From the LSU Neuroscience Center, Louisiana State
University Medical Center School of Medicine, New Orleans, Louisiana
70112 and the
Howard Hughes Medical Institute, University of
Massachusetts Medical Center, Worcester, Massachusetts 01605
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ABSTRACT |
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Glutamate receptors modulate multiple signaling
pathways, several of which involve mitogen-activated protein (MAP)
kinases, with subsequent physiological or pathological consequences.
Here we report that stimulation of the
N-methyl-D-aspartate (NMDA) receptor, using
platelet-activating factor (PAF) as a messenger, activates MAP kinases,
including c-Jun NH2-terminal kinase, p38, and extracellular
signal-regulated kinase, in primary cultures of hippocampal neurons.
Activation of the metabotropic glutamate receptor (mGluR) blocks this
NMDA-signaling through PAF and MAP kinases, and the resultant cell
death. Recombinant PAF-acetylhydrolase degrades PAF generated by
NMDA-receptor activation; the hetrazepine BN50730 (an intracellular PAF
receptor antagonist) also inhibits both NMDA-stimulated MAP kinases and
neuronal cell death. The finding that the NMDA receptor-PAF-MAP kinase
signaling pathway is attenuated by mGluR activation highlights the
exquisite interplay between glutamate receptors in the decision making
process between neuronal survival and death.
Glutamate receptors participate in neural development, plasticity,
learning, memory, and pathology, (e.g. excitotoxicity and neurodegenerative diseases.) Overactivation of the glutamate ionotropic receptors leads to excitotoxic cell death.
NMDA1 receptor antagonism
results in prominent neuroprotection in vivo and in
vitro. The signals generated by these receptors activate the
stress-sensitive MAP kinases JNK and p38 that are implicated in
neuronal apoptosis (1). A role for JNK in the excitotoxic death of
hippocampal neurons in vivo has been recently illustrated using JNK3 We used primary rat hippocampal neuronal cultures to determine whether
the injury/inflammatory messenger PAF is a mediator of NMDA activation
of JNK, p38, and ERK MAP kinases. Furthermore, we have explored whether
metabotropic receptor activation affects NMDA receptor signaling to
stress-sensitive MAP kinases and have examined the consequences of
glutamate receptor interplay through PAF signaling on neuroprotection.
Primary Rat Hippocampal Neuronal Cultures--
Culture plates
were coated with Matrigel (Collaborative Research). Hippocampi from
1-3-day old rat pups were pooled in oxygenated Liebovitz's (L-15)
medium plus 0.05% bovine serum albumin. Hippocampi were then moved to
a solution of 0.1% papain in L-15 + bovine serum albumin for 20 min
(under oxygen) and then triturated with a Pasteur pipette. Cells were
plated at 500,000 cells/ml in minimal essential medium plus 10% fetal
calf and horse serum supplemented with insulin, transferrin, and
selenium and with glucose and glutamine and then maintained in
37 °C, 5% CO2 incubators (5). After 1 day in
vitro, cultures were treated with 10 Preparation of Astrocytes--
Cultures were grown in flasks and
were not treated with Ara-C. Repeated exchanges of culture media
containing fetal calf and horse serum were carried out on the
cultures weekly. Once culture flasks reached confluence, cells were
lifted from the flasks and dissociated with trypsin/EDTA treatment,
centrifuged to a cell pellet, and then diluted in culture medium and
replated for experimental use. These cultures were then washed
repeatedly with serum-containing medium until reaching confluence
before using for experiments as described previously (31).
