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*
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, and
Neurotoxicology Laboratories, Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and Fas ligand treatment. These results indicate that
glutathione peroxidase-sensitive reactive oxygen species play an
important role in regulation of cell death during cerebral I/R by
modulating intrinsic neuronal sensitivity as well as brain inflammatory
reactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ROS produced by the immune system are well-recognized toxic metabolites that can directly cause damage to brain cells. It has become apparent that ROS have a much broader role in the regulation of the immune response itself. For example, ROS might serve as endogenous signals, released by injured tissue to trigger activation of the immune system. ROS are important regulators of many intracellular signaling pathways, leading to the release of inflammatory mediators as well as those that represent reaction of different cell types to the mediators. The signaling pathways involve G proteins, protein tyrosine kinases, protein tyrosine phosphatases, Janus kinases, mitogen-activated kinases, caspases, a variety of transcription factors, etc. (3).
Many studies of the involvement of ROS in regulatory pathways have relied on the use of exogenous sources of ROS and therefore could not completely support their physiological role. To address the role of ROS, particularly those sensitive to intracellular glutathione peroxidase (GPx1) activity in brain I/R, we used transgenic mice overexpressing human GPx1 (4). Experiments with GPx1 transgenic mice subjected to intraluminal blockade of the middle cerebral artery (MCA) as a model system have shown that they are protected against brain I/R damage (5). Our GPx1 animals displayed a significant decrease in infarct volume (48%) compared with nontransgenic mice. Recently, in a model of kidney I/R we were able to show a significant inhibition of activation of neutrophil-attracting chemokines in those animals (6). The current report demonstrates that the increased level of GPx1 activity in transgenic mice modulates inflammatory response in focal brain I/R model of stroke and decreases the sensitivity of brain cells to induced cell death. The data obtained suggest that the development of therapeutic approaches aimed at inhibition of ROS production may be beneficial not only for stroke but also for other inflammation-related neurodegenerative diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of transgenic mice with human GPx1 genes in a C57BL/6 x CBA/J background was previously reported (4, 7). The mouse line GPE23 (containing 200 copies of the human GPx1 gene) was used for these studies. To obtain nontransgenic and heterozygous transgenic animals for the experiments, transgenic founders were bred with (C57BL/6 x CBA/J)F1 mice.
Focal cerebral ischemia
The experimental procedure was previously described (5, 8). These conditions provide reproducible I/R injury in brain of normal and transgenic mice. In brief, 6-mo-old normal or transgenic males, weighing 35 ± 5 g, were subjected to focal I/R using the intraluminal suture method. Mice were deeply anesthetized using 2–3% isoflurane in a 30% oxygen/70% nitrous oxide gas mixture. Body temperatures were monitored and maintained at 37 ± 0.5°C with a feedback-regulated heating pad. After exposing the left carotid artery, 12 ± 1 mm of 5-0 monofilament nylon suture was inserted into the internal carotid artery through the external carotid artery stump just beyond the MCA branch. The animals underwent MCA occlusion for 1 h and were subjected to reperfusion by removing the nylon suture. After recovering from the anesthesia, mice were maintained in an air-conditioned room at 24°C during reperfusion periods. Mice were sacrificed at different time points after surgery. Brains were immediately removed after perfusion with cold PBS and rapidly frozen in liquid nitrogen for obtaining extracts and RNA analysis.
MPO activity assay
Myeloperoxidase (MPO) activity was measured spectrophotometrically at 630 nm, as described previously (9). The 1-ml assay mixture contained 100 µl of heat-inactivated extract, 80 mM phosphate buffer (pH 5.4), and tetramethylbenzidine and H2O2 in final concentrations of 1.6 and 3.0 mM, respectively. MPO activity was expressed in units, where 1 U represents the amount of enzyme degrading 1 µmol H2O2/min, and was standardized with the protein content of the extract (units per milligram of protein).
