From the
Trinity College Institute of
Neuroscience, Department of Physiology, Trinity College, Dublin 2, Ireland and
the ¶Department of Human Anatomy and Physiology,
Conway Institute, University College Dublin, Earlsfort Terrace, Dublin 2,
Ireland
Received for publication, March 12, 2003 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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It has emerged in several experimental models that increased JNK
phosphorylation is associated with deficits in synaptic function; for
instance, increased activation of JNK has been reported in the hippocampi of
aged rats (9,
10), rats exposed to whole
body irradiation (11), and
rats injected with the proinflammatory cytokine, interleukin (IL)-1
(12) or lipopolysaccharide
(13), and in all cases
glutamate release was decreased. In each of these experimental conditions,
long term potentiation (LTP), a model of synaptic plasticity, was markedly
impaired, and this impairment was coupled with an increased hippocampal
concentration of IL-1
.
A number of groups have reported that A administration exerts an
inhibitory effect on LTP. For instance, A
peptides
(1416)
and naturally secreted A
oligomers
(17) inhibited LTP in the CA1
region in vivo, and A
peptides also inhibit LTP in dentate
gyrus and the CA1 in vitro
(1821).
Similarly, a deficit in LTP was reported in aged mice that overexpress amyloid
precursor protein and in which deposition of A
was observed
(22). In this study, we
investigated the signaling events induced by A
(140)
that might explain its impact on LTP and report that activation of JNK is a
pivotal event in A
-induced inhibition of LTP and in A
-induced cell
death.
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EXPERIMENTAL PROCEDURES |
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AnimalsGroups of young male Wistar rats (200300 g;
Bio Resources Unit, Trinity College, Dublin 2, Ireland), maintained at an
ambient temperature of 2223 °C under a 12 h light-dark schedule,
were used in this experiment. The rats were anesthetized by intraperitoneal
administration of urethane (1.5 mg/kg) and were injected
intracerebroventricularly with either sterile water (5 µl) or
A(140). 6 h post-injection, the rats were killed by
decapitation, the brains were rapidly removed on ice, and area CA1 was
dissected free. The tissue was cross-chopped (350 x 350 µm) and
frozen in Krebs solution containing 10% Me2SO as previously
described (23) until required
for analysis.
Analysis of JNK Phosphorylation, c-Jun Phosphorylation, Cytosolic
Cytochrome c Expression, Bax Expression, FasL Expression, and PARP
CleavageJNK phosphorylation, c-Jun phosphorylation, and expression
of Bax, cytosolic cytochrome c, PARP, and FasL were analyzed in
samples prepared from CA1 tissue using a method previously described
(13). In the case of JNK,
c-Jun, FasL, and PARP, tissue homogenates were diluted to equalize for protein
concentration, and aliquots (100 µl, 2 mg/ml) were added to 100 µl of
sample buffer (0.5 mM Tris-HCl, pH 6.8, 10% glycerol, 10% SDS, 5%
-mercaptoethanol, 0.05% bromphenol blue (w/v)), boiled for 5 min, and
loaded onto 10% SDS-PAGE gels. In the case of cytochrome c, cytosolic
fraction was prepared by homogenizing slices of hippocampus in lysis buffer
(20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
pepstatin A, 2 µg/ml leupeptin, 2 µg/ml aprotinin), incubating for 20
min on ice, and centrifuging (15,000 x g for 10 min at 4
°C). The supernatant (i.e. cytosolic fraction) was suspended in
sample buffer to a final concentration of 300 µg/ml, boiled for 3 min, and
loaded (6 µg/lane) onto 12% SDS-PAGE gels. The pellet (i.e.
mitochondrial fraction) was resuspended in sample buffer to a final
concentration of 300 µg/ml, boiled for 3 min, and loaded (6 µg/lane)
onto 12% SDS-PAGE gels. Bax expression was assessed in the mitochondrial
fraction. In all experiments the proteins were separated by application of 32
mA constant current for 2530 min, transferred onto nitrocellulose
strips (225 mA for 90 min), and immunoblotted with the appropriate antibody.
