bnip3 upregulation and endog translocation in delayed neuronal

BNIP3 Upregulation and EndoG Translocation in DelayedNeuronal Death in Stroke and in Hypoxia

Zhengfeng Zhang, PhD; Xuefen Yang, MD; Surong Zhang, PhD; Xiuli Ma, MSc; Jiming Kong, PhD

Background and Purpose—Delayed neuronal death is a hallmark feature of stroke and the primary target ofneuroprotective strategies. Caspase-independent apoptosis pathways are suggested as a mechanism for the delayedneuronal injury. Here we test the hypothesis that one of the caspase-independent apoptosis pathways is activated byBNIP3 and mediated by EndoG.

Methods—We performed immunohistochemistry, Western blotting, cell transfection, subcellular fractionation, and RNAinterfering to analyze the expression and localization of BNIP3 and EndoG in degenerating neurons in models of strokeand hypoxia.

Results—BNIP3 was upregulated in brain neurons in a rat model of stroke and in cultured primary neurons exposed tohypoxia. The expressed BNIP3 was localized to mitochondria. Both forced expression of BNIP3 by plasmid transfectionand induced expression of BNIP3 by hypoxia in neurons resulted in mitochondrial release and nuclear translocation ofEndoG and neuronal cell death. Knockdown of BNIP3 by RNAi inhibited EndoG translocation and protected againsthypoxia-induced neuronal death.

Conclusions—BNIP3 plays a role in delayed neuronal death in hypoxia and stroke and EndoG is a mediator of theBNIP3-activated neuronal death pathway. The results suggest that BNIP3 may be a new target for neuronal rescuestrategies. (Stroke. 2007;38:000-000.)

Key Words: BNIP3 � EndoG � hypoxia � mitochondria � stroke

Stroke results in acute loss of neurons in the ischemic coreregion.1 The acute neuronal damage is followed by a

second round of neuronal injury that occurs hours to daysafter brain ischemia, called delayed neuronal death, in theneighboring areas.2 Evidence suggests that the delayed celldeath occurs primarily through an apoptosis mechanism.3 Thecaspase family of cysteine proteases plays an indispensablerole in the signal transduction and execution of apoptosis.4

However, a growing number of studies supports that a largeproportion of the delayed neuronal death in stroke is mediatedby caspase-independent pathways.5–7

BNIP3 is a member of a unique family of death-inducingmitochondrial proteins.8 Normally, BNIP3 is not expressed inneurons.9,10 Expression of BNIP3 is increased under prolongedhypoxic conditions primarily by the transcriptional factor hyp-oxia inducible factor 111 and causes cell death in a variety ofcells.8,11 The BNIP3-induced cell death is characterized bymitochondrial damage but is independent of caspase activation.9

BNIP3 is also implicated in autophagic cell death.12

Recently, the mitochondrial protein EndoG has beenshown to be involved in caspase-independent cell deathpathways.13 This signal peptide is cleaved off on enteringmitochondria and the mature EndoG can be released frommitochondria during apoptosis.13 Once released from mito-

chondria, EndoG is able to translocate to the nucleus andcleave chromatin DNA into nucleosomal fragments indepen-dently of caspases.13

Here we have tested the hypothesis that BNIP3 activates acaspase-independent form of neuronal cell death in stroke andhypoxia and EndoG is a mediator of the BNIP3-activated celldeath pathway. Our results suggest that BNIP3 may be a newtarget for neuronal rescue strategies.

Materials and MethodsFocal Brain Ischemia/ReperfusionAll animal protocols were approved by the University of ManitobaAnimal Care Ethics Committee. Male Sprague-Dawley rats weigh-ing 250 to 300 g (University of Manitoba Central Animal CareBreeding Facility, Winnipeg, Canada) were anesthetized with halo-thane. A 4–0 nylon suture with silicon-coated tip (Doccol Co) wasinserted through the left external carotid artery to occlude the leftmiddle cerebral artery (MCAO). The suture was withdrawn 90minutes after MCAO to allow reperfusion. Sham-operated ratsunderwent only the dissection of the left carotid tree. At 12, 24, 48,and 72 hours after MCAO, the animals were examined by T2-weighted MRI using a Bruker Biospec MSL-X 7/21 spectrometerand then euthanized for further analyses.

