These authors contributed equally to the work.
BNIP3 mediates pre-myelinating oligodendrocyte cell death in hypoxia and ischemia
Version of Record online: 5 JUN 2013
© 2013 International Society for Neurochemistry
Journal of Neurochemistry
Volume 127, Issue 3, pages 426–433, November 2013
How to Cite
J. Neurochem.(2013) 127, 426–433
- Issue online: 20 OCT 2013
- Version of Record online: 5 JUN 2013
- Accepted manuscript online: 20 MAY 2013 11:37AM EST
- Manuscript Accepted: 15 MAY 2013
- Manuscript Revised: 7 MAY 2013
- Manuscript Received: 9 APR 2013
- Canadian Stroke Network
- Natural Science Foundation Project of ChongQing CSTC of China. Grant Number: 2009BB5320
- International Science & Technology Cooperation Program of China. Grant Number: 2010DFB30820
- oligodendrocyte progenitor cell (OPC)
Developing oligodendrocytes, collectively termed ‘pre-myelinating oligodendrocytes’ (preOLs), are vulnerable to hypoxic or ischemic insults. The underlying mechanism of this vulnerability remains unclear. Previously, we showed that Bcl-2⁄E1B-19K-interacting protein 3 (BNIP3), a proapoptotic member of the Bcl-2 family proteins, induced neuronal death in a caspase-independent manner in stroke. In this study, we investigated the role of BNIP3 in preOL cell death induced by hypoxia or ischemia. In primary oligodendrocyte progenitor cell (OPC) cultures exposed to oxygen–glucose deprivation, we found that BNIP3 was upregulated and levels of BNIP3 expression correlated with the death of OPCs. Up-regulation of BNIP3 was observed in preOLs in the white matter in a neonatal rat model of stroke. Knockout of BNIP3 significantly reduced death of preOLs in the middle cerebral artery occlusion model in mice. Our results demonstrate a role of BNIP3 in mediating preOLs cell death induced by hypoxia or ischemia, and suggest that BNIP3 may be a new target for protecting oligodendrocytes from death after stroke.
Pre-myelinating oligodendrocytes (preOLs) are known to be highly vulnerable to ischemic insults. It remains unclear, however, how preOLs die. This study shows that BNIP3, a proapoptotic member of the Bcl-2 family proteins, is a mediator of hypoxia/ischemia-induced preOLs death. The BNIP3 cell death pathway may therefore be a new target for protecting oligodendrocytes from death after stroke.
Bcl-2⁄E1B-19K-interacting protein 3
chemically defined medium
myelin basic protein
middle cerebral artery occlusion
oligodendrocyte progenitor cell
platelet-derived growth factor receptor-α
sodium dodecyl sulfate polyacrylamide gel electrophoresis
terminal-deoxynucleoitidyl transferase-mediated nick end labeling
Ischemic stroke causes cerebral ischemic damage by means of inflammation, excitotoxicity, mitochondrial dysfunction, and oxidative stress (Martin et al. 1998; Lakhan et al. 2009; Pandya et al. 2011). Oligodendrocytes (OLs), the myelin-forming cells of the central nervous system, are vulnerable to ischemic damage (Deng et al. 2004). The loss of myelin adversely influences the connectivity and function of neurons. Therefore, protection against ischemic damage of OLs is important to attenuate loss of neurological functions after a stroke (Arai and Lo 2009).
Oligodendrocytes arise from oligodendrocyte progenitor cells (OPCs) through several developmental stages, namely, the pro-oligodendrocyte, the immature oligodendrocyte. Collectively, developing oligodendrocytes are referred to as pre-myelinating oligodendrocytes (preOLs) (Volpe et al. 2011). Immature white matter predominantly consists of preOLs. The adult brain contains a reservoir of OPCs to replace damaged oligodendrocytes (Horner et al. 2000; Dawson et al. 2003). Increased evidence shows that the vulnerability of OL lineage cells to ischemic injury is maturation-dependent and that preOLs are highly vulnerable to noxious stimuli like oxidative stress and/or glutamate excitotoxicity (Deng et al. 2003). However, the molecular pathway involved in this process is still unknown.
