Potential Role of PUMA in Delayed Death of Hippocampal
CA1 Neurons After Transient Global Cerebral Ischemia
Kuniyasu Niizuma, MD; Hidenori Endo, MD; Chikako Nito, MD, PhD;
D. Jeannie Myer, PhD; Pak H. Chan, PhD
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Background and Purpose—p53-upregulated modulator of apoptosis (PUMA), a BH3-only member of the Bcl-2 protein
family, is required for p53-dependent and -independent forms of apoptosis. PUMA localizes to mitochondria and
interacts with antiapoptotic Bcl-2 and Bcl-XL or proapoptotic Bax in response to death stimuli. Although studies have
shown that PUMA is associated with pathomechanisms of cerebral ischemia, clearly defined roles for PUMA in
ischemic neuronal death remain unclear. The purpose of this study was to determine potential roles for PUMA in
cerebral ischemia.
Methods—Five minutes of transient global cerebral ischemia (tGCI) were induced by bilateral common carotid artery
occlusion combined with hypotension.
Results—PUMA was upregulated in vulnerable hippocampal CA1 neurons after tGCI as shown by immunohistochemistry.
In Western blot and coimmunoprecipitation analyses, PUMA localized to mitochondria and was bound to Bcl-XL and
Bax in the hippocampal CA1 subregion after tGCI. PUMA upregulation was inhibited by pifithrin-␣, a specific inhibitor
of p53, suggesting that PUMA is partly controlled by the p53 transcriptional pathway after tGCI. Furthermore, reduction
in oxidative stress by overexpression of copper/zinc superoxide dismutase, which is known to be protective of
vulnerable ischemic hippocampal neurons, inhibited PUMA upregulation and subsequent hippocampal CA1 neuronal
death after tGCI.
Conclusions—These results imply a potential role for PUMA in delayed CA1 neuronal death after tGCI and that it could
be a molecular target for therapy. (Stroke. 2009;40:618-625.)
Key Words: PUMA 䡲 cerebral ischemia, global 䡲 apoptosis 䡲 superoxide dismutase 䡲 oxidative stress
T
he Bcl-2 protein family is a principal regulator of
mitochondrial membrane integrity and function and is
classified into 3 subgroups according to structural homology (Bcl-2 homology [BH] domains): the antiapoptotic
proteins (Bcl-2, Bcl-XL, Mcl-1, BCL-W), the proapoptotic proteins (Bax, Bak), and the BH3-only proteins (Bim, Bad, Bid,
Bik, p53-upregulated modulator of apoptosis [PUMA],
NADPH oxidase activator, Hrk). BH3-only proteins share
sequence homology with other Bcl-2 proteins in the BH3
region only1 and are involved in the mechanisms of cytochrome c release in neuronal apoptosis.2
According to a recent hierarchy model, BH3-only proteins are subdivided into activator or inactivator proteins.3
Activator BH3-only proteins can interact with both antiapoptotic Bcl-2 proteins and proapoptotic Bcl-2 proteins.
Among these activator proteins, PUMA was initially identified as a gene activated by p53 in cells undergoing
p53-induced apoptosis.4,5 In p53-induced cell death, PUMA
was shown to localize to mitochondria; interact with Bcl-2,
Bcl-XL, and Bax; and induce cytochrome c release, thereby
activating caspases-9 and -3.3–5 However, the roles of PUMA in
cerebral ischemia remain unclear.
To determine these roles, we used as our model 5
minutes of transient global cerebral ischemia (tGCI),
which induces delayed neuronal death in the hippocampal
CA1 subregion in rats.6 We investigated expression of
PUMA and the interaction between PUMA and Bcl-XL,
Bcl-2, and Bax in the hippocampal CA1 subregion after
tGCI. To investigate the regulation of PUMA by p53 after
tGCI, we administered pifithrin-␣ (PFT), a specific inhibitor of p53. To demonstrate the effects of oxidative stress
on PUMA expression after tGCI, we used copper/zinc
superoxide dismutase (SOD1) transgenic (Tg) rats, which
have neuroprotection against ischemia because of reduced
oxidative stress.7
Materials and Methods
Global Cerebral Ischemia
Five minutes of tGCI was induced by bilateral common carotid
artery occlusion combined with hypotension according to a
Received April 28, 2008; final revision received June 26, 2008; accepted July 15, 2008.
