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The Journal of Neuroscience, January 1, 1998, 18(1):214-226
In Vivo Expression and Regulation of Elk-1, a Target
of the Extracellular-Regulated Kinase Signaling Pathway, in the
Adult Rat Brain
Véronique
Sgambato1,
Peter
Vanhoutte1,
Christiane
Pagès1,
Monique
Rogard1,
Robert
Hipskind2,
Marie-Jo
Besson1, and
Jocelyne
Caboche1
1 Laboratoire de Neurochimie-Anatomie, Institut des
Neurosciences-Unité de Recherche Associée 1488, Université Pierre et Marie Curie, 75005 Paris, France, and
2 Institut de Génétique Moléculaire,
Unité Mixte de Recherche 5535, Centre National de la Recherche
Scientifique, 34033 Montpellier, France
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ABSTRACT |
The transcription factor Elk-1, a nuclear target of
extracellular-regulated kinases (ERKs), plays a pivotal role in
immediate early gene induction by external stimuli. Notably, the degree of phosphorylation of Elk-1 is tightly correlated with the level of
activation of transcription of c-fos by proliferative
signals. No data yet indicate the role of Elk-1 in the adult brain
in vivo. To address this question, we have analyzed in
the present work (1) Elk-1 mRNA and protein expression in the adult rat
brain, and (2) the regulation of Elk-1 (i.e., its phosphorylation
state) in an in vivo model of immediate early gene (IEG)
induction: an electrical stimulation of the cerebral cortex leading to
c-fos and zif268 mRNA induction in the
striatum. Using in situ hybridization, we show that
Elk-1 mRNA is expressed in various brain structures of adult rat, and
that this expression is exclusively neuronal. We demonstrate by
immunocytochemistry using various specific Elk-1 antisera that the
protein is not only nuclear (as shown previously in transiently
transfected cell lines) but is also present in soma, dendrites, and
axon terminals. On electrical stimulation of the glutamatergic
corticostriatal pathway, we show a strict spatiotemporal correspondence
among ERK activation, Elk-1 phosphorylation, and IEG mRNA induction.
Furthermore, both activated proteins, analyzed by immunocytochemistry,
are found in cytosolic and nuclear comparments of neuronal cells in the
activated area. Our data suggest that the ERK signaling pathway plays
an important role in regulating genes controlled by serum response
element sites via phosphorylation of Elk-1 in vivo.
Key words:
SRE; Elk-1; transcription factor; mRNA expression; subcellular distribution; phosphorylation; ERK; electrical stimulation; striatum; immediate early gene induction
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INTRODUCTION |
In the CNS excitatory
neurotransmission can elevate calcium concentration in neuronal cells
and can trigger activation of transcription of a class of genes known
as immediate early genes (IEGs) (Sheng and Greenberg, 1990 ; Ginty et
al., 1992; Ghosh and Greenberg, 1995). The characterization of calcium
regulation of the prototypical IEG c-fos has provided a
structural framework useful for understanding how this second messenger
ion governs early nuclear events that affect neuronal plasticity.
Within the promoter of c-fos, there are at least two
critical response elements with activity that is regulated by calcium
signaling (for review, see Ginty, 1997): the
cAMP-Ca2+ responsive element (CRE) and the serum
response element (SRE).
The SRE, located at position  310 in the c-fos promoter,
was initially described as a protein-binding site required for the induction of c-fos expression by serum (Treisman, 1992). It
serves as the site of assembly of multiprotein complexes that include a
dimer of serum response factor (SRF) (Treisman, 1986; Norman et al.,
1988; Schröter et al., 1990) together with ternary complex factor
(TCF) (Shaw et al., 1989; for review, see Treisman, 1992, 1995). A
major regulatory input received by the SRE can be attributed to TCF
phosphorylation (Hill et al., 1993). The TCFs represent a subgroup of
the ETS domain transcription factor family that comprises at
least three members, Elk-1, SAP1, and SAP2/ERP/Net (Hipskind et al.,
1991; Dalton and Treisman, 1992; Giovane et al., 1994; Lopez et al.,
1994). Among TCFs, the most attention has been directed toward Elk-1,
the first TCF to be identified (Hipskind et al., 1991). Elk-1 is
rapidly phosphorylated on Ser383 and
Ser389 in its C-terminal region in response to
activation of the extracellular-regulated kinases (ERKs) of the
mitogen-activated protein (MAP) kinase family. Phosphorylation at these
sites increases its ability to form a ternary complex with SRE and SRF
(Gille et al., 1992, 1995) and to potentiate definitively its ability
to activate c-fos transcription strongly (Hill et al., 1993;
Marais et al., 1993; Janknecht et al., 1994; Zinck et al., 1995;
Hipskind et al., 1994a,b).
In the CNS, ERK proteins can be activated after glutamate receptor
stimulation (Bading and Greenberg, 1991; Fiore et al., 1993b; Kurino et
al., 1995). However, no clear link has been established among ERK
activation, Elk-1 phosphorylation, and gene regulation on glutamate
activation or in any in vivo model.
In this study we first analyzed the expression of Elk-1 mRNA and the
corresponding protein in adult rat brain. Using in situ hybridization, we confirmed that Elk-1 mRNA is strongly expressed in
adult brain (Price et al., 1995) and observed that it is restricted to
neuronal cells. Immunocytochemistry revealed that Elk-1 protein is
nuclear as expected (Janknecht et al., 1994; Pingoud et al., 1994) and
surprisingly associated with the cytosol. In a second step we used an
in vivo stimulation of cortical cells inducing c-fos and zif268 mRNA in the projection field of
the stimulated cortical area: the lateral striatum. The
hyperphosphorylation of both ERK and Elk-1 was found in a strict
spatiotemporal correspondence with c-fos and
zif268 mRNA induction in the activated striatal area. The
presence of phosphorylated signaling components in both nuclear and
cytoplasmic compartments suggests that the activation of both ERK and
Elk-1 can occur locally, near the calcium influx source, and thus
provides novel insights on the mechanisms by which calcium influences
gene regulation.
