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The Journal of Neuroscience, June 15, 2000, 20(12):4563-4572
The MAPK/ERK Cascade Targets Both Elk-1 and cAMP Response
Element-Binding Protein to Control Long-Term Potentiation-Dependent
Gene Expression in the Dentate Gyrus In Vivo
Sabrina
Davis1,
Peter
Vanhoutte2,
Christiane
Pagès2,
Jocelyne
Caboche2, and
Serge
Laroche1
1 Laboratoire de Neurobiologie de l'Apprentissage, de
la Mémoire, et de la Communication, Centre National de la
Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR)
8620, Université Paris Sud, 91405 Orsay, France, and
2 Laboratoire de Neurochimie-Anatomie, Institut des
Neurosciences, CNRS UMR 7624, Universtité Pierre et Marie Curie,
75005 Paris, France
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ABSTRACT |
The mitogen-activated protein kinase/extracellular signal-regulated
kinase (MAPK/ERK) signaling cascade contributes to synaptic plasticity and to long-term memory formation, yet whether MAPK/ERK controls activity-dependent gene expression critical for long-lasting changes at the synapse and what the events underlying transduction of
the signal are remain uncertain. Here we show that induction of
long-term potentiation (LTP) in the dentate gyrus in
vivo leads to rapid phosphorylation and nuclear translocation
of MAPK/ERK. Following a similar time course, the two downstream
transcriptional targets of MAPK/ERK, cAMP response element-binding
protein (CREB) and the ternary complex factor Elk-1, a key
transcriptional-regulator of serum response element (SRE)-driven gene
expression, were hyperphosphorylated and the immediate early gene
zif268 was upregulated. The mRNA encoding MAP kinase phosphatase MKP-1
was upregulated at the time point when MAPK/ERK phosphorylation had
returned to basal levels, suggesting a negative feedback loop to
regulate deactivation of MAPK/ERK. We also show that inhibition of the
MAPK/ERK cascade by the MAPK kinase MEK inhibitor SL327
prevented CREB and Elk-1 phosphorylation, and LTP-dependent gene
induction, resulting in rapidly decaying LTP. In conclusion, we suggest
that Elk-1 forms an important link in the MAP kinase pathway to
transduce signals from the cell surface to the nucleus to activate the
genetic machinery necessary for the maintenance of synaptic plasticity
in the dentate gyrus. Thus, MAPK/ERK activation is required for
LTP-dependent transcriptional regulation and we suggest this is
regulated by two parallel signaling pathways, the MAPK/ERK-Elk-1
pathway targeting SRE and the MAPK/ERK-CREB pathway targeting CRE.
Key words:
hippocampus; LTP; MAPK/ERK; Elk-1 phosphorylation; zif268; transcriptional regulation
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INTRODUCTION |
At many synapses, brief bursts of
tetanic stimulation trigger calcium influxes into the postsynaptic
neuron and induce a persistent increase in synaptic strength. This form
of synaptic plasticity, known as long-term potentiation (LTP), is
widely accepted as a candidate cellular mechanism for the storage of
information (Bliss and Collingridge, 1993 ). In the hippocampus, a brain
structure implicated in several forms of learning, LTP can last for
many hours or as long as days or weeks (Barnes, 1979; Doyère and
Laroche, 1992). The longer-lasting phases of LTP require the
transcription of genes and the synthesis of proteins during a critical
period (Otani et al., 1989; Nguyen et al., 1994; Frey and Morris,
1997). Within the first few hours of LTP, there is activation of
specific immediate early genes (IEGs) (Cole et al., 1989; Wisden et
al., 1990) encoding transcription factors that interact with promoter regulatory elements of a host of downstream effector genes. A crucial
event in signal transduction leading to gene regulation in neurons is
the activation of protein kinases. Several kinases, including PKC, PKA,
 calcium/calmodulin-dependent kinase II (CaMKII) and the
tyrosine kinases have been implicated in LTP (Soderling and Derkach,
2000). Recent work suggests that the mitogen-activated protein
kinase/extracellular-regulated kinase (MAPK/ERK) cascade, a complex
kinase cascade implicated in cell differentiation and proliferation, is
essential for long-term synaptic plasticity (English and Sweatt, 1996,
1997) and for certain types of learning (Atkins et al., 1998; Blum et
al., 1999). Moreover, there is abundant cross-talk between kinase
pathways, suggesting that MAPK/ERK may be a point of convergence
integrating PKC, PKA, and CaMK signals (Impey et al., 1998a; Vanhoutte
et al., 1999; Roberson et al., 1999), in addition to the activity of
individual signaling systems. In cell lines, MAPK/ERK translocates to
the nucleus once it has been activated in which it can regulate
transcriptional activity of many IEGs (for review, see Treisman,
1996).
In this study, we tested whether MAPK/ERK is activated in LTP in
vivo and required for LTP-induced gene expression and investigated how MAPK/ERK controls activity-dependent transcriptional regulation. Recent experiments have suggested that transactivation of the cAMP
response element-binding protein (CREB) by MAPK/ERK via the CREB kinase
ribosomal protein S6 kinase (Rsk2) plays a role in synaptic plasticity
and memory formation (for review, see Impey et al., 1999). For example,
using CRE-LacZ transgenic mice, it was shown that LacZ expression was
upregulated in the CA1 slice by the induction of LTP (Impey et al.,
1996) and during contextual learning (Impey et al., 1998b). The other
strong potential candidate, however, is the ternary complex factor
Elk-1, a prime nuclear substrate of the MAPKs c-Jun N-terminal
protein kinase (JNK), p38, and ERK (Treisman, 1995), which plays
a pivotal role in IEG induction by various extracellular signals
(Hipskind et al., 1991; Marais et al., 1993, 1994). In cell cultures,
phosphorylation of Elk-1 by MAPK/ERK strongly potentiates its ability
to activate transcription through a ternary complex assembled on the
serum response element (SRE), a DNA sequence motif present within the upstream regulatory region of many IEGs (Wasylyk et al., 1998), including zif268 which is strongly upregulated in
LTP. Although Elk-1 is expressed in hippocampal neurons (Sgambato et
al., 1998a), to date there is no evidence implicating Elk-1 as a
mediator of transcriptional induction in LTP.
