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The Journal of Neuroscience, October 1, 1998, 18(19):7953-7961
Brain-Derived Neurotrophic Factor and Basic Fibroblast Growth
Factor Downregulate NMDA Receptor Function in Cerebellar Granule
Cells
Cinzia
Brandoli1,
Angela
Sanna1,
Maria A.
De Bernardi2,
Paolo
Follesa1,
Gary
Brooker2, and
Italo
Mocchetti1
1 Department of Cell Biology, Division of Neurobiology,
Georgetown University, School of Medicine, Washington, DC 20007, and
2 Department of Biology, Johns Hopkins University,
Baltimore, Maryland 21218
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ABSTRACT |
Evidence has accumulated to suggest that the NMDA glutamate
receptor subtype plays an important role in neuronal degeneration evoked by hypoxia, ischemia, or trauma. Cerebellar granule cells in
culture are vulnerable to NMDA-induced neuronal excitotoxicity. In
these cells, brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF2) prevent the excitotoxic effect of NMDA.
However, little is known about the molecular mechanisms underlying the
protective properties of these trophic factors. Using cultured rat
cerebellar granule cells, we investigated whether BDNF and FGF2 prevent
NMDA toxicity by downregulating NMDA receptor function. Western blot
and RNase protection analyses were used to determine the expression of
the various NMDA receptor subunits (NR1, NR2A, NR2B, and NR2C) after
BDNF or FGF2 treatment. FGF2 and BDNF elicited a time-dependent
decrease in the expression of NR2A and NR2C subunits. Because NMDA
receptor activation leads to increased intracellular
Ca2+ concentration
([Ca2+]i), we studied the
effect of the BDNF- and FGF2-induced reduction in NR2A and NR2C
synthesis on the NMDA-evoked Ca2+ responses by
single-cell fura-2 fluorescence ratio imaging. BDNF and FGF2 reduced
the NMDA-mediated [Ca2+]i increase
with a time dependency that correlates with their ability to decrease
NR2A and NR2C subunit expression, suggesting that these trophic factors
also induce a functional downregulation of the NMDA receptor. Because
sustained [Ca2+]i is believed to be
causally related to neuronal injury, we suggest that BDNF and FGF2 may
protect cerebellar granule cells against excitotoxicity by altering the
NMDA receptor-Ca2+ signaling via a downregulation
of NMDA receptor subunit expression.
Key words:
BDNF; FGF2; glutamate; NMDA receptor subunits; NR1; NR2A; Ca2+
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INTRODUCTION |
Neurotrophic factors
are polypeptides that induce differentiation and maturation of neuronal
cells (Mocchetti and Wrathall, 1995 ). The spectrum of action of
neurotrophic factors has expanded in recent years to include protection
against glutamate-mediated excitotoxicity (Mattson and Scheff, 1994),
because glutamate is thought to play an important role in neuronal cell
death in a variety of acute and chronic neurodegenerative diseases
(Wielock, 1985; Rothman and Olney, 1986; Choi, 1988; Coyle and
Puttfarcken, 1993). Among various neurotrophic factors, brain-derived
neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF2)
have been shown to prevent glutamate-mediated neuronal cell death in
several neuronal populations both in vitro (Mattson et al.,
1989; Fernandez-Sanchez and Novelli, 1993; Lindholm et al., 1993;
Zirrgiebel et al., 1995; Kume et al., 1997; Marini et al., 1997a) and
in vivo (Freese et al., 1992; Frim et al., 1993; Peterson et
al., 1996). However, the molecular mechanisms by which BDNF and FGF2
prevent glutamate excitotoxicity are still poorly understood.
Cerebellar granule neurons in culture express a number of glutamate
receptor subtypes (Burgoyne and Cambray-Deakin, 1988; Cox et al., 1990;
Van der Valk et al., 1991). Consequently, these cells have been used to
study possible mechanisms of excitotoxicity evoked by excessive
activation of glutamate receptors and, in particular, the NMDA
receptor subtype (Favaron et al., 1988; Novelli et al., 1988; Marini
and Paul, 1992; Resink et al., 1994). Exposure of cerebellar granule
cells to FGF2 and BDNF for at least 6 hr increases neuronal survival
after glutamate- or NMDA-mediated neurotoxicity (Fernandez-Sanchez and
Novelli, 1993; Lindholm et al., 1993; Marini et al., 1997a), indicating
that these cells are also a good in vitro model for studying
the mechanisms of neuroprotection against excessive stimulation of NMDA
receptors.
The NMDA receptor (NR) is an ion channel formed by distinct subunits.
The obligatory subunit termed NR1 contains the binding site for ligands
(Moriyoshi et al., 1991). The NR2 regulatory subunit consists of four
homologous isoforms (NR2A-D) (Kutsuwada et al., 1992; Monyer et al.,
1992). When coexpressed with NR1, each of the NR2 subunits can form an
ion channel (Meguro et al., 1992; Ishii et al., 1993) characterized by
high Ca2+ conductance (MacDermott et al., 1986).
