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Transforming Growth Factor-αα Attenuates NMDA Toxicity in Cortical Cultures by

Preventing Protein Synthesis Inhibition through an Erk1/2-dependent Mechanism.

Valérie Petegnief1, Bibiana Friguls1, Coral Sanfeliu1, Cristina Suñol2 and Anna M. Planas1

1Departament de Farmacologia i Toxicologia; 2Departament de Neuroquímica, Institut

d’Investigacions Biomèdiques de Barcelona, CSIC-IDIBAPS. Barcelona, Spain

Running title: TGF-α protects against NMDA by recovering protein synthesis

Corresponding author:

Dr Valérie Petegnief

Tel:34 93 363 83 00 ext 359

Fax:34 93 363 83 01

E-mail: vpefat@iibb.csic.es

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 27, 2003 as Manuscript M300661200 by guest on D

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SUMMARY

Transforming growth factor-α (TGF-α), a ligand of the epidermal growth factor receptor, reduces

the infarct size after focal cerebral ischemia in rat but the molecular basis underlying the

protection are unknown. Excitotoxicity and global inhibition of translation are acknowledged to

significantly contribute to the ischemic damage. Here, we studied whether TGF-α can rescue

neurons from excitotoxicity in vitro and how it affects calcium homeostasis, protein synthesis,

and the associated Akt and extracellular signal-regulated kinase 1/2 (Erk1/2) intracellular

signaling pathways in mixed neuron-glia cortical cultures. We found that 100 ng/ml TGF-α

attenuated neuronal cell death induced by 30-min exposure to 35 µM NMDA (as it reduced

lactate dehydrogenase release, propidium iodide staining, and caspase-3 activation) and

decreased the elevation of intracellular Ca2+ elicited by NMDA. TGF-α induced a prompt and

sustained phosphorylation of Erk1/2, and prevented the loss of Akt-P induced by NMDA 3hr

after exposure. The protective effect of TGF-α was completely prevented by PD 98059, an

inhibitor of the Erk1/2 pathway. Studies of incorporation of [3H]leucine into proteins showed that

NMDA decreased the rate of protein synthesis and TGF-α attenuated this effect. TGF-α

stimulated the phosphorylation of the eukaryotic initiation factor 4E (eIF4E), but did not affect

eIF2α, two proteins involved in translation regulation. PD 98059 abrogated TGF-α effect on

eIF4E. Our data demonstrate that TGF-α exerts a neuroprotective action against NMDA-toxicity,

in which Erk1/2 activation plays a key role, and suggest that the underlying mechanisms involve

recovery of translation inhibition, mediated at least in part by eIF4E phosphorylation.

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TGF-α is protective in models of permanent and transient focal ischemia induced by occlusion of

the middle cerebral artery in the rat (1,2). Though TGF-α is known to promote neuronal survival

(3) and to induce proliferation and differentiation of astrocytes in vitro (4), little is known about

its effects on neural cells. TGF-α binds to the epidermal growth factor receptor (EGFR), which

stimulation induces its dimerization, autophosphorylation on tyrosine residues and triggers a

cascade of reactions that require the contribution of adapter proteins and kinases. EGFR activates

several signal transduction pathways, amongst others phosphatidylinositol-3 kinase/protein

kinase B (PI-3K/Akt) and the p44/p42 mitogen-activated protein kinase also referred to as

extracellular signal-regulated kinases 1/2 (Erk 1/2) (5).

Amongst the very early consequences of energy depletion after an ischemic insult to the brain are

membrane depolarization that induces excitatory amino acid release and inhibition of global

protein synthesis, which both significantly contribute to the development of brain infarct (6-8).

Indeed, glutamate receptor overactivation results in an increase in intracellular calcium and

extensive neuronal death by excitotoxicity (9). Likewise, persistent blockade of protein synthesis

is associated to brain damage (10), and neuronal survival after focal ischemia may depend on the

recovery of protein synthesis (11). Intracellular Ca2+ overload by activation of glutamate

receptors (12) or disturbances of endoplasmic reticulum (ER) Ca2+ homeostasis decrease protein

synthesis (13-16). Protein synthesis is mainly regulated at the initiation step. Initiation of

translation is a complex process that requires an initiator methionyl-tRNA and the eukaryotic

initiation factors (eIF) for the assembly of mRNA and the ribosome subunits (17). Availability

and activity of some of these factors, which depends on their phosphorylation state, will

determine the efficiency of translation (18). Phosphorylation of eIF2α is well known to lead to a

decrease in protein synthesis and increasing evidence accumulates to show the critical

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involvement of phospho-eIF4E in translation (18). Mnk1, a kinase activated by Erk1/2 and p38

(19) phosphorylates eIF4E. Cellular stresses such as excitotoxicity or hydrogen peroxide in

neurons (12,20,21), oxygen and glucose deprivation (22) and serum deprivation (23) in PC12

cells down-regulate protein synthesis. On the opposite, growth factors and neurotrophins enhance

translation in cultured neurons but different agents regulate specific translation factors through

activation of Akt and/or Erk1/2 signaling pathways (24–26).

In order to better understand how TGF-α exerts its neuroprotective action in vivo, we examined

the effect of TGF-α in a model of excitotoxicity in cortical cultures. Thereafter, we studied some

of the transduction signaling pathways activated by TGF-α and its effect on NMDA-induced

increase in cytosolic Ca2+, the rate of amino acid incorporation into proteins, and the

phosphorylation of eIF2α and eIF4E.