Here we addressed two questions: first, are MAPK/ERK and Elk-1
activated in a coordinated manner after induction of LTP, and second,
is MAPK/ERK activation necessary for Elk-1 phosphorylation and
LTP-induced SRE-driven transcription. We demonstrate that induction of
LTP in the dentate gyrus in vivo results in a strong and
transient phosphorylation of MAPK/ERK in dendrites and nuclei of
granule cells and phosphorylation of both CREB and Elk-1 in strict
spatiotemporal correspondence with MAPK/ERK activation. Inhibition of
MAPK/ERK phosphorylation and nuclear translocation prevents Elk-1 and
CREB phosphorylation and LTP-induced transcriptional activation of
zif268, resulting in rapidly decaying LTP.
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MATERIALS AND METHODS |
Electrophysiology. Male Sprague Dawley rats
(n = 73) weighing 300-400 gm were anesthetized with
urethane carbamate (1.5 mg/kg), placed in a stereotaxic frame, and
maintained at a constant body temperature of 37°C. Unilateral
implantation of electrodes were performed using standard stereotaxic
procedures (Laroche et al., 1989). Recording electrodes were lowered
into the dentate gyrus (bregma  4.2 mm, 2.5 mm from midline) under
electrophysiological control. Stimulating electrodes were implanted in
the angular bundle of the perforant path (bregma 8.0 mm, 4.4 mm from
midline) to evoke a positive-going response in the hilus of the dentate gyrus. Low-frequency test pulses (100 µsec, 0.033 Hz) to the
perforant path were delivered via a photically isolated constant
current unit. Individual stimulus intensities were selected to give a population spike amplitude between 1 and 2 mV. After the response in
the dentate gyrus had stabilized, a 30 min baseline period was
recorded, followed by delivery of a tetanus or a pseudotetanus. Tetanic
stimuli consisted of six trains of pulses (400 Hz, 20 msec), delivered
at a 10 sec interval and repeated six times at an interval of 2 min.
Pseudotetanus consisted of six pulses, delivered at a 10 sec interval,
repeated six times with an interval of 2 min, to match the tetanus
without inducing LTP. To ensure maximal stimulation of the fibers
during the tetanus or pseudotetanus, the stimulus intensity was
increase during this period. Evoked responses were stored as averages
of four for off-line analysis. The maximum slope of the EPSP and
amplitude of the population spike were measured as described previously
(Laroche et al., 1989). In specified experiments, SL327 (100 mg/kg in
100% DMSO) or DMSO (100%) was injected intraperitoneally 1 hr before
the tetanus. Experimental procedures were conducted in accordance with
the guidelines of Centre National de la Recherche Scientifique and the
French Agricultural and Forestry Ministry (decree 87848, license number A91429).
Tissue preparation. Rats were killed at specified times
after receiving a tetanus or a pseudotetanus. For in situ
hybridization and immunocytochemistry, brains were fixed by
intracardiac perfusion of 4% paraformaldehyde (PFA) in 0.1 M
Na2HPO4/NaH2PO4
buffer, pH 7.5 (phosphate buffer). Brains were removed and post-fixed in the same fixative solution for 2 hr, washed overnight in 0.1 M phosphate buffer containing 15% sucrose, and
then frozen in isopentane (1 min at 25°C). Sections (20 µm) were
cut on a microtome and then kept in a solution containing 30% ethylene
glycol, 30% glycerol, 0.1 M phosphate buffer,
0.1% diethyl pyrocarbonate (Sigma) at 20°C until processed
for in situ hybridization or immunohistochemistry. For
Western blots, rat brains were rapidly removed, and the dorsal dentate
gyrus was dissected on ice and lysed in solubilization buffer (10 mM Tris-Cl, 50 mM NaCl, 1%
Triton X-100, 30 mM sodium pyrophosphate, 50 mM NaF, 5 µM
ZnCl2, 100 µM
Na3VO4, 1 mM DTT, 5 nM okadaic acid,
2.5 µg of aprotinin, 2.5 µg of pepstatin, 0.5 µM PMSF, 0.5 mM
benzamidine, and 2.5 µg of leupeptin). Insoluble material was removed
by centrifugation (13,000 rpm for 20 min at 4°C). Cell lysates [30
or 10 µg per lane for the detection of phospho-Elk1 (p-Elk-1)
and phospho-ERK (p-ERK), respectively] were separated by 10% SDS-PAGE
before electrophoretic transfer onto polyvinylidene difluoride membrane
(ICN Biochemicals, Orsay, France).
Immunocytochemistry and Western blotting. The
immunohistochemical procedure for detecting active ERK and Elk-1
proteins were performed as described previously (Sgambato et al.,
1998a). Briefly, free-floating sections were rinsed in Tris-buffered
saline (TBS) (0.25 M Tris and 0.5 M NaCl, pH 7.5), incubated for 5 min in TBS containing 3% H2O2 and
10% methanol, and then rinsed three times for 10 min each in TBS (0.1 mM NaF was included in all buffers and incubation
solutions). After 15 min incubation in 0.2% Triton X-100 in TBS,
sections were rinsed three times in TBS. These were incubated with the
primary antibody (see below) for 72 hr at 4°C. After three rinses in
TBS, sections were incubated for 48 hr at 4°C with the secondary
biotinylated antibody (anti-IgG) using a dilution twice that of the
first antibody in TBS. After washing (three times in TBS), sections
were incubated overnight in avidin-biotin-peroxidase complex solution
(ABC solution; final dilution 1:50; Vector Laboratories, Burlingame,
CA). Sections were then washed two times in TBS and two times in TB
(Tris 0.25 M, pH 7.5), 10 min each, placed in a
solution of TB containing 0.1% 3-3' diaminobenzidine (50 mg/100 ml)
and developed by adding
H2O2 (0.02%). After
processing, tissue sections were mounted onto gelatin-coated slides and
dehydrated through alcohol to xylene for light microscopic examination.
