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Introduction
Glutamate is an excitotoxic neurotransmitter in the
central nervous system. Its effects are mediated through the
activation of glutamate receptors, which can be either coupled
to G proteins or ionic channels that open upon binding of
glutamate[1]. Of the latter, the
N-methyl-D-aspartate (NMDA) receptors have been described as being
associated with several pathological changes involving excessive
stimulation of glutamate receptors[2]. A deregulated
increase in the intracellular Ca2+ concentration caused by
the overactivation of glutamate receptors is considered to
trigger neuronal death through the activation of
Ca2+-activated proteases, such as calpains or by indirectly activating
the apoptotic-related caspases[3]. Glutamate neurotoxicity
is known to be associated with numerous neurodegenerative
disorders, including Alzheimer's disease (AD), and is
considered to be a key factor in the pathogenesis of
AD[4]. Some studies have shown that amyloid
β protein (Aβ) inhibits glutamate uptake and causes an increase in extracellular
glutamate[5]. There are also some reports that
Aβ enhances the toxicity induced by
excitoxicity[6], indicating that Aβ-induced cytotoxicity might be mediated through glutamate
cytoxicity to some extent. It has been shown that
in vitro, glutamate induces different types of neuronal disorders,
including apoptosis and necrosis. Therefore, the potential
role of apoptosis in glutamate-induced toxicity suggests
that its regulation may slow acute and chronic
neurodegen-erative processes.
Sodium ferulate (SF), extracted from a traditional Chinese
herbal medicine, has potent
antioxidant[7] and anti-inflammatory
activities[8]. It has recently been reported that the
long-term administration of ferulic acid protects mice from
learning and memory deficits induced by centrally administered
β-amyloid[9]. The primary action site of ferulic acid could be
the microglia[10] and
astrocytes[11]. A recent report showed
that ferulic acid inhibited the formation of Aβ fibrils and destabilized preformed fibrillary
Aβ[12]. Sultana et al reported that ferulic acid ethyl ester significantly inhibited
Aβ1-42-induced cytoxicity, intracellular reactive oxygen species
accumulation, lipid peroxidation, and the induction of
induci-ble nitric oxide synthase (iNOS) in primary hippocampal
cultures[13]. In addition, ferulic acid attenuates iron-induced
oxidative damage and apoptosis in cultured
neurons[14] and reduces the expression of inducible NOS and cyclooxygenase
activity following exposure to
lipopolysaccharides[15]. Our previous study showed that SF had protective effects from
Aβ-induced neurotoxicity through the suppression of p38
mitogen-activated protein kinase and the upregulation of
extracellular signal-regulated kinase (ERK) and
Akt[16,17]. In the present study, we investigated the roles of the ERK
pathway and phosphatidylinositol 3-kinase (PI3K) pathway in
the neuroprotection by SF from glutamate toxicity.
Materials and methods
Materials SF, a colorless powder with >99% purity, was
obtained from Suzhou Changtong Chemical Co (Suzhou,
China). Glutamate (Sigma Chemical Co, St Louis, MO, USA)
and SF was dissolved with Dulbecco's modified Eagle's
medium (DMEM, Sigma Chemical Co, USA).
Phospho-mitogen-activated protein kinase kinase (MEK)1/2 (Ser217/221,
No 9121), phospho-ERK1/2 (Thr202/Tyr204,
No 9101), cleaved caspase-3
(No 9661), polymerase (PARP, No 9542),
cleaved PARP (ASP330, No 9541), and calpain 1 large
subunit (Mu-type, No 2556) antibodies, horseradish peroxidase
(HRP)-linked secondary antibody, the biotinylated protein
ladder detection pack (No 7727), and U0126
(No 9903) were purchased from Cell Signaling (Beverly, MA, USA).
