Báo cáo khoa học: The proapoptotic member of the Bcl-2 family Bcl-2 / E1B-19K-interacting protein 3 is a mediator of caspase-independent neuronal death in excitotoxicity pot

The proapoptotic member of the Bcl-2 family Bcl-2 / E1B-19K-interacting protein 3 is a mediator of caspase-independent neuronal death in excitotoxicity Zhengfeng Zhang 1,2 , Ruoyang Shi 1 , Jiequn Weng 1 , Xingshun Xu 3 , Xin-Min Li 1 , Tian-ming Gao 4 and Jiming Kong 1,4 1 Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada 2 Department of Orthopedics, Xinqiao Hospital, TheThrid Military Medical University, Chongqing, China 3 Institute of Neuroscience, Soochow University, Suzhou, Jiangsu Province, China 4 Department of Anatomy and Neurobiology, Southern Medical University, Guangzhou, China Introduction Excessive activation of glutamate receptors results in excitatory neuronal cell death, a process called excito- toxicity, which has been shown to be a contributory factor to neuronal cell loss in neurodegenerative diseases [1,2]. The mechanisms responsible for neuro- excitotoxicity include neuronal Ca 2+ overload [3,4], mitochondrial depolarization [3,5–8], and opening of mitochondrial permeability transition pores, through which mitochondrial solutes with molecular masses up to 1.5 kDa can pass [9]. Members of the Bcl-2 family are important regulators of apoptotic cell death [10–12]. Antiapoptotic members of the Bcl-2 family, including Bcl-2 and Bcl-X L , prevent apoptosis by preserving mitochondrial integrity [11]. Keywords apoptosis; Bcl-2 ⁄ E1B-19K-interacting protein 3 (BNIP3); caspase-independent cell death; excitotoxicity; neuron Correspondence J. Kong or T. Gao, Department of Human Anatomy and Cell Science, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba R3E 0W3, Canada; Department of Anatomy and Neurobiology, Southern Medical University, Guangzhou 510515, China Fax: +1 204 789 3920 Tel: +1 204 977 5601; +011 86 20 6164 8216 E-mail: kongj@cc.umanitoba.ca; tianminggao@tom.com (Received 3 June 2010, revised 1 September 2010, accepted 25 October 2010) doi:10.1111/j.1742-4658.2010.07939.x Caspase-independent neuronal death has been shown to occur in neuroexci- totoxicity. Here, we tested the hypothesis that the gene encoding Bcl- 2 ⁄ E1B-19K-interacting protein 3 (BNIP3) mediates caspase-independent neuronal death in excitotoxicity. BNIP3 was not detectable in neurons under normal condition. BNIP3 expression was increased dramatically in neurons in both in vivo and in vitro models of excitotoxicity. Expression of full-length BNIP3 in primary hippocampal neurons induced atypical cell death that required protein synthesis but was largely independent of caspase activities. Inhibition of BNIP3 expression by RNA interference protected against glutamate-induced neuronal cell death. Thus, BNIP3 activation and expression appears to be both necessary and sufficient for neuronal apoptosis in excitotoxicity. These results suggest that BNIP3 may be a new target for neuronal rescue strategies. Abbreviations BNIP3, Bcl-2 ⁄ E1B-19K-interacting protein 3; CL, contralateral; CNQX, 6-cyano-7-nitroquinaloxine-2,3-dione; EGFR, enhanced green fluorescent protein; GST, glutathione-S-transferase; KA, kainic acid; NMDA, N-methyl- D-aspartate; RNAi, RNA interference; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. 