Lipid peroxidation assay
Lipid peroxidation in the brain was assessed by measurement of malondialdehyde and 4-hydroxy-2(E)-nonenal, using a lipid peroxidation kit (Calbiochem, San Diego, CA) according to the manufacturer’s protocol. Lipid peroxidation was normalized to the protein content of the supernatant.
Preparation of RNA and RNase protection assay
Total RNA was isolated from brain samples using TRIzol reagent
(Life Technologies, Gaithersburg, MD) according to manufacturer’s
protocol. The RNA concentration was measured by a spectrophotometer.
RNase protection assay was performed using a Riboquant kit and an
mCK-3, mCK-5, and mAPO-3 Multiprobe Template Set (BD PharMingen, San
Diego, CA) according to the manufacturer’s protocol. The mCK-3
Multiprobe Template Set contains RNA probes for the mouse chemokines
lymphotactin, RANTES, eotaxin, macrophage-inflammatory protein
(MIP)-1
, MIP-1, MIP-2, inducing protein 10 (IP-10), monocyte
chemoattractant protein (MCP)-1, and TCA-3; the mCK-5 Multiprobe mCK-5
Template Set contains RNA probes for mouse cytokines TNF-,
leukotriene-, TNF-, IFN-
, IFN-, TGF-1, and TGF-2, and
the mAPO-3 Multiprobe Template Set contains RNA probes for mouse TNFR
superfamily proteins Fas ligand (FasL), Fas, Fas-associated death
domain protein (FADD), Fas-associated phosphatase, Fas-associated
protein factor (FAF), TNF-related apoptosis-inducing ligand (TRAIL),
TNFR p55, TNFR-associated death domain protein (TRADD), receptor
interacting protein (RIP), and caspase-8. RNA probes and for
positive controls (L32 and GAPDH) were generated in vitro using a T7
polymerase transcription kit and [-32P]UTP
as a label. A standard curve was made using undigested probes as
markers, and the identities of RNase-protected bands were established.
To compare RNA amounts in the protected bands, the films were scanned
with an imaging densitometer (GS-670; Bio-Rad, Hercules, CA), and final
values were factored relative to GAPDH levels.
Western blotting
Proteins were analyzed by Western blot analysis as reported previously (6). In brief, tissue samples were homogenized in RIPA buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml leupeptin, aprotinin, pepstatin, and phosphatase inhibitors (10 mM NaF, 1 mM NaVO4, 1.5 mM Na2MoO4, 1 mM benzamidine, 20 mM glycerophosphate, and 20 mM p-nitrophenyl phosphate)). SDS-PAGE (5x) sample buffer was added to the final concentrations of 50 mM Tris-HCl (pH 6.8), 2% SDS, 100 mM DTT, 0.006% bromophenol blue, and 10% glycerol. Lysates were centrifuged at 13,000 x g for 20 min, and supernatants were used for 10% PAGE. After electrophoresis gels were electroblotted onto a polyvinylidene difluoride membrane and analyzed using anti-Fas (sc-1023) and anti-FasL (sc-6237) Abs (both from Santa Cruz Biotechnology, Santa Cruz, CA), and phospho-SAPK/Erk kinase (SEK1/MKK4) (Thr261) and phospho-c-Jun (Ser63) Abs (Cell Signaling Technology, Beverly, MA). Proteins were detected with the Phototope-HRP Western Blot Detection kit (New England Biolabs, Beverly, MA).
Cortical cell cultures, experimental treatments, and quantification of neuron survival
Primary neuronal cultures were prepared as described by
Berezovska et al. (10). In brief, cells were isolated from
neocortex of embryonic day 16–17 normal and transgenic mice and placed
in Neurobasal medium (Life Technologies) with 10% FBS. The neurons
were plated on 0.025% poly-D-lysine-coated plates. After
1 h the medium was replaced with serum-free Neurobasal medium
containing 2% B27 supplement. The cultures were maintained at 37°C
with 5% CO2 in a tissue culture incubator.
