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The Journal of Neuroscience, December 1, 1998, 18(23):9695-9702
Specific Agrin Isoforms Induce cAMP Response Element Binding
Protein Phosphorylation in Hippocampal Neurons
Ru-Rong
Ji,
Christian M.
Böse,
Christian
Lesuisse,
Dike
Qiu,
Justin C.
Huang,
Qin
Zhang, and
Fabio
Rupp
Department of Neuroscience, School of Medicine, The Johns Hopkins
University, Baltimore, Maryland 21205
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ABSTRACT |
The synaptic basal lamina protein agrin is essential for the
formation of neuromuscular junctions. Agrin mediates the postsynaptic clustering of acetylcholine receptors and regulates transcription in
muscles. Agrin expression is not restricted to motor neurons but can be
demonstrated throughout the CNS. The functional significance of
agrin expression in neurons other than motor neurons is unknown. To
test whether agrin triggers responses in neurons that lead to the
activation of transcription factors, we have analyzed phosphorylation of the transcriptional regulatory site serine 133 of the transcription factor CREB (cAMP response element binding protein) in primary hippocampal neurons. Our results indicate that the neuronal (Ag4,8), but not the non-neuronal (Ag0,0), isoform of agrin induces CREB phosphorylation in hippocampal neurons. The kinetics of agrin- and
BDNF-induced CREB phosphorylation are similar: peak levels are reached
in minutes and are strongly reduced 2 hr later. Neuronal responses to
agrin require extracellular calcium, and, in contrast to tyrosine
kinase inhibitors, the specific inhibition of protein kinase A (PKA)
does not affect agrin-evoked CREB phosphorylation. Our results show
that hippocampal neurons specifically respond to neuronal agrin in a
Ca2+-dependent manner and via the activation of tyrosine kinases.
Key words:
agrin isoforms; CREB phosphorylation; hippocampal
neurons; PKA; receptor tyrosine kinases; synapses; calcium
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INTRODUCTION |
Many functional properties of
synaptic transmission depend on the precise localization and
stoichiometry of specialized molecules present on pre- and postsynaptic
membranes. The high concentration of neurotransmitter receptors at
postsynaptic sites is a general feature of synapses. The search for
molecules able to regulate synaptic differentiation led to the
discovery of agrin, an extracellular molecule capable of inducing the
aggregation of acetylcholine receptors (AChR) on the surface of
myotubes (McMahan, 1990 ; Burden, 1998). A critical role for agrin in
the formation of neuromuscular junctions (NMJ) has been demonstrated
in vitro and in vivo. Blocking the activity of
neuronal agrin with specific antibodies in motor neuron/myotube
cocultures prevents AChR aggregation at contact sites (Reist et al.,
1992). Moreover, homologous recombination experiments revealed that
agrin expression is essential for embryonic development and that NMJ do
not form in homozygous agrin-deficient embryos (Gautam et al.,
1996).
Agrin mediates AChR clustering in part via the activation of the
receptor tyrosine kinase muscle-specific kinase (MuSK) (Jennings et
al., 1993; Glass et al., 1996). MuSK phosphorylation in myotubes occurs
rapidly after agrin addition, NMJ formation is similarly affected in
MuSK-null mice and in agrin-deficient mice, and agrin does not induce
AChR clustering in MuSK-deficient myotubes (DeChiara et al., 1996;
Fuhrer et al., 1997; Hopf and Hoch, 1998).