For JNK phosphorylation, the proteins were immunoblotted with an antibody that
specifically targets phosphorylated JNK (1:300 in Tris-buffered saline
(TBS)-Tween (0.05% Tween 20) containing 0.1% BSA; Santa Cruz Biotechnology
Inc.) for 2 h at room temperature. The blots were stripped and stained for
total JNK. Nitrocellulose strips were probed with a mouse monoclonal
IgG1 antibody (1:200; Santa Cruz Biotechnology Inc.) raised against
a recombinant protein corresponding to amino acids 1384 representing
full-length JNK1 of human origin. To assess phosphorylation of c-Jun, we
immunoblotted with a mouse monoclonal IgG1 antibody (1:400 in
PBS-Tween (0.1% Tween 20) containing 2% nonfat dried milk) raised against the
peptide corresponding to a short amino acid sequence of phosphorylated c-Jun
of human origin (Santa Cruz Biotechnology Inc.). To assess cytoplasmic
cytochrome c, a rabbit polyclonal antibody (1:250 in PBS-Tween
containing 2% nonfat dried milk; Santa Cruz Biotechnology Inc.) raised against
recombinant protein corresponding to amino acids 1104 of cytochrome
c was used. In the case of FasL, we immunoblotted with a rabbit
polyclonal antibody (1:500 in TBS-Tween containing 1% BSA; Santa Cruz
Biotechnology Inc.) raised against a peptide corresponding to a short amino
acid sequence at the N terminus of FasL of human origin. Bax expression was
assessed in the mitochondrial fraction using a mouse monoclonal
IgG1 antibody (1:200 in TBS-Tween containing 1% BSA; Santa Cruz
Biotechnology Inc.). To assess the cleavage of PARP, we immunoblotted with an
antibody (1:500 in PBS-Tween (0.1% Tween 20) containing 2% nonfat dried milk)
raised against the epitope corresponding to amino acids 7641014 of PARP
of human origin (Santa Cruz Biotechnology Inc.). All of the nitrocellulose
strips were reprobed for actin expression to ensure equal loading of protein
on all SDS-PAGE gels. Actin expression was assessed using a mouse monoclonal
IgG1 antibody (1:300 in PBS-Tween containing 2% nonfat dried milk)
corresponding to an amino acid sequence mapping at the C terminus of actin of
human origin (Santa Cruz Biotechnology Inc.). Immunoreactive bands were
detected as follows: peroxidase-conjugated anti-mouse IgG (Sigma) and
Supersignal chemiluminescence (Pierce) for JNK, c-Jun, Bax, and actin and
peroxidase-conjugated anti-rabbit IgG (Sigma) and Supersignal (Pierce) for
cytochrome c, FasL, and PARP.
Induction of LTP in CA1 in VivoMale Wistar rats
(175200 g; Biomedical Facility, University College, Dublin, Ireland)
were anesthetized with urethane (1.5 mg/kg), placed in a stereotaxic frame,
and assessed for LTP as described previously
(16). Small holes were drilled
in the skull to allow insertion of a guide cannula to facilitate
intracerebroventricular injection and to allow insertion of the reference,
stimulating, and recording electrodes. The recording electrode was positioned
in the stratum radiatum of area CA1 (3 mm posterior and 2 mm lateral to
bregma), and a bipolar stimulating electrode was placed in the Schaffer
collateral/commissural pathway distal to the recording electrode (4 mm
posterior and 3 mm lateral to bregma). The cannula was positioned above the
lateral ventricle in the opposite hemisphere to that of the electrodes (1 mm
posterior and 1.2 mm lateral to bregma). Test shocks (0.033 Hz) were delivered
to the Schaffer collateral/commissural pathway, and base-line excitatory
postsynaptic potentials (EPSPs), recorded at 3540% of maximal response,
were sampled for at least 30 min to allow the response to stabilize. Rats were
then injected intracerebroventricularly with either
A(140) (1nmol in 5 µl), the membrane soluble JNK
inhibitor D-JNKI1 (1 nmol in 5 µl), combined A
(140)
and D-JNKI1 (1 nmol of each in 5 µl) or vehicle (5 µl sterile water);
and base-line recordings were monitored for a further 60 min before delivery
of a series of high frequency stimuli (HFS; 3 x 10 trains of 10 stimuli
at 200Hz; intertrain interval, 20 s). Responses to test shock stimulation were
recorded for a further 5 h post-HFS, and deep body temperature was maintained
at 36.5 ± 0.5 °C using heating pads. Paired pulse facilitation with
an interstimulus interval of 50 ms was also examined preinjection, 1 h
post-injection of drug/vehicle (prior to HFS), and 5 h following HFS. Deep
body temperature was maintained at 36.5 ± 0.5 °C using heating
pads. Extracellular field potentials were amplified (x100), filtered at
5 kHz, digitized, and recorded using MacLab software. The EPSP slope was used
to measure synaptic efficacy. EPSPs are expressed as percentages of the mean
initial slope measured during the first 10 min of the base-line recording
period.