EXPERIMENTAL PROCEDURES

All products for culture except MEM were purchased from Invitrogen (Paisley. Scotland. UK).

Chemicals and reagents, unless otherwise stated, were obtained from Sigma-Aldrich

(Alcobendas. Spain). Recombinant human TGF-α was from Oncogene Research Products

(Boston. USA), tyrphostin AG 1478, Ac-DEVD-AMC and Ac-DEVD-CHO from Alexis

(Laufelfingen. Switzerland). PD 98059 and the mouse antibody against eIF4E were from BD

Transduction Laboratories (Heidelberg. Germany). Antibodies against NeuN and anti-eIF2α

were purchased from Santa Cruz Biotechnology (Heidelberg. Germany). Anti-mouse, anti-goat

and anti-rabbit HRP-conjugated antibodies were from Amersham (Buckinghamshire. England).

Vectastain ABC reagent and mouse biotinylated-HRP secondary antibody were from Vector

Laboratories (Brulingame. CA. USA). Complete, the protease inhibitor cocktail, and antibody

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against β-tubulin were from Boehringer Mannheim (Germany). Mouse anti-phospho-p44/p42

MAPK (i.e. anti-pErk1/2) (Thr 202/Tyr 204), rabbit anti-phospho-Akt (Ser 473), rabbit anti-

phospho-eIF4E (Ser 209), rabbit anti-phospho-eIF2α (Ser 51) and rabbit anti-p44/p42 (Erk1/2)

were obtained from Cell Signaling Technology (Beverly, MA. USA). L-[4,5-3H] leucine (S.A. =

73 Ci/mmol) was purchased from Amersham (Buckinghamshire. England). Phase Lock GelTM

heavy tubes were a generous gift of Eppendorf Iberica (Barcelona, Spain). Microcon 30 was

purchased from Amicon-Millipore (Bedford, USA) and DE52 cellulose was from Whatman

(Maidstone, USA).

Cell cultures

Mixed primary cortical cultures of neurons and glia were prepared from 18-day-old Sprague-

Dawley rat embryos (IFA-CREDO, Lyon. France) as previously described (27). Cells were

resuspended in Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum and

100 µg/ml gentamycin and seeded onto poly-L-lysine (5 µg/ml)-precoated 24-well plates (Nunc,

Roskilde. Denmark) at a density of 3680 cells/mm2. Medium was partly changed on 4, 7 and 10

day-in vitro (DIV) with MEM supplemented with B27. For immunocytochemistry, cells were

plated on poly-L-lysine-coated glass coverslips. For the determination of intracellular Ca2+, cells

were plated at a density of 3180 cells/mm2 on 48-well plates. For the determination of leucine

specific activity (SA) in [3H]leucyl-tRNA, cells were plated at a density of 3470 cells/mm2 on 6-

well plates.

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Treatments

Excitotoxic lesion was performed on 12 DIV by treating cultures for 30 min with NMDA.

Medium was thereafter replaced with MEM supplemented with B27 and gentamycin. Unless

otherwise stated, recombinant human TGF-α was added twice to the culture medium: on 7 DIV

and immediately after the 30 min-NMDA incubation on 12 DIV. PD 98059 at 40 µM was added

30 min before NMDA and was also present in the medium after NMDA washout. Tyrphostin AG

1478 at 10 µM was added after NMDA removal 10 min before TGF-α on 12 DIV.

Measurement LDH activity

Cell death was estimated 24hr after the lesion by measuring the activity of lactate dehydrogenase

(LDH) released in the medium according to a modification of the method of Wroblewski and

LaDue (28). Briefly, the decrease in 0.75 mM NADH absorbance at 340 nm was followed in a

phosphate buffer (50 mM, pH 7.4) in presence of 4.2 mM pyruvic acid as substrate. Serial

dilutions of medium from 0.2% Triton X-100 lyzed cells were used to construct a standard curve,

and cell death was expressed as the percentage of maximal death in the dose-response study.

In all other experiments, the value of LDH activity in control cultures was subtracted from values

of treated cultures and 35 µM NMDA values were normalized to 100% (maximum cell death) as

described in Bruer et al. (29). Results of LDH release were expressed in % of NMDA and

presented as mean ± SEM.

Propidium iodide nuclear staining

Cultures pre-treated or not with TGF-α were lesioned with NMDA. Sixteen hr after NMDA

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lesion, cultures were incubated with 7.5 µM of propidium iodide (PI) for 30 min at 37ºC

protected from light. After 2 washes with PBS, cells were fixed for 30 min with 4%

paraformaldehyde at 4°C and washed with PBS. PI positive cells were counted in 8 fields per

well and the sum of the 8, corresponding to a total area of 1.1776 mm2 was calculated. Results

were expressed as % of control.

Measurement of caspase-3 activity

The caspase-3 activity enzymatic assay was performed according to Valencia and Moran (30)

using 100 µg of proteins and 25 µM of Ac-DEVD-AMC as the substrate. Fluorescence of AMC,

generated by cleavage of Ac-DEVD-AMC (excitation/emission 380/460 nm), was monitored

every 5 min for 30 min in a CytoFluor 2350 Millipore scanner. Enzymatic activity was calculated

as ∆ fluorescence/mg prot/min and expressed as % of control. We checked that 50 µM Ac-

DEVD-CHO, the caspase-3 selective inhibitor completely blocked NMDA-induced caspase-3

activity.

