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The Journal of Neuroscience, July 1, 2001, 21(13):4668-4677
Activation of the Nuclear Factor-
B Is a Key Event in
Brain Tolerance
Nicolas
Blondeau,
Catherine
Widmann,
Michel
Lazdunski, and
Catherine
Heurteaux
Institut de Pharmacologie Moléculaire et Cellulaire, Centre
National de la Recherche Scientifique, Unité Mixte de Recherche
6097, Sophia Antipolis, 06560 Valbonne, France
 |
ABSTRACT |
The transcription factor nuclear factor-
B (NFB) is an
ubiquitously expressed inducible regulator of a broad range of genes and plays a pivotal role in cell death and survival pathways. Three
models of brain tolerance (ischemic, epileptic, and polyunsaturated fatty acid-induced preconditioning), known to confer resistance to
neurons against ischemia or status epilepticus, were used to determine
whether NFB mediated the late preconditioning. A sublethal 3 min
ischemia, a dose of 5 mg/kg kainic acid (KA5) or 500 nmol of linolenic
acid (LIN500) led to a rapid increase of NFB DNA-binding activity
and nuclear translocation of p65 and p50 subunits of NFB in neurons.
Pretreatment with the NFB inhibitor diethyldithiocarbamate or B
decoy DNA blocked the increased DNA-binding activity and the nuclear
translocation of NFB and abolished the neuroprotective effects of
different delayed preconditionings against severe ischemia or epilepsy.
The inhibition of NFB observed in rats preconditioned with 3 min
ischemia, KA5 or LIN500 treatments compared with ischemic or epileptic
controls was correlated with the prevention of the inducible
degradation of the inhibitory protein IB
. Preconditioning probably inhibits the activation of NFB by interfering with a pathway that leads to the direct transcriptional activation of IB
by NFB itself. The present work provides evidence that activation of
NFB is a crucial step in the signal transduction pathway that underlies the development of brain tolerance and may open new strategies in the prevention of cerebral diseases, such as ischemia or epilepsy.
Key words:
NFB; brain preconditioning; ischemia; kainic acid; excitotoxicity; polyunsaturated fatty acids
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INTRODUCTION |
Noxious stimuli applied at doses
close to but below the threshold of cell injury induce adaptative
responses that protect the brain against additional stress from the
same (tolerance) or other (cross-tolerance) stimuli. Among different
stresses, hypoxia (Gidday et al., 1994
), ischemia (Kitagawa et al.,
1990; Kirino et al., 1991; Liu et al., 1992; Simon et al., 1993;
Glazier et al., 1994; Heurteaux et al., 1995; Matsushima and Hakim,
1995; Toyoda et al., 1997), seizures (Plamondon et al., 1999), anoxia (Perez-Pinzon et al., 1996), spreading depression (Kawahara et al.,
1994; Matsushima et al., 1996), heat (Chopp et al., 1989; Kitagawa et
al., 1991b), oxidative stress (Ohtsuki et al., 1992), polyunsaturated
fatty acids (PUFAs) treatment (our unpublished data), and
inhibitors of oxidative phosphorylation (Riepe et al., 1997) induce
tolerance to subsequent cerebral (focal or global) ischemia.
Similarly, kainic acid (KA)- or bicuculline-induced seizures
and hippocampal kindling reduce the injurious impact of a second
epileptic challenge (Kelly and McIntyre, 1994; Sasahira et al., 1995;
Plamondon et al., 1999). The existence of early and delayed phases of
preconditioning against brain injuries is now as well established as it
is in cardiac preconditioning (Millar et al., 1996). Clearly, the first
phase occurs within minutes and is transient (Perez-Pinzon et al.,
1997), whereas the delayed phase takes hours to become apparent and
lasts for days (Kitagawa et al., 1990; Kirino et al., 1991). Recent
studies show that the initial signals responsible for triggering the
development of both preconditionings involve the opening of
ATP-sensitive K+ channels
(KATP channels) via the activation of adenosine
A1 receptors (Heurteaux et al., 1995; Reshef et
al., 1998a,b; Plamondon et al., 1999; Blondeau et al., 2000). However,
delayed preconditioning requires de novo synthesis of
proteins, promoting neuronal survival, including heat shock protein
70 (Kitagawa et al., 1990, 1991a; Liu et al., 1993; Nishi et
al., 1993; Simon et al., 1993; Blondeau et al., 2000), Bcl-2 (Shimazaki
et al., 1994), and superoxide dismutase (MnSOD) (Toyoda et al., 1997).
It seems likely that the signaling pathway of late preconditioning
would include the activation of transcription factors that drive the
expression of proteins responsible for neuroprotection.
One of the transcription factors that could activate gene expression in
response to epileptic or ischemic preconditioning is the nuclear
factor-B (NFB). This oxidative responsive transcription factor
plays a pivotal role in neuronal survival and plasticity (for review,
see Mattson et al., 2000). NFB is activated by various intercellular
signals, including cytokines, neurotrophic factors, and
neurotransmitters. Oxidative stress and elevation of intracellular calcium levels are particularly important inducers of NFB
activation. Stimulation of glutamate receptors and membrane
depolarization lead to activation of NFB in hippocampal pyramidal
neurons and cerebellar granule neurons (Guerrini et al., 1995;
Kaltschmidt et al., 1995). NFB activity is increased in neurons and
glial cells in both neurodegenerative disorders (Hunot et al., 1997; Kaltschmidt et al., 1997; Lukiw and Bazan, 1998) and models of stroke,
cardiac arrest, or epilepsy (Rong and Baudry, 1996; Clemens et al.,
1997; Gabriel et al., 1999). The involvement of NFB in the
inhibition of apoptosis is now well established (Mattson et al., 2000).
NFB plays a central role in the induction of neuroprotective antiapoptotic gene products, such as MnSOD and Bcl-2 that are known to
contribute to ischemic tolerance (Toyoda et al., 1997). NFB is made
up of two prototypical subunits of 50 kDa (p50) and 65 kDa (p65; RelA)
that belong to the Rel family. The most usual form of NFB is a
heterodimer of p65 and p50, which normally exists in the cytoplasm in a
dormant form bound to one of a member of inhibitory proteins called
IB, IB
, IB
, p105, and p100 (for review, see
Verma et al., 1995; Baeuerle and Baltimore, 1996). During activation,
NFB dissociates from I and translocates as a p50/p65 dimer to
the nucleus as a result of the complete proteolytic degradation of
I proteins or the partial degradation of p105 and p100
precursors. Phosphorylation by a protein kinase complex I kinase
and ubiquitination of I are necessary for dissociation of I
from the transcription dimer, which binds to consensus sequences
in the enhancer region of -responsive genes and then can initiate
gene transcription (Chen et al., 1995; Verma et al., 1995; Baeuerle and
Baltimore, 1996).