The AChR-aggregating activity of agrin is regulated by alternative
splicing. Splicing at one particular location of the agrin mRNA,
referred to as the Z position, produces isoforms that differ by
~1000-fold in their ability to cluster AChR on myotubes in vitro (Ferns et al., 1992, 1993; Ruegg et al., 1992; Rupp et al., 1992). The alternative splicing of agrin mRNA is regulated during development and is tissue-specific (Hoch et al., 1993; O'Connor et
al., 1994; Ma et al., 1995; Stone and Nikolics, 1995; N. Cohen et al.,
1997). Highly active agrin isoforms are found exclusively in
neurons, whereas non-neuronal tissues, including muscle, express isoforms with low AChR-aggregating activity (Hoch et al., 1993; Ma et
al., 1993; O'Connor et al., 1994; Stone and Nikolics, 1995; N. Cohen
et al., 1997). Besides motor neurons, agrin is expressed in many
different types of neurons throughout the CNS, including CA1 and CA3
pyramidal cells in the hippocampus and granule cells in the dentate
gyrus, where agrin expression is regulated during development and by
neuronal activity (O'Connor et al., 1995; N. Cohen et al., 1997).
Despite its widespread expression in the nervous system, the role of
agrin in neurons remains undetermined. Preliminary anatomical
examination of agrin isoform-deficient mice does not reveal any gross
abnormalities, suggesting that defective agrin expression does not
compromise early aspects of neural development such as neurogenesis and
axon guidance (Gautam et al., 1996). Although NMJ are absent in
agrin-mutated mice, it is not known whether agrin mediates synapse
formation in the CNS. Along with its effect in synapse formation, it
has been proposed that agrin may function as a neural adhesion molecule
and as a stop signal for sensory and motor neurons (Campagna et al.,
1995; Chang et al., 1997; Martin and Sanes, 1997).
Recently, it has been shown that agrin induces specific changes of gene
expression in muscle. Muscle and neuronal agrin increase the expression
of utrophin in cultured myotubes, and agrin regulates the expression of
AChR subunits in vivo (Jones et al., 1996; I. Cohen et al.,
1997; Meier et al., 1997, 1998; Gramolini et al., 1998). Defective
transcription in muscle fibers also has been observed in
agrin-deficient mice (Gautam et al., 1996). To determine whether agrin
isoforms induce specific responses in neurons that lead to the
activation of transcription factors, we have analyzed the
phosphorylation of the cAMP response element binding protein (CREB) in
primary hippocampal neurons. CREB is a transcription factor that
regulates gene expression in many cell types, including neurons (Ginty,
1997; Tamai et al., 1997). Phosphorylation of CREB at serine 133 is
required for CREB-mediated transcription (Ginty et al., 1993; Montminy,
1997). CREB can be phosphorylated via the activation of many kinases,
including RSK2, a member of the pp90(RSK) family, protein kinase A
(PKA), and Ca2+-calmodulin-dependent kinase IV (CaMK
IV) (Sheng et al., 1991; Ginty et al., 1994; Xing et al., 1996;
Montminy, 1997). Therefore, CREB phosphorylation is considered an
integrative component of the cellular response underlying a variety of
physiological processes (Tamai et al., 1997). In hippocampal neurons,
for example, CREB phosphorylation is induced in response to trophic
factors and by patterns of activity that lead to changes of synaptic
efficacy (Bito et al., 1996; Deisseroth et al., 1996; Martin and
Kandel, 1996). Our data show that specific agrin isoforms are able to induce CREB phosphorylation in hippocampal neurons, that this response
requires extracellular calcium, and that it is mediated by tyrosine kinases.
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MATERIALS AND METHODS |
Primary cell cultures. Four to five hippocampi of 2- to 3-d-old Sprague Dawley rats were dissected rapidly (<40 min).
CA1-CA3 regions were isolated, sliced into four to five small pieces
each, washed once in Hank's buffer, and incubated for 3-5 min at room temperature with trypsin (3 mg/ml) (Sigma, St. Louis, MO) and DNase
(0.6 mg/ml) (Sigma) in digestion solution [containing (in mM) 137 NaCl, 5 KCl, 7 Na2HPO4, 25 HEPES, and 4 NaHCO3, pH 7.4). Tissues were dissociated by gentle
pipetting in Hank's buffer supplemented with 0.3% BSA, 12 mM MgSO4, and 0.6 mg/ml DNase. Approximately 350,000 hippocampal neurons in 1 ml of Neurobasal medium
(Life Technologies, Grand Island, NY) were cultured per well in
12-well plates precoated with poly-D-lysine. Arabino
cytokine (5 µM; Sigma) was added on the second day of culture.