After overactivation of this channel, a sustained rise in cytosolic
Ca2+ concentration
([Ca2+]i) occurs that is
believed to trigger neurotoxicity (Garthwaite et al., 1986; Choi, 1987;
Rothman et al., 1987; Hahn et al., 1988; Anegawa et al., 1995). Thus,
preventing elevated [Ca2+]i after NMDA
receptor activation may limit the neuropathological processes
associated with the excitotoxic effects of glutamate.
FGF2 has been shown to decrease the glutamate-mediated surge in
[Ca2+]i in hippocampal neurons
(Mattson et al., 1989), suggesting that the neuroprotective properties
of this and potentially other neurotrophic factors may rely on their
ability to reduce [Ca2+]i. However,
the mechanisms underlying the effect of FGF2 on
[Ca2+]i remain primarily unknown.
Using cerebellar granule cells, we tested the hypothesis that FGF2 and
BDNF exert their neuroprotective effect by decreasing NMDA receptor
subunit expression and Ca2+ signaling. We report
that BDNF and FGF2 selectively decrease the expression of NR2A and NR2C
subunits and reduce receptor sensitivity to NMDA activation.
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MATERIALS AND METHODS |
Cerebellar granule cell preparation. Granule cells
were prepared from postnatal day 8 Sprague Dawley rat pups (Taconic,
Germantown, NY) as described previously (Longone et al., 1993; Marini
et al., 1997b). Neurons were plated onto poly-L-lysine-
(1%) precoated 100 mm plastic dishes at a density of 2.5 × 106 cells/ml and were grown in Basal Medium Eagle
(Life Technologies, Gaithersburg, MD) containing glutamine (2 mM), fetal calf serum (10%), KCl (25 mM),
gentamycin (100 µg/ml), and penicillin/streptomycin (10,000 U/ml).
Cells were maintained at 37°C in 5% CO2/95% air. Cytosine arabinoside (10 µM) was added 24 hr after cell
plating to inhibit glial proliferation. Neurons were exposed to BDNF
(50 ng/ml), FGF2 (50 ng/ml), or vehicle (0.19% BSA in PBS) in
serum-free growth medium starting on day 8 in vitro (8 DIV)
for all experiments.
Probe preparation. Plasmids pFPR2AR, pFPR2BR, and pFPR2R
contain cDNA fragments for the rat NR2A, NR2B, and NR2C subunits, respectively, inserted into the polylinker region of pAMP1 cloning vector (Follesa and Ticku, 1995). These plasmids were linearized with
EcoRI, and SP6 RNA polymerase was used to generate
[32P]CTP-labeled cRNAs. cRNAs used in the
protection assay for detection of NR2A, NR2B, and NR2C mRNAs were 662, 530, and 404 bases long, respectively, including 92 bases of the
polylinker region of the cloning vector. The cRNA for cyclophilin was
in vitro transcribed with SP6 polymerase from
EcoRI-linearized plasmid pIG15 (Follesa et al., 1994). This
probe was used as a standard control to correct for potential artifacts
caused by RNA extraction and gel loading (Follesa et al., 1994;
Mocchetti et al., 1996).
RNase protection assay. The RNase protection assay was
performed as described previously (Follesa et al., 1994; Mocchetti et
al., 1996). In brief, total RNA extracted from cerebellar granule cells
was dissolved in hybridization solution containing
32P-labeled NR2A, NR2B, and NR2C cRNAs (150,000 cpm of
each; specific activity, >1 × 108 cpm per
µg of RNA) and pIGl5 cRNA, used at a lower specific activity (~1 × 106 cpm per µg) to balance the
relatively high abundance of the corresponding mRNA. Hybridization was
performed at 50°C overnight. RNA was digested with RNase A (1 U/ml)
and T1 (200 U/ml) for 30 min at 37°C. The reaction was stopped, RNA
was precipitated with isopropanol [1:1 (v/v)], and the pellet,
containing the RNA:RNA hybrids, was dissolved in loading buffer (80%
formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, and 2 mM EDTA), boiled at 95°C, and separated on a 5%
polyacrylamide/urea sequencing gel. The gel was dried, and the
protected fragments were visualized by autoradiography on x-ray film
with the use of an intensifying screen (Hyperscreen; Amersham,
Arlington Heights, IL). Autoradiographic bands were analyzed with a
densitometer (Hoefer GS 300; Hoefer Scientific, San Francisco, CA). The
content of mRNA was calculated in arbitrary units expressed as a ratio between the densitometric area of the NMDA receptor subunit band and
that of the cyclophilin band as described previously (Follesa et al.,
1994; Mocchetti et al., 1996).
Western blot. Cells were washed twice in PBS, harvested in
50 mM Tris HCl, pH 7.0, and pelleted. Pellets were
homogenized in the same buffer with a polytron, centrifuged at
100,000 × g for 20 min, resuspended in 1 ml of Tris
HCl, and stored at  20°C. After removal of cellular debris by
centrifugation, protein levels in the lysates were measured by the
Bradford Coomassie blue colorimetric assay (Bio-Rad, Hercules, CA).