NeuN immunocytochemistry

After exposure for 5 days to 100 ng/ml TGF-α, mixed cultures were washed with 10 mM cold

phosphate buffered saline (PBS) and fixed for 30 min with 4% paraformaldehyde at 4°C. Cells

were then washed and endogenous peroxidase activity was inhibited by a 2-min incubation in 1%

H2O2 diluted in Methanol/PBS (30:70). Blocking was then performed for 30 min at room

temperature in PBS containing Triton X-100 and 7% horse serum. Cells were then incubated for

1hr with the primary antibody (anti-NeuN 1:100) washed and incubated with a mouse

biotinylated-HRP secondary antibody. After a wash, cells were incubated with the ABC reagent

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according to the instructions of the manufacturer. The reaction was developed in 0.5 mg/ml DAB.

Three control wells and 3 TGF-α-treated wells within a same 24-well plate were analyzed. NeuN

immunoreactive cells were counted in 3 fields per well and the sum of the 3, corresponding to a

total area of 0.44 mm2 was calculated. The result was expressed in NeuN positive cells per mm2.

A t-test was performed for statistical analysis.

Measurement of cytosolic [Ca2+]

Cells were loaded with 15 µM of the Ca2+-sensitive dye Fluo-3/AM (Molecular Probes. Leiden.

The Netherlands) for 1hr at 37ºC, washed three times with HEPES buffer (10 mM HEPES pH

7.4, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.62 mM MgSO4, 6 mM glucose) and incubated

with 35 µM NMDA for 10 min at room temperature. Fluorescence of Fluo-3/AM

(excitation/emission 485/530 nm) was monitored at different times from 0.5 to 10 min with a

CytoFluor 2350 Millipore scanner. Results were expressed as % of control at each time-point.

Western blot

Cultures were washed with cold PBS and harvested at several time points in RIPA lysis buffer

(10 mM PBS, 1% Igepal AC-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate)

supplemented with a protease inhibitor cocktail (Complete) and 1 mM sodium orthovanadate.

Protein content was determined by the Bradford assay (Bio-Rad Laboratories. Munchen.

Germany). Proteins were separated by electrophoresis on 10 or 12% polyacrilamide gels in

denaturing conditions and transferred to polyvinylidene difluoride Immobilon-P membranes

(Millipore). After 1hr blocking in 20 mM Tris-HCl, 150 mM NaCl, 0.1 % Tween 20 (T-TBS)

containing 5% albumin bovine and 5% non-fat dry milk, membranes were incubated overnight at

4ºC with the following primary antibodies: mouse anti-phospho-p44/p42 MAPK (Thr 202/Tyr

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204), rabbit anti-phospho-Akt (Ser 473), rabbit anti-phospho-eIF4E (Ser 209), rabbit anti-

phospho-eIF2α (Ser 51), rabbit anti-p44/p42, mouse anti-eIF4E diluted 1:1000 and goat anti-

eIF2α diluted 1:100. After 2 washes in T-TBS, membranes were then incubated for 1hr at room

temperature with either anti-rabbit, anti-mouse or anti-goat horseradish peroxidase-conjugated

antibody at 1:2000. The reaction was visualized using a chemiluminescence detection system

based on the luminol reaction. Reprobing the membranes with an antibody against β-tubulin

diluted 1:15000 was carried out to check equal loading in the lanes.

Incorporation of [3H]leucine into proteins

A time-course of leucine uptake was performed in order to determine the time at which

[3H]leucine steady state between the medium and the cells was reached. Culture medium was

withdrawn and cells were incubated in 300 µl of MEM/B27 (commercial MEM contains 0.052

g/L of leucine) in the presence of 4 µCi/ml [3H]leucine for different times ranging from 1 to 30

min at 37ºC. Medium was then removed, cells were washed once with 0.5 mg/ml non-radioactive

leucine in PBS, harvested in lysis buffer (20 mM Tris HCl, pH 7.6, 10 mM potassium acetate, 1

mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1mM benzamidine,

0.25% Igepal) and processed as described in Alcazar et al. (13). Briefly, lysates were spun for 30

min at 12000 X g at 4°C. Supernatants were collected and an aliquot was taken for protein

determination before addition of trichloracetic acid (TCA) 10%. Proteins were precipitated by a

30 min centrifugation at 12000 X g at 4°C and the supernatants (TCA soluble fraction) were

separated from the pellet (TCA precipitable fraction), The TCA precipitable fraction was

resuspended in 0.2N NaOH and radioactivity was measured to determine [3H]leucine

incorporation into proteins. Radioactivity was also measured in the TCA soluble fraction to

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calculate intracellular soluble radioactivity (dpm/mg of protein) at each time point. According to

the uptake results showing that steady state was already reached by 20 min, in further

experiments we chose to study the effect of treatments on the incorporation of [3H]leucine into

proteins (dpm/mg prot/ min) after 30 min incubation with the radiotracer.

The content of intracellular free leucine (nmol) was measured by HPLC in a separate experiment

as follows: after treatments, medium was replaced with MEM/B27 without [3H]leucine and 30

min later cells were washed with leucine-free PBS and processed as above. Five µl of the TCA

soluble fraction were brought to pH 10 with 1 M NaOH and mixed with 15 µl of the derivatizing

reagent o-phthaldialdehyde at 1 mg/ml. Separation of endogenous leucine was carried out in a

reverse-phase C18 column (Tracer Nucleosil C18, 5 µm particle size, 10x0.4 cm; Teknokroma,

Spain) using the following mobile phase: solution A (100 mM sodium acetate, 5.5 mM

triethylamine, 11% acetonitrile, pH 5.5) and solution B (acetonitrile) mixed with a gradient

program from 100% to 30% of solution A within 60 min at 0.8 ml/min. Leucine content was

calculated after fluorimetric detection (excitation/emission: 360nm/450 nm) using an external

standard method according to Suñol et al. (31).