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MATERIALS AND METHODS |
Tissue preparation for in situ
hybridization and immunohistochemistry. Adult male Sprague Dawley
rats (250-300 gm) were housed under standard conditions. After
intraperitoneal injection of sodium pentobarbital (0.2 mg/gm total body
weight), rat brains were fixed by intracardiac perfusion of 4%
paraformaldehyde (PFA) in 0.1 M
Na2HPO4/NaH2PO4
buffer, pH 7.5 (phosphate buffer), delivered with a peristaltic pump at
50 ml/min during 10 min. Brains were removed and post-fixed in the same
fixing 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, and 0.1% diethyl pyrocarbonate
(DEPC; Sigma, St. Louis, MO) at 20°C until processed for in
situ hybridization or immunohistochemistry.
Probe synthesis. The antisense (complementary to cellular
mRNA) and control sense (identical to cellular mRNA) probes used in
this study were 33P-radiolabeled riboprobes. Elk-1 RNA
probes were produced by in vitro transcription of a 3
region of a mouse 475 bp cDNA subcloned into Bluescript (Stratagene, La
Jolla, CA). For the antisense riboprobe, the recombinant plasmid
containing the Elk-1 insert was linearized with XhoI and
transcribed with T3 RNA polymerase; for the sense probe, the plasmid
was linearized with BamHI and transcribed with T7 RNA
polymerase. For c-fos antisense riboprobe a human c-Fos cDNA
subclone (pBFH480) corresponding to exon 4 was linearized after
HindIII digestion and transcribed with T7 RNA polymerase.
The sense probe was obtained after linearization with EcoRI
and transcribed with T3 RNA polymerase. For zif268 antisense
riboprobe of a murine zif268 cDNA subclone (Bluescript) corresponding
to 1.6 kb was linearized after HindIII digestion and
transcribed with T7 RNA polymerase; the sense probe was obtained after
linearization with EcoRI digestion and transcribed with T3
RNA polymerase. Transcription reactions contained 1 µM
[ -33P]UTP (3000 Ci/mmol, Isotopchim), 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.
In situ hybridization. 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
treatments: 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 on sections, which were then covered with GelBond film (FMC
Bioproducts, Rockland, ME). The hybridization mixture contained 200 ng/ml (4 ng/section) of 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 Escherichia
coli tRNA, 0.1 M dithiothreitol (DTT), and 50%
formamide. Hybridization was performed at 60°C in humid chambers for
16 hr. After removing the GelBond coverslips in 4× SSC 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. The slides were then coated with NTB3
emulsion (Kodak) and exposed for 3 weeks. After development slides were
counterstained with cresyl violet and mounted with Eukitt (Calibrated
Instruments).
Immunohistochemistry. The immunohistochemical procedure was
adapted from protocols previously described. On day 1, 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 10 min each in TBS. After a 15 min incubation in
0.2% Triton X-100 in TBS, sections were rinsed three times in TBS,
preincubated for 30 min in TBS containing 30% normal goat serum, and
washed three times for 10 min in TBS. Sections were then incubated for
72 hr at 4°C with the following primary antibodies. Rabbit polyclonal
antisera raised against synthetic peptides (Santa Cruz Biotechnology,
Santa Cruz, CA; and Upstate Biotechnology, Lake Placid, NY)
encompassing the regions listed (as amino acids) in each protein, (1)
407-428 of Elk-1, (2) 123-137 of CRE-binding protein (CREB), and (3)
686-709 of signal transducers and activators of transcription (STAT3), were used at a dilution of 1:100 in TBS containing 1% goat serum and
1% rat serum. Rabbit polyclonal antibodies raised against recombinant
proteins containing N- or C-terminal regions of human Elk-1 were used
at a dilution of 1:500 in the same buffer. On day 2, after three rinses
in TBS, sections were incubated 24 hr at 4°C with the secondary,
biotinylated antibody (rabbit anti-IgG) using a dilution twice that of
the first antibody in TBS containing 1% normal rat serum and 1%
normal goat serum. On day 3, after washing the sections three times in
TBS, they were incubated for 90 min in avidin-biotin-peroxidase
complex solution (Vector Laboratories, Burlingame, CA; final dilution,
1:50). Sections were washed two times in TBS and two times in Tris
buffer (TB; 0.25 M Tris, 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 H2O2 addition (0.02%) for 60 min. After processing, tissue sections were mounted onto gelatin-coated
slides and dehydrated through alcohol to xylene for light microscopic
examination. For confocal microscopic analysis, the same procedure was
applied on day 1. On day 2, sections were incubated with the
Cy3-labeled goat anti-rabbit IgG (Amersham, Les Ulis, France) at a
dilution 1:200 in TBS containing gelatin (1:40). After five washes in
TBS and one wash in distilled water, sections were mounted onto
gelatin-coated slices with Vectashield (Vector) mounting medium.
Confocal analysis. Confocal data were obtained using a
Sarastro 2000 confocal microscope (Molecular Dynamics, Sunnyvale, CA) at 63× (1.4 numerical aperture) magnification. Excitation was achieved
with the 514 nm ray of an argon ion laser, and the emitted light was
selected by using appropriate filters. The contrast was enhanced, and
the background was reduced by applying a 3 × 3 × 3 Gaussian
filter on the series of digitized optical sections. Maximum intensity
projections were computed with the Image Space software (Molecular
Dynamics).