Equal amounts of proteins were loaded onto an 8.5% SDS-polyacrylamide
gel. Proteins were transferred on a nitrocellulose membrane and blocked
with Tris-buffered saline (TBS-T) (25 mM Tris and 1%
Tween) containing 5% milk powder. Blots were incubated with rabbit
affinity-purified polyclonal antibodies anti-NR2A (AB1555; Chemicon,
Temecula, CA) or anti-NR1 (AB1516; Chemicon), both at a dilution of
1:2500 in TBS-T plus 5% milk. After several washes with TBS-T,
blots were then incubated with the secondary antibody,
peroxidase-conjugated anti-rabbit IgG (dilution 1:10,000; Boehringer
Mannheim, Indianapolis, IN). Immunoreactivity was detected by the use
of an enhanced chemiluminescence system (Amersham).
Fluorescence Ca2+ imaging. The
[Ca2+]i was measured by
single-cell fura-2 fluorescence ratio imaging as described previously
(De Bernardi et al., 1996). For this purpose, cells were plated onto 25-mm-round, 1-mm-thick glass coverslips (Fisher Scientific, Houston, TX) precoated with poly-L-lysine (1%). At the end of the
trophic factor treatment, cells were loaded with the cell-permeable
fura-2 AM (2-5 µM; Molecular Probes, Eugene, OR)
at 37°C and in an atmosphere of 5% CO2. Loading was
performed in either serum-free growth medium or Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM
NaHCO3, 2.3 mM CaCl2,
1.2 mM MgCl2, 5.6 mM
glucose, and 15 mM HEPES, pH 7.4) for 30 and 45 min,
respectively. Cells were then washed with Mg2+-free
Locke's solution and allowed to sit for 10 min to complete fura-2
de-esterification. Ca2+ imaging was performed at
room temperature while neurons were bathed in
Mg2+-free Locke's solution. Neurons were imaged
using an Attofluor RatioVision digital fluorescence microscopy system
(Atto Instruments, Rockville, MD) equipped with a Zeiss Axiovert 135 microscope and a F-Fluar 40×, 1.3 numerical aperture
oil-immersion objective, as described previously (De Bernardi et al.,
1996). Briefly, fura-2 was excited at 334 and 380 nm with its emission
monitored at 510-530 nm; the 334/380 nm excitation ratio increases as
a function of the [Ca2+]i. Each day
before the experiments, the instrument was calibrated (calibration was
done in vitro with fura-2 pentapotassium salt in the
presence of a high concentration of Ca2+ or EGTA),
and the 334/380 nm excitation ratio was converted to [Ca2+]i nM values
(Grynkiewicz et al., 1985). For each coverslip, 50-99 neurons were
simultaneously imaged in a given microscopic field, and single-cell
Ca2+ responses were collected. Flat
[Ca2+]i traces from cells
nonresponding to NMDA (1-5%) both in control and neurotrophic
factor-treated cells were identified and not included in the
calculation of the mean Ca2+ response.
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RESULTS |
To test our working hypothesis that BDNF and FGF2 protect
cerebellar granule cells from glutamate toxicity by downregulating NMDA
receptor expression and function, we exposed these neurons to either
neurotrophic factor for different times. The levels of mRNA and/or
protein for NR1, NR2A, NR2B, and NR2C subunits, as well as the
NMDA-evoked Ca2+ responses, were analyzed. NR2D, the
other known isoform of the NR2 subunit component, was not included in
our study because this subunit appears not to be expressed in rat
cerebellar granule cells (Ishii et al., 1993; Monyer et al., 1994).
Lack of effect of BDNF and FGF2 on NR1 subunit protein levels
Cerebellar granule cells were exposed to BDNF and FGF2 at the
concentration of 50 ng/ml that has been shown previously to protect
these cells against glutamate-induced cell death (Fernandez-Sanchez and
Novelli, 1993; Lindholm et al., 1993; Marini et al., 1997a). NR1
subunit protein levels were determined by Western blot analysis using a
specific antibody that recognizes only one NR1-immunoreactive band
(Fig. 1A). Time course
studies performed at 3, 6, 24, and 48 hr after the addition of BDNF,
FGF2, or vehicle (0.19% BSA in PBS) to the serum-free culture medium
revealed that neither neurotrophic factor changed NR1 subunit levels
(Fig. 1B). To eliminate the possibility that the
expression of NR1 subunit cannot be regulated under our culture
conditions, cerebellar granule cells were also exposed to a subtoxic
concentration of NMDA (100 µM) that has been shown to
downregulate NR1 expression in these cells (Resink et al., 1995, 1996).
NMDA elicited a time-dependent decrease in NR1 immunoreactivity
starting at 6 hr and lasting up to 48 hr (Fig.
1A,B). This effect was blocked by
the NMDA-receptor antagonist MK-801 (1 µM) (data not
shown), confirming findings by other investigators (Resink et al.,
1995, 1996). Thus, it appears that the mechanism by which BDNF and FGF2
exert a neuroprotective effect in cerebellar granule cells does not
involve an alteration in NR1 subunit protein levels.
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Figure 1.