Stimulation protocols. Agrin containing supernatant
(conditioned media) were prepared as described (Ferns et al., 1993).
The amounts of agrin isoforms in conditioned medium were normalized by
Western blotting to standardized agrin quantities. Both the Ag0,0-conditioned and Ag4,8-conditioned media were used at 1:300 dilution, corresponding to ~50 pM agrin. Control medium
from untransfected cells also was used at the same dilution.
Ag4,8-conditioned medium (1:300) was tested for AChR clustering
activity by using C2 myotubes as described (Ferns et al., 1993).
Brain-derived neurotrophic factor (BDNF; kindly provided by Dr. David
Ginty, Johns Hopkins University, Baltimore MD) was used at 40 ng/ml. Conditioned medium containing the 50 pM Ag4,0
isoform was tested also. Various conditioned media and BDNF were added
to hippocampal cultures for 10 min. All pharmacological agents were
added to the medium 20-30 min before stimulation. Cell morphology was
evaluated after treatments; no alterations in processes and/or cell
bodies were observed. TTX, CNQX, genistein, herbimycin A, forskolin,
and Rp-8-cAMP were purchased from Calbiochem (San Diego, CA); MK801 was
purchased from RBI (Natick, MA).
Immunostaining. At 10 min after the stimulation the cells
were fixed in 4% paraformaldehyde for 30 min and processed for
immunocytochemistry according to the ABC method, as previously
described (Ji and Rupp, 1997). Briefly, cultures were incubated with
anti-pCREB (Polyclonal antibody, 1:2000; Upstate Biotechnology, Lake
Placid, NY) at 4°C overnight. Then the cells were incubated with
biotinylated secondary antibody and subsequently with ABC complex. The
reaction product finally was visualized with DAB/hydrogen peroxide in
acetate buffer containing ammonium nickel sulfate. Synaptophysin
staining occurred as follows: cells were fixed in 4%
paraformaldehyde/4% sucrose for 30 min at 4°C. Fixed cells were
permeabilized in 1% BSA/0.4% saponin (Sigma) for 1 hr at 4°C.
Synaptophysin-specific monoclonal antibody SY38 (Boehringer Mannheim,
Indianapolis, IN) and FITC-conjugated goat anti-mouse antibodies
(Jackson Laboratories, Bar Harbor, ME) were used as suggested by the manufacturers.
Western blot. Hippocampal cultures were lysed after
treatment in 100 µl of boiling lysis buffer (100 mM
Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 10% 2-mercaptoethanol, and
0.1% bromophenol blue) per well. Lysate (20 µl) was loaded onto
SDS-PAGE gradient gel (4-15%, Bio-Rad Laboratories, Melville, NY) and
transferred onto nitrocellulose filters. Filters were incubated with
anti-pCREB antibodies (1:3000; Upstate Biotechnology) overnight at
4°C. pCREB-immunoreactive bands were visualized via the ECL detection
kit (Amersham, Arlington Heights, IL) and exposed onto X-ray
films (XAR-5, Kodak, Rochester, NY) for 5-20 min. Subsequently,
the blots were submerged in stripping solution (62.5 mM
Tris-HCl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol), incubated at 50°C for 30 min, and probed with anti-CREB antiserum (1:2000; New England Biolabs, Beverly, MA) to detect the total level of
CREB expression.
Quantification. pCREB-positive neurons were counted in each
well. Counting was performed by a person unaware of the treatment protocol. Neurons with distinct nuclear staining, which were obviously different from the background, were counted as pCREB-positive. Five
optic fields per 2.5 cm diameter well (one at the center and four at
~0.5 cm from the edge of the well) were selected for counting at
20× magnification. The total number of neurons ranged from 70 to 230 per optic field. The percentage of pCREB-immunoreactive nuclei per
optic field was calculated. The percentages of five fields were
averaged as a mean, and four to six experiments in duplicate were
included in each treatment. The data were assessed by an ANOVA test.