Phospho-ERK and phospho-Elk-1 immunocytochemistry. Sections
were processed as described above with the following modifications. NaF
(0.1 mM) was included in all buffers and incubation
solutions, and all steps that included normal goat serum and normal rat
serum were omitted. These were incubated with commercial available
antisera produced by immunizing rabbits with synthetic dual-specific
anti-active MAP kinase (MAPK; Promega, Madison, WI) corresponding to
Thr183 and Tyr185 in p42 MAPK
(diluted 1:100) and phospho-Ser383 peptide
corresponding to residues 379-392 of human Elk-1 (New England Biolabs,
Beverly, MA) (diluted 1:80).
Double labeling by in situ hybridization and
immunocytochemistry. Free-floating sections were processed for
phospho (P-Elk) Elk-1 immunocytochemistry as described above, except
that all buffers used were autoclaved and diluted with DEPC-treated
water. Sections were mounted onto SuperFrost Plus slides and air-dried for at least 60 min, followed by zif268 in situ
hybridization as indicated above.
Tissue preparation for Western blot analysis. Tissue samples
were rapidly extracted from the brain 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 (10 µg/lane) were separated by 10% SDS-PAGE before electrophoretic
transfer onto a polyvinylidene difluoride membrane (ICN Biochemicals).
The blots were blocked (1 hr room temperature) with 5% nonfat dry milk
and incubated (overnight at 4°C) with a rabbit polyclonal antiserum
raised against peptide 407-428 of Elk-1 (anti-Elk-1, 1:200; Santa
Cruz). The blots were subsequently incubated for 2 hr at room
temperature with goat anti-rabbit horseradish peroxidase-conjugated
antibodies before exposure to the ECL substrate. For the detection of
the active forms of the proteins, the same procedure was applied except
for the following modifications. For P-Elk-1 detection, 30 µg of
protein/lane was loaded on the SDS-PAGE. The saturation step was
performed in BSA (fraction V, Sigma), 5% (for P-ERK) and 8% (for
P-Elk-1). The antibody specifically directed against the phosphorylated proteins was first applied (P-ERK, 1:2500; P-Elk-1, 1:100), and the
revelation was processed as described above. Then the blots were
stripped (glycine-HCl, pH 2.8, two times for 20 min each at 55°C,
followed by saturation in 5% nonfat dry milk) and incubated with the
antibodies corresponding to the nonactivated proteins as described
above. The efficacy of the stripping step was assessed by omitting the
first antibody and verifying the lack of signals on the blot.
Corticostriatal stimulations. After anesthesia with
pentobarbital (Sanofi; 6 mg/kg, i.p.), cortical stimulations were
applied in animals placed in a conventional stereotaxic apparatus. Care was taken to minimize uncontrolled sources of stimulation; the skin
around the points of stereotaxic contention and areas of incision were
numbed with xylocaine. Body temperature was monitored throughout the
experiment and kept at 37-38°C with a homeothermic blanket. A small
craniotomy was made over the orofacial area of the motor cortex
according to previously described stereotaxic coordinates (Paxinos and
Watson, 1986). Orofacial cortical stimulations were applied through
pairs of wires (Ni-chrome, 0.2 mm diameter) 1.5 mm apart and inserted
at a depth of 1.5 mm from the cortical surface. Electrical stimuli
consisted of trains of pulses of 200 µA intensity and 50 msec
duration delivered at frequencies of 250 Hz, repeated at 4 Hz for 15 min. The localization of the jaw area was precisely identified by
observing the motor effects evoked by these electrical pulses. The
polarity of electrodes was reversed every 30 sec to avoid lesion of
cortical tissue. After the end of stimulation, brains were processed
for either neuroanatomical (in vivo perfusion with 4% PFA)
or biochemical (rapid extraction of lateral or medial part of the
striatum) studies. Sham-operated rats were treated identically, except
that no electrical stimulation was delivered.
Kainic acid lesion of striatal cells. Kainic acid (0.8 µl,
2 µg/µl; Sigma) was delivered unilaterally in the lateral part of
the striatum by means of pressure injections through a Hamilton syringe
fitted with a glass micropipettes (internal tip diameter, 50 µm). The
injection was made over 10 min, and the needle was left in place for 15 min before withdrawal. After a postoperative period of 10 d,
animals were deeply anesthetized and perfused transcardially as
described above.
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RESULTS |
Elk-1 mRNAs are differentially expressed in adult rat
brain structures
To identify the regions of Elk-1 expression in the brain, sections
were hybridized with Elk-1 antisense and sense 33P-cRNA
probes. To label Elk-1 mRNAs specifically and to avoid detection of
mRNAs encoding the other ets-related proteins, we chose a
probe corresponding to the 3 region of the mouse Elk-1-cDNA, which
presents very few sequence homologies with other ets-related cDNAs (Wasylyk et al., 1993). The sense Elk-1 33P-cRNA
probe showed no detectable signal on autoradiograms (Fig. 1A, inset). The
antisense Elk-1 33P-cRNA probe revealed a wide and
heterogeneous expression of Elk-1 mRNA in the whole rostrocaudal
extension of the rat brain (Fig. 1A-I).
Quantification by densitometry showed a very high level of Elk-1 mRNA
expression in the granular layer of olfactory bulbs, the pyriform
cortex, the dentate gyrus of the hippocampus, and the granular layer of
the cerebellum. The labeling was relatively strong in the nucleus
accumbens, the caudate-putamen, various nuclei of the thalamus, the CA
layers in the hippocampus, the substantia nigra pars compacta, the
pedonculopontine nucleus, and throughout the whole cerebral cortex. A
low level of expression was observed in the lateral and medial septal
nuclei, the substantia nigra pars reticulata (SNr), and the external
segment of the globus pallidus (GPe). No significant hybridization
signals were obtained in structures corresponding to white matter. To
visualize hybridization of Elk-1 mRNA at the cellular level,
high-resolution emulsion autoradiograms of sections were processed
after cresyl violet counterstaining to allow the identification of
neuronal and glial cells. The sense probe gave very weak, uniformly
distributed hybridization signals in all brain sections (data not
shown). In all brain structures examined, cells of neuronal subtypes
showed strong Elk-1 mRNA-specific hybridization signals (Fig.