Experiments were performed in 10- to 12-day cultures; >95% of the
cells in the cultures were neurons, as judged by cell morphology and
immunostaining with Abs against neurofilaments. Neurons at this age
become vulnerable to hypoxia and glucose deprivation (11, 12). Cells in serum-free medium without glucose were exposed to
hypoxia in a modular incubator chamber (Billups-Rothenberg, Del Mar,
CA) by flushing the chamber with humidified 95%
N2/5% CO2 as described
previously (13). A progressive drop in
pO2 was measured with a Clark-style probe (model
DO-166; Lazar Research Laboratory, Los Angeles, CA) installed in the
chamber and placed in the culture dish with medium.
pO2 usually dropped to approximately 5 torr by
the end of 4-h hypoxia. At the end of hypoxia, medium was changed to
normal growth medium with or without TNF-/FasL (recombinant murine
TNF- and SUPERFAS ligand were obtained from R&D Systems
(Minneapolis, MN) and Alexis (San Diego, CA), respectively), and
cultures were returned to the incubator in air and 5%
CO2. Neuronal cell viability was assessed by the
release of lactate dehydrogenase (LDH) into the culture medium. LDH
activity was measured by using a commercial kit (Cyto Tox 96
Cytotoxicity Assay; Promega, Madison, WI). The percent cytotoxicity was
expressed as (experimental LDH release/maximum LDH release)
x 100.
Preparation of nuclear extracts and EMSA
Nuclear extracts were prepared from sham-operated normal and
transgenic mice as well as animals after ischemia and 60 min of
reperfusion, as previously described (6). Brains were
dissected to isolate tissues from ipsilateral and contralateral
cortexes. Aliquots of the extract were stored at -80°C. Protein
content was assayed using Bio-Rad protein reagent. The following
oligonucleotides were used in EMSAs: 5'-CGCTTGATGAGTCAGCCGGAA-3'
containing AP-1 consensus site; several oligonucleotides bearing the
potential AP-1 site were deduced from the mouse FasL promoter sequence:
-282 to -252
(5'-CGCTGACTTGCTGAGTTGGACTCAGGCAGGC-3'),
-233 to -215 (5'-TCTCAGTTAGCACAGAGAC-3'), and -119
to -103 (5'-AAGTGAGTGGCTGTCTC-3'). For each
oligonucleotide, the two complementary strands were synthesized using
an Applied Biosystems automated DNA synthesizer (Foster City, CA).
These oligonucleotides were end-labeled using polynucleotide kinase and
[-32P]ATP (NEN Life Science Products,
Boston, MA), annealed, and purified by PAGE. For EMSA, 3 µg of
extract was incubated in a reaction mixture containing 20 mM HEPES (pH
7.9), 60 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.2 mM
EDTA, 0.5% Nonidet, 1 µg poly(dI-dC), and 8% glycerol in a final
volume of 20 µl for 20 min at 4°C. After preincubation,
105 cpm radiolabeled DNA probe was added, and the
incubation was continued for 20 min at room temperature. The
DNA-protein complexes were separated on native 5% polyacrylamide gels
in 0.25x Tris-borate-EDTA buffer. Supershift assay was conducted after
incubation of the nuclear extracts with Abs (0.5 µg of anti-mouse
c-Fos, c-Jun, ATF-2, p50, p65 Abs; sc-52X, sc-45X, sc-6233, sc-372X,
sc-7178X, respectively, from Santa Cruz Biotechnology) for 20 min at
4°C followed by EMSA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the presence of neutrophils in brain infarcts we measured MPO activity. It was reported by Matsuo et al. (14) that the increase in brain MPO activity after transient focal ischemia correlates closely with the neutrophil infiltration and that this process is implicated in postischemic brain injury. According to our measurements in normal mice, the level of MPO activity increases in infarct regions compared with noninfarcted contralateral cortex by 70% at 24 h after I/R (10.7 ± 1.7 vs 6.3 ± 0.8 U/g; p < 0.05; n = 6). In contrast, almost no changes in MPO activity were observed in GPx1 transgenic mice (6.2 ± 1.0 vs 6.5 ± 1.1 U/g, infarct and contralateral side, respectively; n = 6). Data from detailed histopathologic analysis and quantitative neutrophil counts in brain sections from normal and transgenic mice after I/R are well correlated with MPO measurements and are currently included in a manuscript submitted for publication.5 Therefore, the results indicate that overexpression of glutathione peroxidase (GP) inhibits neutrophil transmigration after focal I/R.