The criterion for statistical significance was p < 0.05.
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RESULTS |
Agrin induces CREB phosphorylation in hippocampal neurons
The 3-d-old primary neurons obtained from postnatal day 3 (P3) rat
hippocampi were exposed to conditioned media containing an equal amount
(~50 pM) of neuronal (Ag4,8) and non-neuronal (Ag0,0)
agrin isoforms. CREB phosphorylation at serine 133 (pCREB) was detected
by using a specific polyclonal serum that recognizes CREB
phosphorylated, but not CREB unphosphorylated (Ginty et al., 1993).
pCREB immunoreactivity was analyzed by Western blotting with protein
extracts prepared from hippocampal neurons incubated with agrin
isoforms and compared with the response elicited by BDNF (40 ng/ml), a
strong inducer of pCREB in neurons (Montminy, 1997). Exposure to Ag4,8
and BDNF, but not to Ag0,0 or conditioned medium from untransfected COS
cells, induced an increase in CREB phosphorylation (Fig.
1A,B). To quantify this
response more precisely, we determined the number of
pCREB-immunoreactive cells. A significant increase in the percentage of
cell nuclei immunoreactive for pCREB, as compared with conditioned
medium, was detected in cells exposed to Ag4,8 and BDNF, but not to
Ag0,0 (82% p < 0.01, 159% p < 0.01, and  1.4% p > 0.05, respectively) (Fig.
2B). The pCREB response to both Ag4,8 and BDNF is transient, reaches its maximum within 10 min,
and has decreased significantly at 2 hr (Fig. 2C). A larger number of hippocampal neurons respond to BDNF than to the Ag4,8 isoform.
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Figure 1.
The neuronal agrin isoform (Ag4,8),
but not the non-neuronal isoform (Ag0,0), can induce
CREB phosphorylation (pCREB) in P3-P5
hippocampal neurons. A, Detection of pCREB
immunoreactivity in 3-d-old cultures. BDNF (40 ng/ml), the agrin
isoforms Ag0,0 (50 pM) and Ag4,8 (50 pM), and
conditioned medium from untransfected COS cells
(CM) were added for 10 min. Cells were stained
with the pCREB-specific antiserum. For illustration purposes, fields of
particularly high cell density were chosen. Scale bar, 50 µm.
B, Western blot analyses of 3-d-old P3-P5 hippocampal
neurons. Neurons were incubated for 10 min with conditioned medium,
Ag0,0, Ag4,8, and BDNF (lanes 1-4, respectively), and
the extracts were probed with pCREB-specific antiserum (top
panel). After the blots were stripped, they were probed
with a CREB-specific antiserum to assess the total amount of CREB
expression (bottom panel).
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Figure 2.
A, Profile of synaptophysin
immunoreactivity in 3- and 10-d-old cultures. A shift from diffuse to
punctate staining is observed between these two time points, indicating
the absence and presence of synapses, respectively. Ph,
Phase contrast, Syn, synaptophysin. B,
Quantification of agrin-induced pCREB in 3- and 10-d-old cultures. The
agrin isoform Ag4,8 and BDNF, but not Ag0,0, induce an increase in the
percentage of pCREB-positive neurons both in 3- and 10-d-old cultures
(**p < 0.01 as compared with control medium,
ANOVA; n = 4). C, Agrin induces a
transient increase in pCREB-immunoreactive cells. Incubation with Ag4,8
or BDNF was analyzed at 0, 10, 30, and 120 min, respectively. Both the
Ag4,8 and BDNF responses reach a maximum within 10 min and are reduced
within 2 hr. Despite the difference in magnitude, the BDNF- and
Ag4,8-induced CREB phosphorylation essentially follows similar
kinetics. CM, Control medium.
