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The Journal of Neuroscience, May 15, 2000, 20(10):3529-3536
Developmentally Regulated NMDA Receptor-Dependent
Dephosphorylation of cAMP Response Element-Binding Protein (CREB) in
Hippocampal Neurons
Carlo
Sala,
Sheila
Rudolph-Correia, and
Morgan
Sheng
Department of Neurobiology and Howard Hughes Medical Institute,
Massachusetts General Hospital and Harvard Medical School, Boston,
Massachusetts 02114
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ABSTRACT |
Developmental changes in the signaling properties of NMDA receptors
have been proposed to underlie the loss of plasticity that accompanies
brain maturation. Calcium influx through postsynaptic NMDA receptors
can stimulate neuronal gene expression via signaling pathways such as
the Ras-MAP kinase (MAPK) pathway and the transcription factor cAMP
response element-binding protein (CREB). We analyzed MAPK (Erk1/2) and
CREB activation in response to NMDA receptor stimulation during the
development of hippocampal neurons in culture. At all stages of
development NMDA stimulation induced a rapid phosphorylation of CREB on
Ser-133 (phospho-CREB). However, the time course of decline in
phospho-CREB changed dramatically with neuronal maturation. At 7 d
in vitro (7 DIV) phospho-CREB remained elevated 2 hr
after strong NMDA stimulation, whereas at 14 DIV phospho-CREB rose only
transiently and fell back to below basal levels within 30 min.
Moreover, at 14 DIV, but not at 7 DIV, NMDA receptor stimulation
induced a dephosphorylation of CREB that previously had been
phosphorylated by KCl depolarization or forskolin, suggesting an NMDA
receptor-dependent activation of a CREB phosphatase. There was no
developmental change in the time course of phospho-CREB induction that
followed KCl depolarization or PKA activation, nor was there a
developmental change in the time course of phospho-Erk1/2 induced by
NMDA receptor activation. We suggest that, during neuronal maturation,
NMDA receptor activation becomes linked specifically to protein
phosphatases that act on Ser-133 of CREB. Such a developmentally regulated switch in the mode of NMDA receptor coupling to intracellular signaling pathways may contribute to the changes in neural plasticity observed during brain development.
Key words:
MAP kinase; ERK1/2; synaptic plasticity; protein
phosphatase-1; gene expression; critical period
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INTRODUCTION |
The NMDA class of ionotropic
glutamate receptor is critical for activity-dependent synaptic
modifications that underlie plasticity of the brain. Changes in the
abundance and functional properties of NMDA receptors therefore can
have important effects on synaptic plasticity. Developmental changes in
the signaling properties of NMDA receptors have been proposed to
underlie the loss of plasticity that accompanies brain maturation. For
instance, during cortical development, changes in the gating properties
of NMDA receptors correlate with maturational changes in the plasticity
of visual cortex (Carmignoto and Vicini, 1992 ; Fox et al., 1999).
NMDA receptors are composed of NR1 subunits in combination with
NR2(A-D) subunits, the latter conferring different properties on the
heteromeric receptor (McBain and Mayer, 1994; Dingledine et al., 1999).
The developmental change in gating properties of NMDA receptors is
likely to result from a switch in subunit composition during cortical
maturation, in which NR2A subunits replace or supplement
NR2B-containing receptors (Monyer et al., 1994; Sheng et al., 1994;
Quinlan et al., 1999; Tovar and Westbrook, 1999).
Although a change in subunit composition and hence in gating properties
of NMDA receptors may contribute to the developmental regulation of
NMDA receptor activity, it is probably not the only mechanism for
tuning NMDA receptor signaling. Recently, it has become recognized that
some aspects of NMDA receptor signal transduction depend on receptor
interactions with intracellular signaling proteins mediated by the
scaffold protein postsynaptic density-95 (PSD-95) or other NMDA
receptor-binding proteins (Craven and Bredt, 1998; Migaud et al., 1998;
Sprengel et al., 1998; Sheng and Pak, 2000). In gene targeting
experiments in mice, deletion of the C-terminal tail of NR2 subunits
that binds to PSD-95, or disruption of PSD-95 itself, results in
defective NMDA receptor signaling and altered synaptic plasticity
(Migaud et al., 1998; Mori et al., 1998; Sprengel et al., 1998).