The purpose of the present study is to test the hypothesis that the
delayed preconditioning is mediated by NFB and to characterize the
mechanisms that trigger the translocation of NFB during
preconditioning in three models of brain tolerance (ischemic,
epileptic, and polyunsaturated fatty acid-induced preconditioning)
known to confer resistance to hippocampal neurons against neuronal
injury associated with ischemia or status epilepticus.
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MATERIALS AND METHODS |
Animals. Experiments were performed on male Wistar
rats weighing 250-300 gm (Charles River Laboratories, St. Aubin,
France). The animals, maintained on a 12 hr light/dark cycle,
were given food and water ad libitum. They were acclimatized
for at least 1 week before drug treatments or surgery. All animal
experiments were performed in accordance with NIH Guide for the
Care and Use of Laboratory Animals. All efforts were made to
minimize the number of animals used and their suffering.
Preconditioning procedures. Two different paradigms of brain
injury were used: the four-vessel occlusion model of transient global
forebrain ischemia (Pulsinelli and Brierley, 1979) and KA-induced
(seizure-mediated) excitotoxic damage (Lothman and Collins, 1981) to
hippocampal neurons. Vehicle-injected or sham-operated rats were used
as negative controls for ischemia and epilepsy. Preconditioned groups
were as follows. Ischemic preconditioned rats received a 3 min
sublethal global ischemia (I3) before severe 6 min ischemia (I6);
KA5-preconditioned animals were intraperitoneally injected with a 5 mg/kg dose of KA 3 d before a second KA7.5 challenge (7.5 mg/kg).
PUFA-preconditioned rats were intravenously injected with linolenic
acid (LIN500, 500 nmol/kg body weight) 3 d before KA7.5 injection
or 6 min ischemia. Vehicle-injected or sham-operated animals treated
3 d before KA7.5 injection or 6 min ischemia were used as controls
for the preconditioned groups. Rats were killed at 1, 24, and 72 hr
after the last treatment (n = 6 per time per group).
Transient complete forebrain ischemia. Transient complete
forebrain ischemia was performed by four-vessel occlusion as described previously (Pulsinelli and Brierley, 1979; Heurteaux et al., 1995). Briefly, rats were deeply anesthetized by inhalation of 2% halothane mixed with 30% oxygen and 70% nitrous oxide. The vertebral arteries were irreversibly occluded by electrocoagulation, and a small-diameter silicon tubing was looped around the carotid arteries to facilitate subsequent occlusion. On the following day, both carotid arteries were
clamped with microvascular clamps for 3 or 6 min in awake and
spontaneously ventilating animals. Rats lost their righting reflex
within 1 min of carotid clamping. Cessation of electroencephalographic activity was confirmed during ischemic insults. Heartbeat, arterial blood pressure, and core temperature were continuously monitored during
surgery, ischemia, and drug administration. The body temperature of
rats was supported with a heating blanket during and in the hours after
surgery, ischemia, and reperfusion. No difference in body temperature,
heartbeat, and arterial blood pressure has been seen among the
different groups.
Drug treatments. All drugs were freshly mixed on the day of
experimentation. KA (Sigma, St. Louis, MO) was dissolved in NaCl 0.9%
solution and injected intraperitoneally (5 and 7.5 mg/kg, i.p.). All
rats that were treated with KA exhibited seizures within the first hour
after injection. LIN was first dissolved in ethanol at a molar
concentration and then diluted in NaCl 0.9% solution to reach a final
concentration of 500 µM. LIN500 (500 nmol/kg body weight, i.v.) was injected 3 d before KA (7.5 mg/kg, i.p.) injection or 6 min ischemia. The pH of the different solutions was
adjusted to 7.0. The diethyldithiocarbamate (DTTC) (Sigma) was
dissolved in NaCl 0.9% solution and injected intraperitoneally (150 mg/kg, i.p.) 15 min before LIN500 or KA5 administration or sublethal 3 min ischemia.
Administration of B decoy DNA. As
reported previously (Smith-Swintosky et al., 1994; Yu et al., 1999),
double-stranded B decoy DNA was prepared by annealing complementary
single-stranded oligonucleotides of the following sequences:
5'-GAGGGGACTTTCCCT-3' and 5'-AGGGAAAGTCCCCTC-3'. Control DNA with a
scrambled sequence was prepared by annealing oligonucleotides of the
following sequences: 5'-GATGCGTCTGTCGCA-3' and
5'-TGCGACAGACGC1ATC-3'. Stocks of double-stranded DNA were
prepared at a concentration of 2 mM in saline.
B decoy and control scrambled DNA (60 µg) were infused
intracerebroventricularly at a rate of 0.5 µl/min for 20 min (two
injections at 24 and 2 hr before sublethal 3 min ischemia or KA or
LIN500 treatment) via a stainless steel cannula (23 gauge)
stereotaxically implanted with a Kopf stereotaxic apparatus (David Kopf
Instruments, Tujunga, CA) into the right lateral ventricle
(dorsoventral, 
3.6 mm below the cortical surface; mediolateral, +1.4
mm from bregma; and anteroposterior, 0.8 mm from bregma)
(Smith-Swintosky et al., 1994; Yu et al., 1999).
Histological procedures. At the end of the experiment,
animals were killed at designated time points, and brains were frozen quickly in isopentane at 40°C. Coronal sections (10 µm) were cut
on cryostat (Leica, Nussloch, Germany) and post-fixed by immersion in
4% paraformaldehyde-10
2
M PBS for 30 min. Slides were then
dehydrated in ethanol baths (50, 70, and 100%), air dried, and stored
at 70°C until use. For each brain studied (n = 6 per time point and treatment), two sections were placed on
3-aminopropylethoxysilane-coated slides, and 10 slides (randomly
chosen) per rat were stained with cresyl violet. The neuronal density
of hippocampal CA1 subfield, known to be the most vulnerable to
ischemia, was determined by the method of Kirino (1982) on
coronal sections of the dorsal hippocampus corresponding to brain
sections located between 3.14 and 4.16 mm posterior to bregma (Paxinos
and Watson, 1986). The total linear length of the CA1 sector was
measured using a digitizer. The number of living neurons in the stratum
pyramidale within the CA1 subfield was counted using a Leica Aristoplan
photomicroscope at a magnification of 400×. Neurons that had shrunken
cell bodies with surrounding empty spaces were excluded. The neuronal
density of CA1 sector, i.e., the number of intact pyramidal cells per 1 mm linear length of the CA1 stratum pyramidale observed in each 10 µm
section, was quantified. Thus, a mean value for each hippocampal CA1
substructure was obtained from 10 bilateral measurements on two
sections per slide and 10 slides per rat, for the six animals in each
of experimental group. The neuronal density for a given animal
represents the average of both right and left hippocampal neuronal cell
densities. Neuronal density values were expressed as mean ± SEM.
