial pathway after cytochrome c release from the mitochondria in
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Fig. 1. Inhibition of glutamate-induced apoptotic cell death by BIP. (A) Quantitative analysis of cell viability measured by MTT assay. CGNs were exposed to 10–200 M glutamate (Glu) for 24 h without replacing the culture medium. Data are shown as the fold decrease of the absorbance in the control (con) (n = 3). (B) The mode of cell death switched from apoptosis to necrosis between 20 and 50 M Glu as judged by Hoechst 33342/PI double-staining. Cells with blue intact nuclei (green arrows) were determined as viable cells, whereas those with blue condensed/fragmented nuclei (yellow arrows) were as early apoptotic cells. Cells with pink intact nuclei (white arrows) were considered necrotic cells, whereas cells with pink condensed/fragmented nuclei (red arrows) were late apoptotic cells. Scale bar, 20 m. (C) The neuroprotective effect of BIP was dose-dependent. CGNs were preincubated with different concentration of BIP (+BIP), 200 M NCP (+NCP), or 5 M MK-801 for 1 h, and then exposed together with 20 M Glu for 24 h. Cell viability was measured by MTT assay. Data are expressed as percentage of the control of three independent experiments (n = 8 for each group). * p < 0.05 and ** p < 0.01 versus control in (A), versus NCP in (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
neuronal apoptosis [18]. In this study, the activities of caspases 3 and 9 were significantly increased by glutamate, and were suppressed by BIP pretreatment (Fig. 2C and D). These results strongly suggest that Bax is closely involved in glutamate-induced apoptosis via the mitochondrial pathway. To evaluate the temporal profile of the neuroprotective activity of BIP, CGNs were treated with BIP at various time points before and
after glutamate exposure. BIP maintained significant neuroprotective effect not only at 1 h before but also at 15 min after glutamate exposure. However, BIP was no longer effective when added 30 min after glutamate exposure (Fig. 3). We further investigated the changes in the subcellular distribution of Bax and cytochrome c (Fig. 4A and B). In the control, basal Bax immunoreactivity was detectable in the cytosol and to
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Fig. 2. Protective effect of BIP against glutamate-induced apoptosis. (A) Quantitative analysis of the apoptotic rate measured by Hoechst 33342 staining after Glu exposure. (B) Quantitative analysis of the rate of TUNEL-positive cells 24 h after Glu exposure. (C and D) BIP prevents the increase of activities of caspase 3 (C) and caspase 9 (D) induced by glutamate. These activities were measured at 24 h after addition of 20 M Glu in the presence of 200 M BIP or NCP. Data are obtained from 3 independent experiments (n = 3 per experiment). * p < 0.05, ** p < 0.01 versus control, # p < 0.01 versus NCP.
a lesser extent in the mitochondria, and glutamate exposure dramatically translocated Bax to the mitochondria at 24 h. In addition, cytochrome c immunoreactivity was exclusively detected in the mitochondrial fraction in the control, but upon glutamate exposure for 24 h, cytochrome c was detected mainly in the cytosolic fraction. These changes were observed even at 2 h after glutamate exposure (data not shown). BIP pretreatment elicited the inhibitory effect against the translocation of both Bax and cytochrome c. In our experiments, contrary to some previous reports suggesting that Bax
Fig. 3. BIP elicits time-dependent neuroprotective effects against glutamateinduced cell death. CGNs were exposed to 200 M BIP or NCP at various time points before and after 20 M Glu administration (−60, −30, 0, +15, +30, and +60 min). Cell viability was measured by MTT assay at 24 h after glutamate. Three independent experiments were performed (n = 8 per experiment). ** p < 0.01 versus NCP.