In Vitro Immunocomplex Protein Kinase Assay--
Cell cultures
were exposed to agonists and/or antagonists in Locke's solution
without MgCl2 and supplemented with 1 µM
glycine to optimize the activation of the NMDA receptor. Exposure of
cells to PAF was carried out in serum-free culture medium as indicated in the figure legends. To terminate exposure, medium was removed; cells
were then washed with cold PBS, scraped in homogenization buffer,
homogenized, and centrifuged at 2800 × g for 15 min at 4 °C and the supernatants used for the assay. Plasmids containing GST-cJun and GST-ATF2 were prepared as described previously. Triton-X soluble protein lysates were pre-cleaned with protein A-agarose (Boehringer Mannheim) and then incubated with 1:100 dilutions of
anti-JNK1 antibody, anti-p38 antibody, and anti-ERK1 or ERK2 antibody
for 1-2 h at 4 °C. Antibody against brain-selective JNK3 was
purchased from Upstate Biotechnology. The immunoprecipitates were
washed twice with homogenization buffer and once with kinase assay
buffer (25 mM HEPES, pH 7.4, 20 mM
MgCl2, 20 mM glycerophosphate, 10 mM sodium orthovanadate, and 2 mM
dithiothreitol). Aliquots of 5-µg immunoprecipitates were incubated
for 30 min at 30 °C using either 10 µg of GST-cJun fusion protein,
10 µg of ATF-2 cJun fusion protein, or 10 µg of myelin basic
protein (MBP, Uptstate Biomedical, New York), and 20 µM
ATP (3 µCi, [32P]ATP at 10 Ci/mM, Amersham Pharmacia
Biotech) in 40 µl of kinase buffer as described previously (32). The
reactions were terminated by spotting 20 µl of sample onto P81 filter
paper squares (Whatman) and simultaneously adding 10 µl of Laemmli
buffer to the remaining samples and placing on ice. The filter papers
were washed twice with 10 ml per square of 1% phosphoric acid followed
by one wash with water. After counting using a scintillation counter,
phosphorylation of substrate proteins by SDS-PAGE analysis was
quantified using a phosphoimager (Bio-Rad).
Cytotoxicity Assays--
Treatments were at room temperature in
Locke's solution without MgCl2 and supplemented with 1 µM glycine. Cells were treated with agonists (diluted in
Locke's solution) or control (Locke' s solution alone) for 15 min.
Cells were then replaced in minimal essential medium containing
Earle's salts and no phenol red (37 °C). Cultures were returned to
the CO2 incubator (37 °C) overnight. 18-20 h later,
minimal essential medium-cell supernatant was removed from each well,
the cells remaining were lysed with water, and 50 µl of cell
supernatant and cell membranes were assayed for LDH activity to
calculate total LDH release using Sigma Kit (228-UV), with the aid of a
Beckman DU-640 spectrophotometer, and percent neuroprotection was
calculated (33).
DAPI Staining--
After the 18-20-h recovery period, cells
were loaded with 2 µM DAPI and viewed under phase and UV
microscopy. Digital images were analyzed using Image Pro Plus 3.0 software (Media Cybernetics; Silver Spring, MD). By extracting blue
channel signal, DAPI positive cells were counted under each condition.
Using primary rat hippocampal neuronal cultures in the absence of
MgCl2 and in the presence of 1 µM glycine
(see "Experimental Procedures"), NMDA (100 µM)
prominently activates ERK (10-fold), JNK (8-fold), and, p38 (7.5-fold)
(Fig. 1, A and B).
At submaximal concentrations, NMDA (50 µM) promotes the
rapid activation of these MAP kinases, peaking at 15 min. p38 MAP
kinase and JNK activation are transient, whereas ERK activation is
somewhat sustained after removal of NMDA stimulus (Fig. 1B).
The non-competitive NMDA receptor antagonist MK801 (300 nM), effectively blocked NMDA stimulation of the protein
kinases (Fig. 1A). To test the involvement of PAF as a
mediator of NMDA signaling, the synthetic hetrazepine, BN50730 (1 µM), an intracellular PAF receptor antagonist (22, 34), was used and was found to block NMDA stimulation of the protein kinases
to an extent similar to that of MK801 (Fig. 1A).
INTRODUCTION
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Abstract
Introduction
References
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mice (2). However, the specific messengers and potential antagonist drugs involved in these signaling pathways are not
understood. The bioactive phospholipid, platelet-activating factor
(PAF), is a candidate mediator of excitatory amino acid signaling
because it is a retrograde messenger of long-term potentiation (LTP)
(3, 4) that enhances glutamate release (5), is generated by NMDA
receptor activation (6), and participates in memory formation (7-9).