Leucine specific activity in [3H]leucyl-tRNA

Under steady-state conditions, the rate of protein synthesis is a function of the SA of the

precursor pool for the incorporation of amino acids into proteins (32-34). The immediate

precursor pool is not free amino acid in the intracellular space, but the corresponding aminoacyl-

tRNA. For this reason we determined in a separate group of experiments the SA of leucyl-tRNA

for certain conditions. Isolation of aminoacyl-tRNA and deacylation was performed according to

a modification of the protocol described by Keen et al (32). Medium from control, TGF-α- or

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NMDA-treated cultures was removed and cells were incubated in 1 ml of MEM/B27 containing

10 µCi/ml [3H]leucine for 30 min at 37ºC. Medium was then removed and the cells were washed

once with 3 ml leucine-free PBS and kept at -80°C. Cells from 6 wells per condition were

resuspended in a 3.5 ml final volume of buffer (250 mM NaCl, 10 mM MgCl2, 1 mM Na2EDTA,

0.4 mg/ml bentonite, 10 mM sodium acetate, pH 4.5,) and vigorously mixed with 3.5 ml of

saturated phenol at pH5 for 1 hr at 4°C. The mixture was centrifuged for 5 min at 1500 X g at

room temperature in Phase Lock Gel heavy tubes and the aqueous phase containing total RNAs

was collected and chromatographed in a 0.7 x 10 cm DE52 DEAE cellulose column with buffer

(250 mM NaCl, 10 mM MgCl2, 1 mM Na2EDTA, 10 mM sodium acetate, pH 4.5) at a flow rate

of 4 ml/min. One min fractions were collected for a total of 60 ml. Aminoacyl-tRNAs were

eluted with a higher salt concentration (700 mM NaCl, 10 mM MgCl2, 1 mM Na2EDTA, 10 mM

sodium acetate, pH 4.5) in 0.7 min fractions for a total of 30 ml. Radioactivity was measured in

0.5-ml aliquots and the peak fraction containing the [3H] leucyl-tRNA was further processed to

deacylate the aminoacyl-tRNA. pH was adjusted to 9 by addition of 400 mM sodium borate (pH

9.5) and the samples were incubated at 37°C for 90 min. Free leucine was separated from tRNA

in Microcon 30 ultrafiltration systems by spinning at 14000 X g for 25 min at 15°C. The

ultrafiltrate was concentrated in a speed-vac system and analyzed by HPLC as described above.

The content of free leucine (nmol) was measured and the associated radioactivity (dpm) was

determined to calculate [3H]leucine SA (dpm/nmol) derived from the corresponding leucyl-

tRNA.

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Statistical analysis

Results are expressed as mean ± SEM and the number of replicates (n) is indicated in the legend

of each figure and table. Unless indicated, one-way analysis of variance (ANOVA) with the

Bonferroni post-hoc test was performed to determine which groups were significantly different.

The Kruskall Wallis analysis followed by the Dunn's test for multiple comparisons was used to

compare groups with non-homogenous variance. Two-way ANOVA was performed to compare

changes in intracellular calcium (Fig. 1E) and the effect of NMDA and TGF-α on neuronal death

(Fig. 1B). One symbol indicates P<0.05, two P<0.01 and three P<0.001.

RESULTS

TGF-α reduces NMDA-induced excitotoxicity

A NMDA dose-response study was undertaken to estimate the concentration that kills 50% of

NMDA-sensitive neurons, as assessed by LDH release. LDH release assay, measuring cell

permeability, was previously shown to correlate with cell death (35). EC50 was 37.9 µM ± 7.2

(Fig. 1A), so we chose to perform further experiments with 35 µM NMDA. TGF-α at 1, 30 or

100 ng/ml was added 5 days before and after the NMDA lesion, as indicated in Experimental

Procedures. According to LDH release, TGF-α reduced NMDA-induced neuronal death by 40%

(P<0.05) at the highest concentration (Fig. 1B). This result was further validated with propidium

iodide staining that showed attenuation of NMDA-induced neuronal loss by TGF-α (Fig. 1C).

Furthermore, in agreement with previous reports (36-38), NMDA induced activation of caspase-

3, which again was prevented by TGF-α (Fig. 1D).

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Additionally, we checked that addition of 100 ng/ml TGF-α on 7 DIV did not alter the number of

neurons. After exposure for 5 days to TGF-α, mixed cultures were fixed on 12 DIV and stained

with the antibody against NeuN, a marker of neuronal nuclei. Control and TGF-α-treated cultures

showed 892 ± 132.2 and 936 ± 130.5 NeuN positive cells per mm2, respectively, and no

statistically significant difference was found between the two groups.

Since excitotoxicity induces disturbances in calcium homeostasis, we studied changes in

cytosolic calcium in our model. Free intracellular calcium in cultures exposed to NMDA raised to

2-3-fold over control values (Fig. 1E). The effect was observed as early as 30 sec and was

maintained for at least 10 min. TGF-α reduced the NMDA-induced increase in intracellular

calcium by about 25% at all time points (P<0.05).