During brain maturation the expression of many NMDA receptor-associated
proteins increases and their distribution becomes localized to synapses
(Cho et al., 1992; Rao et al., 1998; Wyszynski et al., 1998). We
hypothesized that the mechanisms of NMDA receptor signaling can be
altered during neuronal development as a result of maturational changes
in the NMDA receptor-associated protein complex. We focused on NMDA
receptor signaling to MAP kinase (MAPK or Erk) and to CREB, because
NMDA receptor stimulation is well established to activate pathways
leading to the phosphorylation and activation of these proteins in
neurons (Shaywitz and Greenberg, 1999). Here we report that strong NMDA
receptor stimulation of mature hippocampal neurons in culture (14 DIV
or older) results in only a transient phosphorylation of CREB on
Ser-133 in contrast to the prolonged CREB phosphorylation seen in
immature neurons. The transience of the increase in phospho-CREB
results from an NMDA receptor-dependent activation of a phosphatase
that acts selectively on CREB and that is coupled to NMDA stimulation
only in neurons beyond a critical stage of development. These findings suggest that synaptic plasticity can be regulated developmentally not
only by modulating NMDA receptor gating kinetics but also by changing
the signal output of NMDA receptors.
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MATERIALS AND METHODS |
Hippocampal neuron culture. Hippocampal neuron
cultures were prepared from embryonic day (E) E18-E19 rat embryos
dissociated with trypsin and plated on 12-well plates or 18-mm-diameter
coverslips coated with poly-L-lysine (1 mg/ml in 100 mM borate buffer, pH 8.5) in MEM containing 10% fetal calf
serum (FCS), 25 µg/ml insulin, 100 µg/ml transferrin, 1 mM pyruvate, and 0.6% glucose. At 4 DIV Ara-C (5 µM) was added to the medium, and at 7 DIV (and
subsequently once a week) one-half of the medium was changed with fresh
medium without FCS.
Cell stimulation. Cells were treated with glutamate (100 µM), NMDA (20 or 100 µM), forskolin (75 µM), or KCl (55 mM) at 3, 7, 14, or 21 DIV.
Tetrodotoxin (1 µM) was added to cultures 12 hr before
stimulation to reduce endogenous synaptic activity. For NMDA receptor
stimulation the neurons were pretreated for 20-30 min with CNQX (40 µM) and nimodipine (5 µM). Neurons
depolarized with KCl were pretreated with CNQX and
aminophosphonopentanoic acid (AP-5; 100 µM). Forskolin
treatment was in the presence of CNQX, AP-5, and nimodipine. In some
case AP-5 or nimodipine was left out to allow multiple treatments like
NMDA plus KCl or forskolin plus NMDA (see Results). All of the drugs
were added directly to the medium, and during the incubation the
neurons were kept in the 5% CO2 incubator.
Western blotting and quantitation. After stimulation the
neurons were extracted with 80 µl of SDS sample buffer per well and boiled for 10 min. The lysates ( of the total) were
separated by 10% SDS-PAGE and transferred onto nitrocellulose filters.
The filters were incubated with polyclonal anti-phospho-CREB (1:1000;
Upstate Biotechnology, Lake Placid, NY) or polyclonal anti-phospho-ERK1/2 (1:1000; New England Biolabs, Beverly, MA) and were
visualized by chemiluminescence. Then the filters were stripped and
reprobed with anti-CREB (1:250; Upstate Biotechnology) or anti-ERK1/2
(1:1000; New England Biolabs) to detect total CREB and ERK1/2. For
quantitation of immunoblot signals, the band intensity was measured
with a Kodak Digital Science 1D program (Rochester, NY). Because CREB
antibodies and Erk1/2 antibodies (either phospho-specific or total)
recognize a major doublet of bands on immunoblots, we measured the sum
of both by densitometry. Each phospho-specific band intensity was
equalized to the total CREB and ERK1/2 signals in the same lane. The
increase in phosphorylation of CREB and ERK was normalized to the basal
level and expressed as fold increase. The results were plotted with
Microsoft Excel.