Effect of GP overproduction on the level of inflammatory mediators
It is known that the inflammatory response is mediated by a series
of regulatory proteins such as cytokines. To measure the expression of
inflammatory genes we took advantage of Riboquant RNase protection kits
and Multiprobe Template Sets mCK-3, mCK-5, and mAPO-3 (Fig. 1
). To compare RNA amounts of the
protected bands, exposed films were scanned by an imaging densitometer
and final values were factored relative to GAPDH levels. Results are
shown in Table I.
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, MIP-1, MIP-2, IP-10, and MCP-1.
Among other cytokines, TNF-, IL-6, and FasL were most affected.
Another death-inducing ligand, TRAIL, as well as a series of adapter
proteins (FADD, TRADD, RIP, and FAF) were also inhibited.
Interestingly, the expression of transmembrane receptors, Fas and TNFR
p55, which are capable of triggering cell death in neurons and other
brain cells, decreased >2.5 times.
Tissue extracts from two brain regions, the core of the infarct and the
pyriform cortex, containing cells predominantly dying by necrosis and
apoptosis, respectively, were analyzed by Western blotting to further
characterize expression of FasL/Fas on a protein level. Fas and FasL
proteins were dramatically lower in brains of GPx1 transgenic mice
compared with normal animals at 8 and 24 h after I/R (Fig. 2). Importantly, the overall level of
proteins in both regions was not very significantly different,
indicating that mechanisms other than ligand/receptor levels are
responsible for the character of the neuronal death pathway, necrotic
or apoptotic. These mechanisms may involve levels of other mediators,
differences in intrinsic cell sensitivity from those brain regions, or
significantly deeper oxygen deprivation and ATP depletion in a core
region of the infarct compared with the penumbral zone. It was recently
reported that both TNF-- and FasL-induced pathways could lead to
apoptosis as well as necrosis (15, 16), and in at least
one model system (17) it has been reported that cellular
ATP concentration is a critical parameter in the decision between the
two cell death pathways.
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Intracellular ROS produced during I/R as well as those triggered
by ligand/receptor TNF superfamily proteins are active players in the
execution of apoptosis and necrosis. Therefore, one of the primary
mechanisms that might be involved in GPx1 protection is modulation of
cell sensitivity to induction of cell death. According to our
previously reported measurements, GP activity was more than two times
higher in purified cortical neurons from GPx1 animals compared with
that in normal mice.5 The ability of antioxidants such as
N-acetylcysteine to protect neuronal cells against
TNF--induced apoptosis in cell culture has been reported
(18). To address this issue we exposed cortical neurons
from normal and transgenic mice to an in vitro model of I/R,
anoxia/glucose depletion following reoxygenation/high glucose
treatment, as well as different concentrations of exogenous TNF- and
FasL. Neuronal viability decreased significantly at 18 and 24 h of
normoxia following hypoxic insult (Fig. 3A). Nevertheless, cortical
neurons from transgenic mice revealed almost 2-fold greater resistance
to anoxia/glucose depletion compared with cells from normal animals.