For Western blotting, blots were treated as described previously
(Vanhoutte et al., 1999). Briefly, they were saturated (1 hr at room
temperature) with BSA (Fraction V; Sigma) 8% (p-Elk-1) or 5% (p-ERK
and p-CREB) and incubated (overnight at 4°C) with the anti-active
antibodies. On the second day, the blots were incubated for 2 hr at
room temperature with goat anti-rabbit-horseradish
peroxidase-conjugated antibodies before exposure to the ECL substrate.
Then the blots were stripped (glycine-HCl, pH 2.8, two times for 20 min
each at 55°C) and saturated overnight in 5% nonfat dry milk.
On the third day, the blots were then incubated with the nonactive
antibodies (see below). The efficacy of the stripping step was assessed
by omitting the first antibody and verifying the lack of signals on the
blot. Anti-active antibodies were polyclonal antibodies raised against
the double-phosphorylated Thr/Glu/Tyr region within the catalytic core
of the active form of p44/ERK1 and p42/ERK2
(anti-phospho-Thr183
-Tyr185 ERKs; New England Biolabs,
Beverly, MA), a phospho-Ser383 peptide
corresponding to residues 379-392 of Elk-1 (New England Biolabs), and
a phospho-Ser133 peptide corresponding to
residues 129-137 of CREB (Upstate Biotechnology, Lake Placid, NY). The
dilutions used for immunostaining were 1:200 for p-ERK antiserum; 1:100
for p-Elk-1 antiserum, and 1:200 for p-CREB. For Western blot analysis,
the dilutions were 1:2500 for p-ERK antiserum, 1:500 for p-CREB
antiserum,1:200 for p-Elk-1, and 1:750 for p-CREB antiserum. For
Western blot analysis, the nonactive antibodies used were anti-ERK
antibody (1:4000; Tebu, Le Perray en Yvelines, France) and anti-Elk-1
antibody (1:1000, rabbit polyclonal antibody raised against a
recombinant protein corresponding to the C-terminal region of human
Elk-1) (Janknecht et al., 1994).
In situ hybridization. The antisense (complementary to
cellular mRNA) probes were
33P-radiolabeled riboprobes. For
zif268, and MAP kinase phosphatase (MKP) riboprobes, murine
cDNA subclones were used. Zif268 insert corresponding to 1.6 kb was linearized after HindIII digestion and
transcribed with T7 RNA polymerase. The MKP-1 (663 bp) was transcribed
with T7 RNA polymerase after linearization with PstI. Transcription reactions contained 1 µM
33P-UTP (3000 Ci/mmol;
Isotopchim, Peyruis, France), 250 µM ATP, CTP,
and GTP, and unlabeled UTP (10.5 µM), and were
incubated at 39°C for 2 hr. After DNase I digestion, the labeled RNA
was purified by phenol/chloroform/isoamyl alcohol (25:24:1) extraction and ethanol precipitation. Gel electrophoresis showed the transcripts to be predominantly full-length. Free-floating sections were mounted on
SuperFrost Plus slides (Menzel-Gläser) in RNase-free conditions. Once dried, mounted sections were rinsed in PBS and treated for 10 min
with 0.1 M glycine in 0.1 M
Tris-HCl, pH 7.4. Sections were rinsed for 5 min at 37°C in 0.1 M Tris-HCl, pH 8, and 50 mM
EDTA, and treated for 15 min at 37°C with 1 mg/ml proteinase K in the
same buffer. Before hybridization, sections were subjected to the
following treatment: post-fixation for 15 min in 4% PFA and 5 mM MgCl2 in PBS at room
temperature, acetylation for 20 min in acetic
anhydride/triethanolamine, pH 8, at room temperature, and stepwise
dehydration in alcohol. The following hybridization solution was
applied to sections, which were then covered with GelBond Film (FMC
Bioproducts, Rockland, ME). The hybridization mixture contained 200 ng/ml (4 ng/section) 33P-RNA probe in 20 mM Tris-HCl, pH 8, 300 mM
NaCl, 5 mM EDTA, 10% dextran sulfate, 1×
Denhardt's solution (0.02% Ficoll, 0.02% polyvinyl pyrolidone, and
10 mg/ml BSA), 0.5 mg/ml E. coli tRNA, 0.1 M DTT, and 50% formamide. Hybridization was
performed at 60°C in humid chambers for 16 hr. After removing the
GelBond coverslips in 4× SSC (1× SSC is 0.15 M
NaCl-0.015 M Na citrate) and 10 mM DTT, the slides were washed in the same
solution for 1 hr at room temperature and then in 50% formamide, 10 mM Tris-HCl, pH 8, 75 mM
NaCl, and 2.5 mM EDTA. Sections were treated with
RNase A (20 µg/ml; Sigma) in 400 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 50 mM EDTA for 1 hr at 37°C and then rinsed for 15 min at 60°C in 2× SSC, followed by 0.1× SSC. After dehydration,
sections were air dried and exposed with Biomax MR films (Eastman
Kodak, Rochester, NY) for 3 d (zif268) or 6 d (for
MKP-1 probes).
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RESULTS |
Activation of MAPK/ERK and Elk-1 after the induction of LTP
We induced LTP in the dentate gyrus of adult rats, using a
protocol of repeated high-frequency stimulation of the perforant path
similar to that used previously to elicit LTP lasting several days in
awake animals (Laroche et al., 1989). As expected, the tetanus induced
a rapid and stable increase in both the slope of the EPSP (29.04 ± 2.29%) (Fig. 1a) and the
population spike (392.12 ± 60.66%), whereas there was no change
in synaptic efficacy in control rats receiving a pseudotetanus (Fig.