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Hippocampal neurons express many types of agrin isoforms, and secreted
full-length agrin preferentially associates with the plasma membrane
(Campanelli et al., 1991; O'Connor et al., 1995; N. Cohen et al.,
1997). In motor neurons, agrin is secreted at axon terminals where it
is incorporated into the synaptic basal lamina (Magill-Solc and
McMahan, 1990; Reist et al., 1992). In fully differentiated NMJ both
 -dystroglycan, the major agrin-binding protein in muscle, and MuSK,
a signaling component of the agrin receptor, are concentrated at
synaptic sites (Hall and Sanes, 1993). This suggests the possibility
that synapse formation may lead to a synaptic concentration of agrin,
agrin-binding proteins, and putative neuronal agrin receptors. To
determine whether synapse formation affects the neuronal response to
agrin in hippocampal neurons, we analyzed pCREB induction in 10-d-old
cultures. The formation of functional synapses in these cultures occurs
after 6-7 d of plating (Banker and Goslin, 1988; Fletcher et al.,
1994). Because of the different cellular densities used in these
studies, we performed immunocytochemistry to determine the pattern of
expression of the synaptic vesicle protein synaptophysin in 3- and
10-d-old cultures. The appearance of punctate synaptophysin
immunoreactivity is used routinely as an indicator of synapse
formation. Synaptophysin immunoreactivity in 3-d-old cultures
predominantly is localized diffusely to the soma and throughout
processes. A few immunoreactive puncta also can be detected. In
contrast, in 10-d-old cultures the staining profile is different. The
characteristic punctate pattern of synaptophysin immunoreactivity is
evident on the soma and on neuritic processes (Fig.
2A). Similarly to neurons cultured for 3 d in
which functional synaptic contacts are absent, a robust pCREB induction
is observed with BDNF and Ag4,8, but not with Ag0,0 and control
conditioned medium (Fig. 2B). The magnitude and the
kinetics of the Ag4,8-induced and BDNF-induced CREB phosphorylation are
similar among 3- and 10-d-old cultured neurons (data not shown). The 50 pM Ag4,0 isoform did not induce significant pCREB both in
3- and 10-d-old neurons (1% p > 0.05 and 0.9%
p > 0.05, respectively, as compared with control
conditioned medium; n = 3). These data show that
hippocampal neurons selectively respond to the same highly active agrin
isoforms as muscle fibers and that synapse formation does not affect
the neuronal responses to agrin.
Agrin-induced CREB phosphorylation depends on
extracellular calcium
Having established the ability of the Ag4,8 isoform to induce
pCREB specifically in hippocampal neurons, we next examined the
signaling pathways involved and whether this response is regulated similarly to the AChR clustering mediated by agrin at NMJ. The agrin-induced clustering of AChR on muscle fibers is dependent on
extracellular Ca2+ (Henderson et al., 1984; Wallace,
1988). To test the role of extracellular Ca2+ for
the agrin-induced induction of pCREB, we treated 3-d-old hippocampal
neurons with the calcium chelator EGTA (4 mM) for 20 min
before agrin treatment. EGTA pretreatment completely blocked pCREB
induction by Ag4,8 but did not affect the BDNF response (Fig.
3A,B). It has been shown
previously that extracellular Ca2+ is not required
for BDNF-induced CREB phosphorylation (Finkbeiner et al., 1997).
Extracellular Ca2+ may be required for agrin binding
to its receptor or may regulate CREB phosphorylation as a consequence
of opening of Ca2+-permeable ion channels after
depolarization (Ginty, 1997). Agrin may cause depolarization by
mediating an acute release of glutamate in the media. To distinguish
among these possibilities, we stimulated 3-d-old hippocampal neurons
with Ag4,8 in the presence of the Na+ channel
blocker tetrodotoxin (TTX; 10 µM) or MK801 (10 µM) and CNQX (10 µM), which are specific
antagonists of the NMDA and AMPA subfamilies of glutamate receptors,
respectively. The presence of TTX or MK801 and CNQX in the medium
partially reduces (21 and 23%, respectively) agrin-induced pCREB (Fig.