From the Departments of Neurosurgery, Neurology and Neurological Sciences, and the Program in Neurosciences, Stanford University School of
Medicine, Stanford, Calif.
Correspondence to Dr Pak H. Chan, Neurosurgical Laboratories, Stanford University, 1201 Welch Road, MSLS #P314, Stanford, CA 94305-5487.
E-mail [email protected]
© 2009 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.108.524447
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method described previously,7 with some modifications.6 Male
Sprague-Dawley rats (300 to 350 g) were anesthetized with 2.0%
isoflurane in 70% nitrous oxide and 30% oxygen via face mask.
Rectal temperature was controlled at 37°C during surgery with a
homeothermic blanket. The femoral artery was catheterized with
a PE-50 catheter to allow continuous recording of arterial blood
pressure. After heparinization, blood was quickly withdrawn via
the jugular vein. When the mean arterial blood pressure became
30 mm Hg, both common carotid arteries were clamped with
surgical clips. Blood pressure was maintained at 30 mm Hg
during the ischemic period. After 5 minutes of ischemia, the clips
were removed and the blood was reinfused. Regional cerebral
blood flow was monitored by laser Doppler flowmetry as previously described.7 Sham-operated animals underwent exposure of
the vessels without blood withdrawal or clamping of the carotid
arteries. The animals were maintained at 20°C with ad libitum
access to food and water. All animals were treated in accordance
with Stanford University guidelines, and the animal protocols
were approved by Stanford University’s Administrative Panel on
Laboratory Animal Care.
anti-Bax (#2772, Cell Signaling Technology) and protein G–Sepharose
for 2 hours at 4°C. The negative control was prepared with protein
G–Sepharose without an antibody. Whole-brain extract was included as
a positive control. The 14 000g pellets were washed 3 times and
analyzed as the samples bound to each antibody by Western blotting
with anti-PUMA (1:1000, #4976; Cell Signaling Technology), anti–
Bcl-XL (1:1000), anti–Bcl-2 (1:1000), or anti-Bax (1:1000).
Drug Treatment
Immunofluorescence Staining
To examine the effect of a specific p53 inhibitor on PUMA
expression after tGCI, we administered PFT (P4359; Sigma-Aldrich,
St. Louis, Mo), dissolved in dimethyl sulfoxide and phosphatebuffered saline (PBS). This drug (4 mg/kg in dimethyl sulfoxide in
PBS) or vehicle (dimethyl sulfoxide in PBS) was injected via the left
jugular vein just after reperfusion as described previously.8
SOD1-Tg Rats
Heterozygous SOD1-Tg rats with a Sprague-Dawley background
carrying human SOD1 genes were derived from founder stock and
were further bred with wild-type (Wt) Sprague-Dawley rats to
generate heterozygous rats, as previously described. The phenotype
of the SOD1-Tg rats was identified by isoelectric focusing gel
electrophoresis as described.7 There were no observable phenotypic
differences in brain vasculature between the SOD1-Tg rats and their
Wt littermates.7
Western Blot Analysis
The hippocampal CA1 subregion was removed after 1, 4, 24, or
72 hours of reperfusion. Protein extraction of the cytosolic,
mitochondrial, and nuclear fractions was performed with a
multiple centrifugation method as described previously.9 Equal
amounts of samples were loaded per lane and analyzed by sodium
dodecyl sulfate–polyacrylamide-gel electrophoresis on a 10% to
20% Tris-glycine gel (Invitrogen, Carlsbad, Calif) and then
immunoblotted. Anti-PUMA (1:1000, #4976; Cell Signaling Technology, Beverly, Mass), anti–p-53 (1:1000, #554147; BD Biosciences, San Jose, Calif), anti– cytochrome c (1:1000, #556433; BD
Biosciences), anti– caspase-9 (1:1000, sc-8355; Santa Cruz Biotechnology, Santa Cruz, Calif), anti–␤-actin (1:10 000, A5441; SigmaAldrich), anti-cytochrome oxidase subunit IV (COX IV; 1:5000,
A21348; Invitrogen), or anti-TFIID (transcription factor II D; 1:200,
sc-204; Santa Cruz Biotechnology) primary antibody was used. After
incubation with horseradish peroxidase– conjugated secondary antibody, the antigen was detected by chemiluminescence Western
blotting detection reagents (Amersham, Buckinghamshire, UK). The
image was scanned with a GS-700 imaging densitometer (Bio-Rad
Laboratories, Hercules, Calif), and the results were quantified by
Multi-Analyst software (Bio-Rad).