Phospho-Akt (Thr308), phosphorylated ribosomal protein
S6 protein kinase (p70S6K; Thr389), and Bcl-2 antibodies
were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). The β-actin antibody and Hoechst 33258,
O-dianisidine tetrazotized, and β-naphthyl acid phosphate were
purchased from Sigma Chemical Co (USA). SeeBlue Plus2
prestained standard (Catalog No LC5925), B27 supplement,
and neurobasal medium were obtained from Invitrogen Life
Technologies (Carlsbad, CA, USA). LY294002 and PD98059
were obtained from Promega Corporation (Madison, WI,
USA). Wortmannin was purchased from Calbiochem (La Jolla,
CA, USA). The enhanced chemiluminescence kit was from
Pierce Biotechnology (Rockford, IL, USA).
Cell cultures Primary cultures were obtained from the
cerebral cortex of 0_24 h-old Sprague_Dawley rats
according to the procedures described
previously[18]. Briefly, after removal of the meninges and white matter, the brain cortex
was collected and resuspended in Hanks' solution without
Ca2+ and Mg2+ (D-Hanks). The cortex was then mechanically
fragmented, transferred to D-Hanks' solution containing
0.125% trypsin, and incubated for 15 min at 37
oC. Follow-ing trypsinization, the cells were washed twice with DMEM
containing 10% heat-inactivated fetal bovine serum and
resuspended in neurobasal medium supplemented with 2%
B27 supplement, 5% heat-inactivated horse serum, 50 U/mL
penicillin, and 50 mg/mL streptomycin. Aliquots of
1×106 cells/mL were seeded onto tissue culture plates precoated
with poly-L-lysine (0.1 g/L) and kept at 37
oC in a humidified atmosphere with 5%
CO2 and 95% O2. Forty eight hours
after plating, cytosine arabinofuranoside was added to
maintain a final concentration of 5 µmol/L to inhibit the
proliferation of non-neuronal cells. At d 3, one-half of the medium
was removed and replaced by the same volume of fresh
medium. Only mature cultures (10_13 d in
vitro) were used for the experiments. Our preliminary experiments using
microtubule-associated protein immuno-staining and glial
fibrillary acidic protein (GFAP) immuno-staining indicated that
about 95% of the cells were neurons after arabinofuranoside
treatment for 3 d.
Treatment of the cultures The rat neurons cultured
for 10_13 d were incubated with 50 µmol/L glutamate for
either 30 min or 24 h, with or without SF treatment (100,
200, and 500 µmol/L, respectively). In co-incubation
experiments, SF was added to the neurons 30 min prior to
incubation with glutamate. LY294002 or wortmannin
(inhibitors of PI3K phosphorylation) and PD98059 or
U0126 (inhibitors of MEK phosphorylation) were added
to the cell cultures 1 h prior to SF treatment. The medium
was gently removed at the indicated times, and the attached
cells were fixed for the morphological assessment of
apoptosis. In addition, total and cytosolic protein fractions
were extracted for Western blotting.
Quantification of apoptosis by nuclear morphological
changes Apoptosis was assessed by an analysis of nuclear
morphology. Briefly, the neurons were fixed with 4%
formaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 10
min at room temperature, incubated with Hoechst 33258 dye
at a concentration of 5 mg/L in PBS for 10 min in the dark, and
then washed with PBS. Fluorescence was visualized using
an Axioskop fluorescence microscope (Carl Zeiss GmbH, Jena,
Germany). Fluorescent nuclei were scored and categorized
according to the condensation and staining characteristics
of chromatin. Normal nuclei showed non-condensed
chromatin dispersed over the entire nucleus. Apoptotic nuclei
were identified by condensed chromatin contiguous to the
nuclear membrane and by nuclear fragmentation and apoptotic bodies. Both the apoptotic and the normal nuclei
were counted under a fluorescent microscope with a
magnification of 40× of the field lens. On each culture slide, the
nuclei in 4 randomly selected microscopic fields were
counted. For each group, the nuclei in the 4 slides were
counted (altogether, the nuclei in 16 microscopic fields were
counted in each group). The mean values were calculated
and the data were expressed as the percentage of the
apoptotic nuclei among the total nuclei.