3C). In all conditions, however, agrin did produce a
significant increase of pCREB-positive cells (38 and 43%,
respectively). The magnitude of the agrin response was comparable when
depolarization was prevented or glutamate receptors were blocked. The
response to BDNF was not influenced by TTX or antagonists of glutamate
receptors (Fig. 3C). These results demonstrate that the
neuronal response to agrin requires extracellular
Ca2+ and that a component of this response is partly
attributable to the stimulation of glutamate release or increased
receptor sensitization (see below).
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Figure 3.
Agrin-induced pCREB in hippocampal neurons depends
on extracellular Ca2+. A,
B, Agrin-induced pCREB is inhibited significantly by
EGTA (4 mM). ***p < 0.001, ANOVA
(n = 4), as compared with agrin response without
EGTA. The BDNF-induced pCREB is not affected. C,
Preventing depolarization and blocking glutamate receptors partially
reduce agrin responses in hippocampal neurons. The pCREB-positive
neurons induced by agrin in the presence of TTX or a combination of the
NMDA receptor antagonist dizocilpine maleate (MK-801)
(10 µM) and the non-NMDA antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM) are reduced by 21% (p < 0.05, ANOVA; n = 5) and 23%
(p < 0.05, ANOVA; n = 4), respectively, when compared with the agrin response. However, a
comparison between responses to agrin and conditioned medium in the
presence of TTX or glutamate receptor antagonists still shows a
significant increase in pCREB-positive cells of 38%
(**p < 0.01, ANOVA; n = 5) and
48% (**p < 0.01, ANOVA; n = 4), respectively. Scale bar, 50 µm.
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Agrin does not activate the PKA signaling pathway
CREB phosphorylation can occur via the activation of
cAMP-dependent PKA after the induction of adenylyl cyclase activity and the subsequent rise of intracellular cAMP (Montminy, 1997). To test
whether agrin activates this signaling pathway, we used the cyclic
nucleotide analog Rp-8-CPT-cAMP, a specific PKA inhibitor, to test the
involvement of PKA (Fig. 4). The
efficiency of Rp-8-CPT-cAMP inhibition in 3-d-old hippocampal neurons
was tested with forskolin, a PKA activator. As described previously,
the application of forskolin (75 µM) induces CREB
phosphorylation (Gonzalez and Montminy, 1989). This response is
inhibited significantly (95%) by Rp-8-CPT-cAMP (50 µM).
In contrast, the Ag 4,8-induced and BDNF-induced responses are not
affected significantly by Rp-8-CPT-cAMP, suggesting that agrin-induced
pCREB in hippocampal neurons does not result from the activation of
PKA.
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Figure 4.
Top. Agrin does not activate the PKA
signaling pathway. The specific PKA inhibitor Rp-8-CPT-cAMP (50 µM) does not affect the agrin- and the BDNF-induced
pCREB. However, Rp-8-CPT-cAMP greatly inhibits (95%) pCREB induced by
the PKA activator forskolin (75 µM);
**p < 0.01, ANOVA; n = 5.
Figure 5.
Bottom. Agrin-induced pCREB
is inhibited significantly by tyrosine kinase inhibitors. The protein
tyrosine kinase inhibitors genistein (100 µM) and
herbimycin A (1.5 µM) greatly reduce the agrin- and the
BDNF-induced CREB phosphorylation in day 3 hippocampal neurons.
**p < 0.01, ***p < 0.001, ANOVA; n = 4, as compared with agrin induction.
+++p < 0.001, ANOVA;
n = 4, as compared with BDNF.