For Figure 7B the following antibodies were used: PP-1
rabbit polyclonal (Westphal et al., 1999) at 1:2000 and  -tubulin
mouse monoclonal (Sigma, St. Louis, MO) at 1:2000.
Immunostaining. After stimulation the neurons were fixed
immediately in 4% paraformaldehyde and 4% sucrose for 15 min. After permeabilization with 0.3% Triton X-100 the cells were incubated with
anti-phospho-CREB (1:1000; Upstate Biotechnology) overnight at 4°C in
1× GDB buffer (30 mM phosphate buffer, pH 7.4, containing 0.2% gelatin, 0.5% Triton X-100, and 0.8 M NaCl),
followed by FITC-conjugated secondary antibodies (Jackson Laboratories,
Bar Harbor, ME) for 1 hr. The fluorescent images were acquired by using
an interline cooled CCD camera (Princeton Instruments, Trenton, NJ) and
prepared for publication with Adobe Photoshop.
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RESULTS |
A developmental change in the time course of NMDA
receptor-induced phospho-CREB
The activation of CREB and Erk1/2 can be monitored by
immunoblotting with antibodies that specifically recognize phospho-CREB (phosphorylated on Ser-133) and phospho-Erk1/2 (phosphorylated on
Thr-202 and Tyr-204). As previously reported (Bading and Greenberg, 1991; Ginty et al., 1993; Xia et al., 1996),
Ca2+ influx mediated by NMDA receptor
activation or KCl depolarization induces phosphorylation of CREB and
Erk1/2 in cultured hippocampal neurons prepared from E18-E19 rat
embryos (Fig. 1). The time course of
induction and maintenance of phospho-CREB and phospho-Erk1/2 was
studied after various treatments of hippocampal cultures grown for 3, 7, 14, and 21 DIV. NMDA receptors were activated by 100 µM NMDA in the presence of 40 µM CNQX and
10 µM nimodipine, whereas calcium influx through
voltage-gated Ca2+ channels (VGCCs) was
stimulated by depolarization with 55 mM KCl in the presence
of 40 µM CNQX and 100 µM APV.
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Figure 1.
Differential time courses of CREB and Erk1/2
phosphorylation and dephosphorylation in response to NMDA stimulation
or KCl depolarization in cultured hippocampal neurons at 7 and 14 DIV.
Representative immunoblot experiments are shown for CREB
(A) and Erk1/2 (B).
Phospho-CREB and phospho-Erk1/2 were immunoblotted with
phospho-specific antibodies; total CREB and Erk1/2 were
immunoblotted with specific antibodies that are not sensitive to a
phosphorylation state. The x-axis shows time in minutes
after the onset of stimulation. The y-axis shows the
fold increase over basal. Results from several experiments
(n = 6) are quantified in graph form for
phospho-CREB (C) and phospho-Erk1/2
(D). Values are normalized to baseline. Error
bars indicate ± SEM.
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In hippocampal neuron cultures at 7 DIV, levels of CREB phosphorylated
on Ser-133 rose rapidly after NMDA receptor stimulation, peaking within
2-5 min and remaining elevated for at least 2 hr (Fig.
1A,C). Erk1/2 phosphorylation also increased rapidly
in response to NMDA stimulation; it peaked by 2 min but returned close
to basal levels at 2 hr (Fig. 1B,D). The same results
were obtained after stimulation with glutamate (100 µM) in the presence of blockers of AMPA
receptors and VGCCs and in hippocampal cultures at 3 DIV (data not shown).
In more mature hippocampal cultures (14 or 21 DIV) a markedly different
time course was observed for NMDA-induced phospho-CREB levels (Fig.