Increased protection in response to TNF- and FasL was also observed
in GPx1 cell cultures (Fig. 3B). Moreover, cortical neurons
overexpressing GPx1 were more resistant to combined treatment by
anoxia/glucose depletion and TNF- or FasL (Fig. 3C). A
weak additive effect of both types of cell injury inducers was
observed, probably due to the use of the same or similar
death-executing mechanisms, which were saturated under experimental
conditions. The data indicate that an increased level
of GP in neurons interferes with the mechanisms of direct cellular
toxicity involved in brain I/R injury.
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Transcriptional regulation of FasL and Fas genes is mediated by
several transcription factors, including AP-1, NF-
B, NF-AT, ATF-2,
Egr3, etc. Most studies performed to date focus on mechanisms of
activation involved in lymphocyte homeostasis. Two studies reported
stress-induced activation of FasL expression via induction of the
following sequences: c-Fos/c-Jun-specific AP-1 site or
c-Jun/ATF-2-specific site (19, 20). Because there is
significant evidence to indicate importance of AP-1 subunits in neural
homeostasis and during focal I/R injury in particular (21, 22), we analyzed the DNA binding activity of AP-1 consensus
oligonucleotides as well as published and potential AP-1 binding sites,
identified using a TRANSFAC search in the mouse FasL promoter
region (http://transfac.gbf.de/TRANSFAC/; Braunschweig, Germany).
Results are shown in Fig. 4. All four
tested oligonucleotides revealed significantly enhanced binding in
damaged brain regions of nontransgenic mice after I/R. Induction was
very low in corresponding samples from GPx1 mice. An Ab supershift
assay was used to analyze transcription factors involved in complex
formation. One of the bands in three oligonucleotides (consensus AP-1,
-282 to -252 and -119 to -103) contained c-Fos and c-Jun, whereas
two bands in DNA binding with oligonucleotide -233 to -215 contained
c-Jun and ATF-2, but not c-Fos. Because oligonucleotide -119 to -103
also contains the binding site of NF-B identified by Matsui et al.
(23) as a factor critically involved in FasL activation in
T cells, we tested anti-p65 and p50 Abs. As shown in Fig. 4C, the results eliminated the possibility of involvement of
this transcription factor in complex formation with oligonucleotide
-119 to -103. The existence of two major complexes in DNA binding
with oligonucleotide -233 to -215 is in agreement with the data
reported by Faris et al. (19), who first identified this
stress-responsive element and showed its binding with c-Jun and ATF-2,
but not c-Fos. None of the described complexes was supershifted with
normal serum (Abs; see Fig. 4). The specificity of the binding was
verified in the presence of competitive and noncompetitive
oligonucleotides (data not shown). Several additional bands not shifted
with tested Abs (Fig. 4, A and C) are currently
under investigation.
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, and probably TRAIL, which
induce neuronal cell death. Because the level of activation of those
transcription factors was sensitive to GP overexpression in GPx1
transgenic mice, we analyzed phosphorylation of SEK1, which is upstream
of the JNK/SAPK, as well as the level of phospho-c-Jun by Western blot
using Abs specific for phosphorylated forms of the proteins. Results
are shown in Fig. 5. The data indicate
that the level of phospho-c-Jun was significantly diminished in brain
regions of transgenic mice. The level of phosphorylated SEK1 was
dramatically decreased in the core region of the infarct and was only
slightly affected in pyriform cortex. This result might indicate
differences in sensitivity of various levels of SAPK/JNK signal
transduction mechanisms in brain regions undergoing predominantly
apoptotic or necrotic cell death in transgenic mice after I/R compared
with normal animals. It is important to note that this effect was
observed very early, before the development of histologically evident
cell damage. This result is in agreement with a role of ROS in
activation of the JNK/SAPK cascade, which is well documented
(26).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In wild-type mice brain injury was accompanied by infiltration by
neutrophils. In contrast, brains of transgenic mice showed decreased
neutrophil migration. One possibility for this effect is the difference
between transgenic and wild-type mice in the ability to produce
chemoattractant factors. Neutrophils in brain ischemia respond to a
variety of chemoattractant signals, including chemokines from injured
cells. We observed dramatic differences in the production of several
key CXC and CC chemokines (such as MIP-2, MIP-1, MIP-1, IP-10,
and MCP-1) attracting neutrophils and macrophages in Gpx1 transgenic
and normal mice as early as 3 h of reperfusion. This effect
preceded the appearance of migrating leukocytes in tissue parenchyma.