NMDA but not BDNF or FGF2 elicits a time-dependent
decrease in NR1 subunit protein levels. Cerebellar granule cells (8 DIV) were exposed to NMDA (100 µM), BDNF (50 ng/ml), or
FGF2 (50 ng/ml) for 3, 6, 24, and 48 hr. Cells were harvested, and the
content of the NR1 subunit protein was determined by Western blot
analysis using NR1 antibody. A, Representative blot
showing NR1 immunoreactivity in cerebellar granule cells 24 hr after
various treatments. Molecular weight markers are in kilodaltons.
B, Time course of the effect of BDNF, FGF2, and NMDA on
the NR1 subunit protein levels. Levels of NR1 were calculated by
densitometric analysis of the NR1-immunoreactive band. Data, expressed
as percent of control, are the mean ± SEM of three separate
preparations of granule cells (n = 6).
*p < 0.05; **p < 0.01 versus
control (ANOVA and Dunnett's test). Similar results were obtained
using cells at 12 DIV.
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BDNF and FGF2 decrease NR2A subunit protein levels
Western blot analyses were then used to determine whether BDNF and
FGF2 alter the levels of NR2A, one of the four known isoforms of the
NR2 subunit involved in the functional assembly of the NMDA receptor
channel. Time course studies revealed that BDNF evoked a time-dependent
decrease in NR2A protein levels beginning at 6 hr (30% decrease),
peaking at 24 hr (50% decrease), and returning to control levels by 48 hr (Fig.
2A,B).
FGF2 also induced a decrease in NR2A levels, but its effect was
transient (only at 6 hr) and overall weaker than that elicited by BDNF
(Fig. 2B). Moreover, each trophic factor failed to
change the total protein content, determined either by Bio-Rad assay on
cell lysates or Coomassie staining on Western blots (data not shown).
Because NMDA has been shown to alter the expression of the NR2A subunit
(Resink et al., 1995, 1996), cerebellar granule cells were also
incubated with NMDA for 24 hr. NMDA (100 µM) decreased
NR2A protein levels to an extent greater than that elicited by BDNF
(Fig. 2A).
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Figure 2.
BDNF and FGF2 decrease NR2A subunit protein
levels. Cerebellar granule cells were exposed to BDNF, FGF2, or NT-3
(all at 50 ng/ml) for 1, 3, 6, 24, and 48 hr or to NMDA (100 µM) for 24 hr. Lysates were prepared, and Western blot
analysis was performed using NR2A antibody. A,
Representative blot showing NR2A immunoreactivity of cells 24 hr after
various treatments. Molecular weight markers are in kilodaltons.
B, Time course of the effect of BDNF, FGF2, and NT-3 on
the NR2A protein levels. Protein content was calculated by
densitometric analysis of the NR2A-immunoreactive band. Data, expressed
as percent of control, are the mean ± SEM of three separate
experiments (n = 6), using different preparations
of granule cells. *p < 0.05;
**p < 0.01 versus control (ANOVA and Dunnett's
test).
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BDNF binds primarily to the receptor tyrosine kinase TrkB, the only
known high-affinity neurotrophin receptor functionally expressed in rat
cerebellar granule cells at 8 DIV (Marini et al., 1997a). To eliminate
the possibility that the BDNF-mediated reduction of the NR2A subunit
occurs via a nonspecific mechanism, we then exposed neurons to a member
of the neurotrophin family known to activate TrkC receptors (NT-3).
NT-3 (50 ng/ml) failed to change NR2A levels at any time tested (Fig.
2B), indicating that the BDNF effect is specific.
BDNF and FGF2 reduce NR2A and NR2C subunit mRNA levels
To determine whether the decrease in NR2A protein content might
result from a decrease in mRNA levels, steady-state NR2A mRNA levels
were analyzed after treatment with either BDNF or FGF2. To test for
specificity of the effect, we performed the RNase protection assay
using simultaneously three distinct cRNAs encoding for NR2A, NR2B, and
NR2C subunits. Analysis of RNA from cerebellar granule cells revealed
that all three subunits are expressed in these neurons, with the NR2A
mRNA being the predominant species (Fig.
3), in accordance with earlier findings
(Ishii et al., 1993; Monyer et al., 1994). BDNF (50 ng/ml) evoked a
time-dependent reduction in NR2A mRNA content, beginning at 6 hr (50%
reduction) and lasting at least up to 24 hr (Fig.
4A). FGF2 (50 ng/ml)
elicited a faster (within 3 hr) decrease in NR2A mRNA levels (Fig.
4A), although this decrease was transient because
NR2A mRNA returned to control levels by 6 hr (Fig.
4A). These results, by showing that the decrease in
NR2A mRNA content precedes the decrease in protein levels, suggest that
FGF2 and BDNF affect NR2A subunit synthesis.
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Figure 3.
Reduction of NR2A and NR2C subunit mRNA levels by
BDNF. RNase protection assay of 25 µg of total RNA extracted from
cerebellar granule cells exposed to serum-free medium in the absence
(CT) or presence of BDNF (50 ng/ml) for 6 hr.
DP, Digested probe; P, aliquot of the
hybridization solution containing the cRNA probes for NR2A
(R2A), NR2B (R2B), NR2C
(R2C), and cyclophilin (cyc). The
cyc probe was labeled at a lower specific activity.