Coimmunoprecipitation
A sample of mitochondrial fractions was prepared as described in the
Western blotting method. The procedure for precipitation was performed as described previously.8 After protein extraction, the mitochondrial samples were incubated with protein G–Sepharose (Amersham)
for 1 hour at 4°C, and this mixed sample was then centrifuged. The
supernatant was incubated with 2 ␮g of anti–Bcl-XL (#2762, Cell
Signaling Technology), anti–Bcl-2 (#610538, BD Biosciences), or
Cresyl Violet Staining and Immunohistochemistry
of PUMA
Anesthetized animals were perfused with 10 U/mL heparin saline
and subsequently with 4% formaldehyde in PBS after 1, 4, 24, or
72 hours of reperfusion. Brains were removed, postfixed for 24
hours, and sectioned at 50 ␮m with a Vibratome. For histologic
assessment, the sections were stained with cresyl violet. For
immunohistochemistry of PUMA, sections were reacted with
anti-PUMA (1:50, #4976; Cell Signaling Technology). Immunohistochemistry was performed with the avidin-biotin technique,
and nuclei were counterstained with methyl green solution.
To evaluate colocalization of PUMA and COX IV with neuronspecific nuclear protein (NeuN), Bcl-XL, or Bax, we performed
double immunofluorescence. For double immunofluorescence of
PUMA and COX IV or Bax, the sections were reacted with
anti–COX IV (1:100, #4844; Cell Signaling Technology) or
anti-Bax (1:50, sc-526; Santa Cruz Biotechnology), followed by
fluorescein isothiocyanate– conjugated anti-rabbit monovalent
Fab fragments of a secondary antibody (Jackson ImmunoResearch, West Grove, Pa) for labeling and blocking of COX IV or
Bax. Then the sections were incubated with anti-PUMA (1:50,
#4976; Cell Signaling Technology) followed by Texas Red–
conjugated anti-rabbit IgG (Jackson ImmunoResearch). For double immunofluorescence of PUMA and NeuN or Bcl-XL, sections
were immunostained with anti-PUMA (Cell Signaling Technology) followed by Texas Red– conjugated anti-rabbit IgG. The
sections were then incubated with anti-NeuN (1:50, MAB377;
Chemicon International, Temecula, Calif) or anti–Bcl-XL (1:50,
#610209; BD Biosciences), followed by fluorescein isothiocyanate– conjugated antimouse IgG (Jackson ImmunoResearch). The
sections were covered with Vectashield mounting medium with
4⬘,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, Calif) and examined under an LSM510 confocal laser
scanning microscope or an Axioplan 2 microscope (Carl Zeiss,
Thornwood, NY).
In Situ Detection of Superoxide Anion Production
Early production of superoxide anions after tGCI was investigated with the use of hydroethidine as previously described.10
Hydroethidine is diffusible into the central nervous system
parenchyma after intravenous injection and is selectively oxidized
to ethidium by superoxide anions. Hydroethidine solution (200
␮L of 1 mg/mL in 1% dimethyl sulfoxide with saline) was
administered intravenously 15 minutes before ischemia induction.