Western blot analysis Western blot was performed to
analyze the expression of phospho-MEK1/2,
phospho-ERK1/2, phospho-Akt/ protein kinase B (PKB),
phospho-p70S6K, caspase-3, m-calpains, PARP, cleaved PARP, and Bcl-2. The
cells were washed once with ice-cold PBS, harvested, and
centrifuged at 600×g for 10 min at 4
oC. The pellets were lysed in ice-cold lysis buffer [1% Triton,
0.1% SDS, 0.5% deoxycholate, 1 mmol/L EDTA, 20
mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 10 mmol/L NaF, and 0.1 mmol/L
phenylmethyl-sulfonyl fluoride (PMSF)]. The nuclear fractions were first
isolated by centrifuging the homogenates at
7500×g for 30 min at 4 oC. The supernatant was further centrifuged at 12
000×g for 20 min at 4 oC to remove insoluble materials. The
protein concentrations were quantified by the method of
Lowry. The samples were equalized for protein
concentra-tions. The proteins were separated with 10%_12%
SDS-PAGE and transferred onto nitrocellulose membranes. The
membranes were blocked with 3% bovine serum albumin in
Tris-buffered saline (TBS, pH 7.6) for 1 h and incubated
overnight at 4 oC with suitably diluted primary antibodies. After
extensive washed with TBS-Tween (0.5% Tween 20), the
membranes were incubated with antirabbit IgG, a HRP-linked
antibody, and an antibiotin antibody for 1 h at room
temperature. The blots were detected using the enhanced
chemiluminescence (ECL) reaction. After visualization by
ECL, all of the nitrocellulose strips were reprobed with the
β-actin antibody to ensure equal loading of the proteins on all
SDS-PAGE gels. The immunoreactive blots were incubated
with the alkaline phosphatase-conjugated antimouse IgG
antibody for 1 h. Finally, the blots were developed with the
alkaline phosphatase substrate O-dianisidine tetrazotized
along with β-naphthyl acid phosphate. Quantification of
protein bands was achieved by densitometric analysis
using Chemimage 5500 software (UVP, Pittsburgh, PA, USA).
Statistical analysis All data were presented as mean±SD.
Statistical analysis was carried out with one-way ANOVA,
followed by least significant difference (LSD)'s
post-hoc test, which was provided by SPSS 11.5 statistical software (SPSS,
Chicago, IL, USA). The level of significance was accepted
as P<0.05.
Results
SF prevents glutamate-induced apoptosis in cultured
cortical neurons Cultured cortical neurons
were incubated with 50 µmol/L glutamate for 24 h and examined for the
characteristic morphological nuclear changes associated with
apoptosis. After 24 h of incubation, about
40%±9% of cells displayed apoptotic morphology, characterized by
chromatin condensation. SF (100, 200, and 500 µmol/L, respectively)
significantly reduced the number of apoptotic cells induced
by glutamate in a dose-dependent manner (Figure 1).
The activation of caspase-3 is a hallmark of apoptosis
and precedes changes in nuclear morphology. The activated
caspase-3 of 20 kDa was observed with the Western blot
analysis. The results showed a remarkable increase of the
active caspase-3 fragment in the neurons incubated with
glutamate compared with the controls. The pretreatment
with SF almost reduced the glutamate-induced increase in
activated caspase-3 compared to the control values (Figure
2A, 2B).
During apoptosis, poly (ADP-ribose) PARP is one of the
earliest targets for caspase-3 cleavage which induces the
formation of a 89 kDa C-terminal fragment containing the
catalytic domain and a 24 kDa fragment that binds DNA
ends[19]. As shown in Figure 2C, the level of intact PARP (116 kDa)
was significantly decreased in the cortical neurons
incubated with glutamate and was associated with the
appearance of the 89 kDa fragment of PARP. In SF-pretreated
neurons, the expression level of the intact PARP (116 kDa)
was up-regulated while the expression of the 89 kDa
fragment was lower than those in the neurons incubated with
glutamate alone (Figure 2C_2E).
Taken together, these finding suggest that
glutamate-mediated apoptosis in the rat cortical neurons is markedly
inhibited by SF.
SF inhibits µ-calpain upregulation in cortical neurons
exposed to glutamate The activation of calpains is now
considered to play a key role in excitotoxic neuronal
damage[20,21]. Therefore, in order to investigate whether SF prevents
glutamate-induced µ-calpain activation, we used Western
blotting to determine the activation of µ-calpain in rat
primary cortical neurons after SF treatments. We found that
compared with untreated cortical neurons, both 80 and 76
kDa µ-calpain bands were significantly increased in cortical
neurons following exposure to glutamate for 24 h.