1J). In contrast, glial cells, characterized by a
strong nuclear coloration with cresyl violet coloration, showed very
low labeling (the astrocytes in the cerebral cortex; Fig.
1J) or none, as exemplified by the very weak signals
in the corpus callosum (Fig. 1K).
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Figure 1.
In situ detection of Elk-1 mRNA in
rat brain. Rat cerebral sections were hybridized with
33P-cRNA-Elk-1 sense (A, inset) and
antisense (A-I) probes. Coronal sections (20 µm thick) were taken in the whole rostrocaudal extension of the rat
brain. The cerebellum was taken according to a sagittal plane.
OT, Olfactory tubercles; Cx, cerebral
cortex; Acb, accumbens nucleus; CC,
corpus callosum; CPu, caudate-putamen;
Spt, septum; GP, globus pallidus;
Thal, thalamus; CA, Amon corn layer of
the hippocampus; DG, dentate gyrus of the hippocampus;
SC, superior colliculi; SNr, substantia
nigra pars reticulata; PAG, periaqueducal gray layer;
PN, pedonculopontine nuclei; Cb,
cerebellum. J, High magnification (500×) of Elk-1 mRNA
hybridization in the CA1 layer of hippocampus. The cresyl violet
counterstaining allows the characterization of glial (small
arrows) and neuronal (large arrows) cells. Note that hybridization signals are always observed on neuronal cells. Scale
bar, 17 µm. K, Elk-1 hybridization in the corpus
callosum (glial cells, small arrows).
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Immunocytochemistry reveals nuclear and cytoplasmic localization of
Elk-1 protein
Expression of Elk-1 protein was assessed in three different
structures: the striatum, the CA1 layer of the hippocampus, and the
Purkinje cell layer of the cerebellum (Fig.
2), using an antiserum specific for the
Elk-1 C terminus (COOH antibody). Its specificity was confirmed using
Western blots, which yielded a band of 62 kDa (apparent molecular
weight) for each cerebral structure (Fig. 2A). In the
striatum, the immunocytochemical labeling of Elk-1 was very dense in
the neuropil. Neuronal cells were labeled in both nuclear and somatic
compartments (Fig. 2B, arrow), along with some
dendritic processes (Fig. 2B, arrowheads). A
subpopulation of striatal cells also showed an exclusively somatic and
dendritic immunoreactivity (see Fig. 4). In the hippocampus, the
dendritic arborization of the pyramidal cells of the CA1 layer was also very densely labeled by this antiserum (Fig. 2B,
arrowheads) together with the nucleus and the cytoplasm. In the
cerebellum, Elk-1 immunoreactivity was concentrated within the cell
bodies and dendrites of Purkinje cells (Fig. 2, arrowhead).
In addition, the molecular layer showed a diffuse reactivity of
moderate intensity throughout the molecular layer. No labeling of glial
cells was observed (data not shown). In all cases, the
immunocytochemical labeling of Elk-1 was blocked by preadsorbing the
serum with the immunizing peptide (data not shown).
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Figure 2.
Expression of Elk-1 protein in various brain
structures. A, Western blot analysis of Elk-1 expression
in the striatum, the hippocampus, and the cerebellum. The COOH antibody
yields a band with an apparent molecular weight of 62 kDa (protein
molecular weight standards). B, Immunocytochemical
detection of Elk-1 using the COOH antibody. Shown are the striatum, the CA1 layer of
hippocampus, and the Purkinje cell layer of the cerebellum. Elk-1 COOH
antibody was used on rat brain sections processed in parallel. Note
that in all three structures, the protein is present in the nucleus and
the cytosol, as well as dendritic processes (arrowhead).
Arrow, Heavy immunoreactivity for Elk-1 in a subpopulation of striatal cells. Scale bar, 17 µm.
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Elk-1 localizes in the nucleus in transiently transfected HeLa cells
and is associated predominantly with the nuclear fraction in these
cells (Pingoud et al., 1994). The antisera used for this localization,
those directed against the entire C-terminal region (C-terminal
antibody) or the whole N-terminal Ets-domain (Ets antibody) specifically label a 62 kDa endogenous protein that is
indistinguishable from Elk-1 by multiple criteria (Pingoud et al.,
1994). We compared the pattern of Elk-1 expression obtained with the
various Elk-1 antibodies (COOH, C-terminal, and Ets; Fig.
3, top panel) in
neuronal cells. In the pyramidal cell layers of the cerebral cortex
(Fig. 3A), all three antisera stain Elk-1 in the nucleus
(thin arrows), cytosol (arrows), and dendrites (arrowheads). Thus Elk-1 is found in several different
cellular compartments in neuronal cells. To test our conditions of
immunocytochemical detection, we performed a similar characterization
of two other transcription factors, the proteins CREB (Fig.
3B) and STAT3 (Fig. 3C). The subcellular
localization of both factors is well established, and both play a key
role in c-fos activation. Immunolabeling of CREB was
exclusively nuclear, whereas STAT3 was present within the cytoplasm in
neuronal cell bodies, a result consistent with its cytoplasmic
localization before activation (Darnell et al., 1994).
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Figure 3.
Subcellular localization of Elk-1, CREB, and STAT3
proteins in the pyramidal cell layer of the cerebral cortex.
A, Serial sections were processed for
immunocytochemistry with three different Elk-1 antibodies (described in
the diagram at the top): anti-COOH, anti-C-terminal (C-term), and anti-Ets.
Note that all three antibodies show the protein in the nucleus
(thin-headed, long arrows), cytosol (short
arrows), and also in dendritic processes
(arrowheads) of neuronal cells. B, CREB
and C, STAT3 immunocytochemistry in adjacent sections of
cerebral cortex. Note the nuclear localization of CREB (B,
thin-headed, long arrows) and the cytoplasmic localization of
STAT3 (C, short arrows). Magnification, 500×. Scale
bar, 17 µm.