A sample was prepared as described in the immunohistochemistry
method. For fluorescent double staining of the ethidium signal
and PUMA, sections were incubated with anti-PUMA (1:50,
#4976; Cell Signaling Technology), followed by fluorescein
isothiocyanate– conjugated anti-rabbit IgG (Jackson ImmunoResearch). Slides were covered with DAPI (Vector Laboratories)
and observed with a fluorescence microscope.
Cell Death Assay
For quantification of apoptosis-related DNA fragmentation, we used
a commercial enzyme immunoassay to determine cytoplasmic histone–associated DNA fragments (1774425; Roche Molecular Biochemicals, Mannheim, Germany) and to detect apoptotic but not
necrotic cell death.11 A sample was prepared as described in the
Western blotting method. A cytosolic volume containing 20 ␮g of
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Figure 1. A, Representative photomicrographs of immunohistochemistry of
PUMA after tGCI. Cresyl violet staining
shows no neuronal degeneration in the
hippocampal CA1 subregion 1, 4, and 24
hours after tGCI. Seventy-two hours after
tGCI, ⬎80% of the CA1 neurons degenerated, although neurons in the hippocampal CA3 subregion were spared.
Immunoreactivity for PUMA increased,
peaked at 4 hours, and declined by 24
hours. At 72 hours, the CA1 neurons
degenerated, and immunoreactivity could
not be evaluated. C indicates control.
Scale bar⫽300 ␮m (hippocampus),
50 ␮m (CA1). B, Representative photomicrographs of fluorescent double staining
of PUMA (red) and NeuN (green) in the
hippocampal CA1 subregion 4 hours after
tGCI. Nuclei were counterstained with
DAPI (blue). NeuN immunoreactivity
showed the distribution of neurons. Overlapped image demonstrates that PUMApositive cells in the hippocampal CA1
subregion colocalized with neurons.
Scale bar⫽50 ␮m.
protein was used for the ELISA, according to the manufacturer’s
protocol.
Statistical Analysis
Comparisons among multiple groups were performed with ANOVA
followed by a Scheffé post hoc analysis (SigmaStat; Systat Software,
San Jose, Calif). Comparisons between 2 groups were achieved with a
Student unpaired t test. Data are expressed as mean⫾SD, and significance was accepted with P⬍0.05.
Results
PUMA Induction and Selective Neuronal Death
After tGCI
One, 4, or 24 hours after 5 minutes of tGCI, there was no
neuronal degeneration in the hippocampal CA1 subregion, as
confirmed by cresyl violet staining (Figure 1A). However,
⬎80% of the CA1 neurons were degenerated 72 hours after
tGCI, which was compatible with our previous reports.6,7 In
contrast, neurons in the hippocampal CA3 subregion were
spared even at 72 hours. PUMA immunoreactivity increased
after tGCI, peaked at 4 hours, and then started to decline at 24
hours. At 72 hours, the CA1 neurons degenerated and
immunoreactivity could not be evaluated (Figure 1A). Double
immunofluorescence for PUMA and NeuN demonstrated that
PUMA-positive cells in the hippocampal CA1 subregion
colocalized with neurons 4 hours after tGCI (Figure 1B).
These results indicate that PUMA is upregulated in the
hippocampal CA1 neurons after tGCI, which precedes CA1
neuronal death.
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Role of PUMA in Neuronal Death After Ischemia
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Figure 2. Mitochondrial upregulation of
PUMA preceded cytochrome c release
and caspase-9 activation. A, Western blot
analysis showed that immunoreactivity of
PUMA in the cytosolic fraction was only
slightly detectable at any time point. In
contrast, PUMA immunoreactivity significantly increased in the mitochondrial
fraction 4 and 24 hours after tGCI (n⫽4,
*P⬍0.05). ␤-Actin and COX IV analyses
are shown as internal controls. C indicates
control; OD, optical density. B, Western
blot analysis showed that nuclear p53 was
significantly increased 4 hours after tGCI
(n⫽4, *P⬍0.05). Transcription factor II D
(TFIID) analysis is shown as an internal
control. C, Western blot analysis revealed
that cytosolic cytochrome c and cleaved
caspase-9 were significantly increased in
the hippocampal CA1 subregion 24 hours
after tGCI (n⫽4, *P⬍0.05). ␤-Actin analysis
is shown as an internal control.