It is now becoming clear that chemokines and their receptors might have
additional roles during I/R other than chemotaxis, such as gliosis,
regulation of neuronal sprouting, etc. (29). The role of
ROS in chemokine activation is well recognized (30).
Another possible target for regulation by GP-sensitive pathways
includes adhesion molecules, which play key roles in leukocyte
recruitment and are known to be sensitive to oxidative stress
(30, 31).
Chemokines may diffuse through the neuropil to induce neutrophil margination in blood vessels; however, such attraction may not be sufficient to induce neutrophils to traverse the vascular wall. For example, direct intracerebral injection of neutrophil-activating protein-1/IL-8 caused neutrophil margination, but no infiltration into the perivascular space, indicating an intact vascular barrier (32). To enter the brain parenchyma, inflammatory cells must either traverse the endothelial cells cytoplasm or pass between cells, a process impeded by endothelial tight junctions. In wild-type mice, compromise of vascular integrity was indicated by the appearance of perivascular space containing edema fluid, as we reported previously (5). Transgenic animals manifested less vascular permeability, possibly due to an increased constitutive ability of GPx1-overexpressing cells to withstand oxidative stress and toxic metabolites. A dramatic effect of GP overexpression on leukocyte behavior has been previously reported in two other inflammation-related injury models, kidney I/R and LPS-induced septic shock (6, 7). Significant resistance to injury in both models correlated with the level of key inflammatory mediators.
Measurement of mRNA for several cytokines after brain I/R indicates
significant effects of GP on the regulation of their expression. Levels
of TNF-, FasL, and especially IL-6 mRNA as well as receptors for
TNF- and FasL (TNF Rp55 and FasL) were down-regulated after 3 h
of reperfusion. There is strong evidence implicating several
inflammatory cytokines and their receptors in exacerbation of focal I/R
damage, although protective effects for TNF- and IL-6 also were also
reported. Levels of IL-6 had a strong correlation with neurologic
function and infarct size in animal experimental ischemia as well as in
stroke patients (33, 34). It was also demonstrated that
the IL-6 system plays an important role in local inflammatory reactions
by amplifying leukocyte recruitment and changing endothelial
permeability (35). The ability of GPx1 to modulate
the level of IL-6 is most likely due to the inhibitory effect on ROS
production, which is known by several investigators to stimulate
transcriptional activation of IL-6 (36, 37).
Increased production of TNF- during permanent and transient ischemia
in experimental models and clinical studies is well documented
(38). Its inhibition by pharmacological agents,
neutralizing Abs, or soluble receptors has a protective effect. Despite
several reports of beneficial effects of this cytokine from in vitro
studies and knockout mice, endogenous production of TNF- in I/R
brain models is deleterious (38). TNF- is a potent
activator of necrosis and/or apoptosis through TNF Rp55 receptor
depending on the cell type and/or the intracellular ATP concentration
(15).