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In some cases, neurons showed an exclusively somatic and dendritic
Elk-1 immunoreactivity. This was confirmed using confocal microscopic
imagery (Fig. 4). In confocal sections of
two striatal neurons (Fig. 4A1,B1), the fluorescent
signal (predominated) in the somatic compartment (arrows),
whereas the nuclei were devoid of staining. Dendritic immunoreactivity
(Fig. 4A2,B2, arrowheads) was clearly
visible in maximum intensity projections.
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Figure 4.
Visualization of Elk-1 immunoreactivity with
confocal microscopy. Two examples of Elk-1-immunoreactive striatal
neurons with COOH antibody. In these two cells, the fluorescent signal
predominates in the somatic (arrows) and dendritic
(arrowheads) cytoplasm, and the nuclei
(n) are not stained. A1, B1,
Confocal sections; A2, B2, maximum intensity projection
(25-28 sections, respectively). Scale bar, 5 µm.
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Other regions, such as the GPe and the SNr, also showed very dense
labeling for Elk-1 protein (Fig.
5A,C) despite low levels of
Elk-1 mRNA expression (Fig. 1D,G). In fact, this
labeling represented a very small subset of cells within a dense
neuropil (Fig. 5A,C). To determine whether this
immunoreactive neuropil represented axon terminals of striatal neurons,
the major afferent structure of both GPe and SNr, we performed a
unilateral kainic acid lesion of striatal neurons. This lesion led to a
complete disappearance of striatal neurons ipsilaterally to the
injection but not contralaterally. In the GPe and the SNr ipsilateral
to the lesion, a marked loss of Elk-1 immunoreactivity was observed in
the neuropil in the projection field of lesioned striatal neurons (Fig.
5B,D). By comparison, Elk-1 immunostaining remained intense
in the neuropil in the GPe and in the SNr contralateral to the striatal
lesion (Fig. 5A,C). These results indicate that Elk-1 is
present not only in striatal cell bodies but also in striatopallidal
and striatonigral afferent terminals.
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Figure 5.
Elk-1 protein detection in striatopallidal and
striatonigral axon terminals. After unilateral kainic acid lesion of
striatal cells, Elk-1 immunodetection was processed on sections
corresponding to the external segment of the globus pallidus
(GPe) and the substantia nigra pars reticulata
(SNr) levels. Note in both cases the clear decrease (*)
of the neuropil immunoreactivity in the GPe (B)
and the SNr (D) ipsilateral to the lesion when
compared with the contralateral side of the lesion (A,
C, respectively).
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In vivo hyperphosphorylation of Elk-1 on
corticostriatal stimulation
The striatum receives a massive glutamatergic excitatory input
originating from the whole cerebral cortex. In anesthetized rats, the
sustained unilateral electrical stimulation of the cortex leads to Fos
protein induction in the striatum, and this induction is
topographically related to the stimulated cortical area (Sgambato et
al., 1997). In particular, the stimulation of the sensorimotor cortex,
which projects to the lateral part of the striatum, leads to Fos
induction specifically in this region (Sgambato et al., 1997). This
induction could already be observed 1 hr after the end of stimulation,
indicating a robust and rapid activation of c-fos. Because
Elk-1 plays a key role in SRE-dependent activation of IEGs, we wished
to evaluate whether it is targeted by the signals activated in this
in vivo model of sensorimotor cortex stimulation. Activation
of the ERK signaling pathway and phosphorylation of Elk-1 just precedes
the appearance of mRNAs for IEGs such as c-fos and
zif268, an IEG which contains four SRE sites in its promoter (Zinck et al., 1993; Hipskind et al., 1994a,b). We thus optimized experimental conditions to allow us to visualize IEG mRNAs and phosphorylation of ERK and Elk-1 concomitantly. These turned out to
involve immediately sacrificing rats after a stimulation period of 15 min.
Model of IEG mRNA induction
In sham-operated (implanted but not stimulated) rats (Fig.
6, left panels), in
situ hybridization shows that c-fos and
zif268 mRNAs are induced in the cortex ipsilateral to the
electrode implantation. In the striatum, c-fos is not
detectable, whereas zif268 shows a moderate basal
expression. In stimulated rats (Fig. 6, right panels), both
RNAs are slightly induced in the cortex ipsilateral to the stimulation
site. However, they show a strong and restricted induction bilaterally
in the lateral part of the striatum, corresponding to the projection
area of the stimulated cortical region, especially in comparison with
the nonactivated striatal medial area.
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Figure 6.
c-fos and
zif268 mRNA induction after unilateral electrical
stimulation of the corticostriatal pathway. Adjacent brain sections were in situ-hybridized with
33P-c-fos and
33P-zif268 antisense probes. Left
panels, In sham-operated (Sham) rats,
c-fos and zif268 mRNAs are induced in the
cortex ipsilateral to the electrode implantation. In the entire
striatum, whereas c-fos is not detectable, a
constitutive expression of zif268 is observable.
Right panels, In stimulated (Stim) rats,
c-fos and zif268 mRNAs are slightly
induced in the cortex ipsilateral to the stimulation. In the lateral
part of the striatum, a restricted bilateral induction is strongly
observed for both messengers, when compared with the medial area.
iCx, Ipsilateral cortex; cCx, contralateral cortex to the electrode implantation; mSt,
lSt, medial and lateral parts of the striatum,
respectively.
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Hyperphosphorylation of ERK and Elk-1 spatiotemporally correlates
with IEG mRNAs induction
We have shown that Elk-1 is a nuclear and cytoplasmic
transcription factor in the adult rat brain. We wondered whether Elk-1 might be activated in these various neuronal subcellular compartments after glutamatergic stimulation that leads to IEG mRNAs induction.
