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: ;

(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.
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