Neuronal Culture and HypoxiaPrimary cortical neuronal cultures were prepared as describedpreviously.14 Cells were plated in neurobasal medium (Invitrogen)

Received October 11, 2006; final revision received November 29, 2006; accepted December 19, 2006.From Department of Human Anatomy and Cell Science, University of Manitoba Faculty of Medicine, Winnipeg, Manitoba, Canada.Correspondence to Jiming Kong, PhD, Department of Human Anatomy and Cell Science, University of Manitoba, 730 William Avenue, Winnipeg,

Manitoba, R3E 0W3, Canada. E-mail [email protected]© 2007 American Heart Association, Inc.

Stroke is available at http://www.strokeaha.org DOI: 10.1161/STROKEAHA.106.475129

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supplemented with 5 mmol/L HEPES, 1.2 mmol/L glutamine, 10%fetal bovine serum, 2% B27, and 25 �g/mL gentamicin at a densityof 1�104 cells/cm2 on plates or cover slips coated with poly-D-lysine.The medium was replaced with neurobasal without fetal bovineserum after 24 hours. After 7 days, glutamine was removed from themedium and neurons were then exposed to hypoxia for up to 72hours in a hypoxia chamber (Billups-Rothenberg Inc) flushed with apreanalyzed gas mixture of 5% CO2 and 95% N2.

Cell TransfectionPlasmids pcDNA3-huBNIP3 and pcDNA3-huBNIP3�TM were giftsfrom the late Dr A.H. Greenberg (University of Manitoba, Winnipeg).8

The pCDNA3-haBNIP3 carrying the hamster BNIP3 was a generousgift from Dr R.K. Bruick (University of Texas Southwestern MedicalCenter, Dallas, Tex).11 For preparation of pEGFP-C2-rBNIP3, ratBNIP3 (rBNIP3) cDNA was isolated from primary neuronal culturesexposed to hypoxia for 36 hours by reverse-transcription polymerasechain reaction. The huBNIP3 and huBNIP3�TM were prepared bypolymerase chain reaction from pcDNA3-huBNIP3 and ligated topGEM-T (Promega) by T-A cloning. After sequencing, the BNIP3fragments were subcloned into pEGFP-C2 vector (Clontech). Neuronswere transfected on day 4 in vitro using Lipfectamine 2000 (Invitrogen).

shRNA Lentiviral VectorsThe shRNA sequences were designed using Invitrogen’s BLOCK-iTRNAi Designer. The oligonucleotides were synthesized, annealed togenerate double-stranded oligos, and cloned to pENTR/U6 vectors(Invitrogen). After cotransfection with BNIP3 plasmids intoHEK293 cells, the inhibition efficiencies of the shRNAs weredetermined by immunofluorescence microscopy and quantitativeWestern blot analysis. Selected shRNA sequences were inserted intoInvitrogen’s BLOCK-IT Lentiviral RNAi Expression system. Len-tiviral stocks were produced using ViraPower Packaging Mix (In-vitrogen). Transduction of neurons was performed by adding theviral vectors (multiplicity of infection �20) to medium 1 day beforehypoxia.

Western BlottingProtein samples were separated on 10% polyacrylamide gels andtransferred to polyvinylidene diflouride membranes. The primaryantibodies included: polyclonal BNIP3 (1:1000),11 monoclonalBNIP3 (1:500),8 EndoG (1:800; Prosci Inc.), Cox IV (1:10 000;Abcam), Histone H1 (1:500; Prosci Inc.), and �-actin (1:2000;Sigma). The secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (1:10 000; AmershamPharmacia Biotech). Immunoblotting was detected by ECL (Amer-sham) and imaged on a FluorChem 8900 imager (Alpha Innotech).Western blot bands were quantified using the NIH ImageJ software.

ImmunohistochemistryBrain sections or cells on coverslips were labeled with primaryantibodies (BNIP3, 1:200; NeuN, 1:500; or EndoG, 1:100) followedby appropriate secondary antibodies (1:200; Jackson). Nuclei werestained by Hoechst 33342 (Calbiocam). Degenerating neurons werelabeled with Fluoro-Jade C (FJC; Chemicon). Fluorescence pictureswere taken on a Nikon TE2000-E microscope equipped with aRETIGA camera (QImaging).