TGF-α neuroprotection is mediated through EGFR by activation of Erk1/2

TGF-α is an endogenous ligand of EGFR. Here we found that the protective effect of TGF-α was

mediated through EGFR as AG 1478, a specific inhibitor of the tyrosine kinase activity of EGFR,

prevented the protective effect of TGF-α (Fig. 1F). EGFR is coupled to several intracellular

signaling pathways. Here, we studied Akt and Erk1/2 phosphorylation after TGF-α and NMDA

treatments. TGF-α induced a strong phosphorylation of Erk1/2 in a concentration-dependent

manner at 5 min (Fig. 2A). NMDA caused a comparatively moderate Erk1/2 phosphorylation

(Fig. 2A). In the presence of both drugs Erk1/2 activation was higher than after single treatment

(Fig. 2A). However, the effect of NMDA was transient as it was detected at 5 min during NMDA

exposure, but it was no longer found at 1 hr (Fig. 2D) or 3 hr after NMDA removal (Fig. 2B),

whereas Erk1/2 phosphorylation by TGF-α was maintained at these times. We then wanted to

figure out whether Erk1/2 activation was responsible for TGF-α protection. Preincubation with

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PD 98059, an inhibitor of MAPK kinase (MEK1/2), totally blocked Erk1/2 phosphorylation (Fig.

2B). PD 98059, which did not compromise neuronal survival on its own, fully prevented TGF-α

neuroprotective effect but it did not alter NMDA-induced toxicity (Fig. 2C). These data

demonstrate that the MEK/Erk1/2 pathway is involved in TGF-α neuroprotective process. In

addition, we observed that AG 1478 blocked TGF-α-induced phosphorylation of Erk1/2,

showing that this effect is mediated through activation of EGFR (Fig. 2D).

In contrast to the TGF-α-induced Erk1/2 activation, we did not observe any activation of Akt

after treatments (Fig. 2A and D). However, 3 hr after NMDA lesion the level of Akt-P was

reduced in relation to control (Fig. 2B). This effect was not seen at earlier time points, i.e. during

5 min NMDA exposure (Fig. 2A) and at 1 hr after NMDA (Fig. 2D), likely reflecting an early

step in neuronal degeneration. Since TGF-α reduced neuronal death, maintenance of Akt-P in the

NMDA+TGF-α condition is in accordance with the protection. No major effect of PD 98059 on

Akt phosphorylation was seen (Fig. 2B).

[3H]Leucine incorporation into proteins is reduced by NMDA and this effect is prevented by

TGF-α.

Since early and persistent inhibition of protein synthesis has been reported in vivo and in vitro

after ischemia and excitotoxicity, we investigated whether NMDA altered this parameter in

cortical cultures. We first performed a kinetic study of cellular [3H]leucine uptake from the

medium to determine the time at which [3H]leucine reached steady state. [3H]leucine uptake was

linear during the first minutes and reached a plateau from 20 min (Fig. 3A). Therefore, we

decided to incubate cells for 30 min with [3H]leucine for protein synthesis studies. NMDA

inhibited the incorporation of [3H]leucine into proteins in a dose-dependent manner (Fig. 3B) in

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parallel to its neuronal toxicity (Fig. 1A), indicating that the degree of inhibition of [3H]leucine

incorporation correlates with the severity of the lesion. TGF-α prevented the reduction of

[3H]leucine protein incorporation induced by NMDA (Fig. 3C). This effect of TGF-α was not

observed in the presence of the Erk1/2 inhibitor PD 98059 (Fig. 3C). Yet, PD 98059 affected

[3H]leucine protein incorporation by itself (Fig. 3C) suggesting that Erk activity is involved in

maintaining protein synthesis under control conditions. Therefore we cannot conclude that PD

98059 reverses the effect of TGF-α on protein synthesis recovery after NMDA.

Effect of treatments on intracellular free leucine and leucyl-tRNA

The amount of TCA soluble radioactivity was reduced by NMDA in relation to controls (Table

I). Concomitantly, slight reductions in the content of intracellular free leucine were detected after

NMDA (Table I), although differences did not reach statistical significance. Therefore, the

reduced amount of TCA-soluble intracellular radioactivity under steady-state conditions was

associated to a faint reduction in the amount of intracellular free leucine. Yet, this small change

in intracellular free leucine by NMDA was not limiting for protein synthesis, as the SA of leucyl-

tRNA (the actual precursor pool for protein synthesis), was not modified by NMDA (Table I).

This indicated that amino acids were available for protein synthesis and thus the deep inhibition

of amino acid incorporation into proteins caused by NMDA was due to alterations in the process

of translation. This view was supported by the observation that treatment with TGF-α alone, in

spite of not decreasing the incorporation of radioactivity into proteins in relation to controls (Fig.

3C), reduced, as NMDA did, TCA-soluble intracellular radioactivity and it slightly reduced (non

significantly) the content of intracellular leucine (Table I). But again, the SA of leucyl-tRNA was

unchanged after TGF-α (Table I), thus implying that this treatment affected the molecular

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mechanism underlying the process of translation rather than the availability of amino acids for

protein synthesis.

NMDA reduces and TGF-α activates the phosphorylation of eIF4E. eIF2α phosphorylation is not

affected by NMDA or by TGF-α

Since translation is predominantly regulated at the initiation level and some of the eIFs can be

limiting factors, we examined the phosphorylation state of eIF4E and eIF2α. The former is more

efficient after phosphorylation whereas the phosphorylated eIF2α binds to the GDP/GTP

exchanger eIF2B and inhibits it so that a new round of translation initiation is blocked. NMDA

did not induce a phosphorylation of eIF2α (Fig. 4A). This indicates that eIF2α does not mediate

NMDA downregulation of protein synthesis. Acute addition of TGF-α did not modify eIF2α

phosphorylation either (Fig. 4A).