Bcl-2⁄E1B-19K-interacting protein 3 (BNIP3) is a member of a unique subfamily of death-inducing mitochondrial proteins that induce cell death in a caspase-independent apoptosis (Chen et al. 1997; Vande Velde et al. 2000). Our previous studies found that BNIP3 and the mitochondrial protein endonuclease G (EndoG) defined a new pathway for neuronal death after stroke and in cultured neurons exposed to hypoxia (Zhang et al. 2007). Here, we show that the BNIP3 is involved in death of oligodendrocyte lineage cells. In primary OPC cultures exposed to oxygen–glucose deprivation (OGD), BNIP3 was up-regulated and levels of BNIP3 expression correlated with death of OPCs. Up-regulation of BNIP3 was observed in preOLs in the white matter in a neonatal rat model of stroke. Knockout of BNIP3 significantly reduced death of preOLs in the middle cerebral artery occlusion (MCAO) model in mice. Our results suggest that BNIP3 may be a new target for protecting oligodendrocytes from death after stroke.
Materials and methods
All procedures performed were approved by the Animal Care Committee at the University of Manitoba, whose standards meet the Animal Care guidelines of the Canadian Council. BNIP3 knockout mice were obtained from Dr. Spencer Gibson's lab in University of Manitoba. Animals were obtained from Animal Care Center of University of Manitoba, housed in a temperature-controlled environment (24 ± 2°C) with a 12-h-light-dark cycle, and allowed free access to food and water. All efforts were made to minimize animal suffering and reduce the number of animals used.
Models and treatments
OPCs were purified and cultured as previously described (Niu et al. 2010). The purified OPCs were plated into a poly-d-lysine (PDL)-coated flask at a density of 2 × 104 cells/mL and cultured in proliferation medium. For OGD, the cells were incubated with glucose-free Earle's balanced salt solution, placed in a modular incubator chamber, and infused with mixed gas (95% N2 and 5% CO2) at 37°C for 6 h. The culture medium was then switched to chemically defined medium (CDM) and returned to a normoxic incubator for reperfusion.
Neonatal hypoxic–ischemic (HI) rat model was made as described previously (Zaidi et al. 2004). Briefly, seven-day-old (P7) SD rat pups of both sexes (n = 11) were anesthetized with 2.5% halothane. The left common carotid artery was isolated and permanently ligated. The pups were returned to their dam for a 2 h recovery. Then pups were exposed to 2 h of hypoxia (6% O2/92% N2). Thereafter, the pups were returned to their home cage with their dam until they were killed. The sham-operated controls (n = 10) received the same surgical procedures except for the artery ligation. The animals were killed at 48 h, 72 h or 5 days after HI, respectively.
Focal cerebral ischemia was induced by intraluminal MCAO, as described previously (Khayyam et al. 1999). Male wild-type (n = 4) and BNIP3 knockout mice (n = 5) were anesthetized with isoflurane. Body temperature was kept at 37°C using a thermostatically controlled heating blanket until the animals recovered from surgery. The blood gas conditions were kept at constant levels throughout the surgery (PO2, 120 ± 10 mm Hg; PCO2, 35 ± 3 mm Hg). Ischemia was induced by introducing a 6-0 Doccol (Doccol Co., Sharon, MA, USA) monofilament via the external carotid artery into the internal carotid artery to block the origin of the MCA. The filament was left in place for 60 min and then withdrawn. The sham-operated animals received the same operation without middle cerebral artery ligation.
Cell viability assay
Viable cell numbers of OPCs at 0, 24, 48, and 72 h after OGD were measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay as previously described (Niu et al. 2012). Ten microlitre of MTT (5 mg/mL) was added to each well of OPCs, and the cells were incubated at 37°C for 4 h. After removal of the medium, 150 μL of dimethylsulfoxide was added to each well to dissolve the MTT formazan crystals. OD value was measured with a microplate reader (Bio-Rad, Munich, Germany) with 490 nm and a reference wavelength of 650 nm.