Cell Death and Viability AssaysCell death was estimated using trypan blue exclusion. Neuronalviability was estimated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on a WallacVICTOR3

microplate reader (Perkin Elmer Life Sciences).

Statistical AnalysisOne-way ANOVA was used to test for overall statistical signifi-cance. A difference was considered significant at P�0.05.

ResultsBNIP3 Expression Is Upregulated inBrain IschemiaOf 35 rats operated for MCAO, 5 died postoperatively and 6were discarded for insufficient brain ischemia as determined byMRI. Each group consisted of 6 animals for further analysis(3 for immunohistochemistry and 3 for Western blot).

Unilateral MCAO induced severe ischemia of brainareas (supplemental Figure I, available online at http://stroke.ahajournals.org). Immunohistochemical staining with apolyclonal antibody to BNIP39 did not reveal positive stainingin brains of animals euthanized at 0, 12, and 24 hours afterMCAO. In the 48-hour and 72-hour groups, intensive BNIP3staining was found exclusively in the ischemic regions ofbrain, especially in the penumbral region of ischemia (Figure1A). The expressed BNIP3 was primarily found in large cellsand predominately localized to the cytoplasm (Figure 1B). In19.7% of BNIP3-positive cells, BNIP3 was found in both thecytoplasm and the nuclei. Exclusive nuclear localization ofBNIP3 was rarely found. Double labeling with antibodies toBNIP3 and NeuN revealed that most of the BNIP3-positivecells were neurons (Figure 1C). Preincubation of the BNIP3antibody with a BNIP3–GST protein completely blocked theimmunostaining for BNIP3 (Figure 1D). Double labelingwith the BNIP3 antibody and the dying neuron marker FJCshowed that almost all BNIP3 expressing cells were FJC-positive (Figure 1E through 1H). Using a monoclonal anti-body recognizing rat BNIP3,11 we further determined byWestern blotting the BNIP3 levels in brain extracts. Levels ofBNIP3 were low in ischemic brains for up to 24 hours afterMCAO. BNIP3 accumulated at 48 hours after brain ischemia(Figure 1I). High levels of BNIP3 were detected in theipsilateral brain as compared with the contralateral andnormal brains at 72 hours after MCAO (Figure 1K). Quanti-fication of the immunoblotting bands showed that the levelsof BNIP3 were increased by 12-fold and 11-fold, respec-tively, at 48 hours and 72 hours after MCAO (Figure 1J and1L). To determine whether the expressed BNIP3 wasmembrane-bound, brain extracts were separated into heavymembrane pellets (P100) and supernatants (S100) by ultra-centrifugation. The cleanliness of the subcellular preparationswas confirmed by the absence of the mitochondrial mem-brane protein cytochrome c oxidase (Cox IV; Figure 1M) inthe S100 fraction. BNIP3 was primarily found in the mem-brane fraction samples of the ipsilateral brains, indicating thatthe expressed BNIP3 was active because inactive BNIP3 doesnot integrate with cell membranes.9

Hypoxia Induces BNIP3 Expression in PrimaryNeuronal CulturesPrimary rat cortical neurons at day 8 in vitro weresubjected to normoxia or hypoxia for 6, 12, 24, 36, 48, 60,and 72 hours, respectively. Levels of BNIP3 expressionwere determined by Western blotting using a polyclonalBNIP3 antibody described previously.11 In addition to anonspecific band, the antibody recognized two BNIP3bands with molecular weights of 30 kD and 60 kD,respectively (Figure 1N). For control, immunoblotting

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with a monoclonal �-actin antibody was performed and the�-actin bands were used as standards for calculation ofBNIP3 expression. Levels of BNIP3 were low in neuronsexposed to hypoxia for �24 hours and the BNIP3 proteinconsisted of almost entirely the 30-kDa form. After expo-

sure to hypoxia for 36 hours, both forms of the BNIP3protein started to accumulate (4-fold). After 72 hours ofhypoxic exposure, the levels of BNIP3 increased to �10-fold (Figure 1O). BNIP3 was not observed to accumulatein neurons under normoxic conditions.