1A,C). The level of phospho-CREB rose during the first 2 min, but at 5 min the level decreased rapidly and by 10 min had
fallen below the basal level. At 1 hr after NMDA treatment, phospho-CREB was virtually undetectable in 14 DIV cultures (Fig. 1A,C). The change in time course was specific for
phospho-CREB, because the phospho-Erk1/2 response to NMDA in 14 DIV
cultures had the same temporal profile as at 7 DIV (Fig.
1B,D). Similar results were obtained at 21 as at 14 DIV (data not shown). Thus in hippocampal cultures older than 14 DIV,
there was a specific change in the duration of CREB phosphorylation
induced by NMDA receptors. Instead of staying elevated (as in cultures
at 7 DIV), phospho-CREB appeared to be dephosphorylated rapidly after
NMDA stimulation in neurons older than 14 DIV.
We found no differences in the time course of phospho-CREB induced by
KCl depolarization at 7, 14, or 21 DIV. At all developmental stages the
level of phospho-CREB peaked rapidly within 2-5 min and remained
elevated at 2 hr (Fig. 1A,C). KCl induced Erk1/2 phosphorylation that was more sustained than that induced by NMDA, but
as with NMDA stimulation there was no difference between 7 and 14 DIV
in the temporal profile of the phospho-Erk response (Fig.
1B,D). Forskolin (75 µM),
which activates adenylate cyclase and PKA, also induced a persistent
CREB phosphorylation that was similar for both 7 and 14 DIV cultures.
In both cases the phospho-CREB reached a plateau after 5 min and
remained elevated for at least 2 hr (Fig.
2A). Thus the transient
phosphorylation followed by a rapid dephosphorylation of CREB is a
specific response to NMDA receptor activation and occurs only in
cultures 14 DIV or older.
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Figure 2.
Time course of CREB phosphorylation and
dephosphorylation in response to forskolin or low concentration (20 µM) NMDA in cultured hippocampal neurons at 7 and 14 DIV.
Shown in A are a representative immunoblot and graphical
quantification of CREB phosphorylation in response to forskolin, as
shown in Figure 1. The time course of CREB phosphorylation in response
to 20 µM NMDA is shown as an immunoblot and is quantified
in B.
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Interestingly, the transient increase followed by the persistent
decrease in phospho-CREB in 14 DIV neurons was seen only in response to
a strong activation of NMDA receptors. By using 20 instead of 100 µM NMDA, the time course of phospho-CREB at 14 DIV was
similar to that seen at 7 DIV, remaining elevated at 2 hr (Fig.
2B).
We used immunostaining in addition to immunoblotting to monitor CREB
phosphorylation in hippocampal cultures and obtained similar results
(Fig. 3; data not shown). Both NMDA and
KCl stimulation caused a rapid increase in nuclear immunoreactivity for
phospho-CREB in neurons at 14 DIV. At 90 min after NMDA stimulation,
however, phospho-CREB staining in nuclei was below baseline, whereas it remained elevated 90 min after KCl depolarization (Fig. 3). Thus immunocytochemistry corroborates the Western blotting results, confirming that NMDA stimulation leads to a transient phosphorylation followed by a dephosphorylation of CREB in 14 DIV neurons.
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Figure 3.
Differential time course of CREB phosphorylation
and dephosphorylation in response to NMDA and KCl as shown by
immunocytochemistry for phospho-CREB in 14 DIV hippocampal neuron
cultures. Cultures were stained for phospho-CREB at the indicated times
(in minutes) after NMDA stimulation or KCl depolarization.
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NMDA receptor stimulation blocks subsequent CREB phosphorylation
by depolarization
After an initial spike the level of phospho-CREB falls to barely
detectable levels by 1 hr after NMDA stimulation in 14 DIV neurons (see
Fig. 1A,C). Can this dephosphorylated CREB be
rephosphorylated by further stimulation with KCl or forskolin?