134 FEBS Journal 278 (2011) 134–142 ª 2010 The Authors Journal compilation ª 2010 FEBS Upon activation by death stimuli, proapoptotic mem- bers of the Bcl-2 family, including Bad, Bax, Bid, and Bim, permeabilize mitochondrial membranes [13]. Bcl-2 ⁄ E1B-19K-interacting protein 3 (BNIP3) is a member of a unique subfamily of death-inducing mito- chondrial proteins [14,15]. BNIP3-induced cell death has been characterized by early plasma membrane and mitochondrial damage independently of cytochrome c release and caspase activity [16,17]. However, the extent to which BNIP3 is involved in excitotoxicity-induced neuronal cell death is not known. Here, we tested the hypothesis that BNIP3 is a gene that mediates caspase- independent neuronal death in excitotoxicity. Our results show that BNIP3 expression is upregulated in in vivo and in vitro models of neuroexcitotoxicity, that expression of full-length BNIP3 induced an atypical form of cell death, and that inhibition of BNIP3 by RNA interference (RNAi) and expression of a domi- nant negative form of BNIP3 that lacks the functional transmembrane domain protected against glutamate- induced neuronal cell death. Thus, BNIP3 activation and expression appear to be both necessary and suffi- cient for atypical neuronal apoptosis in excitotoxicity. Results Kainic acid (KA) is a specific agonist for the kainate receptor (a subtype of the ionotrophic glutamate recep- tor) that mimics the effect of glutamate. Of the 15 rats that received KA injections, five were used for prepa- ration of brain sections and 10 for biochemical analy- sis. Under control conditions, wer and others were able to only barely detect BNIP3 in brain tissue or hippocampal neurons [17,18]. As a first step to testing the hypothesis that BNIP3 expression plays an impor- tant role in neuroexcitotoxicity, we examined levels of BNIP3 expression by immunohistochemistry in brains of rats injected intrastriatally with KA. Two days after unilateral injection of KA, BNIP3-immunopositive neurons were present in striatal areas adjacent to the site of injection (Fig. 1A). High levels of BNIP3 immuno- staining were found in the cytoplasm of striatal neurons affected by the KA, and almost all of the BNIP3- positive neurons showed signs of DNA damage when stained with Hoescht 33342 (Fig. 1B). BNIP3-immuno- negative neurons showed normal nuclear morphology. DNA fragmentation in KA-induced neuronal cell death was further confirmed by terminal deoxynucleot- idyl transferase dUTP nick end labeling (TUNEL), with TUNEL-positive nuclei being detected only in areas adjacent to sites of KA injection, and not in the contralateral (CL) striatum (Fig. 1C,D). To confirm that the increased expression of BNIP3 after KA administration was caused by activation of kainate receptors, brain tissue was processed from rats that received intrastriatal injections (1 lL) of 2.5 nmol of KA, 5 nmol of 6-cyano-7-nitroquinaloxine-2,3-dione (CNQX), a mixture of 5 nmol of CNQX and 2.5 nmol of KA, or 50 mm Tris ⁄ HCl (pH 7.4). BNIP3 expression was observed only in those rats that received KA alone, and not in those rats receiving CNQX or the buffer (data not shown). To more quantitatively determine the levels of BNIP3 and determine the molecular mass of the BNIP3 expressed, immunoblots were run for samples derived from KA-injected striata, CL uninjected striat- a, Tris ⁄ HCl-injected striata and CL uninjected striata from Tris ⁄ HCl-injected rats. A 60 kDa band was present in KA-injected striata (Fig. 1E); this band was much weaker in CL striata, and was absent in sam- ples from Tris ⁄ HCl-injected rats. To demonstrate the specificity of the BNIP3 immunoblotting, control experiments were performed in which the BNIP3 anti- body was first incubated for 30 min with a BNIP3– glutathione-S-transferase (GST) protein. As shown in Fig. 1E, immunoblotting for BNIP3 was completely blocked by the BNIP3–GST protein. A nonspecific 62 kDa band was detected in all of the striatal sam- ples. Quantification of the bands with the b-actin bands as internal controls revealed that injection of KA upregulated BNIP3 expression nine-fold (Fig. 1F; n = 6). To determine whether KA increased BNIP3 tran- scription as well as translation as described above, brain samples from KA-injected rats were processed by in situ hybridization with an RNA probe specific for BNIP3. Levels