Data analysis was performed by two-factor (experimental condition and
brain region) ANOVA, followed by Tukey's w test for
multiple comparisons. A probability <5% was considered statistically significant.
Preparation of nuclear and cytosolic extracts. Nuclear and
cytosolic extracts from hippocampi were prepared according to the method reported previously with some modifications (Dignam et al.,
1983). Briefly, after being removed from storage at 70°C, hippocampi (n = 6 per experimental group) were
homogenized in four volumes of ice-cold lysis buffer containing 10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 1%
NP-40, 0.5 mM dithiothreitol, and protease
inhibitor cocktail on ice using a Dounce homogenizer. After 10 min at
room temperature, homogenates were centrifuged at 6000 × g for 5 min at 4°C. The supernatant was taken as the crude
cytosolic fraction. The pellet was resuspended in 10 vol of cold
washing buffer (20 mM HEPES, pH 7.9, 25%
glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol, and protease inhibitor cocktail). After 30 min at
4°C, under continuous gentle mixing, the extracted nuclear proteins
were collected by centrifugation at 12,500 × g for 30 min at 4°C. The supernatant was dialyzed 1 hr at 4°C against 10 mM Tris, 10 mM
MgCl2, 100 mM KCl, 1 mM EDTA, 1 mM
dithiothreitol, and protease inhibitor cocktail. This dialyzed
supernatant was again centrifuged at 50,000 × g for 30 min at 4°C. The pellet resuspended in 20 mM
HEPES, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 0.2 mM
dithiothreitol, and protease inhibitor cocktail was aliquoted and
stored at 70°C until use.
The crude cytosolic fractions were mixed with 
vol cytosolic
extraction buffer (0.3 M HEPES, pH 7.9, 0.03 mM
MgCl2, 1.4 M KCl, 0.5 mM
dithiothreitol, and protease inhibitor cocktail) and centrifuged at
100,000 × g for 1 hr at 4°C. The supernatant was
dialyzed, centrifuged, aliquoted, and stored at 70°C as described
above. Protein concentrations of the final nuclear and cytosolic
fractions were determined by using Bradford's method (Bradford, 1976).
Aliquots of the nuclear and cytosolic extracts were stored at 70°C
for gel shifts, supershift assays, and Western blottings.
Electrophoretic mobility shift assay. The gel-shift assay
was performed using a commercial DNA-binding protein detection system (Promega, Charbonniéres, France), as described by the
manufacturer. Briefly, double-stranded oligonucleotide containing the
B consensus sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3') was end-labeled
using [
-32P]ATP (6000 Ci/mmol;
ICN Biochemicals, Orsay, France) and T4 polynucleotide kinase and was
purified by centrifugation through a G-25 spin column (Amersham
Pharmacia Biotech, Saclay, France). DNA binding was performed in
20 µl reaction containing a 20 µg aliquot of extracted nuclear
protein, 10 mM HEPES, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 2.5 mM DTT, 10% glycerol, and 0.05% NP-40. After 10 min of incubation on ice, 3 × 104
cpm of labeled probe was added to the reaction, and the mixture was
incubated for 20 min at 37°C. Then, protein-DNA complexes were
resolved by electrophoresis through 4 or 7% native polyacrylamide gels
(37.5:1 acrylamide/bisacrylamide) in buffer containing 5 mM Tris, pH 8.3, and 38 mM
glycine for 4 hr at 150 V. Gels were dried, autoradiographed on
a Bas-1500 phosphorimager (Fujifilm, Tokyo, Japan) for 4 hr, and
exposed to x-ray film (X-Omat; Eastman Kodak, Le Pontet, France) using
intensifier screens at room temperature for 3 d. The specificity
of the identified -binding proteins in the nuclear extracts was
determined by adding a 50-fold excess of unlabeled competitor to 20 µg of nuclear extract before incubation with the binding mixture
containing buffer and labeled probe. The transcription factor AP2
consensus oligonucleotide (5'-GATCGAACTGACCGCCCG CGGCCCGT-3') was
used as the unlabeled nonspecific competitor. To ensure consistency in
the data analysis, the nuclear and cytosolic extracts of different
experimental groups were run on the same gel.
To measure total NFB activity in the cytosol, cytosolic extracts (20 µg) were treated after the DNA-binding reaction with deoxycholate
(DOC) at a final concentration of 0.5% for 5 min on ice, followed by
incubation in 1% NP-40 for 30 min at room temperature to release
NFB from IB.
For gel supershift experiments, the oligonucleotide-nuclear protein
binding reaction was followed by incubating the mixture overnight at
4°C with 1 µg of either anti-p50 or anti-p65 antibodies (Santa Cruz
Biotechnology, Tebu, France) separately or together. Electrophoretic
mobility shift assay (EMSA) was then performed as described above.
All radioactive gels were quantified by densitometry using a Bas-1500
phosphorimaging system (Fujifilm) and TINA software. Data are
expressed as mean ± SEM (n = 6). Data
analysis was performed by two-factor ANOVA, followed by Tukey's
w test for multiple comparisons. A probability <5% was
considered statistically significant.
Western blotting. Proteins in the nuclear and cytosolic
extracts (20 µg) were separated by SDS-PAGE on 10% SDS-PAGE
gels for 1 hr at 100 mA. Proteins were transferred onto nitrocellulose membrane (Hybond-C) in blotting buffer (156 mM
Tris, 1 M glycine, and PBS) for 2 hr at 50 mA and
blocked with 4% skim milk (Regilait) in PBS for 2 hr at room
temperature. The blotted membrane was incubated for 4 hr at room
temperature with the rabbit polyclonal anti-NFB p65 antibody
(AB1604, diluted 1:2000; Chemicon, Euromedex, Mundolsheim, France) and
overnight at 4°C with the mouse monoclonal antibody raised against
the active form of NFB (MAB3026, diluted 1:150; Chemicon) or the
rabbit polyclonal anti-IB antibody (sc-847, diluted 1:500; Santa
Cruz Biotechnology). After washing with 0.1% Tween 20-PBS
(four times, 15 min each), the blots were incubated for 1 hr at room
temperature with goat anti-rabbit IgG coupled to horseradish peroxidase
(diluted 1:15,000; Jackson ImmunoResearch, Interchim, Montluson,
France) and then washed again in 0.1% Tween 20-PBS. Fluorography was
performed on Kodak X-Omat AR film using Western blotting detection
reagents (Pierce, Rockford, IL) following the enhanced
chemiluminescence technique. To ensure consistency in the data
analysis, the nuclear and cytosolic extracts of the same sample were
run on the same gel (n = 6). In each blot, -tubulin was used as an internal control for the loading of protein level (data
not shown).