Pretreatment of neurons with SF (100, 200, and 500 µmol/L,
respec-tively) significantly reduced the glutamate-induced
production of µ-calpain, both of the inactivated 80 kDa and
activated 76 kDa forms in a concentration-dependent manner.
Furthermore, SF alone significantly downregulated the basal
expression levels of µ-calpain (Figure 3).
SF inhibits glutamate-induced downregulation of Bcl-2
protein levels in cultured cortical neurons The Bcl-2
protein plays a key role in the anti-apoptotic process. The
incubation of neurons with glutamate for 30 min significantly
reduced Bcl-2 protein levels as compared with the control.
Pretreatment with SF (100, 200, and 500 µmol/L, respectively)
significantly inhibited the reduction of Bcl-2 expression
induced by glutamate in a dose-dependent manner (Figure
4), suggesting that Bcl-2 may mediate, to some extent, the
protective effects provided by SF against glutamate toxicity.
In addition, SF alone (500 µmol/L) had no obvious effect on
the basal expression of Bcl-2 in the cultured cortical neurons
(data not shown).
PI3K/Akt/p70S6K pathway is involved in
neuroprotec-tion by SF against glutamate toxicity
To investigate the role of the PI3K pathway in the protection by SF against glutamate
toxicity, the neurons were pre-incubated with either
LY294002, a synthetic bioflavonoid that reversibly binds to
and inhibits p110[22] or wortmannin, a fungal toxin that
covalently binds to and blocks the activity of the catalytic
p110 subunit of PI3K[23]. As assessed by Hoechst 33258
staining, LY294002 (10 µmol/L) and wortmannin (100 nmol/L)
significantly reduced the protective effects of SF against
glutamate cytotoxicity (Figure 5A), suggesting that SF
exerted an anti-apoptotic effect on cortical neurons exposed
to excitotoxic insult through the PI3K/Akt pathway. A
control experiment revealed that 2 PI3K inhibitors used had no
effect on the toxicity of glutamate in the absence of SF. To
test for possible toxic effects of those inhibitors, cortical
neurons were treated with LY294002 and wortmannin alone
at the concentration used in this study and no toxicity was
observed (data not shown). We examined the level of
phosphorylated Akt with hybridizing to the antibody specific
against phospho-Akt (Thr308) using Western blotting. The
basal level of phospho-Akt in cultured cortical neurons was
relatively high. The treatment of neurons with glutamate for
30 min induced a significant decrease in the phosphorylated
level of Akt. SF (500 µmol/L) prevented a glutamate-induced
decrease of phosphorylated Akt. LY294002 (10 µmol/L)
counteracted the effect of SF on phosphorylated Akt (Figure
5B,5C). In addition, SF treatment alone (500 µmol/L) exerted an
increase in phosphorylated Akt expression that did not reach
statistical significance compared with the untreated cortical
neurons (data not shown).
Because p70S6K acts downstream of Akt, we examined
the level of phosphorylated p70S6K detected by an
antibody specific for phospho-p70S6K (Thr389). The results
showed that incubation of the neurons with glutamate for 30
min elicited a significant decrease in the phosphorylated level
of p70S6K. SF (500 µmol/L) prevented a glutamate-induced
decrease in the expression of phosphorylated p70S6K. This
effect on the phosphorylation of p70S6K was blocked by
the application of LY294002, indicating that the SF-induced
phosphorylation of p70S6K depended on PI3K activation
(Figure 5D,5E). Similarly, SF treatment alone did not elicit a
significant increase in phosphorylated p70S6K expression
(data not shown).