1a). Because it is known that LTP in the dentate gyrus can
lead to the rapid induction of several immediate early genes (Cole et
al., 1989; Wisden et al., 1990), we used in situ
hybridization on brain sections from rats killed immediately after the
10 min tetanus (time 0), 15 min or 1 hr later, to examine the temporal
pattern of expression of zif268, an IEG containing four SRE
sites on its promoter (Treisman, 1995). In our conditions, there was a
strong induction of zif268 mRNA expression in the
ipsilateral dentate gyrus in all rats after LTP (Fig. 1b),
which was already observed immediately after the end of the tetanus
(LTP 0) and was sustained for at least 1 hr after LTP. There was only
moderate constitutive expression of zif268 in the dentate
gyrus at any time point in control rats receiving a pseudotetanus (Fig.
1b). Additional experiments showed that levels of the mRNA
was back to baseline levels 3 hr after the induction of LTP (data not
shown). Thus, our results are consistent with previous findings (Cole
et al., 1989; Wisden et al., 1990) and show that increased expression
of zif268 induced by LTP occurs earlier than previously
reported.
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Figure 1.
LTP in the dentate gyrus in vivo
and corresponding upregulation of zif268 mRNA
expression. a, The slope of the EPSP is plotted as a
percentage change against the baseline before high-frequency tetanic
stimulation (LTP) or a pseudotetanus
(control). Each point represents
an average of four consecutive evoked responses. The white
bar indicates the 10 min period in which the tetanus or the
pseudotetanus was delivered. Rats were killed either immediately
after the last train of tetani (LTP 0), or 15 (LTP 15) or 60 (LTP 60)
min after the tetanus. In rats that were killed immediately after the
tetanus, an average of four responses were measured between each
tetani. b, Autoradiograms shows upregulation of
zif268 mRNA at different time points after the induction
of LTP compared with control rats (CT 0). There were
three to four rats in each group at each of the different time
points.
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In the next step, we tested whether the induction of LTP in the dentate
gyrus in vivo leads to MAPK/ERK activation and defines its
time course of activation. Adjacent sections to those used for in
situ hybridization were used for immunocytochemical detection of
activated MAPK/ERK proteins using an antibody to the
double-phosphorylated form of MAPK
(antiphospho-Thr183-Tyr185
ERK1/2). A slight p-MAPK/ERK immunostaining was observed in control rats in both the dentate gyrus and CA layers with no difference between
the stimulated and nonstimulated sides (Fig.
2), showing that phosphorylation of
MAPK/ERK was not influenced by low-frequency stimulation of the
perforant path. In sections taken from rats in which LTP was induced,
we observed a marked increase in p-MAPK/ERK in the molecular layer and
granule cell layer of the dentate gyrus immediately after the induction
of LTP, relative to the control side or to rats receiving a
pseudotetanus (Fig. 2a). This was a transient increase,
because p-MAPK/ERK was no longer detected 15 min later (Fig. 2).
Densitometric analysis revealed a significant increase in p-MAPK/ERK
immediately after the LTP-inducing tetanus (331 ± 62%,
n = 4, p < 0.05 relative to the
control side) (Fig. 2c). High-magnification microscopy
showed numerous p-MAPK/ERK-immunopositive dentate granule cells after
LTP (Fig. 2b). The increased immunostaining was observed in
cytoplasmic compartments and dendrites, suggesting local postsynaptic
activation of the protein in the vicinity of the receptors. Upon
activation, MAPK/ERK translocates to the nucleus (Chen et al., 1992;
Lenormand et al., 1993). The appearance of strong nuclear staining
(Fig. 2b) in numerous granule cells suggests nuclear
translocation of activated MAPK/ERK proteins after the induction of
LTP. Our results showing the transient activation of MAPK/ERK
immediately after the induction of LTP were confirmed biochemically
using Western blot analysis of dorsal dentate gyrus extracts from other
groups of animals in each condition (Fig. 2d). p-MAPK/ERK
antibody yielded two bands of 42 and 44 kDa corresponding to ERKs 2 and
1, respectively. Immediately after the 10 min LTP-inducing tetanus, we
observed a significant increase in p-ERK1 (51%) and p-ERK2 (280%)
relative to low-frequency stimulation, suggesting an early activation
with the first few trains. This increase reflected MAPK/ERK activation,
because total levels of MAPK/ERKs present in tissue extracts were
comparable (Fig. 2d). In concordance with the immunolabeling
(Fig. 2a,b), MAPK/ERK1 and ERK2 activation was no
longer significantly increased 15 min after the end of the tetanus
(Fig. 2d). Thus, as demonstrated previously in area CA1
in vitro (English and Sweatt, 1996), LTP in the dentate
gyrus in vivo results in a rapid and transient activation of
MAPK/ERKs. Activation of the MAPK/ERK1 isoform has not been reported
previously, a difference with the present results that may reflect
structure specificity (dentate gyrus vs CA1) or a difference between
LTP in vivo and in vitro.
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Figure 2.
Increase in p-MAPK/ERK after the induction of LTP.
a, Immunocytochemical images representing p-MAPK/ERK in
the ipsilateral dentate gyrus show that it is increased at LTP 0 compared with the contralateral side. No change was observed in the
controls or LTP 15. b, High magnification (630×) of
images show p-MAPK/ERK labeling in both cell bodies and dendrites of
the granule cells in the ipsilateral dentate gyrus. c,
Densitometric quantification of immunolabeled sections shows that
p-MAPK/ERK is significantly increased only at LTP 0 (p < 0.05). d, Western blots
of p-ERK1 and p-ERK2 confirm immunohistochemical results showing that
the increase in p-MAPK/ERK occurs at LTP 0 (top
panel), and this increase was only observed with the
activated forms (bottom panel).