To address this question, we used our in vivo model of IEG
mRNA induction after corticostriatal stimulation. We concentrated our
analysis on the striatum, because this region showed a specific induction of IEG mRNAs by electrical stimulation. The activation of
Elk-1 protein was examined by using an antibody that specifically and
exclusively recognizes the phosphorylated (and therefore active) form
of Elk-1 (see Materials and Methods). Because Elk-1 represents a major
substrate of the ERK pathway, we also tested for ERK activation by
using an antibody directed against the phosphorylated, active form of
ERK (see Materials and Methods).
P-ERK (Fig. 7A-D) was
analyzed on sections adjacent to those used for IEG mRNA detection (see
Fig. 6). At low magnification, a marked increase in P-ERK
immunolabeling was obvious in the lateral striatum (the activated area)
in comparison to the medial striatum (the nonactivated area) (Fig.
7A), and this activation corresponded spatially with IEG
mRNA induction. Higher-magnification images showed numerous neurons
with a strong P-ERK immunoreactivity in the lateral striatum, with
labeling in the nucleus (Fig. 7C, arrow) as well as the
cytoplasm, dendrites (Fig. 7C, arrowheads), and the neuropil
(Fig. 7, compare C,B). Statistical analysis of P-ERK immunoreactivity indicated a dramatic increase of P-ERK
immunoreactivity in the lateral striatum relative to the medial
striatum (+800%; p < 0.05) (Fig. 7D).
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Figure 7.
Phosphorylation of ERK and Elk-1 on
corticostriatal activation. Adjacent striatal sections from stimulated
rats (n = 3) were processed in parallel for
immunocytochemistry using antibodies specific for phosphorylated forms
of ERK (P-ERK) and Elk-1 (P-Elk-1) proteins. Shown are ipsilateral striata to the stimulation site at low
(40×; A, E) and high (500×; B, C, F, G)
magnification. Note the increase of P-ERK (A-C)
and P-Elk-1 (E-G) in the lateral part of the
striatum. Arrows, Increase of nuclear immunolabeling. Arrowheads, Dendritic immunolabeled processes of
striatal cells. D, H, Optical density measurements of
P-ERK and P-Elk-1 immunoreactivities. Striatal sections were digitized
with an image analyzer (IMSTAR), and precise delineation of medial and
lateral parts was performed on each striatum (4 sections per rat;
n = 3 independent rats). Optical densities were
measured and statistical comparisons were performed between lateral and
medial parts of the striatum by a paired Student's t
test (*p < 0.05; n = 3).
mSt, lSt, Medial and lateral parts of the
striatum, respectively. Scale bar, 530 µm for 16×; 17 µm for 500×
magnification.
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Elk-1 phosphorylation (P-Elk-1) was examined, as above, on adjacent
sections (Fig. 7E-H). As with P-ERK, P-Elk-1
immunoreactivity was increased in the lateral part of the striatum,
again in correspondence with the activated area (Fig. 7E).
At higher magnification, a weak constitutive nuclear labeling appeared
in medial striatal neurons (Fig. 7F). However, a
marked increase in P-Elk-1 immunoreactivity appeared in the nucleus of
lateral striatal cells (Fig. 7G, arrows). In this activated
region, P-Elk-1 was also clearly detectable in cytoplasmic and
dendritic compartments of some neurons (Fig. 7G,
arrowheads). Statistical analysis showed a moderate but
significant increase of P-Elk-1 immunoreactivity in the lateral
striatum in comparison with the medial striatum (+70%;
p < 0.05) (Fig. 7H). Thus Elk-1 is
activated on glutamatergic stimulation, in strong correlation both
spatially and temporally with the activation of ERK and with
c-fos and zif268 mRNA induction.
Biochemical characterization of P-ERK and P-Elk-1 increase in the
lateral striatum
To confirm the P-ERK and P-Elk-1 immunocytochemical labeling
biochemically, Western blots were performed on extracts prepared from
the striata of stimulated rats. The P-ERK antiserum yielded two bands
of 42 and 44 kDa, the expected sizes for ERKs 1 and 2. This
immunoreactivity was clearly increased in the lateral striatal extracts
relative to those from the medial striatum (Fig. 8, left panels). This reflects
increased ERK activation, because comparable levels of ERKs 1 and 2 were present in both extracts (Fig. 8, left panels).
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Figure 8.
Western blot analysis of P-ERK and P-Elk-1
immunoreactivities. The medial and lateral parts of the striatum were
rapidly dissected just after the end of the stimulation period, lysed,
and processed for Western blot analysis. Left panels,
P-ERK and ERK proteins were detected on the same blot after a stripping
procedure. Note the marked increase of phosphorylated 42 and 44 kDa
proteins in the lateral striatal extracts relative to those from the
medial striatum and the equal amount of ERK1 and ERK2 proteins in both extracts. Right panels, The same procedure was applied
for P-Elk-1 and Elk-1 immunodetection. Note the slight increase of 62 kDa phosphorylated protein in the lateral versus medial area with no
variation of Elk-1 in these areas. The data presented are
representative of three independent experiments, which gave similar
results.
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Western blots to detect endogenous P-Elk-1 have proven difficult to
perform in cell lines expressing high levels of Elk-1 (R. A. Hipskind, unpublished observations). Nevertheless, the striatal
extracts yielded a band of 62 kDa that showed a slight increase in
immunoreactivity in the lateral versus the medial area (Fig. 8,
right panels). Once again, equal amounts of Elk-1 protein
were detected in both striatal regions. Thus activation corresponds to
a moderate increase in P-Elk-1.