Analysis of IL-1 ConcentrationIL-1
concentration was analyzed in homogenate prepared from CA1 by enzyme-linked
immunosorbent assay (R & D Systems) and in supernatants prepared from
cultured cells as described below. Antibody-coated (100 µl; final
concentration, 1.0 µg/ml; diluted in PBS, pH 7.3; goat anti-rat IL-1
antibody) 96-well plates were incubated overnight at room temperature, washed
several times with PBS containing 0.05% Tween 20, blocked for 1 h at room
temperature with 300 µl of blocking buffer (PBS, pH 7.3, containing 5%
sucrose, 1% BSA, and 0.05% NaN3), and washed. IL-1
standards
(100 µl; 01000 pg/ml in PBS containing 1% BSA) or samples
(homogenized in Krebs solution containing 2 mM CaCl2)
were added, and incubation proceeded for 2 h at room temperature. Secondary
antibody (100 µl; final concentration, 350 ng/ml in PBS containing 1% BSA
and 2% normal goat serum; biotinylated goat anti-rat IL-1
antibody) was
added and incubated for 2 h at room temperature. The wells were washed, and
detection agent (100 µl; horseradish peroxidase-conjugated streptavidin;
1:200 dilution in PBS containing 1% BSA) was added and incubated continued for
20 min at room temperature. Substrate solution (100 µl; 1:1 mixture of
H2O2 and tetramethylbenzidine) was added and incubated
at room temperature in the dark for 1 h, after which time the reaction was
stopped using 50 µl of 1 M H2SO4.
Absorbance was read at 450 nm, and the values were corrected for protein
(24) and expressed as pg
IL-1
/mg protein.
Preparation of Cultured Cortical NeuronsPrimary cortical
neurons were isolated and prepared from 1-day-old Wistar rats (BioResources
Unit, Trinity College, Dublin 2, Ireland) and maintained in NBM as previously
described (5). The rats were
decapitated, the cerebral cortices were dissected, and the meninges were
removed. The cortices were incubated in PBS with trypsin (0.25 µg/ml) for
25 min at 37 °C. The cortical tissue was then triturated in PBS containing
soy bean trypsin inhibitor (0.2 µg/ml) and DNase (0.2 mg/ml) and gently
filtered through a sterile mesh filter (40 µm). The suspension was
centrifuged at 2000 x g for 3 min at 20 °C, and the pellet
was resuspended in warm NBM, supplemented with heat inactivated horse serum
(10%), penicillin (100 units/ml), streptomycin (100 units/ml), and glutamax (2
mM). The suspended cells were plated at a density of 0.25 x
106 cells on circular 10-mm diameter coverslips, coated with
poly-L-lysine (60 µg/ml), and incubated in a humidified
atmosphere containing 5% CO2:95% air at 37 °C for 2 h prior to
being flooded with prewarmed NBM. After 48 h, 5 ng/ml
cytosine-arabinofuranoside was added to the culture medium to suppress the
proliferation of non-neuronal cells. The media were exchanged for fresh media
every 3 days, and the cells were grown in culture for up to 7 days prior to
treatment. In one set of experiments the neurons were incubated in the
absence/presence of A(140) (2 µM in NBM)
for 72 h with or without caspase-1 inhibitor (100 nM in NBM;
Ac-YVAD-CMK; Calbiochem) or D-JNKI1 (1 µM in NBM; Alexis
Biochemicals). In the case of A
-treated neurones, the supernatant was
removed at 20 h, and IL-1
concentration was assessed. At 72 h, the cells
were rinsed in TBS and fixed in 4% paraformaldehyde in TBS for
immunohistochemical assessment of JNK phosphorylation, caspase-3 activation,
and DNA fragmentation. The cells were incubated in
A
(140) (2 µM) for 18 h for analysis of
changes in gene expression. In a second series of experiments, the neurons
were incubated in the absence/presence of IL-1
(5 ng/ml in NBM) with or
without D-JNKI1 (1 µM in NBM) for 18 h and harvested in lysis
buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1.5
mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1
mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride,
5 µg/ml pepstatin A, 2 µg/ml leupeptin, 2 µg/ml aprotinin) for
assessment of c-Jun phosphorylation and FasL expression.