Immunohistochemistry. Frozen sections (10 µm) were
post-fixed with acetone for 10 min at 20°C. Sections were then
immersed in 0.3%
H2O2-methanol for 30 min,
permeabilized in 0.3% Tween 20-PBS for 15 min, treated for 10 min in
a microwave oven in 0.1 M citrate buffer, pH 6.0, and blocked with 1% horse-goat serum (Vector Laboratories,
Burlingame, CA) for 4 hr at room temperature. Sections were then
incubated with the mouse monoclonal antibody raised against the active
form of NFB (MAB3026, diluted 1:150; Chemicon) overnight at 4°C.
After the primary incubation and three rinses in 1× PBS,
sections were then incubated in biotinylated horse anti-mouse IgG or
biotinylated goat anti-rabbit IgG (diluted 1:1000; Jackson
ImmunoResearch) for 2 hr. NFB labeling was compared with
immunohistochemical stainings obtained with the monoclonal mouse
antibody against the neuron-specific nuclear protein NeuN (neuronal
nuclei) (MAB377, diluted 1:250; Chemicon), the rabbit anti-cow glial
fibrillary acidic protein (GFAP) marker of astrocytes (clone V9,
diluted 1:250; Dako, Trappes, France), the monoclonal mouse
anti-vimentin (Z0334, diluted 1:50; Dako), and the monoclonal mouse
anti-RT1B directed against the Class II major histocompatibility complex (MRC OX6, diluted 1:50; Biosource International, Cliniscience Montrouge, France), markers of activated microglia. Immunohistochemical expressions were visualized by DAB-DAB-Ni staining using the
VectaStain ABC kit (Vector Laboratories). All sections were washed in
distilled water and mounted with Entellan. For double labeling, the
monoclonal mouse antibody against NeuN (MAB377, diluted 1:250;
Chemicon) and the rabbit polyclonal anti-NF-B p65 subunit antibody
(AB1604, diluted 1:150; Chemicon) were used and detected with the Alexa Fluor 488 goat-horse anti-rabbit-anti-mouse IgG antibodies (diluted 1:1000; Molecular Probes, Interchim, Montluson, France). Sections were
analyzed using a laser-scanning confocal microscope (Leitz, Wetzlar, Germany).
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RESULTS |
Effect of preconditioning on NFB DNA-binding activity and
subcellular localization of p65 and p50 subunits (Western blotting and
immunohistochemistry)
Figure 1 demonstrates that the three
different preconditioning stimuli (sublethal 3 min ischemia, KA5
treatment, or LIN500 treatment) were associated with activation of
NFB. The gel-shift analysis (EMSA) of NFB DNA-binding proteins at
1, 24, and 72 hr after ischemia or after treatment in hippocampal
nuclear extracts from rats subjected to 3 min ischemia or injected with
KA5 or LIN500 was compared with that obtained from controls
(sham-operated or vehicle-injected). As illustrated in Figure 1,
A and B, each preconditioning stimulus induced an
intense NFB DNA-binding activity as early as 1 hr after sublethal
ischemia, KA5 treatment, or LIN500 treatment in pooled nuclear
hippocampal tissue. Figure 1B summarizes the
quantification of NFB binding activity in nuclear extracts from the
complete set of rats. For the three preconditioning stimuli, the
increase in NFB DNA-binding activity in the nuclear fraction varied
from 5.4- to 9.7-fold compared with respective control groups. The
specificity of the NFB binding was demonstrated by a competitive
experiment using unlabeled NFB oligonucleotide probe and nonspecific
transcription factor AP2 oligoprobe and performed on nuclear extracts
from rats submitted to 3 min ischemia or treated with LIN500 or KA5.
Competition assays using excess of nonradiolabeled NFB-specific
oligoprobe extinguished the specific retarded NFB band, whereas the
nonspecific competitor AP2 oligoprobe has no effect. Furthermore,
supershift assays showed that the NFB complex was composed
predominantly of p65/p50 heterodimers under all conditions. Figure
1C gives one example of supershift assay obtained with a
preconditioning induced by a sublethal 3 min ischemia. Figure
1C shows the marked slowing down of the migration of the
NFB-oligonucleotide complex (supershift) after the addition of
specific anti-p50 and p65 antibodies to the EMSA reaction mixture. A
combination of anti-p65 and anti-p50 antibodies caused additional gel
retardation and reduced the NFB DNA band (Fig. 1C).
Similar results were obtained with KA5 or LIN500 treatment
preconditioning stimulus (data not shown).
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Figure 1.
EMSA showing the time course of
increased NFB DNA-binding activity induced by the three different
preconditionings (3 min ischemia, KA5 treatment, and LIN500 treatment).
A, Representative gel shifts analysis showing NFB
DNA-binding activity in nuclear protein extracts from hippocampi of
control rats (lane 1) or rats submitted to 3 min
ischemia (lanes 5-9) or treated with KA5 (lanes
2-4) or LIN500 (lanes 10-12) and
obtained at 1, 24, and 72 hr after the different preconditionings.
NFB DNA-binding activity was assayed as described in Materials and
Methods. Competition assays of NFB DNA-binding activity was
performed in the presence of 50-fold excess of unlabeled competitor
NFB (lane 9) and nonspecific competitor AP2
(lane 8) consensus oligonucleotides. The shifted bands
of specific NFB DNA complexes are indicated by the
arrowheads. The right and
left gels correspond to 4 and 7% polyacrylamide
gels, respectively. B, Quantification of NFB
DNA-binding activity in the different experimental groups. The specific
shifted bands were quantified using a phosphorimaging system as
described in Materials and Methods. The values are expressed as a
percentage of control. No significant differences were found between
vehicle-injected and sham-operated rats, and values of these groups
were pooled and termed control. Data represent the mean ± SEM
values. Data are representative of six separate experiments in each
group (n = 6). *p < 0.05 indicates statistical significance when compared with control.