Hippocampal neurons were treated first with 100 µM NMDA in the presence of CNQX for 1 hr, at
the end of which they were stimulated with KCl or forskolin for 5 min
(Fig. 4). In 14-d-old cultures the
dephosphorylated CREB could not be rephosphorylated by KCl or forskolin
treatments; however, Erk1/2 phosphorylation in the same cultures was
stimulated further (two- to threefold) by KCl (Fig. 4). On the other
hand, at 7 DIV, although the level of phospho-CREB remaining 1 hr after NMDA stimulation was still elevated above baseline, it could be increased further by KCl and forskolin. The further phosphorylation of
Erk1/2 and CREB stimulated by KCl is blocked by nimodipine, confirming
that it is mediated by VGCCs. Taken together, these results show that,
after an initial phosphorylation of CREB, strong NMDA receptor
stimulation activates a specific CREB dephosphorylation mechanism in 14 DIV neurons that cannot be overcome by stimulating classical cAMP- or
Ca2+-dependent pathways of Ser-133
phosphorylation.
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Figure 4.
Strong NMDA stimulation specifically results in
dephosphorylation of CREB in 14 DIV neurons, which is refractory to
further rephosphorylation by forskolin or KCl. Top, The
figure illustrates the protocol used for sequential stimulation by NMDA
and forskolin/KCl. Middle, Shown are representative
immunoblotting experiments for CREB phosphorylation
(left) and Erk1/2 phosphorylation (right)
for cultures at 7 and 14 DIV, as indicated. Bottom,
Histograms quantify the relative levels of phospho-CREB or
phospho-Erk1/2 for each condition/lane from several experiments
(n = 5). Asterisk indicates
significance at p < 0.01 in an unpaired,
two-tailed Student's t test when compared with the
value obtained in neurons stimulated additionally with KCl for 5 min or
forskolin for 5 min at 7 DIV.
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NMDA receptor-stimulated CREB dephosphorylation reverses cAMP or
Ca2+-dependent CREB phosphorylation
We next tested whether, after KCl- or forskolin-induced
phosphorylation of CREB, NMDA receptor activation can cause a
dephosphorylation of CREB. Neurons first were treated with KCl in the
presence of CNQX for 1 hr, at the end of which time (when the level of
phospho-CREB is approximately twofold over basal) they were stimulated
with 100 µM NMDA for 5 min (Fig.
5). In 14 d cultures, but not in
7 d cultures, NMDA receptor activation in this protocol caused a dephosphorylation of CREB to basal levels (Fig. 5, left). An
even more profound dephosphorylation of CREB was seen with 10 min
treatments of NMDA or glutamate (data not shown). Significantly, NMDA
did not cause dephosphorylation of Erk1/2 in the same experiments at
either 7 or 14 DIV (Fig. 5, right). NMDA stimulation also
reduced phospho-CREB levels after forskolin stimulation of 14 DIV
cultures (Fig. 6, left), even
as it caused an increase of phospho-Erk1/2 in the same protocol (Fig.
6, right). After either KCl or forskolin the
dephosphorylation of CREB by NMDA was blocked by AP-5, confirming that
this effect is mediated by NMDA receptors (Figs. 5, 6). These data
indicate that NMDA receptor stimulation in more mature neuron cultures
(>14 DIV) activates a pathway that leads specifically to
dephosphorylation of CREB, but not Erk1/2.
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Figure 5.
NMDA stimulation causes dephosphorylation of CREB
that previously was phosphorylated in response to KCl depolarization,
specifically in 14 DIV neurons. Stimulation protocol, immunoblotting
results, and histogram quantification are as shown in Figure 4.
Asterisk indicates significance at p < 0.01 in an unpaired, two-tailed Student's t test
when compared with the values obtained in other conditions in this
experiment.
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Figure 6.
NMDA stimulation causes dephosphorylation of CREB
that previously was phosphorylated in response to forskolin,
specifically in 14 DIV neurons. Stimulation protocol, immunoblotting
results, and histogram quantification are as in Figure 4.