Cell death assay
Cell death or plasma membrane damage was quantitatively assessed by measuring the extent of lactate dehydrogenase (LDH) release using an LDH cytotoxicity detection kit according to the manufacturer's instructions. At indicated time points, the medium was collected and cells were solubilized in a solution of 1% Triton X-100 for 1 h to lyse the cells. The percentage of LDH release was calculated as [(LDH released into media)/(LDH released into media + LDH released from lysed cells)] × 100.
Western blot assessment
Protein lysates were prepared and western blotting was performed using protocols previously described (Zhang et al. 2007). Extracted proteins were separated by 12% SDS-PAGE, transferred, blocked, and incubated with primary antibodies at 4°C overnight. The primary antibodies included anti-myelin basic protein (MBP) (Santa Cruz, Dallas, TX, USA, 1 : 1000), anti-BNIP3 (Abcam, Cambridge, MA, USA, 1 : 2000), antiplatelet-derived growth factor receptor-α (PDGFR-α) (Invitrogen, Grand Island, NY, USA, 1 : 1000). Immunoreactive bands were detected using an ECL plus detection kit (Amersham Biosciences, Buckinghamshire, UK). The protein loading control was performed through a 2-h incubation with corresponding peroxidase-linked secondary antibodies (Dako, Burlington, ON, Canada, 1 : 2000) at 22°C. To normalize immunoreactivity with protein loading, β-actin was used as an internal control, and the optical density ratio of primary antibodies to β-actin was calculated.
For immunofluorescence staining, serial frozen coronal sections (20 μm) of the brains were sliced after perfusion and post-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C overnight. Serial frozen coronal sections of the brain were then kept in 3% sucrose in PBS. The OPCs cultured on glass coverslips were fixed for 30 min in 4% paraformaldehyde. After blocking in solution composed of 0.3% Triton X-100 and 10% normal goat serum for 30 min at 22°C, the sections and cells were incubated with the following primary antibodies: rabbit anti-BNIP3 (Chemicon, Temecula, CA, USA, 1 : 1000), mouse anti-NG2 (Chemicon, 1 : 400), PDGF, and GST, separately, overnight at 4°C. This was followed by a 2-h incubation at 22°C with secondary antibodies conjugated with either Alexa Fluor® 594 or Alexa Fluor® 488 (Invitrogen 1 : 2000) at 22°C. The sections were then rinsed three times with PBS, mounted on gelatin-coated slides, and coverslipped with Dako fluorescent mounting medium (Carpentaria, CA, USA). Hoechst 33342 (Vector labs, Burlingame, CA, USA, 1 : 1000) was used to identify the nuclei. The control specimens underwent the same procedures, but primary antibodies were excluded from these procedures. The immunoreactivity was determined using a 20× objective lens on a fluorescence microscope (Olympus BX-60, Richmond Hill, ON, Canada). Cell counting was conducted on nine randomly chosen fields for each sample. The optical density (OD) of MBP and BNIP3 positive staining were measured using Image-Pro Plus software (version 5.1; Media Cybernetics, Inc., Silver Spring, MD, USA).
Terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) staining
Cell apoptosis was assessed by TUNEL staining using a cell death detection kit (Roche, Indianapolis, IN, USA). The brain sections or OPCs on coverslips were fixed for 30 min in 4% paraformaldehyde at 22°C and then processed for TUNEL staining according to the manufacturer's instructions. The positive signal (green fluorescence) was observed with a fluorescence microscope (Olympus).
All dependent variables were subjected to one-way anovas with follow-up Tukey post hoc tests. Comparisons between the two experimental groups were made using Student's t-test. A probability of p < 0.05 was considered statistically significant.