We then considered eIF4E since it can be phosphorylated after Erk1/2 activation (19). We

observed a slight dephosphorylation of eIF4E after NMDA exposure (Fig. 4B and C), which

might contribute to the inhibition of protein synthesis after NMDA removal. TGF-α induced a

strong phosphorylation of eIF4E, which was detected even after NMDA treatment (Fig. 4B and

C). The level of total eIF4E protein was not affected by TGF-α (Fig. 4B and C). Therefore,

eIF4E phosphorylation might contribute to the maintenance of protein synthesis by TGF-α after

NMDA (Fig. 3C). PD 98059 prevented eIF4E phosphorylation by TGF-α (Fig. 4C), consistently

with its inhibitory effect on TGF-α-induced Erk1/2 activation (see above). Thus, this result

shows that Erk1/2 activation mediates phosphorylation of eIF4E by TGF-α.

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DISCUSSION

In the present paper, we show that TGF-α exerts a neuroprotective effect in cortical cell cultures

subjected to an excitotoxic lesion. Through binding to EGFR, TGF-α reduced neuronal death by

attenuating the calcium influx triggered by NMDA and by persistently activating MEK/Erk1/2

pathway. Subsequently, eIF4E, a downstream target of Erk1/2, was phosphorylated and

facilitated protein synthesis. Transient exposure to NMDA caused persistent inhibition of the rate

of amino acid incorporation into protein without altering the availability of precursor amino acid.

Inactivation of eIF4E, but not eIF2α, was related to this process. TGF-α prevented the collapse

of translation elicited by NMDA and therefore largely contributed to decrease neuronal death. In

addition, our results suggest that phospho-Erk1/2 and phospho-eIF4E play a critical role in the

maintenance of protein synthesis in cortical neurons. In this study, a direct correlation is found

between the recovery of protein synthesis after treatment with a growth factor and its

neuroprotective effect against an excitotoxic injury.

TGF-α increases neuronal survival after an excitotoxic lesion

TGF-α exerts beneficial effects against NMDA-induced toxicity in mixed neuron-glia cortical

cultures. In this experimental condition toxicity is directed towards neurons, as astrocytes in

culture do not express NMDA receptors (39). Treatment with TGF-α attenuated neuronal

calcium overload triggered by NMDA receptor overactivation and reduced neuronal cell death.

Likewise, glial cell line-derived neurotrophic factor (GDNF) was shown to improve neuronal

survival after NMDA by reducing Ca2+ influx in pure cortical neuron cultures (40). A significant

neuroprotective effect of TGF-α against NMDA toxicity was observed at rather high doses (100

ng/ml). Likewise, protection of neurons against oxygen deprivation was found with 100 ng/ml

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EGF (5). We previously reported that 50 ng TGF-α is protective in vivo in the rat brain against

focal cerebral ischemia (1, 2). Yet, glutamate-mediated excitotoxicity is not the only neurotoxic

mechanisms accounting for ischemic neuronal damage, as other disturbances (such as the glial

reaction, inflammation and oxidative stress) are also involved in the generation of infarction. It is

unknown whether TGF-α might exert beneficial effects against the pathogenesis of ischemic

damage, other than reducing NMDA-mediated excitotoxicity

The effect of TGF-α is mediated by activation of the EGFR/Erk1/2 signaling pathway

We then aimed to identify the signal transduction pathways stimulated by TGF-α. We first

confirmed that TGF-α conferred neuroprotection acting on the EGFR, as inhibition of the

tyrosine kinase activity coupled to EGFR with AG 1478 (41) suppressed TGF-α-induced

neuroprotection. Following EGFR activation, the phosphorylated tyrosines act as binding sites

for molecules containing the src homology 2 domain (SH2), such as PLCγ and adapter proteins

such as Grb2 and Shc (reviewed in 5). The adapter proteins function as docking sites for other

signaling molecules such as Cbl that associates to PI-3K, activating in its turn Akt, and

recruitment of Grb2 activates the MAPK pathway through Ras-GTP (5). Thus, we studied the

phosphorylation of Akt and Erk1/2 and observed that Akt was not activated, while Erk1/2 was

highly and persistently phosphorylated after exposure to TGF-α. In accordance with this result,

many effects of TGF-α in the CNS depend on the stimulation of the Erk1/2 pathway (42).

Evidence for the critical involvement of the Erk1/2 pathway in TGF-α function was supported by

the observation that AG 1478 and PD 98059 both abolished Erk1/2 phosphorylation and TGF-α

neuroprotection in our model. The finding that Erk1/2 phosphorylation was important for

neuronal survival is in agreement with previous studies. For instance, estrogen rescues cortical

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neurons from glutamate toxicity through an Erk1/2-dependent mechanism (43). Interestingly, Akt

and Erk1/2 are activated after an ischemic episode (44-47), but the underlying mechanism

remains unknown. While Akt is clearly involved in anti-apoptotic processes (48,49), the

beneficial effect of activated Erk1/2 in neurons is still a matter of controversy. Indeed, though

Erk1/2 signaling regulates synaptic plasticity, long-term potentiation and survival (50), its

inhibition prevents neuronal damage resulting from focal cerebral ischemia (44) and

excitotoxicity in vitro (51). Glutamate receptor stimulation in cortical cultures triggers Erk1/2

phosphorylation (52). In agreement with the latter report, NMDA activated Erk1/2 in our cultures

in a transient manner. However, this activation was not responsible for NMDA toxicity since PD

98059 did not impede NMDA-induced toxicity. In contrast, NMDA caused dephosphorylation of

Akt (in relation to controls), although this was not seen until 3 hr after the lesion. TGF-α

prevented this effect of NMDA in spite that treatment with TGF-α alone did not activate Akt. As

it is likely that dephosphorylation of Akt by NMDA at this time point would impair neuronal

survival, we cannot exclude the possibility that preventing Akt dephosphorylation by TGF-α was

involved in neuroprotection. Yet, the facts that Akt dephosphorylation by NMDA was not an

early event and that TGF-α by itself did not activate Akt suggest that the maintenance of Akt-P

after NMDA and TGF-α was an indicator of neuronal survival.