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Agrin-induced CREB phosphorylation in neurons is regulated
by tyrosine kinases
Activation of the receptor tyrosine kinases is required to induce
AChR clustering at NMJ (Peng et al., 1993; Wallace, 1994; DeChiara et
al., 1996; Ferns et al., 1996; Glass et al., 1996, 1997; Hopf and Hoch,
1998). To investigate the dependency of the agrin-induced
phosphorylation of CREB on the activity of tyrosine kinases, we
incubated 3-d-old hippocampal neurons with the specific protein
tyrosine kinase inhibitors genistein and herbimycin A. Pretreatment (20 min) with either genistein (100 µM) or herbimycin A (1.5 µM) resulted in an inhibition of the Ag4,8 and
BDNF-induced pCREB (Fig. 5). Genistein
treatments equally affected the agrin- and the BDNF-induced responses
by 71 and 69% inhibition, respectively. Herbimycin A was more
effective in suppressing the agrin-induced pCREB (92%) than the
BDNF-induced one (55%). No morphological changes could be observed in
treated cells, suggesting that short-time exposure (30 min) with these
inhibitors did not affect the hippocampal neurons adversely.
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DISCUSSION |
Agrin induces a response in hippocampal neurons that leads to the
phosphorylation of the transcription factor CREB. Several parameters
tested in this study indicate that the neuronal response to agrin and
the agrin-induced aggregation of the AChR at NMJ are regulated
similarly. In particular, only the agrin isoforms with the highest AChR
clustering activity (Ag4,8), but not the ones with low activity
(Ag0,0), induce pCREB in hippocampal neurons. Another similarity is the
role that tyrosine kinases play in mediating agrin responses in muscle
and neurons. The role of the receptor tyrosine kinase MuSK in myotubes
is well defined (Peng et al., 1993; Wallace, 1994; DeChiara et al.,
1996; Ferns et al., 1996; Glass et al., 1996, 1997; Hopf and Hoch,
1998). One of the early events in AChR clustering is the binding of
agrin to MuSK, which becomes phosphorylated at tyrosine residues within
minutes of agrin addition (Glass et al., 1996). Other phosphorylation
events downstream of the initial activation of MuSK also occur.
Tyrosine phosphorylation of the  -subunit of the AChR is concomitant
to AChR clustering, but it does not seem to be necessary for this process (Wallace et al., 1991; Swope et al., 1995; Ferns et al., 1996).
We have demonstrated that, as in myotubes, the activation of tyrosine
kinases is an essential step in the signaling cascade activated by
agrin in neurons.
There is a similar requirement for extracellular
Ca2+ for agrin function in neurons and muscle.
Extracellular Ca2+ is required for both
agrin-induced CREB phosphorylation in hippocampal neurons and for the
agrin-induced aggregation of AChR on myotubes. There are two possible
explanations for the Ca2+ dependence in muscle.
Ca2+ may influence the binding of agrin to its
receptor; it has been reported that in the absence of
Ca2+ agrin does not bind to the myotube surface
(Nastuk et al., 1991; Ma et al., 1993; Gee et al., 1994).