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Two different phosphatases have been implicated in the
dephosphorylation of CREB: calcineurin (PP-2B) and protein
phosphatase-1 (PP-1) (Hagiwara et al., 1992; Bito et al., 1996). We
tested pharmacologically whether these phosphatases play a role in
NMDA-induced dephosphorylation of CREB, using 2 µM
okadaic acid to block PP-1 and two specific antagonists of calcineurin,
cyclosporin A (250 µM) and FK506 (1 µM). We
also used a low concentration of okadaic acid (20 nM) to
distinguish between PP-1 and PP-2A, because PP-2A is inhibited by
okadaic acid in the 10 nM range (Cohen et al., 1990). In
neurons at 14 DIV, only 2 µM okadaic acid, but not 20 nM okadaic acid or cyclosporin A or FK506, was able to
prevent the NMDA receptor-mediated decrease in phospho-CREB after KCl
or forskolin treatment (Fig. 7A). These results are
consistent with PP-1 being involved in the CREB dephosphorylation
mechanism activated by NMDA receptors in >14 DIV neurons. To test
whether the expression of PP-1 increases during development of
hippocampal neurons in culture, we performed Western blot analysis of
cultures at 3, 7, 10, and 15 DIV. Figure 7B shows that, over
this 2 week period during which NMDA receptors become functionally
coupled to CREB dephosphorylation, the levels of PP-1 protein stayed
constant.
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Figure 7.
Protein phosphatases and NMDA receptor-dependent
dephosphorylation of CREB. A, NMDA receptor-dependent
dephosphorylation of CREB is blocked by okadaic acid (2 µM), but not by cyclosporin A (250 µM),
FK506 (1 µM), or okadaic acid (20
nM). Hippocampal cultures (14 DIV) were treated with KCl or
forskolin and then with NMDA +/ - phosphatase inhibitors, as
indicated. Stimulation protocol, immunoblotting results, and histogram
quantification are as in Figure 4. B, Immunoblot of
hippocampal neuron cultures at 3, 7, 10, and 15 DIV for PP-1 levels
(tubulin used as internal control).
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DISCUSSION |
The major conclusions of this paper can be summarized as follows:
(1) The duration of CREB Ser-133 phosphorylation induced by NMDA
receptor activation depends on the developmental maturity of cultured
hippocampal neurons. After 14 DIV, strong NMDA stimulation causes only
a transient increase in phospho-CREB, contrasting with the prolonged
increase in phospho-CREB seen at 7 DIV. (2) This developmental switch
is stimulus- and pathway-specific, in that
K+ depolarization (acting through VGCCs)
induces the same prolonged CREB phosphorylation in young (7 DIV) and
mature (>14 DIV) cultures, and the time course of NMDA
receptor-stimulated phospho-Erk1/2 elevation is unaffected by culture
age. (3) Strong NMDA receptor stimulation in mature neurons actually
causes a robust dephosphorylation of CREB, possibly via
activation of phosphatase PP-1.
The findings of this study imply a pathway-specific developmental
change in the coupling of NMDA receptors to downstream signaling mechanisms. The predominant effect of NMDA receptors on CREB actually was reversed in older neurons (which showed mainly NMDA-mediated dephosphorylation of Ser-133 with strong NMDA stimuli). Significantly, this switch occurred in the absence of any apparent change in NMDA
receptor-stimulated phosphorylation of Erk1/2. These features make it
unlikely that the developmental change in NMDA receptor signaling to
CREB is attributable simply to expression of higher levels of NMDA
receptors or more active NMDA receptors in >14 DIV neurons.
Biphasic regulation of CREB phosphorylation and
synaptic plasticity
The NMDA receptor-dependent CREB dephosphorylation in >14 DIV
neurons depends on the concentration of NMDA. Dephosphorylation of CREB
in older neurons was seen only with 100 µM NMDA, but not with 20 µM NMDA. This suggests the presence specifically
in mature neurons of a biphasic
Ca2+-dependent NMDA receptor-mediated
mechanism, one that has a low threshold for the activation of the
pathway leading to CREB phosphorylation and a higher threshold for the
activation of the phosphatase pathway.