of BNIP3 mRNA were increased by KA (Fig. 1G,H). Positive hybridization signals were found in a group of striatal neurons adjacent to the site of KA injection, whereas neurons in other brain areas showed very low levels of BNIP3 mRNA. To determine the mechanisms by which BNIP3 expression induced by excitotoxicity kills neurons, pri- mary cultures of rat hippocampal neurons were treated with glutamate for 6 h, maintained in Neurobasal medium for 24 h, and stained with trypan blue for membrane integrity. As expected, glutamate increased neuronal cell death in a dose-dependent manner (Fig. 2A); 70% of cells stained positively for trypan blue with 100 lm glutamate, and 10 lm glutamate killed 40% of hippocampal neurons. Expression of BNIP3 was not detectable in the majority of untreated neurons, and less than 15% of the untreated neurons expressed low levels of BNIP3 according to immuno- histochemistry (Fig. 2B). In contrast, more than 70% of cells treated with 100 lm glutamate stained Z. Zhang et al. BNIP3 in excitotoxicity FEBS Journal 278 (2011) 134–142 ª 2010 The Authors Journal compilation ª 2010 FEBS 135 positively for BNIP3 (Fig. 2C). Nuclei in BNIP3-positive neurons showe d a c haracteristic d ysmorp hic a ppearan ce (Fig. 2 D). To determine the time course of BNIP3 expression, protein samples p repared fr om hippocampal neurons were immunoblotted with an antibody against BNIP3. A s ample prepared fr om HEK 293 cells that were transfected with T7-tagged pcDNA3–hBNIP3 was included as a p ositive control. As shown i n Fig. 2E,F, levels of BNIP3 were significantly increased in neurons after exposure to 100 lm glutamate for 36 h, and peaked (seven-fold) at 60 h. Next, we determined the extent to which BNIP3 expression was necessary and sufficient to kill neurons. Primary cultures of hippocampal neurons at day 4 in culture were transfected using LipofectAMINE 2000 with a pcDNA3–hBNIP3 plasmid encoding full-length BNIP3, a pcDNA3–hBNIP3 )163 plasmid encoding the first 163 amino acids of BNIP3, or the empty pcDNA3 plasmid. The transfection efficiency was about 2–8%, on the basis of immunohistochemistry with an anti- body against T7 that recognizes the T7 epitope tag. Transient transfection with pcDNA3–hBNIP3 but not with pcDNA3–hBNIP3 )163 (truncated BNIP3) resulted in DNA condensation and neuronal cell death (Fig. 3). The truncated BNIP3 was diffusely distributed in the cytoplasm, owing to the lack of its transmembrane G 40 H A B 500 μm 500 μm C D 0 4 6 8 10 12 E F 62 kDa BNIP3 BNIP3 antibody BNIP3 antibody +BNIP3–GST 62 kDa BNIP3 BNIP3 expression (fold) ** ** KA CL Ctrl KA CL Ctrl KA CL Ctrl β-actin M 40 M 200 μm 200 μm Fig. 1. BNIP3 expression inbrain increased with excitotoxicity and correlated with mea- sures of ‘apoptotic’ cell death. (A) BNIP3- immunopositive neurons were present adjacent to sites of KA injection. The arrow points to the site of injection. (B) DNA frag- mentation was observed in BNIP3-immuno- positive neurons (arrows). BNIP3- immunonegative neurons showed normal nuclear morphology (arrowheads). (C) ‘Apop- totic’ nuclei, as detected by TUNEL labeling, surrounded sites of KA injection. (D) TUNEL- positive neurons were not detected in normal brain. (E) Immunoblot for BNIP3 demon- strated increased levels of BNIP3 in KA-injected striatum as compared with uninjected CL striatum and normal control rats (Ctrl). Immunopositive blotting for BNIP3 was completely absent when anti- body against BNIP3 was first incubated with a BNIP3–GST protein. (F) Quantification of the western blot bands revealed a nine-fold increase of BNIP3 in KA-injected striatum. There was a 3.5-fold increase in CL striatum of the injected animal as compared with striatum of uninjected animals. (G) Levels of BNIP3 mRNA as demonstrated by in situ hybridization were very low in uninjected CL rat striatum. (H) Levels