Alternatively, engagement of the agrin receptor may trigger a signaling
cascade that leads to Ca2+ influx. Because the
addition of neither the AChR antagonist -bungarotoxin nor the
Na+ channel blocker tetrodotoxin affects
agrin-induced AChR aggregation, neither AChR activation nor
Na+ channel-dependent depolarization must be
required for Ca2+ influx into muscle fibers
(Anderson et al., 1977; Godfrey et al., 1988). A role for a transient
rise of intracellular Ca2+ concentration has been
proposed recently for agrin-induced AChR clustering (Mook-Jung and
Gordon, 1995; Megeath and Fallon, 1998). However, the precise source
responsible for these intracellular calcium transients remains
undetermined. There are other possible roles for
Ca2+ in the neural response to agrin. Again, a
Ca2+ requirement for agrin binding to its
receptor(s) or voltage-gated Ca2+ channels
activation may be involved. In neurons, however, the addition of TTX or
glutamate receptor antagonists reduces the magnitude of the agrin
response even before the establishment of functional synapses. This
suggests that a component of the neuronal response to agrin is
attributable to acute release of glutamate and/or increased glutamate
responsivity. Both cases would result in Na+
channel-dependent depolarization, which would activate voltage-gated Ca2+ channels. How could agrin stimulate
neurotransmitter release? It has been reported that agrin promotes the
differentiation of presynaptic specializations in motor neurons
(Campagna et al., 1997). It is possible that an acute exposure to agrin
may affect the proteins involved in synaptic vesicle release, thus
favoring the fusion of vesicles that are docked either along, or in the proximity of, the plasma membrane of growth cones. Alternatively, agrin
may change the cellular responses to glutamate. This may occur via
sensitization of glutamate receptors, possibly via post-translational modifications, or via stimulation of receptor insertion in the plasma
membrane, as proposed for silent synapses (Malenka and Nicoll,
1997).
Our results show that the neuronal response to agrin is unaffected by
synapse formation. Both the background levels and the magnitude of
agrin-induced CREB phosphorylation are comparable in neurons before or
after functional synapses are formed. This suggests that endogenous
agrin does not saturate agrin receptors and that desensitization of the
receptors occurs. Neuronal agrin receptors may aggregate partially at
synaptic sites, or they may not aggregate at all. This also may explain
the persistent neuronal response to exogenously applied agrin.
Alternatively, agrin expression may decrease after synapse formation to
levels that are no longer able to trigger a response. This hypothesis
is consistent with results showing that agrin expression decreases at
developmental times concomitant with synapse formation in
vivo (Rupp et al., 1991; Thomas et al., 1993; Stone and Nikolics,
1995; N. Cohen et al., 1997; Li et al., 1997) and that a similar
regulation of agrin expression is observed in vitro (Li et
al., 1997). Last, agrin may not be required for synapse formation in
hippocampal neurons but may function as an adhesion molecule. This
hypothesis is supported by data showing that sensory and motor neurons
adhere to agrin and that, in muscle, integrins also can function as an additional agrin receptor (Chang et al., 1997; Martin and Sanes, 1997).
Whether agrin can induce the activation of receptors for adhesion
molecules in hippocampal neurons has not yet been determined.
Finally, it has been shown that CREB phosphorylation in hippocampal
neurons plays an important role in the regulation of gene expression, including the Ca2+-dependent activation
of transcription, which is thought to underlie long-term changes
of synapse strength (Deisseroth et al., 1996; Ginty, 1997). Our
data, along with results showing that agrin expression in neurons is
regulated by synapse formation and by electrical activity (O'Connor et
al., 1995; N. Cohen et al., 1997; Li et al., 1997), are consistent with
the hypothesis that agrin may play a role in the regulation of synaptic
efficacy. Taken together, our data support the hypothesis that
hippocampal neurons and muscle fibers express agrin receptor(s) with
the same specificity for agrin isoforms and that the receptor expressed
in neurons is likely to be a novel member of the receptor tyrosine
kinase family, because MuSK expression is confined to muscle and spleen (Jennings et al., 1993; Glass et al., 1996).
| |
FOOTNOTES |
Received June 18, 1998; revised Sept. 15, 1998; accepted Sept. 18, 1998.
This work was supported by National Institutes of Health (Grant
MH51158) to F.R., the E. A. and J. Klingenstein Fund, the Council
for Tobacco Research to F.R., and the Muscular Dystrophy Association.
We thank David Ginty for the generous gift of brain-derived nerve
growth factor. We are particularly thankful to David Ginty and Richard
Huganir for critically reviewing this manuscript.
Correspondence should be addressed to Dr. Fabio Rupp at the above address.
| |
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Copyright © 1998 Society for Neuroscience 0270-6474/98/18239695-08$05.00/0
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Abstract
Introduction