M, Molecular weight markers
(MspI-digested pBR322). Protected fragments (indicated
by arrows) were visualized by overnight exposure on
x-ray film.
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Figure 4.
BDNF and FGF2 induce a time-dependent decrease in
NR2A and NR2C subunit mRNA content. Cerebellar granule cells were
exposed to BDNF, FGF2, or NT-3 for the indicated times. NR2A
(A), NR2B (B), and NR2C
(C) mRNA levels were determined by RNase
protection assay and were calculated as described in Materials and
Methods. Data, expressed as percent of control, are the mean ± SEM of three independent experiments (n = 6).
*p < 0.05; **p < 0.01 versus
control (ANOVA and Dunnett's test).
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Analysis of NR2B and NR2C mRNA revealed that exposure of cerebellar
granule cells to either BDNF or FGF2 did not alter NR2B mRNA levels
(Fig. 4B), whereas both trophic factors elicited a time-dependent decrease in NR2C mRNA levels similar to that observed for NR2A. As shown in Figure 4C, BDNF caused a 50%
reduction in NR2C mRNA levels by 6 hr, and the effect lasted up to 24 hr. FGF2 elicited a 30% decrease in NR2C mRNA levels by 3 hr
that was over by 6 hr. Again, NT-3 failed to change NR2A, NR2B, or NR2C
mRNA levels at any time point tested (Fig. 4). The levels of
cyclophilin mRNA did not change under our experimental conditions,
suggesting that the BDNF- and FGF2-mediated decrease in NR2A and NR2C
mRNA levels is not attributable to a generalized inhibition of mRNA synthesis.
BDNF and FGF2 reduce NR2A expression via a
receptor-mediated mechanism
The biological activity of BDNF and FGF2 depends on the activation
of tyrosine kinase receptors. To examine whether the reduction of NR2A
synthesis after either BDNF or FGF2 is tyrosine kinase receptor-mediated, we tested whether the blockade of TrkB or FGF2 receptor signaling by the tyrosine kinase inhibitor K252a (Berg et al.,
1992; Kaplan and Stevens, 1994) would inhibit BDNF and FGF2 effects. We
have used this tyrosine kinase inhibitor because it has been shown not
to affect cell viability (Marini et al., 1997a). Cerebellar granule
cells were exposed to K252a (100 nM) 10 min before the
addition of BDNF or FGF2 (each at 50 ng/ml). K252a prevented the
BDNF-mediated decrease in NR2A mRNA subunit at 6 hr (Fig.
5A) and the FGF2- and
BDNF-mediated downregulation of NR2A protein levels observed at 6 and
24 hr, respectively (Fig. 5B). K252a alone did not alter
NR2A mRNA or protein content (Fig. 5A,B) or the NMDA-mediated decrease
in NR2A mRNA (Fig. 5A). These results suggest that the
activation of tyrosine kinase receptor signaling is involved in the
reduced expression of NR2A and, most likely, NR2C subunits evoked by
BDNF and FGF2.
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Figure 5.
K252a prevents the BDNF- and FGF2-mediated
downregulation of NR2A expression. Cerebellar granule cells were
preincubated with K252a (100 nM) 10 min before the addition
of BDNF (50 ng/ml), FGF2 (50 ng/ml), or NMDA (100 µM).
NR2A mRNA (A) and protein
(B) levels were determined by RNase protection
assay and Western blot analysis at 6 and 24 hr, respectively (NR2A
protein levels in FGF2-treated cells were measured at 6 hr). Data,
expressed as percent of control, are the mean ± SEM of three
determinations from three separate cell preparations
(n = 6). *p < 0.05 versus
control; p < 0.05 versus BDNF or FGF2.
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BDNF and FGF2 decrease the NMDA-induced Ca2+
response in cerebellar granule neurons
The decrease in the expression of NR2A and NR2C subunits induced
by BDNF and FGF2 may have a physiological relevance if it is followed
by a reduction in receptor function. Because activation of NMDA
receptor promotes influx of extracellular Ca2+
through its own channel, the functional state of the NMDA receptor can
be assessed by measuring its ability to evoke an
[Ca2+]i increase after stimulation
with a proper ligand. Therefore, we tested whether the decrease in the
expression of NR2A and NR2C subunits induced by BDNF and FGF2 results
in an alteration in NMDA receptor-mediated Ca2+
responses. To this end, time course experiments, similar to those performed for the analysis of NR subunit proteins, were performed with
either growth factor, and the NMDA-induced
[Ca2+]i increase was measured.
Because nerve growth factor, a member of the neurotrophin family
closely related to BDNF, has been shown to increase
[Ca2+]i via either voltage-dependent
Ca2+ channels (Levine et al., 1995) or mobilization
of intracellular Ca2+ (De Bernardi et al., 1996), we
first tested BDNF and FGF2 for any direct effect on the
[Ca2+]i in cerebellar granule neurons.