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The rapid dephosphorylation of MAPK/ERK proteins observed in granule
cells of the dentate gyrus after its initial activation in LTP suggests
a possible negative feedback control arising from a specific
phosphatase. To address this issue, we examined the expression of
MKP-1, described in cell lines in vitro as an IEG that is
rapidly transcribed by mitogens and translated to inactivate MAPK/ERK
(Charles et al., 1993; Sun et al., 1993). Using in situ hybridization, we tested whether MKP-1 mRNA could be upregulated after
the induction of LTP. In control rats, no signal was detectable in the
hippocampus; however, we found an increase in MKP-1 mRNA hybridization
signals in the ipsilateral dentate gyrus 15 min after LTP and to a
greater extent 1 hr after LTP (Fig. 3).
By 3 hr after LTP, the expression of MKP-1 mRNA had returned to basal levels (data not shown). Thus, high-frequency stimulation leads to
rapid and sustained induction of MKP-1 mRNA, which is one potential mechanism that may account for a negative feedback loop underlying the
rapid dephosphorylation of MAPK/ERKs observed 15 min after the
induction of LTP.
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Figure 3.
Expression of MKP-1 mRNA after induction of
LTP in the dentate gyrus. In situ hybridization shows
that MKP-1 is upregulated at LTP 15 and LTP 60 and that the increase is
restricted to the potentiated side of the dentate gyrus. No difference
was observed in the control stimulated rats at either time point. There
were three to four rats in each group.
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In the next experiment, we tested whether the induction of LTP and the
activation of MAPK/ERK resulted in simultaneous activation of the
transcription factor Elk-1. Upon MAPK/ERK activation, phosphorylation of Elk-1 occurs principally on Ser383 and
Ser389 residues (Marais et al., 1993).
Adjacent sections to those used for p-MAPK/ERK immunocytochemistry were
labeled with an antibody that specifically recognizes the
Ser383-phosphorylated form of Elk-1
(antiphospho-Ser383-Elk-1). In control
stimulated animals, constitutive labeling was detectable bilaterally in
the dentate gyrus and CA layers, and no change in p-Elk-1 was
detectable on the side of the dentate gyrus in which low-frequency
stimulation was delivered (Fig. 4). Immediately after the induction of LTP, we observed a marked increase in p-Elk-1 immunolabeling in the granule cell and molecular layers of
the dentate gyrus that was restricted to the side of LTP induction (Fig. 4a) and was still detectable 15 min later (Fig.
4a). Densitometric analysis (Fig. 4c) revealed
that this increase in p-Elk-1 was significant immediately at the end of
the tetanus (203 ± 43%, n = 4, p < 0.05) and 15 min later (130 ± 7%, n = 4, p < 0.05), whereas no change was observed after
control stimulation. High-magnification shows that p-Elk-1 staining was
increased in the nuclei of granule cells and dendrites after LTP (Fig.
4b) compared with the contralateral dentate gyrus in which
nuclei of granule cells showed only constitutive labeling. To confirm
the specificity of anti-active Elk-1 antibody, we performed Western
blots on tissue extracts taken from the dorsal dentate gyrus of control
rats and rats in which LTP was induced. p-Elk-1 antibody, which yielded
one band of 62 kDa, the expected molecular weight for Elk-1, gave
increased signals in extracts taken immediately after the tetanus
(199 ± 60%, n = 6) but not 15 min later
(103 ± 19%, n = 3) relative to the controls
(Fig. 4d), whereas comparable levels of Elk-1 were present
in dentate tissue from all groups (Fig. 4d). At 15 min, the
absence of an increase in p-Elk-1 with Western blots compared with
immunocytochemistry indicates that p-Elk-1 may not be increased in all
cells at this time point. Thus, the results show that Elk-1 is
hyperphosphorylated after high-frequency stimulation of the perforant
path, correlated with the activation and nuclear translocation of the
MAPK/ERKs. In postsynaptic granule cells, phosphorylation of Elk-1
after LTP occurred not only in the nucleus in which it can activate the
SRE-DNA regulatory element of IEG promoters but also in the cytoplasm.
Although this observation seems intriguing, Elk-1 is described in the
adult CNS as a cytoplasmic and nuclear target of activated
MAPK/ERK signaling cascade, and our observations in the dentate gyrus
after LTP are consistent with the previously described localization and
regulation of Elk-1 in the striatum (Sgambato et al., 1998a,b).
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Figure 4.
Increase in p-Elk-1 after the induction of LTP.
a, Immunocytochemical images in the ipsilateral dentate
gyrus shows an increase in p-Elk-1 at LTP 0 and to a lesser extent at
LTP 15 compared with the contralateral side. No change was observed in
the control groups. b, High magnification (630×) of
these images shows p-Elk-1 labeling in both cell bodies and dendrites
in the ipsilateral dentate gyrus. c, Densitometric
quantification of immunolabeling shows that p-Elk-1 is significantly
increased at LTP 0 and LTP 15. d, Western blots of
p-Elk-1 confirm immunohistochemical results showing increased p-Elk-1
at LTP 0 (top panel), and this increase was only
observed with the activated form, with no change in total Elk-1
(bottom panel).
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Inhibition of MAPK/ERK blocks Elk-1 activation and SRE-regulated
gene expression
The data presented so far support the hypothesis that MAPK/ERK
activation and nuclear translocation do have a role to play in
LTP-induced gene expression via the transcription factor Elk-1. If this
hypothesis is correct, then inhibition of MAPK/ERK phosphorylation should result in a blockade of Elk-1 phosphorylation and inhibition of
LTP-induced SRE-dependent gene expression. To test this, we induced LTP
in the presence of an inhibitor of the MAPK kinase MEK, a dual-specific
MAPK/ERK-activating enzyme. The MEK inhibitor SL327 (Favata et al.,
1998) was injected intraperitoneally 1 hr before the induction of LTP
at a dose of 100 mg/kg, conditions that have been reported to abolish
MAPK/ERK activation in CA1 (Atkins et al., 1998). When SL327 was
injected, LTP in the dentate gyrus was induced to the same level as in
drug vehicle (DMSO)-injected controls, but it decayed rapidly with the
slope of the EPSP returning close to baseline levels within 1 hr (Fig.