PAF is synthetized through several pathways (10). The PAF precursor is
enriched in arachidonate at the C2 position. The
Ca2+-dependent PAF remodeling pathway engages a
phospholipase A2, followed by acetylation to give rise to
PAF (11). Neuronal stimulation, such as at the onset of seizures or
from ischemia, promotes the rapid release of arachidonic acid (12, 13),
reflecting synaptic phospholipase A2 activation (14, 15).
The cytosolic form of phospholipase A2 is important in
postischemic neuronal cell death as is shown using knockout
cPLA2 mice (16). Also, a secretory PLA2 may
contribute to excitotoxicity (17, 18). Again, the signaling events are
not clear. In brain ischemia and seizures, there is also PAF
accumulation (19, 20), which in turn, contributes to increased
glutamate release (5) and COX-2 transcription (21, 22), both enhancing
brain injury. Therefore, PAF is a neuronal injury mediator, and
consequently, PAF antagonists elicit neuroprotection (23, 24). Both PAF
(25-27) and glutamate (28-30) activate MAP kinases. These studies
directly implicate PAF as a second messenger in a glutamate-induced
signaling pathway.
EXPERIMENTAL PROCEDURES
5 M
Ara-C. After 4 days in vitro, Ara-C was removed and cells
moved to fresh minimal essential medium lacking serum. All experiments were carried out on cultures after 12 days in vitro.
RESULTS AND DISCUSSION
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Fig. 1.
A, NMDA activates stress-sensitive MAP
kinases in primary cultures of hippocampal neurons. Hippocampi isolated
from 1-3-day old rat pups were isolated and cultured, and MAP kinase
activity was determined after exposure to NMDA as described under
"Experimental Procedures." BN50730 (1 µM) was added
for 30 min, or MK801 (300 nM) was added 10 min prior to
NMDA. Striped bars, ERK; white bars, JNK; and
black bars, p38. Data represent the average of six to eight
independent assays, and standard error is shown. B, typical
time course experiment. After a submaximal NMDA stimulus (50 µM, 15 min), NMDA was removed from cells and kinase
activation calculated. Standard error bars are shown; where error bars
do not appear, data vary less that the height of the symbol. Data
represent the average of duplicates from at least two to three separate
platings. C-H, cells were treated with PAF
(panels C-G) for 1 h at 37 °C in
serum-free medium, and activation of protein kinases was determined
using different substrates as indicated under "Experimental
Procedures." Autoradiogram on each figure is a representative gel.
Line graphs show the quantification of the autoradiograms.
C and E, concentration response curves for PAF
activation of ERK (panel C) and JNK and p38 kinases
(panel E). D and F, antagonism of PAF
activation of MAP kinases. Data have been normalized to activation
elicited by 100 nM PAF, which has been set as 100%. As
indicated, some cells were pretreated with BN50730 (1 µM), CNQX (5 µM), or MK-801 (300 nM). Pretreatment time with antagonists was 30 min for
BN50730 and 10 min for CNQX or MK-801 before addition of PAF.
Antagonists remained during PAF exposure. Activation of ERK, MAP kinase
(panel D), or JNK and p38 kinases (panel F) was
then measured. G, PAF and NMDA activate MAP kinases in
neurons but not in astrocytes. Primary cultures of astrocytes were
prepared as described under "Experimental Procedures." Neurons and
astrocytes were exposed to 100 nM PAF or NMDA at the
indicated concentrations as described for Fig. 2,
A-D. H, antagonism of NMDA activation
of stress-sensitive MAP kinases in hippocampal neurons. Neurons were
treated with NMDA (100 µM) for 15 min at room
temperature. Where indicated, cells were pretreated for 30 min at
37 °C with recombinant PAF-acetylhydrolase (rPAF-AH, 20 µg/ml,
Icos Corp.). Each point was obtained from pooling two wells from five
to seven separate experiments.