Analysis of Bax mRNA and caspase-3 mRNATotal RNA was
extracted from cortical neurones using TRI reagent (Sigma). cDNA synthesis was
performed on 1 µg of total RNA using oligo(dT) primer as per the
manufacturer's instructions (Superscript reverse transcriptase; Invitrogen).
The RNA was treated with RNase-free DNase I (Invitrogen) at 1 unit/µg of
RNA for 15 min at 30 °C. Equal amounts of cDNA were used for PCR
amplification for a total of 28 cycles. Primers were pretested through an
increasing number of amplification cycles to obtain reverse transcriptase-PCR
products in the exponential range. In the case of Bax mRNA expression
following A treatment primers used were as follows: rat Bax, sense
5'-GCAGAGAGGATGGCTGGGGAGA-3', and antisense
5'-TCCAGACAAGCAGCCGCTCACG-3'
(25); rat
-actin, sense
5'-GAAATCGTGCGTGACATTAAAGAGAAGCT and antisense
5'-TCAGGAGGAGCAATGATGATCTTGA-3'. The cycling conditions were 95
°C for 5 min followed by cycles of 95 °C for 75 s, 52 °C for 75 s,
and 72 °C for 90 s. A final extension step was carried out at 70 °C
for 10 min. These primers generated Bax PCR products of 352 base pairs and
-actin PCR product of 360 base pairs. In the case of caspase-3 mRNA
expression following treatment with A
(140) and Bax
mRNA expression following treatment with IL-1
, multiplex PCR was
performed using the Quantitative PCR Cytopress detection kit (Rat Apoptosis
Set 2; BioSource International) generating caspase-3 PCR products of 320 base
pairs, Bax PCR products of 352 base pairs and glyceraldehyde-3-phosphate
dehydrogenase PCR product of 532 base pairs. The cycling conditions were 94
°C for 1 min and 58 °C for 2 min. A final extension step was carried
out at 70 °C for 10 min. The PCR products were analyzed by electrophoresis
on 1.5% agarose gels, photographed, and quantified using densitometry. The
target genes were normalized to expression of
-actin or
glyceraldehyde-3-phosphate dehydrogenase housekeeping genes. No observable
change in
-actin or glyceraldehyde-3-phosphate dehydrogenase mRNA was
observed in any of the treatment conditions
TUNEL StainingApoptotic cell death was assessed using the
DeadEnd colorimetric apoptosis detection system (Promega) according to the
manufacturer's instructions. Briefly, cultured cortical neurones were prepared
from neonatal rats as described above and maintained in NBM for 12 days before
incubating in the absence/presence of A(140) (1
µM in NBM) for 72 h with or without caspase-1 inhibitor (100
nM in NBM) or D-JNKI1 (1 µM in NBM). Biotinylated
nucleotide was incorporated at 3'-OH DNA ends by incubating cells with
terminal deoxynucleotidyl transferase for 30 min at 37 °C. The washed
cells were incubated in horseradish peroxidase-labeled streptavidin and then
incubated in diaminobenzidine chromogen solution, and TUNEL-positive cells
were calculated as a proportion of the total cell number.