C, Supershift analysis of NFB binding proteins
present in nuclear extracts 24 hr after 3 min ischemia. The binding
activity assay was performed in the presence of anti-p65 antibodies,
anti-p50 antibodies, a combination of both, or no antibody. The
arrowheads indicate the bands of specific NFB-DNA
complexes, which were supershifted by anti-p65 antibodies, anti-p50
antibodies, or by their combination.
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Figure 2 depicts the induction of NFB
protein in the hippocampus after the three types of preconditioning
assessed using Western blot analysis (Fig. 2A-C) and
immunohistochemistry (Fig. 3). Figure
2A-C shows representative Western blotting analysis of the p65 (Fig. 2A,B) and p50
(Fig. 2C) content in cytosolic and nuclear extracts from
rats subjected to 3 min ischemia or injected with 5 mg/kg KA or 500 nmol/kg LIN taken at serial times after sublethal or drug treatments.
The p65 and p50 subunits of NFB were rich in the cytosolic fractions
from sham-operated or vehicle-injected rats but undetectable in the
nuclear extracts. In contrast, the protein levels of p65 and p50 in the
three preconditioned groups were significantly enhanced in the nuclear
fraction and concurrently decreased in the cytosol as early as 1 hr,
indicating a rapid translocation of these NFB subunits from the
cytosol to the nucleus.
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Figure 2.
Representative Western blotting analysis of p65
and p50 subunits of NFB in nuclear (A,
C) and cytosolic (B) extracts from
hippocampi of control rats (C) or rats submitted
to 3 min ischemia (I3) or treated with KA5 or LIN500 and obtained at 1, 24, and 72 hr after the different preconditionings.
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Figure 3.
Representative immunohistochemical staining of the
active form of NFB protein in hippocampal CA1 and CA3 substructures
of rats killed 24 hr after each of preconditioning stimuli.
Aa-Ad, 3 min ischemia. Ba,
Bb, Sham-surgery. Ca, Cb,
KA5 treatment. Da, Db, LIN500 treatment.
Aa, Ab, Immunohistochemical staining of
NeuN protein within CA1 and CA3 pyramidal cells after sublethal
ischemia. E, Colocalization of NFB-p65 and the
neuron-specific nuclear protein NeuN (neuronal nuclei) within CA1
(Ea) and CA3 (Eb) pyramidal cells. Brain
sections were immunostained with antibodies against the p65 subunit of
NFB (green) and NeuN (red) for
double-labeling. Scale bars, 20 µm.
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Immunostaining using a monoclonal antibody raised against the active
form of NFB performed at 24 hr after each of different preconditioning triggers (short ischemia, KA5 treatment, or LIN500 treatment) was performed to determine the cellular identity of the
increase in NFB activity associated with preconditioning visualized
above by gel shift and Western blotting analysis (Figs. 1,
2A-C). Immunoreactivity for the activated
form of NFB was not detectable in either sham-operated (Fig.
3Ba,Bb) or vehicle-injected (data not shown)
hippocampus. Sublethal 3 min ischemia (Fig.
3Ac,Ad), KA5 treatment (Fig.
3Ca,Cb), or LIN500 treatment (Fig.
3Da,Db) induced a strong immunoreactivity for
NFB in CA1 and CA3 pyramidal cell layers (Fig.
3Ac,Ad,Ca,Cb,Da,Db),
hilar neurons, and dentate gyrus (data not shown) 24 hr after
preconditioning stimulus. The extent and intensity of immunoreactivity
appeared similar in three treatments. According to morphological
criteria, the positive cells seemed to be neurons. Immunohistochemical
observations with GFAP and vimentin antibodies excluded astrocytes and
microglia as main sources of NFB activation in the three models of
preconditioning tested (data not shown). Immunostainings obtained with
the antibody raised against the neuron-specific nuclear protein NeuN
(Fig. 3Aa,Ab) provided evidence that the
immunoreactivity for the active form of NFB was predominant in the
nucleus. Double labeling with antibodies against the neuron-specific
nuclear protein NeuN and the NFB-p65 subunit indicated nuclear
colocalization (Fig. 3Ea,Eb).
Effects of B decoy DNA and DTTC on NFB DNA-binding activity
and subcellular localization of p65 subunit
B decoy DNA and DTTC have been shown to block NFB
DNA-binding activity in different systems (Schreck et al., 1992;
Smith-Swintosky et al., 1994; Yu et al., 1999). In the present study,
double-stranded B decoy DNA or control double-stranded DNA with a
scrambled sequence was injected intracerebroventricularly (two
injections at 24 and 2 hr before 3 min ischemia, KA5 treatment, or
LIN500 treatment), and rats were killed 1 hr after the preconditioning
stimulus. As illustrated in Figure 4,
A and B, the gel-shift analysis of NFB shows
that the increase in NFB DNA-binding activity induced by each
preconditioning was suppressed in hippocampus of rats injected with
B decoy DNA but was unaffected in animals injected with control
scrambled DNA. Figure 4B summarizes the
quantification of NFB DNA-binding activity in nuclear extracts from
the complete set of rats. For the three preconditioning stimuli, the
decrease in NFB DNA-binding activity in the nuclear fraction varied
from 4.8- to 6.0-fold compared with respective control groups (KA5, LIN500, or I3). The remaining NFB DNA-binding activity corresponded to that of negative controls (vehicle-injected or sham-operated rats).
Administration of B decoy DNA prevented the decrease in p65 content
in the cytosolic extracts (data not shown) and its increase induced by
sublethal ischemia (I3) or LIN500 treatment in the nuclear extracts
analyzed 24 hr after the preconditioning stimulus (Fig. 4C).
A similar result was obtained with KA5 treatment preconditioning
stimulus (data not shown).
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Figure 4.