Involvement of other members of the death receptor family, Fas and
FasL, in active neuron killing during I/R has attracted significant
attention (39, 40). Both molecules were activated during
brain I/R. FasL-induced apoptosis in primary neurons and most
importantly in lpr mice expressing dysfunctional Fas was
more resistant to reversible cerebral artery occlusion (25, 41). According to the first report, FasL in the ischemic
penumbra was exclusively expressed in neurons, whereas the second
report shows positive staining in cells of neuronal and microglial
origin. A role for FasL expression in the control of leukocyte
extravasation as well as maintenance of vascular integrity was also
demonstrated (42). Our data indicate that the expression
of TNF- and TNFR-1 as well as Fas and FasL during I/R was
significantly affected by GPx1 overexpression. Therefore, important
mechanisms of protection in GPx1 transgenic mice may include regulation
of expression of executors of ischemia-mediated neuronal cell death as
well as cellular sensitivity to these molecules. Several transcription
factors were shown to be important for up-regulation of Fas and FasL
expression. Their activation during the cellular response to I/R may be
mediated by several signal transduction mechanisms, including the
JNK/SAPK pathway. The DNA binding activity of AP-1-binding
transcription factors as well as SEK1/MKK4 and c-Jun phosphorylation
were very sensitive to GP overexpression in transgenic mice. Among
other possible direct targets of redox regulation are transcription
factors themselves or other regulatory redox-sensitive proteins, such
as thioredoxin or GST. Proteasome activity, which plays an important
role in transcription factor activation, was shown to be affected by
the level of GPx1 activity in transgenic and knockout mice
(43). Recently, another group (22) reported
the ability of Cu,Zn-superoxide dismutase overexpression to reduce
focal I/R-induced AP-1 activation, although no specific targets of this
regulation were demonstrated.
The behavior of AP-1-related transcription factors was examined in the
present report. As previously noted, involvement of other transcription
factors, especially NF-B, in the mediation of FasL activation was
previously demonstrated. NF-B constitutes an important component of
cellular response to oxidative stress. We as well as others have
previously reported the ability of antioxidant enzymes in transgenic
animals to inhibit activation of this transcription factor during I/R
(6, 44). Characterization of the behavior of other
redox-sensitive transcription factors and the genes that they regulate
is currently in progress.
We also tested whether protection may be provided by the increased antioxidant potential of neurons themselves. In vitro studies show increased protection of purified cortical neurons from transgenic mice to hypoxia/reoxygenation-induced injury, to treatment by death-inducing cytokines, as well as to combined treatment by both insults. These results support the hypothesis that ROS are implicated in Fas-induced apoptosis of neurons, as demonstrated in other type of cells (45, 46).
The mechanism of the GPx1 protective role during brain I/R injury was recently a focus of a report by Crack et al. (47). These authors demonstrated that GPx1 knockout mice display increased infarct size and exacerbated apoptosis compared with normal animals after MCA occlusion and reperfusion. Those data correlate with the high resistance of our GPx1 transgenic mice as well as neurons from those animals in cell culture to inducers of cell death. Importantly, GPx1 knockout mice revealed significant increases in caspase-3 activation as early as 8 h of reperfusion, in contrast to normal mice. Caspase-3 is one of the effector caspases that may be activated by oxidative stress (48). Therefore, the authors suggested that ROS sensitive to GPx1 play an important role in regulation of the apoptosis cascade. The effect of modulation of the level of Cu,Zn-superoxide dismutase on caspase-8 activation in transgenic mice was also reported (49). Interestingly, at 8 h of reperfusion we already observed in our model a significant difference in FasL expression, which represents one of the key initiator of apoptosis. It most likely influences the level of activation of executor apoptotic mechanisms, including caspases. Which of those death-initiating or -executing mechanisms is more sensitive to ROS needs to be further investigated.
In conclusion, GPx1 overexpression in transgenic mice provides significant protection against focal I/R injury by multiple mechanisms. By modulating the activity of cells producing inflammatory mediators and by decreasing the number of injured brain cells, GPx1 significantly attenuated the postischemic inflammatory reaction. On a molecular level, early transcriptional activation of cytokines, including several classes of chemokines and families of death-inducing cytokines and their receptors was a target of GPx1-sensitive ROS. This effect was mediated in part through stress-responsive kinases and transcription factors. The data suggest that GPx1 is able to interfere efficiently not only with major neuronal death pathways, but also with postischemic inflammation. Recent studies with GPx1 mimetic BXT-51072 show a significant neuroprotective effect against oxidative as well as excitotoxic-neuronal cell death (50). Therefore, GPx1 and compounds with similar activity represent promising tools for future therapeutic interventions designed to prevent or regulate pivotal elements of brain damage following ischemic stroke.