Hyperphosphorylated Elk-1 proteins are colocalized in striatal
cells with a strong zif268 mRNA induction
The striatal region in which phosphorylation of Elk-1 was
increased corresponded to that showing IEG mRNA induction (compare Figs. 6, 7). To correlate this more directly, sections were processed for both phosphoimmunostaining and IEG mRNA in situ
hybridization (Fig. 9). In the lateral
striatum, all neurons with a strong P-Elk-1 immunoreactivity also
showed an increase in zif268 mRNA expression (Fig. 9,
large arrows). As in the medial striatum, cells weakly immunoreactive for P-Elk-1 expressed basal levels of zif268
mRNA (Fig. 9, thin arrows). This implicates Elk-1 as an
intermediate in the transcriptional regulation of IEGs controlled by
SREs on glutamatergic stimulation of neurons.
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Figure 9.
Double staining of P-Elk-1 immunoreactivity with
zif268 mRNA in situ hybridization.
Striatal sections from stimulated rats were coprocessed for P-Elk-1
with zif268 mRNA as described above. In both medial and
lateral parts of the striatum, a constitutive expression of
zif268 mRNA is detectable, as well as a weak P-Elk-1 immunoreactivity (thin arrows). In the lateral part of
the striatum, neurons presenting a strong P-Elk-1 immunoreactivity
(large arrows) also show a marked increase in
zif268 mRNA expression. Note the perfect correlation
between strong P-Elk-1 immunoreactivity and zif268 mRNA
expression.
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DISCUSSION |
Three central findings emerge from this study. First, Elk-1 is an
exclusively neuronal transcription factor. Second, this transcription
factor is, in its nonactivated state, present in the soma, dendrites,
and axon terminals of neurons. Third, Elk-1 is phosphorylated on
in vivo glutamatergic stimulation, and this phosphorylation
can occur in various subcellular compartments (e.g., dendrites, soma,
and also the nucleus).
Elk-1 mRNA expression in the CNS of the rat
Evidence for Elk-1 mRNA expression in the brain was variable when
we began this study. Although Rao et al. (1989) had found, by Northern
blot analyses, barely significant levels of Elk-1 mRNA in the adult
mouse brain, more recent data indicated a high level of Elk-1 mRNA
expression in the brain in human, mouse, and rat (Giovane et al., 1994;
Price et al., 1995) (Hipskind, unpublished observations). In
situ hybridization using an antisense cRNA probe allowed us to
identify regional differences in the expression of Elk-1 mRNAs in the
rostrocaudal extension of the rat brain and to characterize neurons as
the main cellular type expressing this transcription factor.
The regional variation in Elk-1 mRNA expression suggests that it may
contribute to the regulation of neuronal function that varies in
different parts of the brain. Elk-1 is one of the major substrates of
the MAP kinase protein family. Among these kinases, ERK (Marais et al.,
1993; Hipskind et al., 1994a,b), Jun N-terminal kinase/stress-activated
protein kinase (JNK/SAPK) (Cavigelli et al., 1995), and
p38MAPK (Raingeaud et al., 1996) have all been shown
from in vivo and in vitro studies to
phosphorylate Elk-1. ERK proteins are not only regulators of cell
proliferation and differentiation but also likely regulators of
hippocampal long-term potentiation (English and Sweatt, 1996; Martin et
al., 1997). On the other hand, JNK/SAPK and p38MAPK
are activated by stress and UV irradiation (Cano et al., 1994; Dérijard et al., 1994; Pombo et al., 1994) and have been
implicated in apoptosis in PC12 cells (Xia et al., 1995). The close
correspondence between ERK2 mRNA (Thomas and Hunt, 1993) and Elk-1 mRNA
expression is consistent with the hypothesis that Elk-1 is an in
vivo substrate for ERK2. By contrast, some differences are found
between JNK/SAPK mRNA (Carletti et al., 1995) and Elk-1 mRNA
expression, as, for example, in the basal ganglia, in which JNK/SAPK
mRNA levels are much lower than those of Elk-1. It would be interesting
to determine whether other MAPKs, e.g., p38MAPK, are
associated with stress-activated pathways in these regions.
Finally, our observation that Elk-1 is expressed principally in
neuronal cell types is interesting, because IEGs, in particular c-fos, can be induced not only in neurons but also in
astrocytes during the early postnatal stages and at adult age in
response to severe injury (axotomy, hypoxia, and hypoglycemia) (for
review, see Arenander and De Vellis, 1995). We have never found
significant levels of Elk-1 mRNA or protein in astrocytes in
vivo, whereas both are abundant in astrocytoma cell lines (Zinck
et al., 1995). The constitutive expression of CREB in this cell type
(Herdegen et al., 1993; Sato-Bigbee and Yu, 1993) implicates it as the
transcription factor governing c-fos mRNA transcription.
Because we have not measured SRF expression levels in astrocytes, the
role of signals targeting this factor (Miranti et al., 1995) cannot be
addressed. Nevertheless, this does not exclude a role for the
TCF-SRF-SRE complexes in c-fos induction in glial cells at
early stages of development and after injuries at adult age. In these
instances, Elk-1 may be transiently induced in astrocytes, which may be
reflected by its strong expression in astrocytoma cells. Alternatively, other TCFs and related proteins could be implicated in SRE-dependent c-fos induction in glial cells.
Subcellular localization of Elk-1 protein in neurons
When overexpressed by transient transfection of HeLa cells
(Janknecht et al., 1994), recombinant Elk-1 was predominantly localized within the nucleus. ERKs, on their activation by growth factors, translocate to the nucleus (Chen et al., 1992), in which they can in
turn phosphorylate Elk-1 in the C-terminal region (Marais et al.,
1993). In sensory neurons of Aplysia, ERKs also translocate to the nucleus during 5-HT-induced long-term facilitation (Martin et
al., 1997).