Immunohistochemical Staining for Phosphorylated JNK and Activated
Caspase-3Cultured cortical neurones were prepared from neonatal
rats as described previously
(5) and maintained in NBM for
12 days before incubating in the absence/presence of
A(140) (1 µM in NBM) for 72 h with or
without caspase-1 inhibitor (100 nM in NBM) or D-JNKI1 (1
µM in NBM). The cells were fixed in 4% paraformaldehyde in TBS,
permeabilized in 0.1% Triton containing 0.2% proteinase K, washed in TBS, and
refixed in 4% paraformaldehyde. The cells were incubated with 2.5% (v/v)
normal goat serum (Vector Laboratories) in TBS. The blocking serum was
removed, and the cells were incubated overnight with either antiactive p-JNK
(1:200 in TBS containing 2.5% normal goat serum; Santa Cruz Biotechnology
Inc.) or antiactive caspase-3 (1:250 in TBS containing 2.5% (v/v) normal goat
serum; Promega) in a humidified chamber. The cells were washed in TBS and
incubated in the dark for 2 h at room temperature in either fluorescein
isothiocyanate-labeled goat anti-mouse IgG or IgM (1:100; Biosource) to
immunolabel p-JNK or L-rhodamine-labeled goat anti-rabbit IgG (1:
100; Biosource) to immunolabel active caspase-3. The cells were washed in TBS,
mounted with an aqueous mounting medium (Vectastain; Vector Laboratories), and
sealed. The slides were examined under a Zeiss fluorescence microscope with
the appropriate filter (fluorescein isothiocyanate: excitation, 495 nm, and
emission, 525 nm; L-rhodamine: excitation, 540 and 574 nm, and
emission, 602 nm).
Statistical AnalysisThe data are expressed as the means ± S.E. A one-way analysis of variance (ANOVA) was performed to determine whether there were significant differences between conditions. When this analysis indicated significance (at the 0.05 level), post hoc Student Newmann-Keuls test analysis was used to determine which conditions were significantly different from each other. A repeated measures ANOVA was used to compare mean EPSP slopes at different time points in the electrophysiological experiments. When comparisons were being made between two treatments, an unpaired Student's t test for independent means was performed.
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RESULTS |
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We argued that this A-induced increase in JNK activation may
contribute to the previously reported A
-induced inhibition of LTP
(16), and to assess this, rats
were injected intracerebroventricularly with A
(140)
alone or in combination with the peptide inhibitor, D-JNKI1.
Fig. 2a shows that, in
control rats tetanic stimulation led to an immediate and persistent increase
in EPSP slope (p < 0.001; ANOVA); treatment with D-JNKI1 (1nmol)
did not significantly affect this change. In contrast, intracerebroventricular
injection of A
(140) inhibited LTP (p <
0.001; ANOVA, Fig.
2b); the effect was observed immediately such that the
mean percentage change in the EPSP slope in the 5 min immediately following
tetanic stimulation was significantly reduced in A
-treated compared with
control rats (p < 0.01; ANOVA;
Fig. 3a). The
A
-associated change persisted so that the mean percentage changes in
EPSP slopes in the final 5-min period of each hour were also significantly
reduced in A
-treated rats compared with control animals (***, p
< 0.001 in all cases; ANOVA; Fig. 3,
bf). Co-injection of D-JNKI1 and
A
(140) reversed the inhibitory effect of
A
(140) (Fig.
2b), but this effect was not apparent until 2 h after
tetanic stimulation (+++, p < 0.001; ANOVA;
Fig. 3, bf).
The mean percentage EPSP slopes at 05 min post-tetanus were
significantly reduced in rats treated with A
and D-JNKI1 compared with
control rats (p < 0.001; ANOVA;
Fig. 2b). The results
from paired pulse facilitation experiments found that there was no significant
change in paired pulse facilitation observed either between groups of animals
or preinjection compared with 1 h post-injection or 5 h following HFS. This
indicates that at the doses used here, A
(140) and
D-JNKI1 do not alter neurotransmitter release, suggesting a postsynaptic site
for the modulation of LTP observed in our experiments.
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The findings of several studies have indicated that enhanced JNK
phosphorylation in the hippocampus is coupled with enhanced IL-1
concentration and impaired LTP
(9,
12,
25); therefore we considered
that the effect of A
(140) might be mediated by
IL-1
. Fig. 4 shows that
the concentration of IL-1
in hippocampus was significantly increased in
hippocampus of A
-treated rats compared with control rats (p
< 0.001; Student's t test for independent means; n =
5).
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Our findings from a previous study
(11) linked activation of JNK
with translocation of cytochrome c from mitochondria, suggesting that
the patency of the mitochondrial membrane was affected by JNK activation. In
an effort to address this question, we assessed expression of the
pro-apoptotic protein Bax in a mitochondrial fraction and cytochrome
c in a cytosolic preparation obtained from control and
A-treated rats. The sample immunoblot shown in
Fig. 5a indicates that
A
(140) increased expression of Bax, and densitometric
analysis revealed that the mean value in samples prepared from A
-treated
rats was significantly enhanced compared with that in control rats (p
< 0.05; Student's t test for independent means; n = 5).