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Mitochondrial Localization of PUMA and
Subsequent Cytochrome c Release in the
Hippocampal CA1 Subregion After tGCI
Western blotting showed that PUMA immunoreactivity was
evident as a single band of molecular mass of 19 kDa (Figure
2A). In cytosolic samples from the hippocampal CA1 subregion, PUMA immunoreactivity was slightly detectable and
showed no significant change after tGCI. In contrast, PUMA
expression was significantly increased in mitochondrial samples, peaking at 4 hours and then decreasing by 72 hours after
tGCI. It was barely detectable in the sham-operated brains
(Figure 2A, n⫽4, P⬍0.05).
To confirm the upstream pathway of PUMA, we investigated nuclear p53 upregulation. Western blotting showed that
nuclear p53 significantly increased 4 hours after tGCI (Figure
2B), presenting a pattern similar to that of mitochondrial
PUMA upregulation. To confirm activation of the mitochondrial apoptotic pathway after tGCI, we examined cytochrome
c release and caspase-9 activation. Cytosolic cytochrome c
and cleaved caspase-9 significantly increased at 24 hours
(Figure 2C, n⫽4, P⬍0.05), which suggests cytochrome c
release to the cytosol and subsequent caspase chain reaction
after tGCI. These results indicate that PUMA increases in the
mitochondria before cytochrome c release and caspase-9
activation.
For further investigation of mitochondrial localization of
PUMA after ischemia, we performed double immunofluorescence for PUMA and COX IV, which was used as a
mitochondrial marker. Double immunofluorescence demonstrated that PUMA colocalized with COX IV in the hippocampal CA1 subregion 4 hours after tGCI (Figure 3).
and 20 kDa, respectively, and showed no significant change
at any time point (data not shown). PUMA expression
precipitated by Bcl-XL increased time-dependently and significantly increased at 24 and 72 hours (Figure 4A, n⫽4,
P⬍0.05). PUMA expression precipitated by Bax also increased 24 hours after tGCI (Figure 4B, n⫽4, P⬍0.01). In
contrast, PUMA expression precipitated by Bcl-2 showed no
significant difference at any time point, although it tended to
increase after tGCI (data not shown).
For further investigation of direct interaction between
PUMA and Bcl-XL or Bax, we performed double immunofluorescence, which demonstrated that PUMA-positive cells
colocalized with Bcl-XL–positive cells (Figure 4C) or Baxpositive cells (Figure 4D) in the hippocampal CA1 subregion
24 hours after tGCI. In combination with the coimmunoprecipitation data, these results indicate that PUMA directly
interacts with Bcl-XL and Bax in the hippocampal CA1
subregion after tGCI.
Interaction Between PUMA and Bcl-XL or Bax
After tGCI
To investigate potential direct interactions between PUMA
and Bcl-XL, Bcl-2, or Bax, we performed coimmunoprecipitations in the mitochondrial fraction from the hippocampal
CA1 subregion. With Western blot analysis, Bcl-XL, Bcl-2,
and Bax immunoreactivity was evident as bands of 30, 26,
Figure 3. Double immunofluorescence of PUMA (red) and COX
IV (green) demonstrated that PUMA-positive cells colocalized
with COX IV–positive cells in the hippocampal CA1 subregion 4
hours after tGCI. Nuclei were counterstained with DAPI (blue).
Scale bar⫽50 ␮m.