of BNIP3 mRNA were increased dramatically following KA injections. Scale bars: (A) 500 lm; (B) 50 lm; (C, D) 200 lm; (G, H) 40 lm. **P < 0.01. BNIP3 in excitotoxicity Z. Zhang et al. 136 FEBS Journal 278 (2011) 134–142 ª 2010 The Authors Journal compilation ª 2010 FEBS domain (Fig. 3B), whereas the full-length BNIP3 showed a pattern of punctate localization (Fig. 3A). Neuronal survival rates after 5 days of transfection with BNIP3 plasmid were decreased (P = 0.0165, n =3)as compared with cells transfected with pcDNA3– hBNIP3 )163 or the pcDNA3 plasmids (Fig. 3C). Among neurons expressing full-length BNIP3, 62% showed DNA condensation. In contrast, DNA damage was observed in only 27% of BNIP3-positive neurons trans- fected with pcDNA3–hBNIP3 )163 . To demonstrate the role of BNIP3 in glutamate neu- rotoxicity, we tested the effects of inhibiting BNIP3 expression by RNA interference. Hippocampal neu- rons were infected on day 1 in vitro with the viral vec- tor pLenti–BNIP3shRNA N167 , designed to express a short hairpin sequence that would target nucleo- tides 167–188 in the BNIP3 mRNA. The vector has been described elsewhere [18], with an inhibitory effi- ciency of at least 98% for BNIP3. On day 8 in vitro , the neurons were exposed to 100 lm glutamate for 48 h, and cell survival rates were measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro- mide assay. As shown in Fig. 3D, inhibition of BNIP3 expression increased neuronal survival by 40% (P = 0.0038, n = 4). To determine whether the BNIP3-mediated cell death pathway in excitotoxicity involved protein syn- thesis, we evaluated the effectiveness of the RNA synthesis inhibitor actinomycin D (1 lgÆmL )1 ) in pre- venting excitatory neuronal cell death. As shown in Fig. 4A, addition of actinomycin D decreased cell death caused by glutamate toxicity by 42% (P < 0.01), whereas actinomycin D alone did not affect cell death rates. F C 0 20 Cell death (%) 40 60 80 612243648 Hours Glu100 μM Glu10 μM D B A Glu 0 h6 h12 h24 h36 h48 h60 h72 hCtrl BNIP3 β-actin E Glu (100 μM) 0 2 4 6 8 10 ** ** ** * BNIP3 expression (fold) 24 h12 h6 h0 h36 h48 h60 h72 h Fig. 2. Glutamate increased BNIP3 expression. (A) Glutamate increased neuro- nal cell death in a dose-dependent manner. (B) Expression of BNIP3 was not detectable immunohistochemically in the majority of untreated neurons; less than 15% of untreated neurons expressed low levels of BNIP3. (C) More than 50% of cells treated with 100 l M glutamate for 6 h stained posi- tively for BNIP3. (D) Nuclei in BNIP3-positive neurons showed a dysmorphic appearance atypical of apoptosis. (E) Time course of BNIP3 expression in neurons exposed to 100 l M glutamate.**P < 0.01; *P < 0.05; n =4. Z. Zhang et al. BNIP3 in excitotoxicity FEBS Journal 278 (2011) 134–142 ª 2010 The Authors Journal compilation ª 2010 FEBS 137 We next examined caspase involvement in BNIP3- mediated neuronal cell death. Primary hippocampal neurons were preincubated with z-VAD-FMK (50 lm) alone or in combination with BOC-D-FMK (50 lm); both of these are potent cell-permeable caspase inhi- bitors. Cell viability was determined by trypan blue exclusion 6 h after application of glutamate or N-methyl-d-aspartate (NMDA). NMDA and glutamate significantly increased neuronal cell death (P < 0.01). z-VAD-FMK alone did not prevent cell death caused by glutamate or NMDA. Coapplication of z-VAD- FMK and BOC-D-FMK resulted in a small (17%) but statistically significant decrease in glutamate-induced cell death (P = 0.045, n = 5; Fig. 4B). Discussion Previously, it was shown for non-neuronal cells that BNIP3 induced cell death distinct from necrosis and apoptosis as defined by classical morphological and molecular criteria [17]. It was also shown that excito- toxicity activates cell death programs that result in atypical neuronal cell death [1,5,19]. However, at present, it is not completely clear whether and which molecular regulators might control such atypical neu- ronal cell death. Accordingly, we tested hypotheses that BNIP3 was an important regulator of neuronal cell death induced by excitotoxic stimuli, that this form of programmed cell death occurred indepen- dently of caspase activation, and that excitotoxic cell death could be prevented if the actions of BNIP3 were blocked. Here, we showed that BNIP3 levels increased dramatically in in vivo and in vitro models of excitotoxicity, that overexpression of full-length BNIP3 decreased the viability of hippocampal neu- rons grown in culture and significantly increased the susceptibility of these neurons to glutamate-induced cell death, that BNIP3-mediated cell death occurred D pcDNA3–hBNIP3 pcDNA3–hBNIP3 –163 AB BNIP3-positive cells with DNA damage C 0 20 40 60 80 100 ** 0 20 40 60 80 100 Glutamate + – + + pLV-N167 – + + – pLV-LacZ – – – + ** Neuronal death (%) Dysmorphic nuclei (%) 20 μ m 20 μ m Fig. 3. BNIP3 expression caused neuronal cell death. (A) Transient transfection of rat hippocampal neurons resulted in DNA condensa- tion and neuronal cell death. (B) Transient transfection of rat hippo- campal neurons with a dominant-negative form of BNIP3 (BNIP3 )163 ) did not cause DNA condensation or localization of BNIP3 to mitochondria; BNIP3 was diffusely distributed in the cyto- plasm. (C) Neuronal survival rates after 5 days of transfection with BNIP3 (n = 6). About 84% of BNIP3-positive neurons in BNIP3- transfected cells showed DNA condensation, as compared with 27% in BNIP3 )163 -transfected cells. (D) Glutamate significantly decreased neuronal survival. Knockdown of BNIP3 by the lentiviral vector pLV-N167 significantly protected neurons from glutamate- induced cell death. **P < 0.01, n = 3). 0 20 40 60 80 Media Lockes Actinomycin D Glumate Glu + actinomycin D ** P = 0.045 A B Cell death (%) 0 20 40 60 80 Cell death (%) Media Lockes Glutamate G l u + Boc-FMK NMDA Boc-D-FMK Glu + z-FMK G l u + Boc-FMK + z-FMK z-VAD-FMK Fig. 4. BNIP3-induced neuronal cell death in excitotoxicity required protein synthesis but was largely independent of caspase activity. (A) Actinomycin D significantly decreased the number of trypan blue-positive cells (P < 0.01) caused by glutamate toxicity to untreated control levels. (B) Inhibition of caspase activity did not prevent cell death caused by glutamate or NMDA. z-VAD-FMK alone did not prevent cell death caused by the excitotoxic toxins. Coapplication of z-VAD-FMK and BOC-D-FMK (FMK) resulted in a small but statistically significant decrease in glutamate-induced cell death (P = 0.045). BNIP3 in excitotoxicity Z. Zhang et al. 138 FEBS Journal 278 (2011) 134–142 ª 2010 The Authors Journal compilation ª 2010 FEBS independently of caspase activation, and that inhibi- tion of BNIP3 by RNAi increased neuronal viability and protected neurons against glutamate-induced excitotoxicity. BNIP3 is a BH3-only proapoptotic member of the Bcl-2 family. However, unlike in other members of the Bcl-2 family, the BH3 domain of BNIP3 is not required for its death-inducing activity. Our results showing that full-length, but not truncated, BNIP3 can result in neuronal cell death are in agreement with pre- vious results, obtained with non-neural cells, that the transmembrane domain of BNIP3 is indispensable for it to cause membrane damage, mitochondrial perme- ability, and DNA fragmentation [17]. These features of BNIP3-induced neuronal cell death are indistinguish- able from those of BNIP3-induced non-neural cell death [17]. BNIP3-regulated cell death appears to be atypical of necrosis, because it is genetically pro- grammed (Fig. 4) and involves mitochondrial perme- ability transition pore opening [17]. Even though BNIP3-induced cell death is genetically programmed, it is