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Footnotes |
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2 Current address: Department of Surgery, Kurume University School of Medicine, Fukuoka, Japan.
3 Address correspondence and reprint requests to Dr. Oleg Mirochnitchenko, Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854. E-mail address: mirochol{at}rwja.umdnj.edu
4 Abbreviations used in this paper: I/R, ischemia/reperfusion; JNK, c-Jun N-terminal kinase; FADD, Fas-associated death domain protein; RIP, receptor interacting protein; TRADD, TNFR-associated death domain protein; SAPK, stress-activated protein kinase; FasL, Fas ligand; FAF, Fas-associated protein factor; SEK1/MKK4, SAPK/Erk kinase; GP, glutathione peroxidase; GPx1, intracellular GP; IP-10, inducing protein 10; LDH, lactate dehydrogenase; TRAIL, TNF-related apoptosis-inducing ligand; MCA, middle cerebral artery; MCP, monocyte chemoattractant protein; MIP, macrophage-inflammatory protein; MPO, myeloperoxidase; ROS, reactive oxygen species.
5 N. Ishibashi, O. Prokopenko, M. Weisbrot-Lefkowitz, K. R. Reuhl, and O. Mirochnitchenko. Glutathione peroxidase inhibits cell death and glial cell activation following experimental stroke. Submitted for publication.
Received for publication October 9, 2001. Accepted for publication December 5, 2001.
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 C. A. Taylor, M. Senchyna, J. Flanagan, E. M. Joyce, D. O. Cliche, A. N. Boone, S. Culp-Stewart, and J. E. Thompson Role of eIF5A in TNF-{alpha}-Mediated Apoptosis of Lamina Cribrosa Cells Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3568 - 3576. [Abstract] [Full Text] [PDF]
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 J. Zitzler, D. Link, R. Schafer, W. Liebetrau, M. Kazinski, A. Bonin-Debs, C. Behl, P. Buckel, and U. Brinkmann High-throughput Functional Genomics Identifies Genes That Ameliorate Toxicity Due to Oxidative Stress in Neuronal HT-22 Cells: GFPT2 Protects Cells Against Peroxide Mol. Cell. Proteomics, August 1, 2004; 3(8): 834 - 840. [Abstract] [Full Text] [PDF]
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 C. M. Maier, L. Hsieh, F. Yu, P. Bracci, and P. H. Chan Matrix Metalloproteinase-9 and Myeloperoxidase Expression: Quantitative Analysis by Antigen Immunohistochemistry in a Model of Transient Focal Cerebral Ischemia Stroke, May 1, 2004; 35(5): 1169 - 1174. [Abstract] [Full Text] [PDF]
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 C.-Y. Kuan, A. J. Whitmarsh, D. D. Yang, G. Liao, A. J. Schloemer, C. Dong, J. Bao, K. J. Banasiak, G. G. Haddad, R. A. Flavell, et al. A critical role of neural-specific JNK3 for ischemic apoptosis PNAS, December 9, 2003; 100(25): 15184 - 15189. [Abstract] [Full Text] [PDF]
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 L. Ding-Zhou, C. Marchand-Verrecchia, B. Palmier, N. Croci, P.-E. Chabrier, M. Plotkine, and I. Margaill Neuroprotective Effects of (S)-N-[4-[4-[(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2-thiophenecarboximid-amide (BN 80933), an Inhibitor of Neuronal Nitric-Oxide Synthase and an Antioxidant, in Model of Transient Focal Cerebral Ischemia in Mice J. Pharmacol. Exp. Ther., August 1, 2003; 306(2): 588 - 594. [Abstract] [Full Text] [PDF]
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