The bioactive phospholipid PAF is a candidate mediator of excitatory amino acid signaling because it is a retrograde messenger of LTP (3, 4) that enhances glutamate release (5), is generated by NMDA receptor activation (6), and participates in memory formation (8). PAF is synthetized through several pathways (10). The PAF precursor is enriched in arachidonate at the C2 position. The Ca2+-dependent PAF remodeling pathway engages a phospholipase A2, followed by acetylation to give rise to PAF (11). Brain stimulation, such as at the onset of seizures or from ischemia, promotes the rapid release of arachidonic acid, reflecting synaptic phospholipase A2 activation (14). This inhibitory effect of BN50730 on NMDA-induced MAP kinase activation suggests that PAF is a messenger in this signaling pathway. Therefore, to test whether PAF activates MAP kinases, we incubated hippocampal neurons with various concentrations of this phospholipid. PAF at 1 and 5 nM potently activated JNK and p38 MAP kinase in hippocampal neurons (Fig. 1, C and E). JNK is nearly maximally activated in the presence of 5-10 nM PAF, whereas p38 MAP kinase activation continues to increase under these conditions. ERK, on the other hand, is activated at 5 nM PAF, and at 50-100 nM of PAF, a 20-fold enhancement in activity over controls was observed. We studied the effect of PAF receptor antagonists. BN52021, an antagonist that displaces PAF binding from presynaptic membranes (34), blocks presynaptic glutamate release (5), and LTP (3), causes weak inhibition of NMDA- and PAF-induced MAP kinase activation (data not shown). However, the intracellular PAF antagonist BN50730 effectively blocks PAF-mediated JNK, p38, and ERK activation. This observation excludes the presynaptic PAF receptor from being directly involved in MAP kinase activation. It is not clear, however, whether postsynaptic membrane PAF receptors are involved to a limited extent because BN52021 is a weak inhibitor of MAP kinase activation by PAF. The NMDA receptor antagonist MK801 and the AMPA-kainate receptor antagonist CNQX were unable to inhibit PAF-induced MAP kinase activation, suggesting that PAF acts downstream of the glutamate receptor (Fig. 1, D and F). Because the cultures used in the present experiments contain approximately 10% astrocytes, the relative contribution of astrocytes to PAF- and NMDA-mediated MAP kinase activation was examined. Fig. 1G shows that astrocytes from the hippocampus, under analogous treatment conditions, do not respond to PAF or NMDA as do neurons. JNK and p38 of astrocytes are not activated by PAF, whereas ERK is enhanced to a considerably lesser extent than in neurons.
NMDA receptor agonists trigger PLA2 activation (35) and PAF synthesis (6). Therefore, if PAF is a messenger in stress-sensitive MAP kinase activation, the hydrolysis of this lipid to the biologically inactive lyso-PAF should inhibit this action of NMDA. PAF acetylhydrolase catalyzes this hydrolysis (36). Plasma-type recombinant PAF acetylhydrolase prevents PAF-induced inflammatory responses in non-neural cells (36, 37) and elicits neuroprotection against NMDA in primary hippocampal neuronal cultures. Preincubation of hippocampal neurons with plasma-type recombinant PAF acetylhydrolase (rPAF-AH) greatly reduced the NMDA-mediated activation of MAP kinases (Fig. 1H).
All of the above findings were made using either NMDA or PAF as an
agonist. Therefore, the effects of glutamate itself were investigated.
It was surmised that glutamate-mediated activation of stress-sensitive
MAP kinases should have at least a component that utilizes PAF as a
mediator. Surprisingly, glutamate was found to activate MAP kinases
through a mechanism insensitive to the PAF receptor antagonist, BN50730
(Fig. 2). MAP kinases from hippocampal astrocytes are weakly activated by glutamate although they are more
responsive to phorbol-12-myristate-13-acetate (TPA), lipopolysaccharide (LPS), and interleukin 1 (IL-1
) (Fig. 2C). The NMDA
receptor antagonist MK801 effectively blocks glutamate activation of
MAP kinases at either 50 or 100 µM. Therefore, although
NMDA activates MAP kinases through a BN50730 sensitive route (Fig.
1A), glutamate, an agonist of the NMDA receptor and non-NMDA
glutamate receptors, does not display BN50730 sensitivity. The failure
of the PAF receptor antagonist BN50730 to block glutamate-stimulated
activation of stress-sensitive MAP kinases (Fig. 2D)
suggests that glutamate and PAF utilize separate pathways for the
activation of these protein kinases, or that glutamate, by activating
the metabotropic receptor is inhibiting the NMDA-stimulated signaling
pathway that involves PAF. To address this dilemma, we investigated the
significance of metabotropic and AMPA/kainate receptors. Fig.