Using immunocytochemical detection of ERK proteins on brain sections,
Fiore et al. (1993a) could localize the proteins in the soma
surrounding the nucleus in neuronal cell bodies and in dendrites. Our
immunocytochemical mapping of Elk-1 protein clearly indicates that, in
addition to nuclear localization, Elk-1 protein is also present in the
soma of cell bodies and dendrites and in some axon terminals. The
significance of this pattern is demonstrated by two important controls
for our immunocytochemical conditions. We found that CREB protein was
exclusively nuclear, a result in agreement with the literature
(Yamamoto et al., 1989), and that STAT3 was localized in the cytoplasm
before stimulation (Darnell et al., 1994). Thus, in neuronal cells
in vivo, Elk-1 may be a cytoplasmic substrate of ERK
proteins, which are activated subsequent to excitatory synaptic inputs
impinging on dendrites and neuronal soma.
The presence of Elk-1 in axon terminals of striatopallidal and
striatonigral afferent terminals is very intriguing. In the SNr, we
have also localized ERKs in axon terminals (data not shown), as
suggested already by Ortiz et al. (1995). Several lines of investigation have suggested that retrograde transport of ERKs could
transduce signals from the nerve terminal to the nerve cell body
(Johanson et al., 1995; Martin et al., 1997). Our observation that
Elk-1 is also localized in nerve terminals suggests that it could also
participate to the retrograde transport of signals.
Hyperphosphorylation of Elk-1 on stimulation of
corticostriatal afferents
In cell culture models, the phosphorylation of Elk-1 plays a key
role in the serum-induced transcriptional regulation of IEGs. Signaling
pathways that lead to Elk-1 phosphorylation in culture cells are also
activated on glutamate receptor stimulation in vitro (Bading
and Greenberg, 1991) or in vivo (Fiore et al., 1993b; Kurino
et al., 1995). Our work is the first attempt to demonstrate in
vivo that Elk-1 can be hyperphosphorylated on glutamatergic electrical stimulation.
In our model of corticostriatal stimulation, ERKs were dramatically
activated in the lateral part of the striatum, as determined by both
immunocytochemical and biochemical criteria. Phosphorylated ERKs were
present in nuclear, cytosolic, and dendritic compartments of neuronal
cells. These data are interesting in light of the nuclear translocation
of ERK in culture cells with serum activation (Chen et al., 1992) and
with 5-HT activation, leading to long-term facilitation in
Aplysia (Martin et al., 1997). The presence of activated ERK
proteins in several different cellular compartments in vivo
is consistent with its role in regulating diverse responses to
extracellular signals (for review, see Seger and Krebs, 1995).
The cortical electrical stimulation also induced a significantly higher
immunoreactivity with phospho-Elk-1 antibody in the projection field of
stimulated striatal afferents. Hyperphosphorylation of Elk-1 was
apparent in the nucleus, in which Elk-1 is likely to play a key role in
the regulation of transcription, and to a lesser extent in other
cellular compartments, i.e., in the cytosol in soma and dendrites. This
observation is, to our knowledge, the first demonstration that Elk-1 is
activated in a non-nuclear compartment. It further supports the notion
that Elk-1 represents, in neuronal cells in vivo, a local
target of activated ERKs and as such could relay the glutamatergic
receptor signal from the membrane to the different intracellular
compartments, including the nucleus.
The activation of NMDA receptors, which has been linked to
SRE-dependent c-fos induction (Bading et al., 1993), is
implicated in striatal Fos induction in our model of electrical
cortical stimulation (Abo et al., 1994). Because cortical afferents
specifically impinge on dendritic spines of striatal neurons (Smith and
Bolam, 1990), and Ca2+ influx through NMDA receptors
remains localized in the proximity of the receptor (Regehr et al.,
1990), we suggest that the activation of ERKs and Elk-1 in dendrites
could relay the signal triggered by this rise of dendritic calcium to
the nucleus. This would be consistent with the recent demonstration
that increased cytoplasmic calcium is linked to gene regulation by the
SRE site, whereas increased nuclear calcium controls CRE-driven gene
expression (Hardingham et al., 1997).
Strong P-Elk-1 immunoreactivity always correlated with
zif268 mRNA induction. Nevertheless, we cannot exclude that
other signaling pathways are also involved in IEG activation in our
model. For example, Ca2+ entry could activate the
SRE via targeting SRF itself (Xia et al., 1996) or CREs via
calcium-calmodulin-dependent protein kinases (CamKs), as well as a
PYK2-Ras-ERK pathway (Lev et al., 1995; Siciliano et al., 1996).
CamKs can lead to CREB activation via its phosphorylation on
Ser133, as can the ribosomal protein S6 kinase
(RSK2), a CREB kinase acting downstream of the ERK
signaling cascade (Xing et al., 1996). Based on our results and those
of Robertson et al. (1995), it seems likely that different combinations
of these signaling systems are involved in IEG induction in the
CNS.
Finally our data suggest that the ERK signaling cascade has an
important role in regulating genes controlled by SREs via
phosphorylation of Elk-1 in vivo. Genes driven by SREs in
different promoter contexts, such as the IEG zif268, are
likely to show differential sensitivity to this signaling pathway
targeting Elk-1, which would support a crucial role of Elk-1 in
synaptic plasticity.
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FOOTNOTES |
Received July 21, 1997; revised Oct. 10, 1997; accepted Oct. 21, 1997.
This work was supported by the University Pierre and Marie Curie, the
Centre National de la Recherche Scientifique, Institut Lilly, and
Biomed Program Grant PL 962215. We thank Drs. D. Stéhelin and D. Grévin for the gift of mouse Elk-1 cDNA, R. Zinck for providing
Elk-1 antisera, and Dr. A. Thriller for confocal analysis.
Correspondence should be addressed to Jocelyne Caboche, Laboratoire de
Neurochimie-Anatomie, Institut des Neurosciences-Unité de
Recherche Associée 1488, Université Pierre et Marie Curie, 9 quai St-Bernard, 75005 Paris, France.
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Top
Abstract
Introduction
Compound via MeSH