5a). Here, we confirm observations made in vitro in the hippocampus that MEK
inhibitors induce rapidly decaying LTP (English and Sweatt, 1997;
Coogan et al., 1999), adding further strength to the role of MAPK/ERK in long-term synaptic plasticity, by demonstrating the same effect in vivo.
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Figure 5.
Inactivation of MEK with SL327 results in
disruption of the downstream activation of p-MAPK/ERK, p-Elk-1, and
zif268 mRNA expression induced by LTP. a,
After the induction of LTP in the presence of SL327, the EPSP declined
to basal levels within 60 min, and there was no effect on LTP after
injections of DMSO. b, Immunocytochemical images of
p-MAPK/ERK in the dentate gyrus at LTP 0 shows that DMSO had no effect
on LTP-induced p-MAPK/ERK, whereas injections of SL327 resulted in
inhibition of p-MAPK/ERK. c, Similarly, p-Elk-1 was also
inhibited by SL327 at LTP 0, with no effect on LTP-induced activation
of p-Elk-1 after DMSO. d, In situ
hybridization images of zif268 at LTP 0 and LTP 60 in
rats injected with either SL327 or DMSO. Zif268 was
upregulated in the potentiated dentate gyrus in the DMSO control group
at both time points, whereas in the SL327 group, LTP-induced
upregulation of zif268 was blocked at both time points.
e, Quantification of densitometric measures of
p-MAPK/ERK and p-Elk-1 at LTP 0 and optical density measures of the
expression of zif268 mRNA at LTP 0 and LTP 60 confirm
blockade of MAPK/ERK and Elk-1 phosphorylation, and of
zif268 induction in the presence of SL327
(asterisks indicate significant difference from DMSO
controls).
|
|
Next, we tested whether SL327 blocks LTP-induced MAPK/ERK and Elk-1
activation on brain sections from rats killed immediately after the
tetanus. We noted that, after the injection of SL327, the level of
p-MAPK/ERK immunostaining was lower that in the contralateral side of
DMSO-treated rats, suggesting a decrease in the constitutive level of
p-MAPK/ERK as reported previously (Atkins et al., 1998). In the LTP
experiments, we found that SL327 strongly inhibited both MAPK/ERK (Fig.
5b) and Elk-1 phosphorylation (Fig. 5c).
Immediately after the tetanus, increases in p-MAPK/ERK and in p-Elk-1
immunolabeling was observed in all rats injected with DMSO, thus
replicating the above results after LTP in noninjected rats. In
contrast, immunostaining of p-MAPK/ERK and p-Elk-1 was strongly
inhibited in rats injected with SL327 (Fig.
5b,c). The densitometric analysis revealed a
significant increase in p-MAPK/ERK (Fig. 5e) and p-Elk-1 (Fig. 5e) staining in the ipsilateral side in DMSO-treated
rats (420 ± 84 and 172 ± 5%, respectively,
n = 3, p < 0.05 in each case), which
was not different from the results obtained after LTP in noninjected
rats, but was significantly different from the SL327 group
(p < 0.05). This result indicates that MAPK/ERK activation is required for LTP-induced Elk-1 phosphorylation. We then
assessed whether SL327 blocks zif268 expression using in situ hybridization on adjacent sections from those used
for immunocytochemistry (immediately after the tetanus, DMSO,
n = 3; SL327, n = 3) and in brains
taken 1 hr after the tetanus (DMSO, n = 3; SL327,
n = 3). Under these conditions, there was no change in
constitutive expression of zif268 mRNA. In accordance with the results shown in Figure 1, a marked increase in the expression of
zif268 was observed immediately and 1 hr after the tetanus in DMSO-injected rats (Fig. 5d). When SL327 was injected, we
found a complete blockade of LTP-induced expression of
zif268 mRNA (Fig. 5d). Surprisingly, not only did
SL327 block zif268 induction, but in two of the three rats
at 1 hr after tetanus, the combination of SL327 injections with the
tetanus resulted in downregulation of zif268 in the
stimulated side (Fig. 5d). Because the phosphorylation state
of both Elk-1 and CREB is linked to a balance between kinase and
phosphatase activity, it is possible that, under SL327, a strong
calcium-induced phosphatase activity induced by LTP leads to
downregulation of zif268. Together, these findings provide a
molecular substrate for the action of MAPK/ERK on long-term synaptic
plasticity by showing direct control on gene regulation, a hypothesis
that has been alluded to in many recent studies (English and Sweatt,
1997; Impey et al., 1998a, 1999) but until now has not been
demonstrated. There were of course some differences between animals.
Most interesting was the observation of a slight residual increase in
p-MAPK/ERK immunoreactivity in some rats after injection of SL327 (Fig.
6a-d), as shown in the
densotimetric analysis (Fig. 5e). Immunocytochemistry showed
that this increase was restricted to dendrites, with very little, if
any, nuclear staining (Fig. 6d). When this occurred, p-Elk-1
was greatly reduced compared with DMSO-treated rats (Fig.
6b-e). Despite the slight activation of MAPK/ERK in
dendrites, there was no transcriptional regulation of zif268
(Fig. 6c-f), suggesting that strong MAPK/ERK
activation and its nuclear translocation is necessary for SRE-driven
gene regulation.
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[in this window]
[in a new window]
|
Figure 6.