3A shows that 1S,3R-ACPD, a selective glutamate metabotropic
receptor agonist, when added together with NMDA, inhibits NMDA-induced
activation of ERK, JNK, and p38. (+)-MCPG, a mGluR antagonist, reverses
the 1S,3R-ACPD effect (Fig. 3A) and
allows for BN50730 to significantly block glutamate-activation of the
MAP kinases (Fig. 2D). Kainic acid also activates the MAP
kinases; however, 1S,3R-ACPD fails to affect this
pathway (Fig. 3B). The 1S,3R-ACPD
effect on ERK and JNK was further confirmed using in-gel kinase assay
(Fig. 3, C and D). Quantitative analysis of
in-gel kinase assays showed that NMDA increases the 42- and 44-kDa ERK
isoforms by 340 and 210%, over controls (Fig. 3C). The JNK
isoforms were increased 240 and 180% for the 54 and 46 kDa,
respectively (Fig. 3D). 1S,3R-ACPD potently inhibited these effects of NMDA, as assayed by in-gel kinase
(Fig. 3, C and D), in agreement with the data
obtained by immunoprecipitation (Figs. 1, 2, and 3, panels
A). We interpret the effect of 1S,3R-ACPD as
a metabotropic receptor-mediated attenuation of the NMDA inductive
signal. By inhibiting mGluR, we postulated that the attenuation exerted
by this glutamate receptor on the NMDA inductive signal might be
removed. To test this prediction, hippocampal neurons were preincubated
with the competitive metabotropic receptor antagonist, (+)-MCPG,
followed by glutamate. Fig. 2D shows that, indeed, under
these conditions, the BN50730-sensitive component of glutamate action
is partially unmasked.
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Glutamate is an endogenous agonist for metabotropic receptors. Because the cumulative MAP kinase signal stimulated by glutamate is presumably the sum of NMDA, AMPA/kainate, and metabotropic receptor activation, we tested whether 1S,3R-ACPD would inhibit MAP kinase signal stimulated by glutamate. Using 50 µM glutamate to stimulate MAP kinases, we found that 50 µM 1S,3R-ACPD inhibited 33.4 ± 0.3%, 29.7 ± 7.4%, and 21.1 ± 4.3% of ERK, JNK, and p-38 activation, respectively. In comparison, using the same platings of hippocampal neurons, we found that MAP kinase activation stimulated by 50 µM NMDA was inhibited by 50 µM 1S,3R-ACPD to the extent of 88.1 ± 0.3%, 86.7 ± 3.7%, and 69.9 ± 7% for ERK, JNK, and p38, respectively. It should also be noted that the metabotropic receptor antagonist (+)-MCPG also slightly inhibited glutamate-stimulated activation of the MAP kinases (Fig. 2D). However, (+)-MCPG is a mixed Group I/II mGlu receptor antagonist, so the inhibition observed in our current studies may be because of inhibition at Group I mGlu receptors, which, when activated, increases neuronal excitation (38).
The major route of MAP kinase activation under the experimental conditions used here is via the NMDA receptor. It should be pointed out that the conditions used during exposure of the hippocampal neurons to either NMDA, glutamate or 1S,3R-ACPD in this work were optimized for the activation of the NMDA receptor, with glycine present and magnesium absent in the Locke's exposure solution (see "Experimental Procedures," Fig. 1A).