atypical of apoptosis because cell death has been shown to occur independently of caspase activation and cytochrome c release [17]. BNIP3 expression has been shown to be induced under conditions of oxidative stress [18] and hypoxia [20,21]. The promoter of the BNIP3 gene contains a functional hypoxia response element [20] that could be a direct target of hypoxia-inducible factors. Excitotox- icity involves Ca 2+ overloading and concomitant gen- eration of reactive oxygen species, which has been shown to trigger hypoxia-induced transcription [22]. Therefore, our studies showing that BNIP3 is both necessary and sufficient for neuronal death in excito- toxicity has wide-ranging implications for the under- standing of mechanisms underlying acute and chronic neurodegenerative disorders and the possible identifica- tion of novel therapeutic interventions. Experimental procedures Animal model Male Sprague–Dawley rats, with body weights ranging between 200 and 250 g, were obtained from the University of Manitoba Central Animal Care breeding facility. All procedures followed Canadian Council on Animal Care guidelines and were approved by the Animal Care Commit- tee at the University of Manitoba. Animals were anesthe- tized with intraperitoneal 74 mgÆkg )1 sodium pentobarbital and placed in a stereotaxic surgery frame. Unilateral intra- striatal injections were performed using the following coor- dinates (in mm); anteroposterior, 9.0; mediolateral, 3.0; and dorsoventral, 4.5 [23]. Drugs were administered over a 5 min period in a volume of 1 lL, using a 10 lL syringe fit- ted with a 30-gauge needle. Following injection, the needle was left in place for 5 min before being slowly withdrawn to allow diffusion of the drug away from the injection site. KA, dissolved in 50 mm Tris ⁄ HCl with the pH adjusted to 7.4 with NaOH, was administered at a dose of 2.5 nmol. Control rats received unilateral injections of 1 lLof50mm Tris ⁄ HCl (pH 7.4). To confirm the role of kainate recep- tors, the receptor antagonist CNQX (dissolved in 0.1 m NaOH with volumes adjusted with 50 mm Tris ⁄ HCl, pH 7.4) was administered at a dose of 5 nmol in a volume of 1 lL, by itself or in combination with 2.5 nmol of KA. Following injection, wounds were sutured, and animals were allowed to recover for periods up to 5 days. From pilot studies, we found BNIP3 expression to be increased from 24 h to 5 days after KA injection (data not included). In the present study, all animals were killed 48 h after intrastriatal injections. Cell culture Primary hippocampal neurons were prepared from 18-day- old embryonic Sprague–Dawley rats as described previously [24]. Briefly, hippocampal tissue was dissociated by gentle tituration in calcium-free Hank’s balanced salt solution, and centrifuged at 1000 g. Cells were resuspended in DMEM ⁄ F12 nutrient mixture containing 10% heat-inacti- vated fetal bovine serum and 1% antibiotic solution (peni- cillin G 104 IUÆmL )1 , streptomycin 10 mgÆmL )1 and amphotericin B 25 lgÆmL )1 ) in 0.9% NaCl (Sigma, St Louis, MO, USA). Hippocampal neurons were plated at a density of 2 · 10 5 cellsÆmL )1 on 12-mm-diameter poly (d-lysine)-coated glass coverslips. Three hours after plating, the medium was replaced with serum-free Neurobasal med- ium containing 1% B-27 supplement (Gibco, Rockville, MD, USA). Immunofluorescent staining for microtubule-associ- ated protein-2 on neurons and glial fibrillary acidic protein in astrocytes showed that cultures were > 98% neurons; the remainder of the cells were predominantly astrocytes. Pharmacological studies To determine the extent to which NMDA-type glutamate receptors are involved in BNIP3 expression and excitotoxic cell death, we used the agonist NMDA (100 lm) in the absence or presence of the NMDA receptor antagonist MK-801 (10 lm). To determine the extent to which caspase