High magnification of phosphorylated MAPK/ERK and
Elk-1, and expression of zif268 at LTP 0 in the presence
of DMSO or SL327. a, In the presence of DMSO, there is
heavy labeling of p-MAPK/ERK in both dendrites and the cell bodies of
granule cells. In addition, there is hyperphosphorylation of Elk-1
(b) and an upregulation of zif268
mRNA (c). d, In some cases, there
was some labeling of p-MAPK/ERK in the SL327-treated rats, but this was
restricted to the dendrites. In these rats, p-Elk-1 was greatly reduced
(e), and there was no upregulation of
zif268 mRNA (f),
suggesting the need for translocation of MAPK/ERK to the nucleus to
phosphorylate Elk-1 and upregulate zif268.
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|
Inhibition of MAPK/ERK also blocks LTP-induced CREB activation
Several studies have demonstrated that MAPK/ERK can also activate
CREB via phosphorylation of the CREB kinase Rsk2 (Xing et al., 1996;
Impey et al., 1998a) and that LTP-driven CRE-LacZ expression in the CA1
slice is blocked by the MEK inhibitor PD98059 (Impey et al., 1998a).
In vivo, activation of CREB was described recently after LTP
in the dentate gyrus (Schulz et al., 1999). Using Western blots,
we have confirmed here that an increase
in phosphorylated CREB occurs after LTP in the dentate gyrus (178 ± 22%, n = 5, p < 0.05 compared with
controls) (Fig. 7c), and the time course was similar to that
observed for Elk-1 phosphorylation. This was also clear from sections
adjacent to those used for p-MAPK/ERK and p-Elk-1 staining and labeled
with p-CREB-specific antibody, showing that p-CREB increases in the
nuclei of granule cells immediately at the end of the tetanus (Fig.
7a). Injection of SL327 before the tetanus strongly
inhibited the LTP-induced increase in p-CREB in granule cell nuclei
(Fig. 7f). Together, the results strongly suggest that
activated MAPK/ERK can regulate gene expression after nuclear
translocation and the phosphorylation of at least two transcription
factors, CREB and Elk-1.
View larger version (85K):
[in this window]
[in a new window]
|
Figure 7.
Phosphorylation of CREB after induction of LTP and
in the presence of SL327. a, b, An
increase in p-CREB labeling was observed on the potentiated side at LTP
0 compared with the contralateral dentate gyrus. c,
d, Western blots confirm that p-CREB is increased only
at this time point compared with control rats, and quantification
showed that this was a significant increase. e,
f, The level of p-CREB was greatly attenuated in
SL327-treated rats compared with DMSO controls.
|
|
| |
DISCUSSION |
Previous studies have indicated a critical role for the MAPK/ERK
cascade in models of neuronal plasticity, such as long-term facilitation in Aplysia sensory neurons (Martin et al., 1997) and LTP
in CA1 of the hippocampus (for review, see Impey et al., 1999). Because
the maintenance of these forms of plasticity requires gene
transcription (Nguyen et al., 1994; Bartsch et al., 1995), we have
addressed the issue of whether and how activation of MAPK/ERK can
mediate gene induction in LTP by measuring the expression of the IEG
zif268, known to be strongly activated in LTP. Our results
support the idea that MAPK/ERK phosphorylation and its subsequent
nuclear translocation is an essential step in mediating gene induction
required for long-lasting LTP in the dentate gyrus in vivo.
We further demonstrate that Elk-1 is a key component between MAPK/ERK
activation and LTP-dependent gene induction. The MAPK/ERK-Elk-1 cascade
has been shown to be activated by electrical stimulation of
corticostriatal fibers (Sgambato et al., 1998a,b), and here we
demonstrate for the first time its role in LTP-dependent gene
induction. Our study shows that MAPK/ERK and the two downstream transcription factors, Elk-1 and CREB, are rapidly phosphorylated in
nuclei of dentate granule cells after the induction of LTP. Inhibition
of MEK, the upstream MAPK/ERK kinase, blocked LTP-induced phosphorylation of MAPK/ERK and, conjointly, the phosphorylation of
Elk-1 and CREB, resulting in a rapidly decaying LTP. These findings
support the model that MAPK/ERK activation and nuclear translocation
are essential to the coactivation of the two transcription factors.
Elk-1 is one of the main nuclear targets of activated MAPK/ERK and has
been shown to be newly phosphorylated on
Ser383 and
Ser389 by growth factors (Marais et al.,
1993), leading to SRE-driven gene transcription in cell cultures (for
review, see Wasylyk et al., 1998). The SRE, together with flanking DNA
sequences, serves as a site of assembly of multiprotein complexes,
including a dimer of the serum response factor (Treisman, 1986; Norman
et al., 1988; Schröter et al., 1990) and a protein of the ternary
complex factor family, such as Elk-1 (for review, see Treisman, 1995),
and this site is present in the promoter region of many IEGs, including zif268. Our results, which show that inhibition of MAPK/ERK
phosphorylation and the resulting inhibition of Elk-1 prevents the
transcriptional activation of zif268, support the model that
activation of the MAPK/ERK signaling pathway targets nuclear Elk-1 to
control SRE-mediated gene expression in LTP. Because the stress-induced
MAPKs p38 and JNK are not affected by the MEK inhibitor, it highlights
the role of the MAPK/ERK subfamily in Elk-1 phosphorylation in LTP. We also found that dendritic Elk-1 was phosphorylated after LTP as has
been reported in the striatum after cortical stimulation (Sgambato et
al., 1998a,b), and this was blocked by inhibition of MEK. Although our
study does not address the function of dendritic Elk-1 protein in LTP,
together the results suggest a role as a local cytoplasmic substrate of
activated MAPK/ERKs, which may be implicated in relaying the
glutamatergic receptor signal to different intracellular compartments, including the nucleus. Interestingly, the fact that, in the presence of
SL327, LTP in the dentate gyrus decayed more rapidly than one would
expect for a cascade involved in the protein synthesis-dependent phase
of LTP, suggests that MAPK/ERK may also contribute to an earlier phase
of LTP and this may well involve one of the many cytoplasmic substrates
of MAPK/ERK, such as dendritic Elk-1.