OGD induces cell death and up-regulation of BNIP3 in OPC cultures
Oligodendrocytes are sensitive to hypoxia and hypoglycemia. Double-labeling showed that the number of TUNEL- and NG2-positive apoptotic OPCs significantly increased after 6 h of OGD treatment (Fig. 1a). To estimate the correlation between OPC injury and BNIP3 expression, we used NG2 and BNIP3 double-labeling. We found that the numbers of BNIP3 and NG2 double-positive cells were significantly increased after OGD, whereas BNIP3 and NG2 double-labeling cells were not detectable in the control (Fig. 1b). Cellular damage was also assayed by quantifying plasma membrane damage or rupture, based on the release of LDH. A dramatic increase in LDH release and decrease in cell viability (about 45%) as shown by MTT assay were found in OPCs after OGD treatment (Fig. 1c–d).
To estimate the correlation of OPCs injury and BNIP3 expression, OPCs were subjected to OGD for 6 h and reperfusion for 24, 48, or 72 h. We found that there is a time-dependent increase in the percentage of OPCs undergoing apoptosis, as shown by TUNEL staining (Fig. 2a and c). BNIP3 expression increased following the reperfusion as determined by immunofluorescence staining (Fig. 2b). Western blot analysis also showed that the levels of BNIP3 were 6-fold, 7.5-fold, 10-fold, and 14-fold increased, respectively, as compared with normal control cells (Fig. 2d and e).
BNIP3 expression is up-regulated in white matter of neonatal hypoxic–ischemic rat model and adult MCAO mice model
Pre-myelinating OLs undergo rapid maturation after birth and are vulnerable to oxidative stress, which may disrupt myelin synthesis and axonal function. Using a neonatal hypoxic–ischemic rat model, of which a pathologic hallmark is selective damage of the white matter with prominent OL loss, we found that BNIP3 expression was significantly increased after 48 and 72 h of HI as determined by western blot analysis (Fig. 3a and b). Double-labeling assay revealed that BNIP3 expression was detected in most of the NG2 positive preOLs and more apoptotic preOLs(NG2+ and TUNEL+) were found in corpus callosum after 48 h of HI (Fig. 3c and d). As a consequence, MBP expression was significantly decreased in the CC of rats at P12, 5 days after HI (Fig. 3e).
Deficiency of BNIP3 protected PreOLs from death induced by ischemia
To determine the role of BNIP3 in ischemic white matter damage, we used BNIP3 knockout (KO) mice in the study. In comparison with wild-type mice, BNIP3-null mice showed significantly less neuronal loss after 3 days of reperfusion following MCAO (data not shown), that was consistent with our previous study. To test the response of OLs to ischemic injury, external capsule regions were analyzed. We found that there were significantly less TUNEL-positive cells in BNIP3-null mice as compared with wide-type mice (Fig. 4a and b) 72 h following MCAO. We also observed more PDGFR-α-positive OPCs and GST-π-positive developing OLs in penumbra of ischemic EC in BNIP3-null mice than that in the wild-type mice (Fig. 4c–e).
To further reveal the role of BNIP3 in preOL damage induced by hypoxia, we investigated the BNIP3 expression in the white matter (external capsule) of BNIP3 KO mice after MCAO. As shown in Fig. 5a, most of PDGFR-α-positive OPCs in ischemic external capsule of wild-type mice were colabeled with BNIP3 (Fig. 5a). More PDGFR-α positive and GST-π-positive preOLs surrounding the core infarct areas were found in BNIP3-null mice (Fig. 4c–e). The western blot analysis demonstrated that the BNIP3-null mice had significantly higher levels of PDGFR-α expression than the control mice. There were no changes of MBP expression in BNIP3-null mice as compared to wild-type mice (Fig. 5b and c). These data suggest that BNIP3 is crucial for induction of preOL cell death in ischemic stroke.