MEK/ERK pathway is involved in neuroprotection by SF
against glutamate toxicity. In order to determine whether
the activation of the ERK pathway was involved in the
neuroprotective effect of SF, we pharmacologically
inhibited MEK, the upstream kinase of ERK, with PD98059 (10
µmol/L) or U0126 (30
µmol/L)[24]. In the presence of
PD98059 or U0126, the neuroprotective effect of SF against
glutamate-evoked cell death was partially abrogated, as observed by
an analysis of nuclear morphology, indicating that the ERK
pathway partially mediates the neuroprotective effect of SF
against glutamate toxicity (Figure 6A). Control experiments
revealed that the 2 MEK inhibitors used had no effect on
glutamate toxicity measured in the absence of SF. To
investigate for possible toxic effects of those inhibi-tors, cortical
neurons were treated independently with PD98059 or U0126
at the concentration used in this study and no toxicity was
observed (data not shown). Then, we determined the effect
of SF on the phosphorylation of ERK1/2 in cultured cortical
neurons by Western blotting. The results showed that the
treatment of neurons with glutamate for 30 min induced a
significant decrease in phosphorylated ERK1/2 expression
(Figure 6D,6E). However, pretreatment with SF prevented
the glutamate-induced decrease in phosphorylated ERK1/2
expression. SF-induced ERK1/2 phosphorylation was
significantly blocked by the simultaneous application of PD98059
(10 µmol/L), suggesting that MEK1/2 activation mediates
the SF-induced increase in ERK1/2 phosphorylation (Figure
6D, 6E). In the extended analysis of the ERK pathway,
phosphorylation of MEK1/2 was detected. The results showed
that treatment of the neurons with glutamate also elicited a
significant decrease in phospho-MEK1/2. SF inhibited the
glutamate-induced decrease in phospho-MEK1. PD98059
(10 µmol/L) did not block the effect of SF on phospho-MEK1
expression (Figure 6B, 6C). SF treatment alone did not elicit
significant effects on basal phospho-MEK and
phospho-ERK1/2 expressions in cultured cortical neurons (data not
shown).
Discussion
The cell death induced by glutamate is believed to be
involved in neuronal loss associated with both acute and
chronic neurodegenerative insults. Thus, the dissection of
glutamate signal transduction may have clinical significance
for neuroprotection. This study demonstrated that
pre-incubation with SF protects cultured cortical neurons against
glutamate toxicity. The activations of the PI3K/Akt/p70S6K
pathway and the MEK/ERK1/2 pathway play important roles
in the protective effect of SF against glutamate toxicity in
cortical neurons. Furthermore, SF upregulated the
expression of the anti-apoptotic protein Bcl-2 and prevented the
upregulation of the µ-calpain protein in rat cortical neurons
after treatment with glutamate, suggesting that the
downregulation of µ-calpain may also account for the
neuroprotective effect of SF.
Calpains represent a superfamily of
Ca2+-activated cysteine-proteases, which are important mediators of apoptosis
and necrosis. In the brain, the presence of µ-calpain was
shown primarily in neurons, whereas µ-calpain is more
prominent in glial cells[25]. The activation of µ-calpain in cortical
neurons specifically linked to Ca2+ entry through the NMDA
receptor[26]. A recent study strongly suggested that calpain
plays a central role as a excitotoxic signal transduction
cascade leading to DNA
fragmentation[20]. The results indicated
that calpain cleaved Bid and that truncated Bid then
translocated to mitochondria and induced mitochondria membrane
permeabilization and the release of DNA fragmentation
factors resulting in
neurodegeneration[20]. In addition, it has
been shown that calpains can cleave Bcl-2, thereby
contributing to the ischemia-induced decrease in Bcl-2 protein
levels thereby triggering the intrinsic apoptotic
pathway[27,28]. Our studies indicated that SF inhibited the upregulation of
µ-calpain in cortical neurons exposed to glutamate. This
inhibitory effect of SF on µ-calpain expression may be partly
responsible for the neuroprotective effects against
glutamate-neurotoxicity. One explanation for the inhibitory effect of SF
on µ-calpain expression could be that SF is a novel
competitive NMDA receptor
antagonist[29]. It is known that
apopto-sis observed in neurons under exposure to glutamate
depends on the hyperactivation of NMDA receptor and hence
excessive Ca2+ influx through NMDA receptor
channels[30]. Dizocilpine (MK-801), a specific antagonist of the NMDA
receptor, was shown to almost completely block cell death
induced by glutamate[31].