What might be the neurobiological significance of the developmental
switch in the mode of NMDA receptor signaling to CREB? In addition to
short-term plasticity lasting from minutes to hours, NMDA receptor
activation can initiate long-term neural plasticity requiring changes
in gene expression. CREB is a transcription factor widely implicated in
synaptic plasticity and memory formation (Frank and Greenberg, 1994;
Ghosh and Greenberg, 1995; Bito et al., 1997). Phosphorylation of CREB
on Ser-133 is critical for activating the transcription of genes
controlled by the CRE element (Montminy, 1997), many of which may be
involved in neuronal growth and plasticity, e.g., BDNF, CaMKIV,
synapsin I, somatostatin, voltage-gated potassium channels, Fos, and
Jun (Sauerwald et al., 1990; Mori et al., 1993; Sassone-Corsi, 1995;
Shieh et al., 1998; Tao et al., 1998).
CREB-responsive genes appear to be activated only when Ser-133
phosphorylation of CREB is prolonged, as occurs during long-lasting synaptic stimulation (Bito et al., 1996, 1997). Our study indicates that an additional factor that influences duration of CREB
phosphorylation is the developmental age of the neurons. Because of the
appearance of an NMDA receptor-coupled phosphatase pathway leading to
CREB dephosphorylation, NMDA receptor stimulation in mature neurons is
less likely to result in persistent CREB activation and hence CRE-directed gene transcription. In older neurons, NMDA receptor activation can even result in the net dephosphorylation of CREB. In
immature neurons, by comparison, NMDA receptor stimulation is favored
to induce CREB-dependent gene expression because of the relative
lack of CREB phosphatase activity and the persistent phosphorylation of
Ser-133. However, CREB phosphorylation by itself is not synonymous with
gene induction (Ginty, 1997), and it remains to be confirmed whether
the regulation of CREB phosphorylation by NMDA receptors correlates
with gene transcriptional responses.
We speculate that the developmental increase in NMDA receptor-mediated
dephosphorylation of CREB may be one of the factors that contributes to
the reduction in synaptic plasticity that normally accompanies nervous
system maturation. Consistent with this, a recent study showed that
CRE-mediated gene expression is downregulated after the critical period
in visual cortex (Pham et al., 1999). Our findings suggest that one
possible mechanism for inhibiting plasticity in mature brain is the
relative loss of the ability of the NMDA receptor to support persistent
phosphorylation of CREB. Prolonged phosphorylation of CREB still can be
achieved in mature neurons, but it may require more stringent
conditions such as sustained synaptic stimulation, which appears to
work in part via an activity-dependent inhibition of CREB
dephosphorylation (Bito et al., 1996; Liu and Graybiel, 1996).
Using hippocampal neurons at 10-14 DIV, Hardingham et al. (1999) also
found that CREB was more transiently phosphorylated in response to NMDA
receptor stimulation than in response to KCl depolarization. These
workers correlated the transience of this CREB phosphorylation with the
ineffective induction of CRE-dependent transcription by NMDA receptors.
By contrast, Hu et al. (1999), using cortical neurons at 5 DIV,
reported that CREB-mediated transcription could be readily stimulated
by 100 µM glutamate in a NMDA receptor-dependent manner.
The apparent discrepancy between these two reports might be explained,
at least in part, by our finding of a developmental switch in NMDA
receptor signaling that allows CREB phosphorylation to persist in
immature neurons and that abbreviates CREB phosphorylation in more
developed neurons. More generally speaking, the NMDA receptor-mediated dephosphorylation of CREB in mature neurons may contribute to the
observation that NMDA receptor stimulation is inefficient in activating
CRE-dependent transcription.
Coupling of NMDA receptors to a phosphatase
In older neurons, NMDA receptor stimulation results in a transient
phosphorylation of CREB, followed by a prolonged dephosphorylation below baseline. After KCl depolarization or PKA stimulation (which induce phosphorylation of CREB), NMDA actually causes dephosphorylation of CREB, while in the same neurons further enhancing Erk1/2
phosphorylation. These observations suggest that NMDA receptors become
coupled to a specific CREB phosphatase activity as neurons mature. That the NMDA receptor-mediated dephosphorylation of CREB is blocked by 2 µM okadaic acid, but not by 20 nM okadaic
acid or 250 µM cyclosporin A or 1 µM FK506,
suggests that PP-1 is more critical in this pathway than PP-2A or
calcineurin. This conclusion is consistent with previous reports
showing that PP-1 is a major phosphatase involved in CREB
dephosphorylation (Hagiwara et al., 1992; Bito et al., 1996).