Electrophoretic mobility shift assay
(A, B) and Western blotting analysis of
p65 subunit of NFB (C) showing the effect of
intracerebroventricular administration of B decoy DNA or
intraperitoneal injection of DTTC on the increased NFB DNA-binding
activity induced by the three different preconditionings (3 min
ischemia, KA5 treatment, and LIN500 treatments). A,
Representative gel shift analysis showing NFB DNA-binding activity
in hippocampal nuclear extracts isolated from rats that had received
intracerebroventricular injections of vehicle, 60 µg of B decoy
DNA (Bdecoy), 60 µg of scrambled control DNA
(ScDNA), or intraperitoneal injection of DTTC (150 mg/kg) before sublethal ischemia (I3) or administration
of either KA5 or LIN500. Rats were killed 1 hr (for B decoy or
scrambled control DNA) or 24 hr (for DTTC) after each type of
preconditioning. NFB activity was assayed as described in Materials
and Methods. The shifted band of specific NFB DNA complexes is
indicated by the arrowhead. B,
Quantification of NFB DNA-binding activity in the different
experimental groups. The specific shifted bands were quantified using a
phosphorimaging system as described in Materials and Methods. Values
are expressed as a percentage of control. Results are expressed as
mean ± SEM. Data are representative of six separate experiments
in each group (n = 6). *p < 0.05 indicates statistical significance when compared with
vehicle-injected rats. C, Representative Western
blotting analysis of p65 subunit of NFB in hippocampal nuclear
extracts isolated from rats that had received intracerebroventricular
injections of vehicle, 60 µg of B decoy DNA
(Bdecoy), 60 µg of scrambled control DNA
(ScDNA) or intraperitoneal DTTC injection (150 mg/kg)
before I3 or LIN500. Rats were killed 24 hr after the conditioning
stimulus. The position of p65 is indicated by the
arrowhead. 1, Vehicle; 2,
DTTC-LIN500; 3, LIN500; 4,
Sham-operated; 5, I3; 6, DTTC-I3;
7, scrambled control DNA-LIN500; 8, B
decoy DNA-LIN500; 9, B decoy DNA-I3;
10, scrambled control DNA-I3.
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In the same manner, administration of DTTC (150 mg/kg, i.p.) 15 min
before 3 min ischemia, KA5 treatment, or LIN500 treatment significantly
blocked the increase in NFB DNA-binding activity in the nuclear
fraction of hippocampi analyzed 24 hr after sublethal ischemia or drug
treatment (KA5 or LIN500) (Fig.
4A,B). Analysis of extracts from
rats pretreated with DTTC and then exposed to the different
preconditioning stimuli showed a greatly reduced level of p65 content
in nuclear fraction compared with hippocampi submitted to 3 min
ischemia or injected with LIN500 in the absence of DTTC (Fig.
4C). A similar result was obtained with KA5 treatment preconditioning stimulus (data not shown).
Effects of B decoy DNA and DTTC treatment on the hippocampal
ischemic tolerance induced by the different preconditionings
The effects of B decoy DNA and DTTC treatment on brain
tolerance were assessed by analysis of the neuronal degeneration in CA1
substructure of rats killed 7 d after the second ischemia. Figure
5, A and C, shows
representative photomicrographs of the CA1 sector in the different
groups, and Figure 5, B and D, reports the
quantitative analysis of neuronal cell density in the respective experimental groups. As expected, control (sham-operated or
vehicle-injected) rats did not show neurodegeneration (Fig.
5Aa). In contrast, typical cell death appeared in pyramidal
neurons of CA1 substructure after 6 min ischemia (Fig.
5Ab,Cb). Compared with control rats (Fig. 5Ba,Da), 21% of CA1 pyramidal cells only
survived (Fig. 5Bb,Db). Preconditioning with
sublethal 3 min ischemia (Fig. 5Ac) or LIN500 injection
(Fig. 5Cc) 3 d before 6 min ischemia totally prevented neuronal death induced by severe ischemia. In these preconditioned groups, 99 and 95% of CA1 cells were preserved, respectively (Fig. 5Bc,Dc). However, the intracerebroventricular
administration of B decoy DNA before each preconditioning stimulus
(3 min ischemia or LIN500 treatment) abolished the ischemic tolerance
and markedly enhanced the hippocampal neurodegeneration (Fig.
5Ad,Cd) compared with rats that were injected
with scrambled control DNA before preconditioning (Fig.
5Ae,Ce). The injection of B decoy DNA alone did not induce any damage in hippocampus (Fig.
5Cg,Dg). In the same manner, the intraperitoneal
injection of DTTC before 3 min ischemia (Fig. 5Af) or
LIN500 treatment (Fig. 5Cf) blocked the ischemic
tolerance induced by each preconditioning. Quantitative analysis of
neuronal damage in CA1 substructure confirmed the marked enhancement of
CA1 pyramidal cell degeneration in rats injected with B decoy DNA
before each preconditioning stimulus (Fig.
5Bd,Dd) compared with rats submitted to 6 min
ischemia (Fig. 5Bb,Db). Seventy and 75% of CA1
pyramidal neurons were destroyed in rats injected with B decoy DNA
before sublethal 3 min ischemia (Fig. 5Bd) or LIN500
injection (Fig. 5Dd), respectively. Similar results were
obtained with epileptic tolerance (data not shown). Together, these
data show that the treatment with B decoy DNA or DTTC prevented the
neuroprotective effects of late preconditionings (sublethal ischemia,
LIN500 treatment, or KA5 treatment) against ischemia or epilepsy and
that the blockade of NFB activation was deleterious in the three
models of brain tolerance used in that work.
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Figure 5.
Effect of B decoy DNA, scrambled control DNA,
and DTTC injections on the ischemic tolerance induced by brief ischemia
or LIN500 treatment. A, C, Representative
photomicrographs highlighting morphological changes in CA1 subfield of
cresyl violet-stained hippocampal sections 7 d after severe 6 min
ischemia in the different experimental groups. I6
corresponds to rats submitted to 6 min ischemia. I3-I6
corresponds to rats submitted to 3 min ischemia 3 d before 6 min
ischemia. decoy I3-I6 or ScDNA I3-I6
corresponds to rats that, respectively, had received
intracerebroventricular injections of 60 µg of B decoy DNA or
scrambled control DNA at 24 and 2 hr before 3 min ischemia. DTTC
I3-I6 corresponds to rats that had received an intraperitoneal
injection of DTTC (150 mg/kg) 30 min before 3 min ischemia. Rats were
killed 7 d after 6 min ischemia. Scale bar, 100 µm.
B, D, Quantification of neuronal density
in the hippocampal CA1 pyramidal layer of different experimental
groups. Results are expressed as mean ± SEM
(n = 6) and represent neuronal densities assessed
in cresyl violet-stained sections per 1 mm linear length of CA1
pyramidal layer. A mean value for each CA1 substructure was obtained
from 10 bilateral measurements on two sections per slide and 10 slides
per animal (n = 6) in each of the experimental
groups. Differences were considered statistically significant when
p < 0.05 (Tukey's test). * indicates
significantly different from control (sham-operated animals). # indicates significantly different from ischemic animals (6 min).
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Changes in NFB DNA-binding activity and subcellular localization
of IB and the active form of p65 in the different groups of
preconditioned brains
Electrophoretic mobility shift assays revealed that NFB
DNA-binding activity was decreased in nuclear extracts of rats
preconditioned with 3 min ischemia, KA5 treatment, or LIN500 treatment
compared with rats submitted to 6 min ischemia or treated with 7.5 mg/kg KA (Fig. 6A).