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Figure 5. The effect of PFT on PUMA expression in the hippocampal CA1 subregion after tGCI. A, Western blot analysis
showed that PUMA expression was significantly decreased in
the PFT-treated animals (PFT) compared with the vehicletreated animals (V) 4 hours after tGCI (n⫽4, **P⬍0.01). COX IV
analysis is shown as an internal control. OD indicates optical
density. B, In an immunofluorescence study, PUMA (red)
expression was decreased in the PFT-treated animals compared
with the vehicle-treated animals 4 hours after tGCI. Nuclei were
counterstained with DAPI (blue). Scale bar⫽50 ␮m.
Figure 4. Interaction between PUMA and Bcl-XL or Bax in the
mitochondrial fraction of the hippocampal CA1 subregion after
tGCI. A, Coimmunoprecipitation analysis of PUMA immunoreactivity precipitated by Bcl-XL showed a significant increase 24
and 72 hours after tGCI (n⫽4, *P⬍0.05). Bcl-XL immunoreactivity precipitated by Bcl-XL was used to show equal precipitation.
C indicates control; IP, immunoprecipitation; IB, immunoblotting;
OD, optical density. B, Coimmunoprecipitation analysis of
PUMA immunoreactivity precipitated by Bax showed a significant increase 24 hours after tGCI (n⫽4, **P⬍0.01). Bax immunoreactivity precipitated by Bax was used to show equal precipitation. C, Representative fluorescent double staining of PUMA
(red) and Bcl-XL (green) in the hippocampal CA1 subregion 24
hours after tGCI. Nuclei were counterstained with DAPI (blue).
Overlapped image demonstrates that PUMA-positive cells colocalized with Bcl-XL–positive cells. D, Representative fluorescent
double staining of PUMA (red) and Bax (green) in the hippocampal CA1 subregion 24 hours after tGCI. Nuclei were counterstained with DAPI (blue). Overlapped image demonstrates that
PUMA-positive cells colocalized with Bax-positive cells. Scale
bars⫽50 ␮m.
PFT Administration
To investigate the regulation of PUMA by p53, we intravenously administered 4 mg/kg of PFT just after reperfusion.
Our previous study indicated that this dose was effective in
inhibiting PUMA expression.8 Western blot analysis showed
that PUMA expression in the mitochondrial fraction from the
hippocampal CA1 subregion was significantly decreased in
PFT-treated animals compared with vehicle-treated animals 4
hours after tGCI (Figure 5A, n⫽4, P⬍0.01). Bax, Bcl-2, and
Bcl-XL expression levels showed no differences between
vehicle-treated and PFT-treated rats (data not shown). An
immunofluorescence study showed that PUMA expression
was decreased in the hippocampal CA1 subregion of the
PFT-treated animals compared with the vehicle-treated animals 4 hours after tGCI, which was compatible with the result
of the Western blot study (Figure 5B). These results indicate
that PFT administration inhibits PUMA upregulation after
tGCI.
SOD1 Overexpression
To confirm that superoxide production is associated with
PUMA induction, we performed double immunofluorescence
of ethidium and PUMA. In the hippocampal CA1 pyramidal
neurons, ethidium signals were shown as small particles in
the cytosol of the nonischemic brains in both the Wt and
SOD1-Tg rats (Figure 6A). Four hours after ischemia, the
hippocampal CA1 neurons showed a marked increase in
punctate and diffuse signals for both ethidium and PUMA in
the Wt rats. However, the increase in signal was less
noticeable in the SOD1-Tg rats. The Western blot analysis
indicated that PUMA expression was significantly decreased
4 hours after tGCI in the SOD1-Tg rats compared with the Wt
rats (Figure 6B, n⫽4, P⬍0.05). Bax, Bcl-2, and Bcl-XL
expression levels showed no differences between the Wt and
Tg rats (data not shown).