The precise mechanism of the protective effect of SF
through the activation of PI3K/Akt/p70S6K pathway is
unclear, but a possible explanation is that the activated
p70S6K phosphorylates Bad. The phosphorylated Bad
appears to be the inactive moiety sequestered in the cytosol
bound to 14-3-3, freeing Bcl-x or Bcl-2 to promote
survival[32]. Another explanation is that PI3K/Akt induces the
upregula-tion of the anti-apoptotic protein Bcl-2. The direct induction
of Bcl-2 by activated PI3K/Akt has been described in some
studies[33]. In the current study, pretreatment with SF
significantly inhibited the decrease of phospho-Akt and Bcl-2
expression induced by glutamate. It has been reported that
infection with the Bcl-2-expressing viral vector protected
cortical cells from glutamate
excitotoxicity[34]. The results indicate that SF protects cortical neurons from
glutamate-induced toxicity by inducing the overexpression of Bcl-2
through PI3K/Akt activation.
In order to investigate whether the activation of the ERK
pathway was involved in the neuroprotective effect of SF,
we inhibited the upstream kinase of ERK (MEK) with PD98059
or U0126. In the presence of the MEK inhibitors, the
neuro-protective effect of SF against glutamate-evoked cell
apo-ptosis was partially abrogated, as observed by an analysis
of nuclear morphology, indicating that the ERK pathway
mediates the neuroprotective effect of SF against glutamate
toxicity. In further support of a role for MEK/ERK1/2 in
SF-induced neuroprotection, SF prevented the
glutamate-induced decrease in phosphorylated level of MEK1/2 and
ERK1/2. The effect of SF on ERK1/2 phosphorylation was
MEK-dependent as the application of PD98059 prevented
this effect, further confirming the role of the ERK
pathway in the neuroprotection by SF. The activation of the
ERK signaling pathway was reported to suppress the
pro-apoptotic activity of the stress-activated c-Jun N-terminal
kinase (JNK)/p38 protein kinase, thus protecting rat
pheochromocytoma cells (PC-12) from nerve growth factor
NGF withdrawal-induced cell death[35]. The MEK/ERK
pathway interferes with apoptosis at the level
of cytosolic caspase activation, downstream of the release of
cytochrome c from
mitochondria[36].
The fact that both PI3K and MEK/ERK contribute to the
protection of cortical neurons by SF from glutamate toxicity
may be explained by the activation of multiple lethal
reactions during excitotoxic cell
death[37,38]. The simultaneous contributions of PI3K and MEK/ERK to the protection of
cortical neurons by SF from glutamate-evoked apoptotic
death may also be due to the cross-talk between the 2
pathways. A recent study showed that
3-phospho-inositide-dependent protein kinase (PDK)1 directly binds and
activates MEK, thereby contributing to the activation of this
pathway[39]. PI3K is also responsible for maintaining
constitutive ERK1/2 activity in different cell lines, in which basal
PI3K and ERK activities are required to prevent cell
death[40]. The activation of the transcription factor cyclic adenosine
monophosphate response element-binding protein (CREB)
has been reported as a consequence of ERK activation. CREB
is not a direct substrate for ERK. Rather, ERK needs to
phosphorylate and activate members of the p90 ribosomal
S6 kinase family, which in turn may phosphorylate and
activate CREB[41]. Interestingly, CREB has also been identified
as a regulatory target for c-Akt[42], suggesting that CREB
may function as a convergence point of 2
survival-promoting signaling pathways. CREB is a key molecule in neuronal
survival.
In conclusion, this study demonstrated that
PI3K/Akt/p70S6K and the MEK/ERK signaling pathway play
important roles in the protective effect of SF against glutamate
toxicity in cortical neurons. Two different signal
transduction pathways, the PI3K/Akt and the MEK/ERK1/2 pathways,
might directly or indirectly inhibit the activation of cell death
mechanism. The results reported here suggest that agents
that selectively target the PI3K and/or the MEK/ERK
pathways may be clinically useful in the protection of neurons
against glutamate toxicity.
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