Treatment with DOC, which dissociates the cytosolic NFB-IB
complex, allowing the detection of inactive NFB, restored NFB
DNA-binding activity in preconditioned hippocampi (Fig.
6A). This indicated that NFB was present but not
active in preconditioned rats. Figure 6B summarizes
the quantification of NFB DNA-binding activity in nuclear and
cytosolic extracts from the complete set of rats. For the three
preconditioned groups, the NFB DNA-binding activity in nuclear
extracts decreased 44-65% compared with respective positive controls.
The restoring of the NFB DNA-binding activity after DOC treatment in
cytosolic extracts reached 90% compared with positive controls
(Fig. 6B). A possible mediator of the inhibitory action of preconditioning on NFB activation is IB, encoded by the
immediate early gene MAD-3 (Haskill et al., 1991). IB degradation is
enhanced by various NFB inducers, which also cause new synthesis of
IB by an inducible autoregulatory pathway (Sun et al., 1993). The
effect of preconditioning on IB and NFB metabolism was examined by immunoblotting with antibodies to IB and active form
of NFB, which recognizes the heterotetrameric protein, consisting of
the two p50 and two p65 subunits. Analysis of nuclear and cytosolic extracts of control, ischemic, or epileptic rats revealed that the loss
of IB correlated with translocation of NFB from the cytoplasm
to the nucleus (Fig. 6C). Severe ischemia and KA treatment (7.5 mg/kg) led to the disappearance of IB and to the presence of
the active form of NFB in cytosolic extracts. In contrast, the
amount of IB protein was notably increased in nuclear extracts of
rats preconditioned with LIN500 treatment (LIN500-I6 or LIN500-KA7.5) and submitted to severe 6 min ischemia or KA7.5 treatment 3 d later (Fig. 6C). A similar result was obtained with rats
preconditioned with 3 min ischemia or KA5 treatment (data not shown).
The profile of preconditioning-induced IB and of the accumulation
of the active form of NFB paralleled that of the inhibition of
NFB DNA-binding activity. Preconditioning with 3 min ischemia, KA5 or LIN500 treatment resulted in retention of p65 in cytoplasm (data not
shown).
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Figure 6.
Changes in NFB DNA-binding activity and
subcellular localization of IB and active form of NFB in the
different groups of preconditioned brains. A,
Representative gel shifts analysis showing NFB DNA-binding activity
in hippocampal nuclear and cytosolic extracts isolated from rats
preconditioned with 3 min ischemia (I3-I6), KA5
treatment (KA5-KA7.5), or LIN500 treatment
(LIN500-I6 or LIN500-KA7.5) and submitted
to 6 min ischemia or KA7.5 treatment 3 d later. Rats were killed
24 hr after the last treatment. Cytosolic extracts were analyzed by
EMSA with DOC treatment after binding as described in Materials and
Methods. The shifted band of specific NFB-DNA complexes is
indicated by the arrowhead. B,
Quantification of NFB DNA-binding activity in the different
experimental groups. The specific shifted bands were quantified using a
phosphorimaging system as described in Materials and Methods. The
values are expressed as a percentage of control. No significant
differences were found between saline-injected and sham-operated rats,
and values of these groups were pooled and termed control. Results are
expressed as mean ± SEM. Data are representative of six separate
experiments in each group (n = 6).
Differences were considered statistically significant when
p < 0.05 (Tukey's test). * indicates
significantly different from control (saline-injected or sham-operated
animals). # indicates significantly different from KA7.5-injected
animals. $ indicates significantly different from ischemic animals (6 min). C, Representative Western blotting analysis of
IB and active form of NFB in hippocampal nuclear and cytosolic
extracts isolated from ischemic (I6) and
epileptic (KA7.5) rats and rats preconditioned with
LIN500 treatment (LIN500-I6 or
LIN500-KA7.5) and submitted to severe 6 min ischemia or
KA7.5 treatment 3 d later. Rats were killed 24 hr after the last
treatment. The positions of IB and active form of NFB are
indicated by the arrowheads.
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DISCUSSION |
Many different mechanisms have been proposed to be involved in the
development of brain tolerance, including adenosine receptor stimulation, KATP channel opening, and delayed
expression of neuroprotective genes. However, little is known on the
nature of events occurring between the early opening of
KATP channels through adenosine receptors and the
induction of neuroprotective genes within the time window of
protection-promoting neuronal survival. The switching "on-off" of
gene expression is the province of transcription factors, which operate
singly or in association with other proteins. The present work provides
the first demonstration of the key role of the NFB transcription
factor in the signal transduction cascade of brain tolerance. The
NFB proteins are a family of inducible transcription factors that
activate a variety of cellular genes involved in control of the
inflammatory response and in regulating cellular growth (Mattson et
al., 2000). The NFB signaling pathway is a major determinant in the
control of neuronal death and/or cell survival. Although NFB
activation occurs in neurons after brain injury, its role in the injury
outcome remains unclear. The assumption that NFB is contributing to
neuronal cell death is documented by many papers reporting increased
NFB activity under pathological conditions in which neurons were
dying (Grilli et al., 1996; Clemens et al., 1997). However, subsequent
in vitro studies provide evidence that the activation of
NFB can protect neurons against amyloid -peptide toxicity (Barger
et al., 1995) and excitotoxic or oxidative stress (Goodman and Mattson,
1996; Mattson et al., 1997). Previous studies, in which injury-induced
NFB activity was suppressed by gene deletion of the p50 subunit (Yu
et al., 1999) or administration of B decoy DNA (Mattson et al.,
1997; Yu et al., 1999) or pharmacological agents (Taglialatela et al.,
1997; Maggirwar et al., 1998), strongly support a neuroprotective role
for NFB activation. NFB consists of a p50 and p65/RelA complex
that is trapped in the cytoplasm by an inhibitory protein IB
(Verma et al., 1995; Baeuerle and Baltimore, 1996). As long as IB
is bound to the p50/p65 complex, its translocation to the nucleus is
prevented. Activation of NFB consists of phosphorylation or
degradation of IB from the complex in the cytoplasm, and during
activation NFB is translocated to the nucleus of the cell (Chen et
al., 1995; Verma et al., 1995; Baeuerle and Baltimore, 1996).
