- Research article
- Open Access
Mitochondrial BNIP3 upregulation precedes endonuclease G translocation in hippocampal neuronal death following oxygen-glucose deprivation
- Shen-Ting Zhao†1,
- Ming Chen†1,
- Shu-Ji Li1,
- Ming-Hai Zhang1,
- Bo-Xing Li1,
- Manas Das1,
- Jonathan C Bean3,
- Ji-Ming Kong1, 2,
- Xin-Hong Zhu1Email author and
- Tian-Ming Gao1Email author
© Zhao et al; licensee BioMed Central Ltd. 2009
- Received: 30 April 2009
- Accepted: 8 September 2009
- Published: 8 September 2009
Caspase-independent apoptotic pathways are suggested as a mechanism for the delayed neuronal death following ischemic insult. However, the underlying signalling mechanisms are largely unknown. Recent studies imply the involvement of several mitochondrial proteins, including endonuclease G (EndoG) and Bcl-2/adenovirus E1B 19 kDa-interacting protein (BNIP3), in the pathway of non-neuronal cells.
In this report, using western blot analysis and immunocytochemistry, we found that EndoG upregulates and translocates from mitochondria to nucleus in a time-dependent manner in cultured hippocampal neurons following oxygen-glucose deprivation (OGD). Moreover, the translocation of EndoG occurs hours before the observable nuclear pyknosis. Importantly, the mitochondrial upregulation of BNIP3 precedes the translocation of EndoG. Forced expression of BNIP3 increases the nuclear translocation of EndoG and neuronal death while knockdown of BNIP3 decreases the OGD-induced nuclear translocation of EndoG and neuronal death.
These results suggest that BNIP3 and EndoG play important roles in hippocampal neuronal apoptosis following ischemia, and mitochondrial BNIP3 is a signal protein upstream of EndoG that can induce neuronal death.
- Hippocampal Neuron
- Neuronal Death
- Nuclear Fraction
- Nuclear Pyknosis
- Permanent Focal Cerebral Ischemia
The hippocampus, a very important structure for learning and memory, is among the most vulnerable brain regions after global cerebral ischemia. The rapid decrease of oxygen and glucose in the ischemic region can trigger delayed neuronal death , and reperfusion may further exacerbate the injury. The delayed cell death occurs primarily through an apoptotic mechanism . Evidence has accumulated that a large proportion of the delayed neuronal death is mediated by caspase-independent pathways [3–5]. However, the signalling mechanisms remain largely unclear. Recent studies have implicated mitochondrial proteins, such as endonuclease G (EndoG) and Bcl-2/adenovirus E1B 19 kDa-interacting protein (BNIP3), as players involved in this pathway in non-neuronal cells .
EndoG is an endonuclease normally localized in the mitochondrial intermembrane space, and translocates to the nucleus when cells are exposed to an apoptosis-inducing stimulus. After moving to the nucleus, EndoG cleaves chromatin DNA into nucleosomal fragments independent of caspases . The compartmentalization of mitochondria plays the major role in EndoG trafficking. The EndoG location site might indicate that this enzyme is not an instrument for immediate response to cell injury .
BNIP3 is a member of a unique family of death-inducing mitochondrial proteins . In neurons, the expression of BNIP3 is undetectable under normal conditions  but can be induced by hypoxia/ischemia and oxidative stress [11, 12]. The BNIP3-induced non-neuronal cell death is characterized by mitochondrial damage but is independent of caspase activation and cytochrome c release . It is presently unclear how BNIP3 initiates neuronal death.
In this study, we profiled BNIP3 and EndoG expression and translocation in cultured hippocampal neurons following oxygen-glucose deprivation (OGD). We demonstrate here that EndoG upregulates and translocates from mitochondria to nucleus in a time-dependent manner. Moreover, the translocation of EndoG occurs hours before the observable nuclear pyknosis. Importantly, we investigated the causal relationship between mitochondrial BNIP3 upregulation and EndoG translocation and nuclear pyknosis by over expressing or knocking down of BNIP3. Our findings strongly support a role for BNIP3 as a signal protein upstream of EndoG leading to the induction of neuronal death.