protein expression induced by glutamate is increased [6,23], total amount of Bax did not change (data not shown). Glutamate has been well known as an important factor in various acute and chronic neurological disorders [7,13]. In the present study using CGN culture, we demonstrated that there is a threshold between apoptosis and necrosis in neuronal cell death induced by glutamate. In addition, we showed that, at lower glutamate concentrations, the neuronal cell death is apoptosis-dominant, which was determined by TUNEL staining, Hoechst staining, and caspases 3 and 9 assays. These results are in agreement with the previous studies demonstrating that mild or low concentrations of excitotoxic agents promote predominantly apoptosis, while intense or high concentrations cause necrotic neuronal cell death [4]. Considering the pathophysiological conditions in stroke, trauma, and some neurodegenerative diseases, the state of excessive extracellular glutamate accumulation persists and neuronal damage evolves over hours to days [16]. A better way to reproduce these phenomena in vitro would be slowly triggered excitotoxicity induced by long glutamate exposure. Therefore, in this study, we used lower concentrations of glutamate to further analyze the intracellular mechanism of excitotoxic neuronal apoptosis. It has been well established that Bax plays a key role in apoptosis induced by various stressors [28], although the insights about the potential involvement of Bax in glutamate-induced neuronal apoptosis remain elusive. While the pathophysiological implications of glutamate control on the expression of Bax have been reported recently [6,23,24], there have been no studies examining the translocation of Bax itself in primary neurons. In this study, where glutamate-induced neuronal cell death was apoptosis-dominant, Bax was translocated from the cytosol to the mitochondria. We
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Fig. 4. BIP prevented glutamate-induced translocation of Bax and the release of cytochrome c. CGNs were treated with 20 M glutamate for 24 h in the presence of 200 M BIP or NCP. After the subcellular fractionation, cell lysates were subjected to immunoblotting. ␣-tubulin and F1 F0 ATP synthase subunit ␣ (F1␣) were used as markers for cytosolic and mitochondrial fractions, respectively. Note that BIP pretreatment significantly prevented the translocation of both Bax (A) and cytochrome c (B).
further demonstrated that BIP was capable of protecting CGNs against glutamate-induced apoptosis by preventing Bax translocation. These findings suggest that the classical Bax-dependent pathway is critically involved in glutamate-induced neuronal apoptosis. Several studies with Bax-null mutant mice have suggested that Bax is dispensable for glutamate-induced neuronal cell death [9,15]. However, it has been well established that both the mechanism and the type of cell death that occurs both in vivo and in vitro depends on both the concentration and the exposure time of glutamate or glutamate agonist [8]. Slowly triggered excitotoxicity, as used in our study, and rapidly triggered excitotoxicity, as in the Bax-null mutant mice experiments, might be mediated via physiologically different pathways or stimulated by different sets of ionotropic glutamate receptors. Clinically, in many cases such as the neonatal hypoxic-ischemic brain damage in which glutamate-induced apoptosis plays a pivotal role [12,13], neuroprotective therapy has no choice but to be initiated after the insult. In our experiments, most importantly, BIP was shown to be still effective even after the glutamate exposure. Therefore, in terms of pharmacological therapy against glutamaterelated diseases, anti-Bax agents might be promising therapies in the future. Acknowledgments We are grateful to C. Luckey for the critical reading of the manuscript. This work was supported in part by a grant from Japan Society for the Promotion of Science. References [1] M. Ankarcrona, J.M. Dypbukt, E. Bonfoco, B. Zhivotovsky, S. Orrenius, S.A. Lipton, P. Nicotera, Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function, Neuron 15 (1995) 961–973. [2] A. Bachis, A.M. Colangelo, S. Vicini, P.P. Doe, M.A. De Bernardi, G. Brooker, I. Mocchetti, Interleukin-10 prevents glutamate-mediated cerebellar granule cell death by blocking caspase-3-like activity, J. Neurosci. 21 (2001) 3104–3112. [3] J. Bilsland, S. Harper, Caspases and neuroprotection, Curr. Opin. Investig. Drugs 3 (2002) 1745–1752. [4] E. Bonfoco, D. Krainc, M. Ankarcrona, P. Nicotera, S.A. Lipton, Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-d-aspartate or nitric oxide/superoxide in cortical cell cultures, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 7162–7166. [5] G. Cao, M. Minami, W. Pei, C. Yan, D. Chen, C. O’Horo, S.H. Graham, J. Chen, Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death, J. Cereb. Blood Flow Metab. 21 (2001) 321–333. [6] R.W. Chen, D.M. Chuang, Long term lithium treatment suppresses p53 and Bax expression but increases Bcl-2 expression. A prominent role in neuroprotection against excitotoxicity, J. Biol. Chem. 274 (1999) 6039–6042.
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