Interestingly, PP-1 activity has been shown to be regulated by synaptic
activity (Mulkey et al., 1994). PP-1 is concentrated in dendritic
spines, perhaps by binding to spinophilin, a scaffold protein
specifically localized in spines (Allen et al., 1997). PP-1 also
interacts with Yotiao, an A-kinase anchoring protein (AKAP) that binds
directly to the NMDA receptor (Lin et al., 1998; Westphal et al.,
1999). Thus PP-1 is in the vicinity of the NMDA receptor, but it
remains to be determined how it is activated after NMDA receptor
stimulation. Alternatively, PP-1 might not be activated locally at the
synapse but, rather, in the nucleus in response to NMDA receptor
signals transduced from the synapse (Bito et al., 1997).
Developmental changes in NMDA receptor signaling
In brain the levels of many excitatory postsynaptic proteins have
been shown to increase during development. For instance, there is an
induction of the NR2A subunit, which is likely to be incorporated into
NMDA receptors via assembly with NR1/NR2B subunits (Tovar and
Westbrook, 1999). A change in the subunit composition of NMDA receptors
can alter electrophysiological and pharmacological properties of the
receptor-channel (Monyer et al., 1994; Dingledine et al., 1999), but
it is hard to see how such biophysical changes by themselves could
specifically switch the mode of NMDA receptor signal transduction to
CREB. We therefore prefer the idea that the developmental switch is
mediated by a quantitative or qualitative change in the
protein-protein interactions that couple NMDA receptors to downstream
signaling cascades.
Expression of PSD-95 and certain PSD-95-associated proteins increases
during postnatal development of the brain (Cho et al., 1992; Lim et
al., 1999; C. Sala and M. Sheng, unpublished observations). In mature
neurons the interaction of NMDA receptors with PSD-95 may link NMDA
receptors to a biochemical pathway that results in the activation of a
CREB phosphatase. Whether interaction with the PSD-95 complex is
required for NMDA receptor coupling to CREB dephosphorylation remains
to be determined, as does the identity of the specific signaling
proteins that mediate this coupling. However, it is intriguing to note
in this regard that the PSD-95 knock-out mouse showed a surprising
enhancement of LTP (Migaud et al., 1998), which was speculated to
involve the loss of a PSD-95-dependent phosphatase (Malenka and Nicoll,
1998). It is also possible that PSD-95-independent interactions of NMDA
receptors mediate the coupling of NMDA receptors to CREB
dephosphorylation. A third possibility is that the NMDA
receptor-dependent CREB phosphatase activity is regulated
developmentally via mechanisms that do not involve protein-protein
interactions, for instance, simply by elevating the expression of the
CREB phosphatase. A pertinent finding here is that the levels of PP-1
do not change during the first 2 weeks of hippocampal neuron culture
(Fig. 7B). Whatever the mechanism, it is clear that NMDA
receptors differ significantly in their downstream effects depending on
the developmental state of the neuron. Further studies on the
maturation of the NMDA receptor signaling complex may shed light on the
mechanisms that control postsynaptic plasticity during brain development.
| |
FOOTNOTES |
Received Dec. 1, 1999; revised Feb. 22, 2000; accepted Feb. 24, 2000.
M.S. is Assistant Investigator of the Howard Hughes Medical Institute.
C.S. was supported by a fellowship from the Harvard Armenise Foundation
(Department of Biological and Technical Research, San Raffaele,
Italy). We thank Haruhiko Bito for helpful comments on this manuscript.
Correspondence should be addressed to Dr. Morgan Sheng, Howard Hughes
Medical Institute (Wellman 423), Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114. E-mail: sheng{at}helix.mgh.harvard.edu.
| |
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