OGD induces EndoG upregulation in primary neuronal cultures
EndoG translocates from the mitochondria to the nucleus after reoxygenation
Nuclear EndoG translocation precedes OGD-induced neuronal death
BNIP3 mediates OGD-induced hippocampal neuronal death
BNIP3 initiates the EndoG translocation
Recent biochemical and genetic studies have revealed that EndoG is an important mitochondrial protein that emanates from the mitochondria during apoptosis and facilitates degradation of nuclear chromatin [7, 13]. The present study, for the first time, strongly implies an essential role of EndoG in post-ischemic hippocampal neuronal cell death in vitro. Western blot analysis and immunocytochemistry demonstrate that EndoG not only upregulates in hippocampal neurons following reoxygenation but also translocates from mitochondria to nucleus. This finding is consistent with previous results on other ischemic models, including cortical neuronal cultures subjected to hypoxia , and cortical neurons of mice subjected to transient or permanent focal cerebral ischemia [14, 15]. Using subcellular fractionation with a detailed time course, we confirmed that EndoG translocation markedly precedes the appearance of biochemical markers of cell death. The good temporal and spatial relationship between EndoG translocation and nuclear pyknosis suggests a causal role of EndoG translocation in OGD-induced hippocampal neuronal death.
The present study also identifies BNIP3 and EndoG translocation as early events in hippocampal neurons subjected to OGD. The increase in mitochondrial BNIP3 expression was observed immediately after reoxygenation. The translocation of EndoG was detectable at 2 h and increased significantly at 6 h after reoxygenation. This is consistent with a previous report in a mouse model of brain ischemia, in which EndoG translocation was observed 4 h after transient focal cerebral ischemia . Although in a previous study we found that BNIP3 and EndoG translocation occurred relatively late in cultured cortical neurons subjected to hypoxia , the mitochondrial translocation of the BNIP3 was before the nuclear translocation of the EndoG in both studies, which strongly supports that BNIP3 acts as an upstream signal of EndoG. The discrepancy might result from either different neuron types examined or different ischemic models used. Compared with hypoxia, OGD is a more severe stress to neurons and re-supply of oxygen and glucose may further exacerbate the injury. In fact, we detected an upregulation of BNIP3 in mitochondria at 0 h after reoxygenation, which might imply that the mitochondrial translocation of BNIP3 occurred and the signalling cascade triggered by BNIP3 started during OGD. The expression of BNIP3 in mitochondria further increased with prolonged reoxygenation, and knockdown of BNIP3 reduced cell death, supporting a role of BNIP3 in activating the cell death program. We also found that forced expression of BNIP3 increased nuclear translocation of EndoG. On the other hand, knockdown of BNIP3 expression reduced OGD-induced EndoG translocation. Therefore, the present findings argue for the importance of mitochondrial BNIP3 upregulation as an upstream modulator of EndoG translocation in ischemic neuronal injury.
In the present study we provide the first detailed description of the time-dependent subcellular localization of EndoG and BNIP3 in the cultured hippocampal neurons following OGD. By exploring the temporal relationship between the neuronal nuclear pyknosis and the translocation of the mitochondrial death-related proteins, BNIP3 and EndoG, we have been able to suggest an important role of mitochondrial BNIP3 upregulation and EndoG translocation in neuronal death. Combined with the findings that forced expression of BNIP3 increases EndoG translocation and neuronal death, and knockdown of BNIP3 decreases EndoG translocation and neuronal death, our results support the role of mitochondrial BNIP3 as a signal protein upstream of EndoG leading to the induction of neuronal death.
Neonatal 1 d Sprague-Dawley rats were provided by Southern Medical University and conditions regarding health and hygiene were confirmed. All experimental procedures in this study were performed within National Institutes of Health guidelines for the care and use of laboratory animals.
Cell culture and OGD
HEK293 cells were cultured in DMEM (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum and 2 mM glutamine. Primary hippocampal neuronal cultures were prepared from neonatal Sprague-Dawley rats (P1) as described previously . Cells were cultured in Neurobasal medium supplemented with 2% (v/v) B27, 2 mM glutamine, and 100 U/ml penicillin/streptomycin (Invitrogen, San Diego, CA). Cultures contain > 95% neurons as routinely controlled by neuronal-specific nuclear protein (NeuN) immunostaining. In 12- to 13-day-old primary neurons, the control culture medium was replaced with Earl's balanced salt solution (EBSS) medium (in mg/L: 6800 NaCl, 400 KCl, 264 CaCl2·2H2O, 200 MgCl2·7H2O, 2200 NaHCO3, 140 NaH2PO4·H2O, and 1000 glucose, pH 7.2). For OGD, glucose-free EBSS medium was purged with N2/CO2/O2 (94%/5%/1%) for 30 min, resulting in an oxygen content of 2-3%. Neurons were then washed three times with this medium and incubated for 4 h in an oxygen-free N2/CO2/O2 (94%/5%/1%) atmosphere. Thereafter, the medium was replaced by standard culture medium (see above). Cells were collected at 0, 2, 6, 12 and 24 h after reoxygenation respectively for western blot analysis, immunocytochemistry and quantification of cell death.