The sample immunoblot and mean data in Fig.
5b indicate that cytochrome c expression in
cytosolic samples prepared from A
-treated rats was enhanced compared
with samples from control rats; comparison of mean values indicated that the
difference was statistically significant (p < 0.05; Student's
t test for independent means; n = 5). A similar pattern was
observed in terms of expression of the intact fragment (116 kDa) of PARP;
Fig. 5c shows one
sample immunoblot and the mean data indicating that
A
(140) significantly decreased expression of 116-kDa
PARP in hippocampal tissue prepared from A
-treated rats (p <
0.05; Student's t test for independent means; n = 5). It is
considered that, in addition to increased Bax expression, cytosolic cytochrome
c expression, and PARP cleavage, expression of FasL is indicative of
apoptotic cell death, and, in parallel with the increases in Bax expression,
cytosolic cytochrome c expression and PARP cleavage,
A
(140) significantly enhanced expression of FasL
(p < 0.01; Student's t test for independent means;
n = 5; Fig.
5d), and in the case of Bax, cytochrome c, PARP,
and FasL, equal protein loading was confirmed by reprobing for actin.
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The A-associated increase in Bax protein was mirrored by an
A
-induced increase in Bax mRNA in cultured cortical neurons as shown by
the sample gel and by analysis of the mean data indicating that the difference
between expression in A
-treated and control cultured cells reached
statistical significance (p < 0.05; Student's t test for
independent means; n = 5; Fig.
6a). Fig.
6c shows that IL-1
induced a similar increase in
Bax mRNA; the increase shown in the sample gel reflected the mean changes,
which revealed a significant IL-1
-induced increase (p <
0.05; Student's t test for independent means; n = 5).
Consistent with the evidence that A
(140) induces cell
death is the finding that it also increased caspase-3 mRNA in cortical cells
(Fig. 6b).
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We addressed the question of whether IL-1 might mediate some of the
actions of A
by investigating the effect of
A
(140) on IL-1
concentration and also on JNK
phosphorylation, caspase-3 activation, and TUNEL staining in cortical neuronal
cells in the presence or absence of the caspase-1 inhibitor, Ac-YVAD-CMK.
Fig. 7a indicates that
incubation of cells in the presence of A
(140)
significantly increased IL-1
in supernatant (p < 0.05;
ANOVA; n = 4), and this effect was inhibited by co-incubation in the
presence of Ac-YVAD-CMK. The cultured cells were stained with an antibody that
identified the phosphorylated form of JNK, and
Fig. 7b shows that
A
(140) induced a marked increase in the number of
cells staining positively for phosphorylated JNK; significantly, this effect
of A
(140) was abolished by co-incubation of cultures
in the presence of A
(140) and the caspase-1 inhibitor.
Similarly, A
(140) markedly increased the number of
cells that stained positively for activated caspase-3
(Fig. 7c) and for
TUNEL (Fig. 7d), and
these changes were abolished by co-incubation of cells in the presence of
A
(140) and the caspase-1 inhibitor, Ac-YVAD-CMK. In an
effort to establish whether JNK activation contributed to the effects induced
by A
(140), neuronal cells were treated with
A
(140) alone or in combination with D-JNKI1.
Fig. 7 (c and
d) indicates that inhibition of JNK abolished the
A
-associated increase in the number of cells that stained positively for
activated caspase-3 and TUNEL.
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To confirm the finding that IL-1 mimics at least some of the effects
of A
(140), cultured cortical neurons were incubated
with IL-1
in the presence or absence of D-JNKI1 and the cell lysates
assessed for activation of c-Jun and for expression of FasL.
Fig. 8a indicates that
IL-1
enhanced c-Jun phosphorylation, and analysis of the mean data
indicated that the IL-1
-induced effect was statistically significant
(p < 0.05; ANOVA; n = 2); the increase in c-Jun
phosphorylation was abrogated by co-incubation in the presence of D-JNKI1.
Similarly, IL-1
significantly increased expression of FasL
(Fig. 8b; p
< 0.05; ANOVA; n = 4), and this effect was also abrogated by
co-incubation in the presence of D-JNKI1. In both cases, equal protein loading
was confirmed by reprobing blots for actin expression.