Results reported in this paper demonstrate that, in the three models of
brain tolerance studied (ischemic, epileptic, and polyunsaturated fatty
acid-induced preconditionings), the activation of NFB was required
for the development of late cerebral preconditioning against severe
ischemia or epilepsy. The three different inducers of preconditioning
(sublethal 3 min ischemia, KA5 treatment, or LIN500 treatment) induced
rapid activation of NFB, as evidenced by its increased DNA-binding
activity and nuclear translocation. The gel-shift analyses revealed
that DNA-binding activity increased as early as 1 hr after sublethal
ischemia, KA5 treatment, or LIN500 treatment, and the shifted band
consisted of p50/p65 heterodimers. The presence of active NFB was
confirmed by nuclear localization of p50 and p65 subunits in Western
blots of hippocampal extracts as early as 1 hr after each
preconditioning stimulus, indicating a rapid translocation of NFB
subunits from cytosol to the nucleus. Immunohistochemical analyses
using p65 antibodies revealed that, 24 hr after each of preconditioning
stimuli, NFB activation occurred in the pyramidal neurons and not in
the glial cells. Pretreatment with the NFB inhibitor DTTC or B
decoy DNA blocked the increased DNA-binding activity and the nuclear
translocation of NFB and, at the same time, abolished the
neuroprotective effects of different delayed preconditionings against
severe ischemia or epilepsy. Previous studies have shown that B
decoy DNA can block activation of B-responsive genes, greatly
increase the vulnerability of neurons to different insults, and promote
neuronal cell death (Mattson et al., 1997; Yu et al., 1999).
Dithiocarbamates, such as DTTC, have also been reported to be potent
inhibitors of NFB activation in various cell types (Schreck et al.,
1992; Sherman et al., 1993; Ziegler-Heitbrock et al., 1993). Together,
these results indicate that activation of NFB is an essential
mechanism whereby sublethal ischemia, KA5 treatment, or LIN500
treatment results in delayed neuroprotection.
The present work also provides evidence that the inhibition of NFB
observed in rats preconditioned with 3 min ischemia, KA5 treatment, or
LIN500 treatment compared with ischemic or epileptic controls was
correlated with the prevention of the inducible degradation of
IB. Treatment of preconditioned extracts with the detergent DOC,
which revealed as much DNA-binding activity in the different preconditioned hippocampi as in the controls, indicated that NFB was
present but not active in the preconditioned extracts. The preconditioning also prevented the release of NFB from IB
after a severe ischemic or epileptic insult. The phosphorylation and degradation of IB is necessary for the activation of NFB and its subsequent appearance in the nucleus (Chen et al., 1995; Verma et
al., 1995; Baeuerle and Baltimore, 1996). Therefore, on activation of
NFB, IB concentrations decrease. Immunoblotting with an antibody raised against IB reveals that the amount of
IB protein increased in the preconditioned hippocampi compared
with ischemic or epileptic cytosolic extracts. An interesting
observation is the presence of IB protein and the active form of
NFB in the nuclear hippocampi of rats preconditioned with 3 min
ischemia, KA5 treatment, or LIN500 treatment compared with ischemic or
epileptic controls, in which IB protein was absent. The increase
in IB abundance in preconditioned rats is probably the result of
increased IB synthesis. This result strongly suggests that the
inhibition of NFB activation induced by preconditioning is mediated
by induction of the IB inhibitory protein, which traps activated
NFB in inactive cytoplasmic complexes. In accordance with previous
work, demonstrating the direct transcriptional activation of IB
by NFB itself (Scott et al., 1993; Sun et al., 1993; Chiao et al., 1994), IB probably newly synthetized after the activation of NFB by preconditioning stimulus will bind free cytoplasmic NFB and inactivate its potential nuclear translocation. Furthermore, excess
IB translocates to the nucleus by an unknown mechanism, in which,
as shown in vitro, it can sequester free NFB (p65), promote net dissociation of DNA-bound NFB, and thereby terminate its
activity (Arenzana-Seisdedos et al., 1995). This nuclear
NFB-IB complex may be transported back to the cytoplasm or
degraded in the nucleus. Recently, it has been shown that
immunosuppression by glucocorticoids may be the direct outcome of the
inhibition of NFB activity through induction of IB synthesis
(Auphan et al., 1995; Scheinman et al., 1995). It is not excluded that
such a mechanism is also involved in the anti-inflammatory response and
neuroprotection induced by sodium salicylate and aspirin (Kopp and
Ghosh, 1994; Grilli et al., 1996).
In conclusion, these findings demonstrate that the transcription factor
NFB is indeed at the crossing of neuronal cell death and survival
pathways and is a crucial component of the signal transduction cascade
of cerebral preconditioning. This paper shows that (1) the three
preconditioning protocols induce a rapid neuroprotective activation of
NFB in hippocampus, (2) the increases in NFB DNA-activity determined by gel shift and Western blot analysis specifically reflect
increases in hippocampal neurons, (3) preconditioning the brain with
sublethal ischemia, KA5 treatment, or LIN500 treatment inhibits the
activation of NFB after the second injury and renders the brain more
resistant to a subsequent potentially lethal ischemic or epileptic
insult, and (4) the inhibition of NFB is mediated by induction of
the IB inhibitory protein, which traps activated NFB in
inactive cytoplasmic complexes. The cellular mechanisms whereby the
three preconditioning treatments activate NFB remains to be
identified. Nitric oxide and free hydroxyl radicals are potential
candidates for the role of NFB activators. Not only they are known
to activate NFB but they are also known to be involved in brain and
heart tolerance (Bolli et al., 1997; Centeno et al., 1999; Xuan et al.,
1999; Rauca et al., 2000). The neuroprotection induced by activation of
NFB in hippocampal neurons related to induction of MnSOD (Mattson et
al., 1997) and the increase of MnSOD activity in brain tolerance
(Toyoda et al., 1997) strongly support the link between NFB and
oxidative stress in cerebral preconditioning. Together, these results
strongly suggest that, whatever the type of preconditioning, the
initial signals elicited by the conditioning stimulus are transduced
into protective changes via an NFB-dependent mechanism. The crucial
involvement of this transcription factor in brain tolerance may open
new ways in the search of therapeutic strategies.
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FOOTNOTES |
Received March 19, 2001; revised April 12, 2001; accepted April 13, 2001.
This work was supported by the Centre National de la Recherche
Scientifique, the Association Française contre les Myopathies, and the Conseil Regional (Provence-Alpes-Cote-d'Azur). We are grateful to G. Jarretou, F. Aguila, and V. Lopez for technical assistance.
Correspondence should be addressed to Prof. M. Lazdunski, Institut de
Pharmacologie Moléculaire et Cellulaire, Centre National de la
Recherche Scientifique, Unité Mixte de Recherche 6097, 660 route
des Lucioles, Sophia Antipolis, 06560 Valbonne, France. E-mail:
ipmc{at}ipmc.cnrs.fr.
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TOP
ABSTRACT
INTRODUCTION