Transient exposure to NMDA causes persistent inhibition of protein synthesis and eIF4E

dephosphorylation

We then addressed suppression of translation, another important aspect involved in neuronal

death (6,8,10). We found a persistent inhibition of amino acid incorporation into proteins

following transient exposure to NMDA, which was prevented by TGF-α. The effect of NMDA

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was not a consequence of reduced amino acid availability as the leucyl-tRNA precursor pool for

protein synthesis after NMDA was not different from control. Yet, a small reduction in the free

leucine intracellular pool was detected after NMDA, but this was not limiting for the formation of

leucyl-tRNA. Treatment with TGF-α alone did not alter leucine incorporation into proteins in

relation to control. However, unexpectedly, TGF-α by itself decreased, as NMDA did, the free

leucine pool, but again this had no effect on the pool of leucyl-tRNA. Therefore, we conclude

that NMDA inhibited protein synthesis by a mechanism other than reducing amino acid

availability, and that TGF-α prevented the downregulation of protein synthesis caused by

NMDA.

In order to find out the molecular mechanism underlying protein synthesis inhibition and

recovery, we focused our study on two translation factors, namely eIF2α and eIF4E. eIF2α is a

critical regulator of protein synthesis as it mediates the binding of the Met-tRNA to the ribosome.

eIF2α has been extensively studied since it is phosphorylated after ischemia and cellular stress

(21,53,54). Depletion in ER Ca2+, that is suspected to occur in various pathologies including

cerebral ischemia, leads to phosphorylation of eIF2α and subsequent inhibition of protein

synthesis (13,15). However, eIF2α was not phosphorylated after NMDA exposure compared to

the control condition. Therefore, the inhibition of protein synthesis observed after treatment with

NMDA is not caused by inactivation of this factor. We also examined the phosphorylation state

of eIF4E after NMDA. eIF4E is the m7G cap mRNA binding protein that associates to eIF4G and

eIF4A to form the eIF4F complex. This complex interacts with cap mRNA and facilitates its

binding to the ribosome. Affinity of eIF4E for the cap is increased after phosphorylation (18).

Here we report that eIF4E suffered dephosphorylation (i.e. inactivation) after transient exposure

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to NMDA, which likely contributed to persistent inhibition of protein synthesis when cells were

no longer exposed to the toxin.

TGF-α promotes protein synthesis recovery and eIF4E phosphorylation

Regarding the mechanism involved in the effect of TGF-α preventing NMDA-induced protein

synthesis inhibition, we observed that TGF-α induced eIF4E phosphorylation after NMDA

exposure and that this was mediated through activation of Erk1/2. Several growth and trophic

factors cause eIF4E phosphorylation after Erk1/2 activation in various cells under different

experimental conditions (19,23). eIF4E was shown to play a pivotal role in the activation of

translation by EGF and NGF in PC12 cells after nutrient deprivation (23). In cortical cultures,

BDNF is a better stimulator of protein synthesis than insulin since it phosphorylates eIF4E

whereas insulin does not (25). In a similar manner, eIF4E phosphorylation by TGF-α might be

involved in the recovery of protein synthesis after NMDA exposure. Besides Erk1/2 activation,

regulation of eIF4E may occur through another pathway. Indeed, some proteins -eIF4E binding

proteins (eIF4E-BP)- associate with eIF4E and impede the formation of the eIF4F complex.

Under the phosphorylated state, they dissociate from eIF4E and no longer block the complex

formation. The mammalian target of rapamycin (mTOR), a downstream kinase of Akt (18), is the

enzyme responsible for eIF4E-BP phosphorylation in cortical neurons (25). Many growth factors,

such as IGF-1 (26), BDNF (25) and EGF (55) activate mTOR and phosphorylate eIF4E-BP.

However, since TGF-α did not activate Akt signaling pathway in our cultures, we assume that

eIF4E-BP did not play a major role in the regulation of eIF4E. Beyond its effect on eIF4E, TGF-

α did not affect eIF2α phosphorylation in the absence or presence of NMDA.

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Altogether our data show that TGF-α unlocked the blockade of translation by activating the

EGFR/Erk1/2 route and reduced the Ca2+ entry elicited by NMDA, thereby promoting neuronal

survival. Though TGF-α has pleiotropic effects, the present results support that the recovery of

NMDA-induced persistent protein synthesis inhibition is critical for the neuroprotective effect of

TGF-α against excitotoxicity. Since disturbances in calcium homeostasis and protein synthesis

occur after an ischemic insult, a partial restoration of these parameters after in vivo TGF-α-

treatment may contribute to its protective effect.

ACKNOWLEDGMENTS:

This work was supported by CICYT SAF 2002-01963, FIS 00/0957 and 01/1318 grants. V.P. and

B.F. are recipients of grants from the Spanish Ministry of Science and Technology and from the

University of Barcelona, respectively. We thank Albert Parull for technical assistance.