Figure 1. Upregulation of BNIP3 in hypoxia and stroke. A, Representative immunohistochemical image of brain section cut from a rateuthanized at 48 hours after MCAO. The dotted line separates the ischemic area from the normal. BNIP3 was not detectable in nonis-chemic regions but upregulated in the ischemic regions (blue, nuclei; green, BNIP3; bar�250 �m). B, The expressed BNIP3, as deter-mined by immunohistochemistry with an antibody to BNIP3, was predominately localized to neuronal cytoplasm (bar�50 �m). C, Dou-ble labeling with antibodies to NeuN and BNIP3 (red, NeuN; green, BNIP3; bar�50 �m). D, Preincubation of the BNIP3 antibody with aBNIP3–GST protein completely blocked the immunostaing for BNIP3 (bar�50 �m). E through H, Representative image showing colo-calization of BNIP3 and FJC in ischemic neurons 48 hours after MCAO. Brain sections were stained for BNIP3 immunohistochemicallyand further labeled with FJC and Hoechst 33342 (bar�50 �m). I and J, Time course of BNIP3 expression after MCAO by Western blotanalysis. K and L, Expression levels of BNIP3 in the ipsilateral brain (lane 1) and the contralateral (lane 2) and normal brains (lane 3) 72hours after MCAO. M, Protein samples prepared from the ipsilateral brain (lane 1) and the contralateral (lane 2) and normal brains (lane3) were separated into membrane (P100) and supernatant (S100) fractions and then Western-blotted. The amount of proteins loaded ineach S100 lane was 30 �g, and in each P100 lane was 9 �g. N and O, Hypoxia-induced BNIP3 expression in primary cortical neurons.Protein samples were Western-blotted. A sample from BNIP3-transfected 293T cells was used as control. Results shown in (O) repre-sent the mean�SD of both the 30-kD and 60-kD BNIP3 bands from 4 independent experiments. **P�0.01.

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Inhibition of BNIP3 by RNAi Protects NeuronsFrom Hypoxia-Induced Cell DeathTo identify a shRNA sequence that is of high-inhibitionefficiency for BNIP3, 12 pairs of oligonucleotides wereinitially designed, synthesized, and cloned into InvitrogenpENTR/U6 vectors. The vectors were cotransfected with theBNIP3-expressing plasmid pcDNA3-haBNIP3 into HEK 293cells. The inhibition efficiencies of BNIP3 varied from noneto almost complete inhibition as determined by immunofluo-rescence microscopy and quantitative Western blot analysis(data not shown). One pair of oligonucleotides (N167,forward, 5�-CACC-GCTTCCGTCTCTATTTATATTCA-AGAGATATAAATAGAGACGGAAGC-3�; backward, 5�-AAAA-GCTTCCGTCTCTATTTATATCTCTTGAATA-TAAATAGAGACGGAAGC-3� (bold, sense and antisensestrands; underlined, loop) targeting the nucleotides 167 to188 in the BNIP3 mRNA sequence (GenBank accessionnumber NM_053420) showed the most potent inhibition(Figure 2). Quantification of the Western blot bandsrevealed that the inhibition efficiency of the N167 forBNIP3 expression was 98.1% as compared with thenontransfected controls. In addition to its inhibition onhamster BNIP3, the N167 was able to inhibit the expres-

sion of mouse and rat BNIP3 genes with similar efficien-cies (data not shown). To inhibit BNIP3 expression inneurons, we developed a lentiviral vector carrying theN167 sequence. Transfection of primary cortical neuronswith the N167 lentiviral vector resulted in completeinhibition of BNIP3 in neurons exposed to hypoxia for 48hours (Figure 2C), whereas no inhibition of BNIP3 wasobserved with a lentiviral vector carrying the LacZ se-quence and a vector carrying a scrambled sequence (S167)that contained the same nucleotide composition as N167.

We next tested the effects of inhibition of BNIP3 onhypoxia-induced neuronal death. Primary cortical neuroncultures were infected with the N167 or the LacZ lentiviralvectors 24 hours before hypoxia. As shown in Figure 3A,hypoxia induced neuronal cell death in a time-dependentmanner. Inhibition of BNIP3 expression by the N167vector reduced hypoxia-induced neuronal death by 15% to23% and the protection was statistically significant at 36and 48 hours of hypoxic exposure. Analysis of cellviability by MTT measurement revealed significant pro-tection of the N167 for neurons exposed to hypoxia for 24,36, 48, and 60 hours. No protective effects of the LacZvector were observed.