Transcriptional regulation in LTP is classically attributed to CREB,
which binds to the CRE site present in the promoter regions of several
IEGs, and CREB appears to be critical for the maintenance of LTP and
the formation of certain forms of long-term memory (Bourtchuladze et
al., 1994). Consistent with a recent report (Schulz et al., 1999), we
show that, after the induction of LTP, CREB was phosphorylated in
dentate granule cell nuclei. In addition, we show here that it follows
a similar time course to that of MAPK/ERK and Elk-1 phosphorylation,
and it is blocked by inhibition of MEK. The data here add a further
element to the previous suggestion of MAPK/ERK-dependent CRE-driven
expression in LTP in CRE-LacZ transgenic mice in vitro
(Impey et al., 1996, 1998a), and this presumably occurs via a CREB
kinase of the Rsk family resulting in phosphorylation of CREB (Xing et
al., 1996; Impey et al., 1998a). Together, we propose that the signal
transduction mechanisms underlying LTP-dependent transcription involves
two parallel signaling pathways, the direct MAPK/ERK-Elk-1 signaling
pathway mediating SRE-dependent transcription and the indirect
MAPK/ERK-Rsk-CREB pathway mediating CRE-dependent transcription. It is
possible that these transcription factors cooperate to activate genes,
at least those carrying both CRE and SRE. The analysis of
c-fos expression in transgenic mice models showing
that the combination of these two DNA-binding elements is crucial for
gene transcription (Robertson et al., 1995) suggests that this may well
be the case. The zif268 promoter contains two putative CRE
sites but multiples SREs and is therefore likely to be strongly
controlled by the MAPK/ERK-Elk-1 pathway. Under normal conditions, it
is possible that the strong activation of zif268 in LTP is
attributable to the combined activation of both Elk-1 and CREB
by MAPK/ERK. This may occur via their interactions with the coactivator
CREB binding protein, which facilitates more efficient transcription
through multiple contacts with the basal transcriptional machinery
(Kwok et al., 1994; Janknecht and Nordheim, 1996) and plays a key role
in calcium-mediated gene transcription in cellular models of plasticity
(Hardingham et al., 1999; Hu et al., 1999).
The fact that MAPK/ERK, CREB, and Elk-1 are only transiently activated
suggests that there may be a mechanism that dephosphorylates these
proteins. As postulated in a recent model of the dynamics of
interactions in signaling systems (Bhalla and Iyengar, 1999), a
feedback loop may involve phosphatase activity. Our findings that the
mRNA encoding the MAPK/ERK phosphatase MKP-1 is overexpressed in
granule cells after LTP is entirely consistent with this prediction. Although not the only possibility, the activation of MKP-1 for between
1 and 3 hr after LTP thus provides one example of a feedback mechanism
that is likely to function in this way, resulting in deactivation of
CREB and Elk-1, and subsequently that of IEG transcription.
In summary, these data provide evidence that, in the transcriptional
events associated with LTP, the MAPK/ERK signaling cascade plays an
important role in regulating genes controlled not only by CRE via
phosphorylation of CREB (Impey et al., 1998a) but also by SRE via
phosphorylation of Elk-1. Together with previous findings, we propose
that the MAPK cascade controls diverse transcriptional responses
induced by LTP through two distinct pathways, one targeting CRE-mediated transcription via the activation of CREB, the second pathway controlling SRE-mediated transcription via phosphorylation of
the transcription factor Elk-1.
| |
FOOTNOTES |
Received Feb. 24, 2000; revised March 30, 2000; accepted March 31, 2000.
This work was supported in part by a grant from Institut Lilly to
J.C. P.V. is a doctoral fellow of the Ministère de
l'Education Nationale et de l'Enseignement Supérieur. We thank
M. Rogard for technical assistance. We are grateful to J. M. Trzaskos, J. L. Hytrek, A. C. Tabaka, J. S. Piecara, and
C. Teleha for the generous gift of SL327.
Correspondence should be addressed to J. Caboche, Laboratoire de
Neurochimie-Anatomie, Institut des Neurosciences, Centre National
de la Recherche Scientifique, Unité Mixte de Recherche 7624, Universtité Pierre et Marie Curie, 9 quai St. Bernard, 75005 Paris, France. E-mail: jocelyne.caboche{at}snv.jussieu.fr.
| |
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M. Mackiewicz, B. Paigen, N. Naidoo, and A. I. Pack
Analysis of the QTL for sleep homeostasis in mice: Homer1a is a likely candidate
Physiol Genomics,
October 8, 2008;
33(1):
91 - 99.
[Abstract]
[Full Text]
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S. C. Pandey, H. Zhang, R. Ugale, A. Prakash, T. Xu, and K. Misra
Effector Immediate-Early Gene Arc in the Amygdala Plays a Critical Role in Alcoholism
J. Neurosci.,
March 5, 2008;
28(10):
2589 - 2600.
[Abstract]
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N. Granado, O. Ortiz, L. M. Suarez, E. D. Martin, V. Cena, J. M. Solis, and R. Moratalla
D1 but not D5 Dopamine Receptors Are Critical for LTP, Spatial Learning, and LTP-Induced arc and zif268 Expression in the Hippocampus
Cereb Cortex,
January 1, 2008;
18(1):
1 - 12.
[Abstract]
[Full Text]
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W. B. Chwang, J. S. Arthur, A. Schumacher, and J. D. Sweatt
The Nuclear Kinase Mitogen- and Stress-Activated Protein Kinase 1 Regulates Hippocampal Chromatin Remodeling in Memory Formation
J. Neurosci.,
November 14, 2007;
27(46):
12732 - 12742.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION