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DISCUSSION |
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Several observations have contributed to the development of the idea that
FasL expression and consequently Fas activation play a role in
neurodegeneration, and we report that increased hippocampal expression of FasL
accompanied the A-induced increases in JNK phosphorylation, cytochrome
c translocation, PARP cleavage, and caspase-3 activation and that
these changes were abrogated by D-JNKI1. These data suggest that in the
hippocampus a causal relationship between these factors exists that has been
shown previously in experimental ischemic injury
(28,
40,
41), Parkinson's disease, and
Down's syndrome
(4244).
Specifically, activation of the JNK cascade has been shown to play a
significant role in FasL expression that mediates cell death in cortical
neurons (6), PC12 cells
(30), and epithelial and
lymphoid cells (45,
46), whereas Fas-Fc, which
prevents Fas binding to FasL, protects cells from apoptotic cell death
(47,
48). Significantly, increases
in JNK and c-Jun phosphorylation and expression of FasL are found in
association with apoptotic neurons that are detected in the AD brain
(2,
4244,
49,
50), suggesting that
activation of the JNK-c-Jun-FasL signaling cascade may mediate A
-induced
neuronal cell death. Indeed the finding that JNK activation is detected in
degenerating neurons in AD brains has led to the hypothesis that JNK
activation plays a key role in neuronal loss in AD
(51,
52).
We demonstrate that A treatment increased in IL-1
concentration
in hippocampus in vivo and cortical neurons in vitro.
Interestingly the A
-induced increase in IL-1
concentration
observed in cortical neurons was blocked by co-incubation of cells in the
presence of the caspase-1 inhibitor Ac-YVAD-CMK. The A
-induced increases
in activation of JNK and caspase-3 and in TUNEL staining were also inhibited
by Ac-YVAD-CMK, consolidating a role for IL-1
in A
-induced
changes. A link between A
and IL-1
has been described in other
studies; for example, A
has been shown to stimulate production of
proinflammatory cytokines, like IL-1
from differentiated human monocytes
and from a microglial cell line
(53), whereas IL-1
was
induced in reactive astrocytes surrounding A
-containing deposits in
14-month-old transgenic mice that overexpress human amyloid precursor protein
(54). Interestingly,
astrocytic overexpression of S100
, a component of A
-containing
plaques, has been reported to be triggered by IL-1
(55). Consistently, several
reports have provided evidence demonstrating a role for IL-1
in the
etiology of AD based largely on the finding that IL-1
expression in
different brain areas in AD and also in the cerebrospinal fluid of AD patients
(5658),
and it has been shown that a common polymorphism in IL-1B (the gene
encoding IL-1
) is associated with a 4-fold increase in IL-1
production and an associated increased risk of the disease
(55). Of particular
significance are our observations that the inhibitory effect of Ac-YVAD-CMK on
A
-induced changes is closely paralleled by similar effects of D-JNKI1
and that D-JNKI1 also inhibited IL-1
-triggered changes in c-Jun
phosphorylation and FasL expression.
Several studies have revealed that increased hippocampal IL-1
concentration, paralleled by increased JNK activation, exerts an inhibitory
effect on LTP (9,
12), and these changes, both
of which are induced by A
, undoubtedly contribute to the deficit in LTP
observed here. Coupled with these changes are the downstream consequences of
enhanced IL-1
and JNK activation on cell viability, and we therefore
conclude that the A
-induced deficit in LTP is a consequence of
activation of cellular cascades induced by IL-1
and JNK that lead to
cell death.
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FOOTNOTES |
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Recipient of a Trinity College Ussher Fellowship.
|| To whom correspondence should be addressed. Tel.: 353-1-608-1770; Fax: 353-1-679-3545; E-mail: lynchma{at}tcd.ie.
1 The abbreviations used are: AD, Alzheimer's disease; A,
amyloid-
; JNK, c-Jun N-terminal kinase; FasL, Fas ligand; IL,
interleukin; LTP, long term potentiation; NBM, neurobasal medium; PARP,
poly-(ADP-ribose) polymerase; TBS, Tris-buffered saline; BSA, bovine serum
albumin; PBS, phosphate-buffered saline; HFS, high frequency stimuli; EPSP,
base-line excitatory postsynaptic potential; TUNEL, TdT-mediated dUTP nick-end
labeling; ANOVA, analysis of variance.
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