Figure 2. Inhibition of BNIP3 by RNAi. A,The predicted short hairpin structureexpressed by the N167 plasmid. B, Inhi-bition efficiency of the N167 on BNIP3expression in 293 cells cotransfectedwith indicated plasmids. Results shownrepresent mean�SD for combined datafrom 3 independent experiments. C, Inhi-bition of hypoxia-induced BNIP3 expres-sion in primary neurons by RNAi. Neu-rons were transfected with lentiviralvectors carrying the N167 or the controlsLacZ and S167 24 hours before hypoxicexposure.

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Hypoxia-Induced BNIP3 Is Localizedto MitochondriaTo determine how the expression of BNIP3 leads to neuronaldeath, we examined the subcellular localization of hypoxia-induced BNIP3. By differential centrifugation, mitochondrial,cytosolic, and nuclear fractionations were prepared and West-ern blotted with a BNIP3 antibody. Controls were performedusing a �-actin antibody for cytosolic, a Cox IV antibody formitochondrial, and a histone H1 antibody for nuclear frac-tions. Hypoxia-induced BNIP3 was primarily localized tomitochondria, particularly at prolonged hypoxia (60 hoursand 72 hours). A small amount of BNIP3 was present in thecytosol and only a trace amount of BNIP3 was observed inthe nuclear fractionation at late stages of hypoxic exposure(Figure 4A and 4B).

EndoG Is a Mediator of the BNIP3-Activated CellDeath PathwayWe next tested the involvement of EndoG in hypoxia-inducedneuronal death. As shown in Figure 5, total amount of EndoGdid not vary with exposure times to hypoxia. EndoG in themitochondrial fraction decreased with exposure times tohypoxia; it started to decrease significantly after hypoxic

exposure for 36 hours and disappeared from mitochondriaafter 72 hours of hypoxia. Meanwhile, EndoG was detected innuclear fraction after hypoxia for 36 hours and accumulatedwith increased times of hypoxia, suggesting that the releasedEndoG from mitochondria was translocated to nuclei. Thistime course correlates well with that of hypoxia-inducedBNIP3 expression (Figure 1).

To evaluate the role of BNIP3 in hypoxia-induced EndoGtranslocation in neurons, we transfected neurons with ourN167 lentiviral vector and then exposed neurons to hypoxiafor up to 72 hours. Western blotting analysis revealed thatinhibition of BNIP3 delayed mitochondrial release and nu-clear translocation of EndoG by 24 hours (Figure 5B and 5C).In the absence of BNIP3, EndoG was present in mitochondriaeven after 72 hours of hypoxic exposure and did not accu-mulate in nuclei until after 60 hours of hypoxia. The resultwas also confirmed by double-labeling immunohistochemis-try with antibodies to BNIP3 and EndoG (Figure 5D);inhibition of BNIP3 by RNAi with the N167 lentiviral vectorprevented EndoG translocation in neurons exposed to hypox-ia for 48 hours. The control LacZ vector showed no inhibitionon BNIP3 expression and no effect on hypoxia-inducedEndoG translocation. As a result, DNA condensation wasobvious in BNIP3-expressing neurons.

To test whether forced expression of BNIP3 inducesEndoG translocation, we transfected neurons with a

Figure 3. Inhibition of BNIP3 by RNAi protects neurons fromhypoxia-induced cell death. Primary cortical cultures wereinfected by the N167 or the LacZ lentiviral vectors 24 hoursbefore hypoxia. Results shown represent the mean�SD forcombined data from 3 independent experiments. A, Cell deathwas evaluated by trypan blue assay. B, Cell viability was esti-mated by MTT activity measurement and expressed as percent-age of the untreated controls. *P�0.05.

Figure 4. BNIP3-induced neuronal death is mitochondria-mediated. A and B, Hypoxia-induced BNIP3 expression wasprimarily localized to mitochondria (mito, mitochondrial fraction;cyto, cytosolic fraction; nuc, nuclear fraction). Levels of BNIP3were normalized as the percentage of the total amount ofBNIP3 at 72 hours.