activation contributes to BNIP3-mediated cell death, hippo- campal cells were incubated in the absence or presence of the broad-spectrum cell-permeable caspase inhibitors z-VAD-FMK (50 lm) and BOC-D-FMK (50 lm); these inhibitors were applied 30 min prior to application of glutamate or NMDA. To determine the role of protein Z. Zhang et al. BNIP3 in excitotoxicity FEBS Journal 278 (2011) 134–142 ª 2010 The Authors Journal compilation ª 2010 FEBS 139 translation in glutamate-mediated toxicity, we used actino- mycin D (1.0 lgÆmL )1 ). Plasmids and cell transfection Rat BNIP3 (rBNIP3) cDNA was prepared by RT-PCR from primary neuronal cultures exposed to hypoxia for 36 h, with sense primer 5¢-GA GAATTC TCG CAG AGC GGG GAG GAG AAC-3¢ and antisense primer 5 ¢-AT GGATCC TCA AAA GGT ACT AGT GGA AGT TG-3¢. The PCR product was ligated to pGEM-T (Promega) by T-A cloning. After the resulting construct had been verified by sequencing, the rBNIP3 fragment was subcloned to pEGFP-C2 (Clontech, USA) to yield green fluorescent pro- tein–rBNIP3. T7-tagged pcDNA3–hBNIP3 and T7-tagged pcDNA3–hBNIP3 )163 plasmids were gifts from the late A. H. Greenberg (University of Manitoba) [14]. Transfection of cells was performed on day 4 in culture with LipofectA- MINE 2000 (Invitrogen, Burlington, Ontario, Canada), according to the manufacturer’s protocol. The transfection efficiency was 2–8% as estimated by enhanced green fluo- rescent protein (EGFP) expression from transfection of pEGFP-C2–rBNIP3 or by immunohistochemistry with a monoclonal antibody against T7 (1 : 200; Novagen, Madi- son, WI, USA) when T7-tagged pcDNA3 plasmids were used. Cells were exposed to glutamate after 9 days in culture for excitotoxicity experiments. Lentiviral vectors expressing short hairpin RNA sequences targeting BNIP3 and LacZ have been described elsewhere [18]. Briefly, complementary DNA oligonucleo- tides targeting rat BNIP3 and LacZ were annealed and ligated into a pENTR ⁄ U6 vector (Invitrogen, San Diego, CA, USA). The U6 RNAi cassette (U6 promoter + dou- ble-stranded oligonucleotides + Pol III terminator) was then transferred to the pLenti6 ⁄ BLOCK-iT-DEST vector (Invitrogen) by an LR recombination reaction. Lentiviral stock was produced by transfecting this plasmid into the 293FT Cell Line, using ViraPower Packaging Mix in DMEM containing 10% fetal bovine serum. The lentiviral stock was titered by counting crystal violet-stained blue col- onies of 293FT cells after incubation for 3 days with selec- tive medium containing different concentrations of blasticidin. For transduction, the vectors (multiplicity of infection = 5) were placed with the neuron in fresh med- ium 1 day before the neurons were exposed to glutamate. Immunohistochemistry and in situ hybridization For immunohistochemistry and in situ hybridization, rats were perfused transcardially with 0.9% saline and then 4% paraformaldehyde. Brains were carefully removed and post- fixed overnight in NaCl ⁄ P i containing 4% paraformalde- hyde. After being rinsed in NaCl ⁄ P i , the brains were placed in NaCl ⁄ P i containing 0.5 m sucrose (pH 7.3) at 4 °C until buoyancy was lost. Eight-micrometer sections were cut on a cryostat (Shandon) and mounted on silane-treated slides. Frozen brain sections cut from KA-injected and control rats were blocked and permeabilized with NaCl ⁄ P i contain- ing 2% BSA, 5% normal goat serum and 0.3% Triton X-100 for 30 min at room temperature. The sections were then incubated overnight at 4 °C with a polyclonal anti- body against BNIP3 (1 : 200), followed by rhodamine- conjugated goat anti-(rabbit IgG) (1 : 200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 2 h at room temperature. The polyclonal antibody against BNIP3 recognizes both human and rat BNIP3, and was also used to detect BNIP3 expression in primary rat hippo- campal neurons after exposure