We then examined apoptosis-related DNA fragmentation
after tGCI to investigate neuroprotection of SOD1 overexpression. DNA fragmentation in the hippocampal CA1 subregion at 72 hours was significantly decreased in the
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Figure 6. Effect of SOD1 overexpression
on PUMA expression, ethidium signals,
and DNA fragmentation after tGCI. A,
Representative photomicrographs show
fluorescent double staining of PUMA
(green) and ethidium (red) in the hippocampal CA1 subregion. Nuclei were
counterstained with DAPI (blue). Ethidium
signals were seen as small particles in
the cytosol in nonischemic brains of both
the Wt and Tg rats. Four hours after
tGCI, hippocampal CA1 neurons showed
a marked increase in ethidium signals in
the Wt rats. However, the signal increase
was less noticeable in the Tg rats 4 hours
after tGCI. In the Wt rats, the signals for
PUMA increased dramatically at 4 hours
compared with the sham-operated rats
and overlapped with the ethidium signals.
In the Tg rats, PUMA expression was less
strong than in the Wt rats at 4 hours. C
indicates control. Scale bar⫽50 ␮m. B,
Western blot analysis showed that PUMA
expression decreased significantly in the
Tg rats 4 hours after tGCI (n⫽4,
*P⬍0.05). COX IV analysis is shown as an
internal control. C, Apoptosis-related
DNA fragmentation assay. DNA fragmentation at 72 hours was significantly
decreased in the hippocampal CA1 subregion of the Tg rats compared with the
Wt rats (n⫽4, *P⬍0.05).
SOD1-Tg rats compared with the Wt rats at the same time
point, which was compatible with the results of a counting
study of cells positive for terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (Figure 6C, n⫽4,
P⬍0.05).7 In combination with the results of immunofluorescence and Western blotting, these results indicate that SOD1
overexpression reduces superoxide production, PUMA upregulation, and subsequent hippocampal CA1 neuronal death
after tGCI.
Discussion
Important roles for PUMA in apoptosis have been explored
under various conditions. Although the role of PUMA in
cerebral ischemia is unresolved, our results suggest an
important role, through cytochrome c release and caspase
activation. We base this conclusion on the following
findings. First, PUMA increased in mitochondria of vulnerable hippocampal CA1 neurons after tGCI. Second,
PUMA induction temporally preceded cytochrome c release and caspase-9 activation. Third, it localized to
mitochondria and interacted with Bcl-XL and Bax. Fourth,
PUMA upregulation was inhibited by PFT administration
or SOD1 overexpression, both of which have neuroprotective effects against cerebral ischemia through inhibition of
cytochrome c release and caspase activation.7,12,13 Finally,
reduction in oxidative stress by SOD1 overexpression
decreased not only PUMA upregulation but also neuronal
death in the hippocampal CA1 subregion after tGCI. Our
findings are supported by studies reporting that PUMA is
extremely effective in inducing apoptosis. In an in vitro
study, PUMA expression induced rapid apoptosis,5 and
PUMA suppression by an antisense oligonucleotide reduced apoptosis.4 Furthermore, PUMA induces apoptosis
through cytochrome c release and caspase activation.4,5
PUMA also plays an important role in neuronal apoptosis.
PUMA-nullizygous neurons are protected against araCinduced apoptosis,14 and forced expression of PUMA was
sufficient to induce apoptosis in primary neurons.15 PUMA
regulated oxidative stress–induced neuronal apoptosis
through cytochrome c release and caspase activation in a
primary mouse neuron culture.16 It was also necessary for
camptothecin-induced neuronal death in a primary culture of
mouse neurons.17 In our study, PUMA expression was upregulated after tGCI as previously described.18 PUMA expression was inhibited by PFT, which can inhibit p53 transcriptional activity and prevent DNA damage–induced
apoptosis.19 Its expression was also inhibited in SOD1-Tg
rats, resulting in significant neuroprotection. Finally, these
results indicate that PUMA has important roles in delayed
and selective CA1 neuronal death after tGCI.