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Figure 5. EndoG translocation in hypoxic neurons. A, Western blot analysis of EndoG in cortical neuronal cultures exposed to hypoxia.B, Western blot analysis of EndoG in cortical neuron cultures transfected with the N167 lentiviral vector 24 hours before hypoxia. C,Inhibition of BNIP3 by RNAi delayed hypoxia-induced EndoG release and translocation for 24 hours. D, Representative images of neu-rons transfected with indicated vectors for 24 hours and then exposed to hypoxia for 48 hours. Green, BNIP3; red, EndoG; blue, DNA.

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pcDNA3-huBNIP3 plasmid encoding the full length of hu-man BNIP3. Immunohistochemical analysis showed that themajority (62%) of BNIP3 expressing neurons had nuclearpresence of EndoG. Neurons transfected with a truncatedform of BNIP3 (pcDNA3-huBNIP3�TM) that did not con-tain the C-terminal transmembrane domain did not induceEndoG translocation (Figure 6A). Cell death rates in BNIP3and BNIP3�TM transfected neurons were 73% and 22%,respectively (Figure 6B).

DiscussionDelayed neuronal death is a hallmark feature of brain ische-mia. This is evidenced by the fact that even if cerebral bloodflow is re-established quickly enough to prevent immediatecell death after brain ischemia, many of the initially survivingneurons still die hours to days after reperfusion.1,5,6 Of themany pathophysiological events that may contribute to thisdelayed injury, apoptotic signaling pathways are, amongothers, an established feature.1 The present study has revealedthe delayed feature of BNIP3 expression in the MCAO modelof stroke and in neurons exposed to hypoxia (Figure 1) andsuggested the involvement of BNIP3 in delayed neuronal celldeath after brain ischemia. This finding is consistent withprevious studies using other types of cells.9,11 In addition, thepresent study has revealed evidences in support of a BNIP3-activated cell death pathway in hypoxia and stroke: (1)BNIP3 is upregulated in the MCAO model of stroke and inneurons exposed to hypoxia; (2) forced expression of BNIP3in neurons induces mitochondrial dysfunction and cell death;and (3) knockdown of BNIP3 protects against neurons fromhypoxia-induced cell death. Furthermore, we have provideddata that EndoG is a mediator of the BNIP3-activatedneuronal death: (1) the time course of mitochondrial releaseand nuclear translocation of EndoG correlates well with thatof BNIP3 expression in neurons exposed to hypoxia; (2)forced expression of BNIP3 induces EndoG translocation;and (3) inhibition of BNIP3 expression significantly delayedEndoG translocation.

The present study demonstrates that BNIP3 is not onlyupregulated in stroke and in hypoxia but also plays a role inactivating a unique cell death program. In our in vivo

experiments, in which MRI was used to verify the extent ofbrain ischemia after MCAO, we show clearly that BNIP3-positive neurons are found only in the ischemic brain regionsand concentrated in the penumbra, a region where pro-grammed cell death usually occurs after brain ischemia.6 Theexpressed BNIP3 is primarily in the cytoplasm, as shown inboth immunohistochemistry and Western blot analyses. Wehave observed nuclear localization of BNIP3 in �20% of theBNIP3-expressing neurons. This is in disagreement with 2recent reports on the distribution of BNIP3 in brain ischemiabased on their immunohistochemistry data.15,16 This discrep-ancy may be resulted from different antibodies used, celltypes examined, and the time course of BNIP3 expressionobserved. However, the functional role of nuclear localizationof BNIP3 in neurons is unclear. In tumor cells, nuclearlocalization of BNIP3 has been suggested as a mechanism ofcell’s escaping of apoptosis.10

In the present study, we report not only the time course ofBNIP3 upregulation that correlates well with hypoxia-induced neuronal death17 but also the active status of theexpressed BNIP3 as shown by its membrane association andmitochondrial localization. The active role of BNIP3 isfurther evidenced by results from the inhibition experimentsusing RNAi (Figure 5). Previous loss-of-function phenotypicstudies on BNIP3 have not been satisfactory because of lackof specific inhibitors for BNIP3 and limited inhibition effi-ciencies with dominant-negative and antisense strategies.18,19

By coupling rational design of shRNAs with their validationin cell transfection, we have identified the N167 shRNAsequence specific for BNIP3 and achieved an inhibitionefficiency of 98.1%. This is so far the most powerful tool forBNIP3 inhibition. With this tool, we found that knockdownof BNIP3 protected 15% to 23% of neurons from hypoxia-induced cell death (Figure 4). It is noticeable that theprotective rates are much lower than the rate of neuronaldeath (73%) caused by forced expression of BNIP3 (Figure6B). This may suggest that other members of the BNIP3family, eg, Nix, may compensate for the loss of BNIP3. Also,the BNIP3 pathway may exist in tandem with other cell deathpathways,6 and inhibition of one pathway may likely causeshift of others. Further research is required to determine theneuroprotective effects by inhibiting simultaneously multiplepathways.