Preparation of the mitochondrial and nuclear fractions
Mitochondrial protein was extracted according to the manufacturer's protocol (PIERCE). Nuclear fractions were prepared as described by Yu et al. with slight modifications . Briefly, the cells were washed and resuspended in isotonic homogenization buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride, 10 mM Tris-HCl, pH 7.4) containing a proteinase inhibitor cocktail (4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatinA, E-64, bestatin, leupeptin, and aprotinin, Sigma). After homogenization, the homogenate was trypsinized with 0.1% trypsin for 5 min, and the unbroken cells were spun down. Then, the nuclei were fractionated at 80 × g for 10 min from the supernatant and washed three times with homogenization buffer containing 0.01% NP-40.
Western Blot analysis
Western blot analysis was performed as previously described . Protein samples (30 μg protein) were separated on 12% polyacrylamide gels and transferred to polyvinylidene diflouride membrane. The membrane was incubated with 5% skimmed milk in Tris-buffered saline for 1 h to block nonspecific binding. The membrane was then incubated overnight at 4°C with anti-EndoG antibody (1:500; Abcam), anti-BNIP3 (1:500; Sigma), anti-COX IV (1:5000; Abcam) or anti-Histone H3 (1:5000; BOSTER, Wuhan, China) and then further incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h. Immunoblotting was detected by ECL (Key GEN, Nanjing, China) and imaged on a FluorChem 8900 imager (Alpha Innotech). Western blot bands were quantified using the FluorChem™ SP V.126.96.36.199 software.
Cultured cells were fixed with 4% formaldehyde in PBS for 15 min and nonspecific binding was blocked for 60 min in PBS containing 0.3% Triton X-100 and 3% BSA. Anti-EndoG antibody (1:50) was applied in PBS containing 0.3% Triton X-100, incubated overnight at 4°C, followed by a Rhodamine (TRITC)-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson) secondary antibody (1:200) for 60 min. Negative controls, in which the primary antibody was omitted, were similarly treated as described above. Nuclear staining was followed with the fluorescent DNA-binding dye 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:1000; Roche) for 15 min. Fluorescence pictures were taken on a Leica TCS SP2 scanning confocal microscope. Neurons with condensed and fragmented nuclei were considered apoptotic. Five randomly chosen visual fields were counted in each sample by an investigator blind to the experimental conditions. About 500 cells from each group were counted.
BNIP3 gene silencing
Interfering plasmids targeting BNIP3 (BNIP3-miRNA) were generated using BLOCK-iT Pol II miR RNAi Expression Vector Kits (Invitrogen) following the instructions of the manufacturer. The miRNA sequences were designed using Invitrogen's BLOCT-iT RNAi Designer. Briefly, the oligonucleotides were synthesized, annealed and inserted to the pcDNA6.2-GW/EmGFP-miR vector. The pcDNA6.2/EmGFP-miR-neg control plasmid (neg-miRNA) was used as a negative control.
Neurons cultured for 7 days in vitro or HEK293 cells were transfected with plasmids pEGFP-C3-rBNIP3, pEGFP-C3, BNIP3-miRNA and neg-miRNA as we described previously  using Lipofectamine 2000 (Invitrogen) according to manufacturer's recommendations.
All data are given as means ± SE if not indicated otherwise. One-way ANOVA was used to test for overall statistical significance. A difference was considered significant at P < 0.05.
This work was supported by grants from NSFC (U0632007), National Basic Research Program of China (2006CB504100), PCSIRT (IRT0731), Key Project of Guangdong Province (06Z007, 2005A30801008, 9351051501000003) to TM Gao, and Natural Science Foundation of Guangdong Province (8451051501000070) to M Chen.
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