As the most vulnerable oligodendroglial lineage cells, preOLs injury is a hallmark feature of white matter damage induced by hypoxia–ischemia (McTigue et al. 2006; Yu et al. 2009; Li et al. 2010). Consistently, we demonstrated in this study that exposure of OPCs to OGD resulted in cell death as evidenced by increased membrane leakage of LDH, and neonatal pups exposed to hypoxia/ischemia resulted more preOLs damage and decreased in myelin production. Interestingly, we found that expression of the death-inducing gene BNIP3 was up-regulated and the levels of BNIP3 up-regulation correlated with death of preOLs after stroke and in cultured OPCs exposed to hypoxia. More importantly, deficiency in BNIP3 protected preOLs from death after stroke. Our results thus, for the first time, demonstrate that the BNIP3 cell death pathway plays a role in mediating death of oligodendroglial lineage cells.
BNIP3 is a unique death-inducing member of the Bcl-2 proteins. Normally, BNIP3 is undetectable. However, gene expression of BNIP3 can be initiated by a classical HIF-1 element and consensus sequences for the transcription factor nuclear factor-κB (NF-κB) and/or E2F-1 (Yurkova et al. 2008). It was found that BNIP3 was up-regulated in neurons 24 h after OGD (Walls et al. 2009), and BNIP3 expression in vivo shows a delayed pattern (Zhang et al. 2007). In our study, although BNIP3 expression in vivo was not increased until 48 h after hypoxia/ischemia in the neonatal rat model of stroke, BNIP3 started to increase in OPCs immediately after OGD, suggesting that preOLs may be more vulnerable than neurons to hypoxic insults, at least in tissue culture conditions. It is not clear if mature OLs exert the same BNIP3 inducibility to oxidative stress as preOLs do. Further study to explore the expression pattern of BNIP3 induced by oxidative stress in different stages of OLs may help to elucidate the underlying mechanism of maturation-dependent vulnerability of oligodendroglia lineage cells.
It has been shown that BNIP3 induces cell death mostly via mitochondrial dysfunction, in that homodimeric BNIP3 inserts into mitochondrial outer membrane (MOM) to increase MOM permeability leading to release of a series of cytotoxic factors, including apoptosis inducing factor (AIF), EndoG, and others (Nakamura et al. 2012). Nuclear localization of BNIP3 sequesters it away from mitochondria in tumor cells and thus blocks cell death or even promotes cell survival (Giatromanolaki et al. 2004; Ishida et al. 2007). In our study, through western blot, we observed that BNIP3 expression was primarily localized to cytoplasm of OPCs or preOLs with molecular weight of 60 KD (Figs 1-3). The data suggest that BNIP3 induces cell death through mitochondrial dysfunction in preOLs. Interesting, BNIP3 has been suggested to activate autophagic cell death by binding to Rheb to activate the mammalian target of rapamycin (mTOR) pathway, which is known to be involved in OL cell death (Li et al. 2007; Tyler et al. 2009). Further investigation is needed to clarify the pattern of BNIP3-mediated cell death induced by hypoxia in OL lineage cells.
By using BNIP3 knockout mice in our study, we demonstrated the contribution of BNIP3 to preOL cell death and white matter damage induced by hypoxia/ischemia. More PDGFR-α-positive OPCs was found surrounding the core infarct areas in BNIP3-null mice after 72 h of reperfusion, this suggested that down-regulation of BNIP3 may provide an effective strategy for oligodendroglial protection.
In summary, our data show that BNIP3 plays a role in mediating preOLs cell death induced by hypoxia or ischemia. The BNIP3-mediated cell death pathway in preOLs helps elucidate the vulnerability of oligodendroglial lineage cells and may be a new target for protecting oligodendrocytes from death after stroke.
The project is supported by Canadian Stroke Network, MHRC and CIHR; and by Natural Science Foundation Project of ChongQing CSTC of China (2009BB5320), and the International Science & Technology Cooperation Program of China (2010DFB30820). The authors wish to thank Jacqueline Hogue for her assistance in preparing the manuscript.
We declare no conflicts of interest.
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