The ubiquitous MAP kinases are components of signal transduction pathways involved in cell proliferation and differentiation. The hippocampal neurons used in the present experiments are post-mitotic; thus, the PAF-mediated protein kinase activation represents cell signaling through which the bioactive lipid elicits other responses. For example, increases in synaptic efficacy in the hippocampus in the form of LTP are accompanied by activation of p42 MAP kinase (39). The NMDA-mediated signaling through PAF leading to stress-sensitive MAP kinase activation may also participate in cell survival. To test this hypothesis, morphological evaluation of neuronal cell injury and nuclear damage with DAPI staining were used (Fig. 4, A-C). Although NMDA-treated hippocampal cultures showed a majority of cell bodies brightly stained in the nucleus with DAPI, control cultures and cultures treated with BN50730 plus NMDA showed far fewer damaged cells. Image analysis over multiple experiments demonstrated a significant protective effect of BN50730 against NMDA-induced cell damage, as measured by DAPI staining (Fig. 4D). These results were correlated in the same cultures used for DAPI staining with measurement of LDH release, which showed neuroprotection by BN50730 against NMDA toxicity (Fig. 4E). Neuroprotection was also observed when 1S,3R-ACPD or rPAF-AH was added (Fig. 4F). The NMDA receptor antagonist MK801 (used as a positive control) was effective in exerting neuroprotection to about 92%. When glutamate was used as an agonist, no neuroprotection by the PAF receptor antagonist BN50730 was observed except in the presence of the metabotropic glutamate receptor antagonist, (+)-MCPG (Fig. 4F).
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In agreement with these observations, agonists of glutamate metabotropic receptors exert neuroprotection in neuron injury models (40). However, other studies have indicated the neurotoxic effects of mGluR agonists (41-43). In this regard, we have evaluated a number of mGluR agonists to 1S,3R-ACPD, comparing the ability to inhibit NMDA-stimulated activation of the MAP kinases. At a concentration of 50 µM, the group I mGlu receptor agonists DHPG and HPG, and the mixed group I and II agonist 1S,3R-ACPD inhibited 50 µM NMDA-stimulated MAP kinase activation with a rank order efficacy of DHPG > 1S,3R-ACPD > HPG, and the resulting inhibition measured at 97% for DHPG, 96% for ACPD, and 57% for HPG. Using antibody to JNK3, the kinase found predominantly in neurons (44), we found that 50 µM NMDA activated both JNK-1 and JNK-3, with activation inhibited by DHPG, 1S,3R-ACPC, and HPG for both forms of the kinase (data not shown). It was of interest to observe that under identical stimulus conditions, 50 µM CPG, an mGluR1 antagonist and mGluR2 agonist, failed to inhibit NMDA-stimulation of MAP kinases (data not shown). Although 1S,3R-ACPD provided significant neuroprotection against NMDA toxicity (Fig. 4F), DHPG and HPG, which inhibited MAP kinases, were not neuroprotective. In fact, at 50 µM, DHPG and HPG were slightly neurotoxic in their own right (30-40% above controls). This reflects the complexity of the multiple subtypes of metabotropic glutamate receptors of different neurons in culture. This is combined with the possible multiple and contradictory effects that these metabotropic receptors may have on ionotropic subtypes of glutamate receptors in neurons.
Previous findings that, in general, activation of group I metabotropic receptors increases neuronal excitability and activation of group II or III diminishes synaptic excitation (38, 45-47) might explain the toxicity of DHPG and HPG in our current work. Thus, group II agonists attenuate excitotoxicity (47-49). Although our results using 1S,3R-ACPD may be consistent with this line of reasoning, Wang et al. (41) have recently described the toxicity of 1S,3R-ACPD in rat striatum. Furthermore, 1S,3R-ACPD attenuated DNA fragmentation induced by NMDA but potentiated that induced by kainate. In the present work, we found no toxic effect of 1S,3R-ACPD, and we found that it had no inhibitory action on KA-stimulated MAP kinase activation (Fig. 3B).
PAF itself increases glutamate release and electrophysiological activity at the synapse (3, 5). Treatment of neuronal cell lines with PAF has been implicated to be neurotoxic; however, unlike in the present study, long-term treatment with PAF was necessary for expression of this toxicity (50). In other studies, PAF antagonists have demonstrated neuroprotection against excitotoxic and ischemic injury (24, 51). Fig. 5A outlines the sequence of events that may be triggered by NMDA-induced Ca2+ influx. PAF synthesis, likely through a phospholipase A2, will lead to MAP kinase activation and cell death. The importance of the cytosolic form of phospholipase A2 has been shown to be important in postischemic neuronal cell death as demonstrated using knockout cPLA2 mice (16). Also, a secretory PLA2 may contribute to excitotoxicity (17, 18). Again, the signaling events are not clear. ERK may potentiate cPLA2 activation through phosphorylation. rPAF-AH neuroprotection (Fig. 4F) may reflect diverting PAF to its biologically inactive metabolite, lyso-PAF (Fig. 5A).