Although our results of PFT administration demonstrated
that PUMA was regulated at least in part by p53, this finding
is controversial. PUMA was first identified as a direct target
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of the p53 oncogene with 2 putative p53 binding sites.4,5
Gene-knockout studies revealed that DNA damage–induced
p53-dependent apoptosis was severely diminished in PUMAdeficient cells in vitro.20,21 In neuronal cell death, PUMA was
shown to be associated with p53. PUMA-deficient neurons
are resistant to p53-induced neuronal apoptosis.15 However,
several studies have reported that PUMA could also be
induced by a p53-independent mechanism.18,20,22 PUMA
mRNA was induced by p53-independent apoptotic stimuli,
including dexamethasone treatment of thymocytes and serum
deprivation of tumor cells.22 Moreover, PUMA induction
directly links the endoplasmic reticulum stress response to the
mitochondrial apoptosis pathway in neurons after tGCI.18
In our study, PUMA induction after ischemia was significantly inhibited by administration of PFT. Although we
cannot exclude the possibility that upregulation of PUMA
after ischemia is facilitated by other mechanisms, such as
endoplasmic reticulum stress after ischemia,18 these results
suggest that PUMA is controlled at least in part by the p53
transcriptional pathway in CA1 neurons after tGCI.
Recent reports have demonstrated that the potency of
PUMA in apoptosis induction is related to its interaction with
anti- or pro-apoptotic proteins.3–5,16,23 The BH3 domain of
PUMA can promiscuously interact with multiple antiapoptotic Bcl-2 family members.23 PUMA was shown to localize to
mitochondria and to bind to Bcl-2 or Bcl-XL through a BH3
domain.4,5 Furthermore, PUMA could interact with Bax as
well as Bcl-2 or Bcl-XL.3,16 In the present study, coimmunoprecipitation showed that PUMA bound to Bcl-XL and Bax in
the mitochondrial fraction after tGCI. A double immunofluorescence study demonstrated that these protein interactions
occurred in vulnerable CA1 neurons. Binding of PUMA to
Bcl-2 also tended to increase, but not significantly. These
results suggest that the interaction between PUMA and
Bcl-XL and Bax has roles in delayed CA1 neuronal death after
tGCI. However, how this interaction is associated with
ischemic neuronal death requires further investigation.
Reactive oxygen species play important roles in the pathogenesis of central nervous system injury. We have reported
that SOD1 is a crucial endogenous enzyme responsible for
eliminating superoxide and that overexpression of SOD1
reduces superoxide production and protects neurons from
death after transient focal cerebral ischemia24 and tGCI.7
Thus, SOD1-Tg animals are very useful tools for investigating the relation between oxidative stress and ischemic neuronal death. In our study, superoxide production and neuronal
death in the hippocampal CA1 subregion after tGCI were
prevented in the SOD1-Tg rats, results consistent with those
of our previous report in the same tGCI model.25 Furthermore, PUMA upregulation after ischemia was significantly
decreased in the SOD1-Tg rats compared with the Wt rats,
suggesting that reduction in oxidative stress by SOD1 overexpression could modulate PUMA upregulation.
In conclusion, PUMA has potential roles in delayed CA1
neuronal death after tGCI and can hypothetically be a
molecular target for therapy, although our study may lack
direct evidence. To confirm the role of PUMA, PUMAknockout mice or an RNA interference technique should be
used in future studies.
Acknowledgments
We thank Liza Reola and Bernard Calagui for technical assistance,
Cheryl Christensen for editorial assistance, and Elizabeth Hoyte for
figure preparation.
Source of Funding
This study was supported by National Institutes of Health grants P50
NS014543, R01 NS025372, R01 NS036147, and R01 NS038653.
Disclosures
None.
References
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Downloaded from http://stroke.ahajournals.org/ by guest on June 18, 2017
Potential Role of PUMA in Delayed Death of Hippocampal CA1 Neurons After Transient
Global Cerebral Ischemia
Kuniyasu Niizuma, Hidenori Endo, Chikako Nito, D. Jeannie Myer and Pak H. Chan
Downloaded from http://stroke.ahajournals.org/ by guest on June 18, 2017
Stroke. 2009;40:618-625; originally published online December 18, 2008;
doi: 10.1161/STROKEAHA.108.524447
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