to glutamate and NMDA. For detection of BNIP3 expression in primary hippocampal neurons after plasmid transfection, a monoclonal antibody against BNIP3 that is specific for human BNIP3 was used at a dilution of 1 : 200. Fluorescent pictures were taken with a Zeiss (Thornwood, NY, USA) microscope equipped with an AxioCamdigital camera (Carl Zeiss, Jena, Ger- many). For in situ hybridization, an RNA probe (specific for BNIP3) was synthesized with a digoxigenin RNA label- ing kit (Roche) according to the manufacturer’s protocol. Brain sections were hybridized with the probe and incu- bated with an alkaline phosphatase-conjugated antibody against digoxigenin, and labeled cells were detected with BCIP ⁄ Nitro Blue tetrazolium. Detection of cell death In vitro cell death was estimated by trypan blue exclusion. Cells were incubated in 0.4% trypan blue solution for 30 min, and then counted under a bright-field microscope. Nonviable cells were distinguished by their dark blue stain- ing. Neuronal viability was also estimated by 3-(4,5-dim- ethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay on a WallacVICTOR 3 1420 Multilabel microplate reader (Per- kin Elmer Life Sciences, Woodbridge, Ontario, Canada). For examination of nuclear morphology, nuclear DNA was stained with Hoescht 33342. DNA fragmentation was detected by TUNEL, using an in situ cell death detection kit with fluorescein (Intergen, Purchase, NY, USA), according to the manufacturer’s recommendations. Morphological characteristics were examined with a Nikon Eclipse TE200 microscope, and fluorescence was examined with a Zeiss Axi- oskop 2. Statistical analyses were perfofmed by ANOVA with Tukey’s post hoc test. Western blots Rats were killed by decapitation, brains were rapidly removed, and striata were dissected out, frozen rapidly on dry ice, and stored at )80 °C. For preparation of protein samples, striata were homogenized in 25 mm phosphate buffer (pH 7.4) containing 1% Triton X-100, 0.1 mm EGTA, 1 mm phenylmethanesulfonyl fluoride, and 5 mm BNIP3 in excitotoxicity Z. Zhang et al. 140 FEBS Journal 278 (2011) 134–142 ª 2010 The Authors Journal compilation ª 2010 FEBS dithiothreitol. After brief centrifugation (1000 g for 10 minutes at 4°C), supernatants were collected. For cultured neurons, cell pellets were resuspended in RIPA lysis buffer (0.01 m Tris ⁄ HCl, 0.15 m NaCl, 1% Triton-X 100, 1% deoxycholic acid, 0.1% SDS, pH 7.4), the lysates were centrifuged at 1000 g in a microcentrifuge for 10 min at 4°C, and supernatants were collected. The protein con- centration was determined by the Bradford method, with BSA as standard. Protein samples were separated by SDS ⁄ PAGE on a 15% polyacrylamide gel, and transferred to poly(vinylidene difluoride) membranes suitable for small molecular mass peptides. Proteins were probed with anti- body against BNIP3 at a dilution of 1 : 500, and immuno- blotting was detected by electrochemiluminescence (Amersham, Piscataway, NJ, USA). Controls were run in the presence of a plasmid-expressed BNIP3 protein. Acknowledgements This work was supported by the Canadian Institutes of Health Research, Canadian Stroke Network and the National Natural Science Foundation of China (Grant numbers: 81070980 ⁄ H0910 to ZZ, 30700245 to XX and U0632007 to TG and JK). J. Kong received a New Investigator award from the Heart and Stroke Founda- tion of Canada. J. Weng received a studentship from the Manitoba Institute of Child Health. References 1 Martin LJ, Al-Abdulla NA, Brambrink AM, Kirsch JR, Sieber FE & Portera-Cailliau C (1998) Neurodegen- eration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis. Brain Res Bull 46, 281–309. 2 Abramov AY & Duchen MR (2008) Mechanisms underlying the loss of mitochondrial membrane poten- tial in glutamate excitotoxicity. 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