Neither growth factor (50-100 ng/ml and up to 30 min) was found to
affect [Ca2+]i either in the presence
or absence of Mg2+ (data not shown). Neurons were
then exposed to BDNF (50 ng/ml for 3, 24, and 48 hr) or FGF2 (50 ng/ml
for 6 and 24 hr), and the [Ca2+]i
increase evoked by NMDA (100 µM) was monitored in
Mg2+-free conditions.
Resting [Ca2+]i showed no
statistically significant differences between control and BDNF- or
FGF2-treated cultures (in nM: control, 38.8 ± 6.6;
BDNF, 44.0 ± 9.1; and FGF2, 34.2 ± 5.4). In neurons exposed
to BDNF for 24 hr (treatment that maximally decreased NR2A protein
levels, see Fig. 2), the NMDA-evoked
[Ca2+]i increase was consistently
reduced by 50% compared with that in the control (Fig.
6A,B).
However, in neurons exposed to BDNF for 3 or 48 hr (treatments that
failed to change NR2A expression), the magnitude as well as the
kinetics of the [Ca2+]i increase
elicited by NMDA was found to be comparable with that in control cells
(Fig. 6B).
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Figure 6.
BDNF elicits a time-dependent inhibition of the
NMDA-evoked [Ca2+]i increase in
cerebellar granule cells. Neurons (8 DIV) were exposed to serum-free
medium for 3, 24, or 48 hr in the absence (control, vehicle-treated) or
presence of BDNF (50 ng/ml). Neurons were then loaded with fura-2, and
Ca2+ imaging was performed in
Mg2+-free Locke's solution. Resting
[Ca2+]i was recorded, and NMDA (100 µM) was applied. Single-cell
[Ca2+]i was measured and analyzed as
described in Materials and Methods. A, Representative of
NMDA-induced Ca2+ response in neurons exposed to
BDNF or vehicle (control) for 24 hr. B, Time course of
the effect of BDNF on the NMDA-induced
[Ca2+]i increase. The single-cell
[Ca2+]i rise after NMDA was measured
in neurons exposed to BDNF for 3, 24, or 48 hr. Data are expressed as
an NMDA-induced fold [Ca2+]i increase
plotted against time after NMDA addition. Results represent the
mean ± SEM of four separate preparations of cerebellar granule
cells (each preparation included at least three coverslips; 50-99
neurons being imaged per coverslip in a single microscopic field).
*p < 0.01 (ANOVA and Dunnett's test).
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A 6 hr exposure of cerebellar granule cells to FGF2 (which also
maximally reduced NR2A protein levels, see Fig. 2) resulted in a
decreased NMDA-induced [Ca2+]i rise
(Fig.
7A,B).
Overall, the extent of the effect exerted by FGF2 on the NMDA-elicited
[Ca2+]i rise was weaker than that
produced by BDNF, in agreement with the results showing that BDNF
inhibits NR2A subunit expression to a greater extent than does FGF2. In
contrast, a 24 hr exposure of the neurons to FGF2 failed to reduce the
magnitude of the NMDA-evoked [Ca2+]i
increase (Fig. 7B), consistent with the lack of effect by
FGF2 at this time point on the NR subunit expression (see Fig. 2). These data, by providing a temporal correlation between the reduction of NR2A expression by BDNF and FGF2 and the NMDA-elicited
[Ca2+]i increase, suggest that the
decreased responsiveness to NMDA in cells exposed to BDNF and FGF2 may
be the result of NMDA receptor downregulation.
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Figure 7.
Inhibitory effect of FGF2 on the NMDA-evoked
[Ca2+]i increase. Cerebellar granule
cells were exposed to serum-free medium for 6 or 24 hr in the absence
(control, vehicle-treated) or presence of FGF2 (50 ng/ml).
[Ca2+]i imaging was performed as
described in the legend of Figure 6. A, Representative
of NMDA-induced Ca2+ response in neurons exposed to
FGF2 or vehicle for 6 hr. B, Time course of the effect
of FGF2 on the NMDA-induced [Ca2+]i
increase. The single-cell [Ca2+]i
increase after NMDA was measured in neurons exposed to FGF2 for 6 or 24 hr. Data are expressed as an NMDA-induced fold
[Ca2+]i increase plotted against time
after NMDA addition and represent the mean ± SEM from a
population of 65 (control) and 70 (FGF2-treated) neurons per coverslip
from four separate preparations of cerebellar granule cells (each
preparation included at least three coverslips). *p < 0.05 (ANOVA and Dunnett's test).
|
|
| |
DISCUSSION |
BDNF and FGF2 are two neurotrophic factors known to prevent and
limit glutamate-induced neuropathological damages in selected neuronal
populations including rat cerebellar granule cells (Fernandez-Sanchez and Novelli, 1993; Lindholm et al., 1993; Marini et al., 1997a). Although the neuroprotective properties of these trophic factors are
rather well established, little is known about the molecular mechanism(s) of neuroprotection. To gain insight into the
neuroprotective effects of BDNF and FGF2, we tested the hypothesis that
these trophic factors prevent excitotoxicity by downregulating NMDA receptor subunit expression and function in cultures of cerebellar granule cells. Neither BDNF nor FGF2 affected the content of the obligatory NR1 subunit protein that contains the binding site for NMDA.