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In brain ischemia and seizures, there is also PAF accumulation (19, 20) and, in turn, PAF accumulation contributes to increased glutamate release (5) and COX-2 transcription (22), both enhancing brain injury. Therefore, PAF is a neuronal injury mediator, and consequently, PAF antagonists elicit neuroprotection (23, 24). Both PAF (25-27) and glutamate (28-30) activate MAP kinases. These studies directly implicate PAF as a second messenger in a glutamate-induced signaling pathway.
Agonists of glutamate metabotropic receptors may attenuate inductive signals generated by another receptor (NMDA) and, as a consequence, lead to the selective termination of NMDA-mediated inductive responses (Fig. 5). In this instance, one signaling route, which is mediated by PAF, is terminated. Initial observations made by Koh et al. (40) that trans-ACPD elicits neuroprotection upon NMDA-induced damage of neurons in culture are thus confirmed here. 1S,3R-ACPD selectively inhibits MAP kinase activation mediated by NMDA but not by Ka (Fig. 5A). Glutamate, by activating NMDA and non-NMDA receptors activates an inhibitory signal acting on the pathway stemming from the NMDA receptor (Fig. 5B). The inhibitory signal is the result of metabotropic receptor activation. Thus, (+)-MCPG unmasks the mGluR receptor action (Fig. 5C).
The use of cells expressing specific receptor subtypes will help to
define whether the inhibition of signals emanating from the NMDA
receptor by mGluR activation originates at the level of the receptor
itself, inhibition of PAF synthesis or enhancement of its degradation,
or at the PAF receptor. Also, other converging and diverging signals,
such as NO, may modulate the events described here. Ultimately, the
activation by PAF of stress-sensitive MAP kinases may lead to
transcriptional activation of genes related to cell survival. For
example, the PAF antagonist BN50730 inhibits COX-2 transcription in
transfected cells (21) as well as in vivo during
kainate-induced hippocampal damage (22). In conclusion, we have
identified a pathway from NMDA through PAF that activates stress-sensitive kinases and leads to hippocampal neuronal cell death.
This pathway contributes to the networks of signals that promote
survival/death. We demonstrate that by activating the metabotropic
glutamate receptor, this signaling pathway is inhibited. We further
show that neuronal cell death involves a mechanism that displays
morphological changes, characteristic of apoptosis. Our results
indicate that mGluR and NMDA receptor interplay is an important
effector in neuronal death signaling. Moreover, agonists of mGluR that
inhibit NMDA signaling through PAF may be useful to ameliorate
excitotoxicity and neurodegeneration as are strategies to selectively
block the PAF receptor or to degrade PAF as it accumulates.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant NS23002.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this paper.
¶ Summer Undergraduate in the Neurosciences (SUN) Program Student, LSU Neuroscience Center.
** To whom correspondence should be addressed: Louisiana State University Medical Center School of Medicine, LSU Neuroscience Center of Excellence, 2020 Gravier St., Suite D, New Orleans, LA, 70112. Tel.: 504-599-0832; Fax: 504-568-5801; E-mail: nbazan{at}lsumc.edu.
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ABBREVIATIONS |
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The abbreviations used are: NMDA, N-methyl-D-aspartate; MAP, mitogen-activated protein; PAF, platelet-activating factor; mGluR, metabotropic glutamate receptor; LTP, long-term potentiation; GST, glutathione S-transferase; DAPI, 4',6-diamidino-2-phenylindol dihydrochloride; rPAF-AH, recombinant PAF acetylhydrolase; DHPG, (S)-3,5-dihydroxyphenylglycine; HPG, (RS)-3-hydroxyphenylglycine; CPG, S(+)-4-carboxyphenylglycine; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; 1S, 3R-ACPD, (1S,3R)-1-aminocytopentane-1,3-dicarboxylic acid; Ara-C, cytosine arabinoside.
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REFERENCES |
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