Another finding of the present study is the identification ofEndoG as a downstream event of the BNIP3-activated celldeath pathway. Previous extensive studies have establishedthat BNIP3-induced cell death is mitochondria-mediated,involving opening of the mitochondrial permeability transi-tion pores, accumulation of reactive oxygen species, and lossof mitochondrial membrane potential.9 The BNIP3 pathwaydiffers from conventional apoptosis in that it appears to beindependent of Apaf-1, caspase activation, cytochrome crelease, and nuclear translocation of apoptosis-inducing fac-tor.9,19–21 Although a previous study with primary cardiacmyocytes supported the involvement of caspase activity inBNIP3-induced cell death and suggested the involvementmight be cell type-specific,22 in primary neuron cultures wefailed to observe any protective effects of caspase inhibitors,alone or in combination, on cell death caused by transient

Figure 6. Expression of BNIP3 results in EndoG translocation.Results shown represent the mean�SD from 5 independentexperiments. **P�0.01. A, Percentage of BNIP3-positive cellswith EndoG nuclear translocation in primary cortical neuronstransfected with the indicated plasmids at 48 hours. B, Celldeath rates determined by trypan blue exclusion in transfectedneurons 48 hours after transient transfection with the indicatedplasmids.



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transfection of BNIP3. In consistent with a previous report,9

we did not detect any apoptosis-inducing factor translocationin BNIP3-expressing neurons. Previous studies support thatBNIP3-induced cell death involves chromatin condensationand DNA fragmentation,9 implicating a caspase-independentDNase in the BNIP3 pathway. The present study providesevidence that expression of BNIP3 results in mitochondrialrelease and nuclear translocation of EndoG, and inhibition ofBNIP3 delayed hypoxia-induced EndoG translocation by 24hours (Figure 5). The 24-hour delay in EndoG translocationshould be considered very significant, taking into consider-ation the presence of many other inducers of EndoG translo-cation in the cells such as caspases23 and other members ofthe BNIP3 gene family that are activated in hypoxia. Once inthe nucleus, EndoG first induces large-scale DNA fragmen-tation, followed by subsequent oligonucleosomal DNA frag-mentation. Recently, Lee et al24 reported early (4 hours afterMCAO) EndoG translocation in a mouse model of brainischemia. This early EndoG release/translocation is not ob-served in our experiments and is certainly not a BNIP3-induced event, because BNIP3 is not activated within 24hours after MCAO. Nevertheless, the role of EndoG inmediating BNIP3-activated cell death needs to be furtherestablished using EndoG-deficient cells and animals.

Sources of FundingThis work was supported by grants from Canadian Institutes ofHealth Research, Heart and Stroke Foundation of Canada andManitoba Health Research Council (to J.K.). Dr Zhengfeng Zhangreceived a Manitoba Health Research Council postdoctoral fellow-ship. Dr Surong Zhang received a postdoctoral fellowship fromManitoba Institute for Child Health. Dr Jiming Kong received a NewInvestigator Award from Heart and Stroke Foundation of Canada.

DisclosuresNone.

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21. Ray R, Chen G, Vande Velde C, Cizeau J, Park JH, Reed JC, Gietz RD,Greenberg AH BNIP3 heterodimerizes with Bcl-2/Bcl-x(l) and inducescell death independent of a Bcl-2 homology 3 (BH3) domain at bothmitochondrial and nonmitochondrial sites. J Biol Chem. 2000;275:1439–1448.

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8 Stroke May 2007


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Figure I. Representative T2-weighted brain MRI taken at 48 hours post-MCAO. Hyperintense areas depict injured tissue and/or edema.



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Zhengfeng Zhang, Xuefen Yang, Surong Zhang, Xiuli Ma and Jiming Kongin Hypoxia

BNIP3 Upregulation and EndoG Translocation in Delayed Neuronal Death in Stroke and

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