This finding suggests that the total number of NMDA receptors capable
of binding the proper ligand is not a likely target for the
downregulation of the NMDA receptor that, according to our working
hypothesis, could account for the neuroprotective activity of BDNF and
FGF2. On the other hand, both trophic factors elicited a time-dependent
decrease in mRNA and protein levels of the NR2A subunit. Importantly,
the decrease in NR2A protein content is preceded by a quantitatively
comparable decrease in NR2A mRNA levels, suggesting that BDNF and FGF2
very likely affect the synthesis of the NR2A subunit rather than the
stability of the protein. In addition, both trophic factors decreased
NR2C (but not NR2B) subunit mRNA with a time course similar to that observed for NR2A mRNA, suggesting that BDNF and FGF2 also reduce NR2C
subunit synthesis. Because homomeric NR1 receptors are less responsive
to NMDA than are heteromeric receptors composed of NR1 and NR2 subunits
(for review, see Hollmann and Heinemann, 1994), it appears that, by
reducing NR2A/NR2C expression, BDNF and FGF2 might change the NR1 to
NR2A/NR2C subunit ratio and, thus, alter the functional state of the
NMDA receptor. Taken together, these findings suggest that BDNF and
FGF2 might modulate the expression of distinct subunits of the NMDA
receptor and support the specificity of the effect of BDNF and FGF2 on
the expression of the various subunits.
Additional findings support the specificity of the reduction in NR2A
subunit synthesis by BDNF and FGF2. The effect of either trophic factor
on the expression of the various NR subunits was compared with that of
NMDA, which has been shown to modulate NR subunit levels in cerebellar
granule cells (Resink et al., 1995, 1996), and NT-3, a member of the
neurotrophin family that is devoid of neuroprotective activity in these
neurons at 8 DIV (Marini et al., 1997a). Although NT-3 failed to change
all the NR2 subunit subtypes tested, NMDA decreased NR1 and NR2A
protein levels, and its effect was specifically blocked by MK-801.
Thus, consistent with a recent report (Resink et al., 1996), NMDA was
able to modulate the expression of NR1 and NR2A subunits, whereas BDNF
and FGF2 were not. The biological effect of modifying NR subunit
composition might be the decreased ability of glutamate to induce cell
death via NMDA receptors. We therefore propose that the neuroprotective activity of BDNF or FGF2 against glutamate may rely on their ability to
reduce the sensitivity of a selected neuronal population to glutamate
by decreasing NR subunit synthesis and function.
A number of reports has shown that NMDA receptors play a key role in
mediating glutamate excitotoxicity in cerebellar granule cells (Favaron
et al., 1988; Novelli et al., 1988; Marini and Paul, 1992; Resink et
al., 1994). It is known that the NMDA receptor is a
Ca2+ channel that, after activation, increases
[Ca2+]i. Abnormally sustained
[Ca2+]i after excessive stimulation of
NMDA receptors has been implicated in neuronal cell death (Garthwaite
et al., 1986; Choi, 1987; Rothman et al., 1987; Hahn et al., 1988;
Anegawa et al., 1995). Both NR2A and NR2C subunits are crucial in the
formation of the NMDA channel and appear to be essential for the
NMDA-mediated Ca2+ influx (Hollmann and Heinemann,
1994). Thus, a decreased synthesis of these subunits by BDNF and FGF2
might result in a decreased NMDA-evoked Ca2+ entry
and, consequently, reduced [Ca2+]i.
Our data indicate that FGF2 and BDNF elicit a time-dependent inhibition
of the NMDA-mediated increase in
[Ca2+]i, suggesting a decreased
"sensitivity" of the NMDA receptor to NMDA activation. This effect
shows a time dependency that correlates with the decreased synthesis of
NR2A and, very likely, NR2C subunits, suggesting that NMDA subunit
downregulation evoked by either trophic factor may account for the
reduced Ca2+ response to NMDA. Although we cannot
completely eliminate the involvement of other cellular mechanisms in
the BDNF- and FGF2-mediated inhibition of
[Ca2+]i by NMDA, such as an effect on
a 71 kDa protein (Mattson et al., 1993) that is believed to be a
necessary component of NMDA receptor signaling (Kumar et al., 1991), or
other routes of Ca2+ entry, the decreased
sensitivity of these neurons to NMDA may explain why BDNF and FGF2
prevent glutamate excitotoxicity.
A decreased Ca2+ response to NMDA after FGF2
treatment is not unique to cerebellar granule cells as has been
reported in hippocampal neurons, which FGF2 protects against glutamate
excitotoxicity (Mattson et al., 1989, 1995; Cheng et al., 1995). Thus,
the cascade of events culminating in a decreased
Ca2+ response to glutamate could represent a general
mechanism by which certain neurotrophic factors can prevent
glutamate-mediated neuronal cell death caused by excessive NMDA
receptor stimulation. Although only speculative at the present time,
the possibility exists that, by reducing the intracellular responses to
glutamate, neurotrophic factors with neuroprotective properties, such
as BDNF and FGF2, might prevent the formation of cytotoxic metabolites such as nitric oxide, peroxidases, and free radicals (Maiese et al.,
1993; Mattson et al., 1995; Kume et al., 1997) that are thought to be
responsible for the final toxic effect of glutamate.
The specific cellular and molecular mechanisms by which BDNF and
FGF2 reduce NR2A and NR2C subunit expression are still under investigation. BDNF and FGF2 exert their biological activity by binding
to specific receptors, FGFR1 and TrkB, respectively, that possess an
intrinsic tyrosine kinase activity (for review, see Mocchetti and
Wrathall, 1995). In addition, BDNF may also activate the nonselective
neurotrophin receptor p75 (Chao and Hempstead, 1995), whose expression
has been detected in cerebellar granule cells (Segal et al., 1995;
Courtney et al., 1997). However, p75 does not activate a tyrosine
kinase pathway (Chao and Hempstead, 1995). Thus, to distinguish which
signaling transduction mechanism is involved in the action of BDNF and
FGF2, we first examined the role of the tyrosine kinase pathway. K252a
is a nonselective kinase inhibitor (Kase et al., 1987), which blocks
TrkB tyrosine kinase signaling (Tapley et al., 1992). K252a per se
failed to affect expression of NR subunit proteins or the NMDA-mediated downregulation of NR2A subunit mRNA. In contrast, K252a blocked the
decrease in NR2A mRNA and protein levels induced by BDNF and FGF2,
suggesting that the activation of the tyrosine kinase signal transduction pathway is required for the BDNF- and FGF2-mediated regulation of NR subunit expression to occur. This is in agreement with
recent findings (Knusen et al., 1997; S. Rabin and I. Mocchetti, unpublished observations) indicating that a prolonged treatment of
cerebellar granule cells with BDNF results in downregulation of TrkB
receptors. Because activation of TrkB is essential for BDNF activity,
the latter finding could explain the short-term effect of BDNF (within
24 hr) on the NR subunit levels and, perhaps, why BDNF neuroprotective
properties are over within 24 hr (Courtney et al., 1997). Importantly
and consistent with our results, a recent report has shown that the
protection against glutamate-induced cell death exerted by BDNF in
cerebellar granule cells is also blocked by K252a (Marini et al.,
1997a). Because tyrosine kinase receptors activate phosphatidylinositol
3-kinase, shown previously to mediate survival of cerebellar granule
cells exposed to insulin-like growth factor I (Miller et al., 1997),
our data support the hypothesis that the neuroprotective effect of BDNF
and FGF2 may involve the activation of a tyrosine kinase signaling
pathway. It remains to be established which tyrosine kinase
substrate(s), either common or specific for FGF2 and BDNF, is involved
in and might account for the neuroprotective effect of these trophic
factors. Future studies will also examine whether these trophic factors
activate a tyrosine kinase-independent pathway known to prevent cell
death in cerebellar granule cell neurons (Courtney et al., 1997).
Glutamate has been suggested to play a major role in acute
neurodegenerative processes, such as those after hypoxic and ischemic neuronal injury. Thus, it is appealing to propose that BDNF and FGF2
may reduce glutamate excitotoxicity after ischemia or traumatic lesion
by downregulating NMDA receptor function in vivo as well. It
has been suggested that neurotrophic factors can be effective neuroprotective agents only when added to neurons in culture several hours before glutamate exposure (Prehn, 1996). By demonstrating that
BDNF and FGF2 downregulate NMDA receptor with a time lag of at least 6 hr, our data may explain why neurotrophic factors prevent NMDA or
glutamate toxicity when delivered several hours before the excitotoxic
insult. If this is the case, the use of neurotrophic factors to prevent
acute neuropathological conditions in humans may indeed be difficult.
On the other hand, FGF2 has been shown to limit secondary injury
processes that occur at a later time after spinal cord trauma (Teng et
al., 1998) and may continue to exacerbate neuronal loss triggered by
the initial traumatic (or ischemic) insult. Therefore, neurotrophic
factors might be potential therapeutic tools in chronic
neurodegenerative diseases.
| |
FOOTNOTES |
Received April 22, 1998; revised June 29, 1998; accepted July 17, 1998.
This work was supported by a Research Career Development Award NS 01675 to I.M., by National Institutes of Health Grants NS 32671 and HL 28940, and by the American Heart Association Nation's Affiliate.
Correspondence should be addressed to Dr. Italo Mocchetti, Department
of Cell Biology, Medical/Dental Building, Georgetown University, 3900 Reservoir Road Northwest, Washington, DC 20007.
| |
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S. J. Rabin, A. Bachis, and I. Mocchetti
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D. M. O'Dell, R. Raghupathi, P. B. Crino, J. H. Eberwine, and T. K. McIntosh
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C. Tiruppathi, W. Yan, R. Sandoval, T. Naqvi, A. N. Pronin, J. L. Benovic, and A. B. Malik
G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells
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A. El Idrissi and E. Trenkner
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Y. D. Teng, I. Mocchetti, A. M. Taveira-DaSilva, R. A. Gillis, and J. R. Wrathall
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A. M. Marini, S. J. Rabin, R. H. Lipsky, and I. Mocchetti
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Top
Abstract
Introduction
Compound via MeSH