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FIGURE LEGENDS

FIG. 1: TGF-αα , through activation of EGFR, reduces NMDA-induced excitotoxicity in

cortical cultures

A, NMDA dose-response in cortical cultures. Cultures were exposed on 12 DIV to increasing

concentrations of NMDA (5-300 µM) for 30 min. NMDA was then washed out and death was

studied 24 hr later by LDH activity. Data are mean ± SEM of results expressed as % of maximum

cell death, a value obtained from Triton X-100-lyzed cultures (n=6 cultures). B, Cultures treated

for 5 days with increasing concentrations of TGF-α were lesioned with 35 µM NMDA on 12

DIV. Thirty min later, the medium was removed and replaced with fresh medium supplemented

with TGF-α (1-100 ng/ml). LDH activity was determined at 24 hr post-lesion. LDH value in

control and NMDA-treated cultures were set to 0% and 100%, respectively. Data were

normalized (expressed as % of NMDA) and presented as mean ± SEM (n=3-4 cultures).

Symbols: open bar for control, filled bar for NMDA. C, Control and TGF-α-pre-treated (100

ng/ml) cultures were lesioned with 35 µM NMDA. Sixteen hr later, cells were incubated with 7.5

µM propidium iodide (PI), and PI stained nuclei were counted under the fluorescence microscope

(see Experimental Procedures). The number of PI-positive nuclei per mm2 was 349 ± 27.6 in

control cultures. NMDA increased the number of PI stained nuclei, an effect that was attenuated

by TGF-α. Data are expressed as % of control (mean ± SEM of n=4 cultures). D, Caspase-3

activity was calculated as ∆ fluorescence/mg protein/min. Data are mean ± SEM of n=8 cultures

from 3 independent experiments of results expressed as % of control. Caspase-3 activity

increased at 16 hr after exposure to NMDA, and this was prevented by TGF-α. E, TGF-α reduces

the NMDA-evoked increase in intracellular Ca2+. Control and TGF-α-pre-treated cultures were

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loaded with 15 µM Fluo 3 AM. Cells were then exposed to NMDA or NMDA-free HEPES

buffer and the fluorescence emitted at 530 nm was monitored from 0.5 to 10 min. Data are mean

± SEM of results expressed as % of control (n=16 cultures from 3 independent experiments).

Symbols: open circle for TGF-α, open triangle for NMDA+TGF-α and filled square for NMDA.

F, EGFR inhibitor AG 1478 completely prevents TGF-α neuroprotection. Cultures treated with

TGF-α were exposed to NMDA; AG 1478 was added after the lesion as indicated in

Experimental Procedures. LDH activity was determined at 24 hr post-lesion. Data are mean ±

SEM of results expressed as % of NMDA (n=10 to 16 cultures from 2 independent experiments).

C: control cultures, N: NMDA, AG: AG 1478, N+T: NMDA+TGF-α, N+T+AG: NMDA+TGF-

α+AG 1478. ***P<0.001, **P<0.01, *P<0.05 indicate a significant difference vs. control,

$$$P<0.001, $$P<0.01, $P<0.05 vs. NMDA and &&&P<0.001 vs. NMDA+TGF-α.

FIG. 2: Activation of Erk1/2 by TGF-αα is involved in neuroprotection against NMDA

toxicity. NMDA induces loss of Akt-P at 3 hr and this effect is prevented by TGF-αα . A,

Cultures were treated for 5 min with NMDA and/or increasing concentrations of TGF-α. Proteins

were then resolved on 10% SDS-PAGE and processed for Western blot. Immunoblotting was

performed with an Erk1/2-P antibody (p42/p44-P) and the membrane was then reprobed for Akt-

P and then β-tubulin. B, Cells cultured for 5 days with TGF-α were treated with PD 98059 and

exposed to NMDA for 30 min as indicated in Experimental Procedures. Erk1/2-P, Akt-P and β-

tubulin Western blot were performed with cell extracts harvested 3 hr after NMDA lesion. PD

98059 inhibits the raise of Erk1/2 phosphorylation in the presence of TGF-α and NMDA.

Phosphorylation of Akt is reduced by NMDA at 3 hr after exposure (panel B), but not at earlier

time points (panel A). C, Cultures were treated as in B and LDH activity was measured at 24hr

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post-lesion. Data are mean ± SEM (n=9-18 cultures from 2 independent experiments) of results

expressed as % of NMDA. ***P<0.001 vs. control, $$$P<0.001 vs. NMDA and &&P<0.01 vs.

NMDA+TGF-α. D, EGFR blockade with AG 1478 prevents TGF-α-induced Erk1/2

phosphorylation. Cultures treated with TGF-α were exposed to NMDA; AG 1478 was added

after the lesion as indicated in Experimental Procedures. Cells were harvested 1hr after the lesion

and processed for Erk1/2-P, Erk1/2 total and Akt-P Western blot. The amount of total protein

was not affected by any treatment. PD: PD 98059, N+PD: NMDA+ PD 98059, N+T+PD:

NMDA+TGF-α+ PD 98059, for other abbreviations see legend Fig. 1.

FIG. 3: NMDA inhibits [3H]leucine incorporation into proteins, and TGF-αα prevents it.

A, Time-course of [3H]leucine uptake. Cultures were incubated with [3H]leucine for 1 to 30 min.

Cells were then processed as described in Experimental Procedures. Data are mean ± SEM (n=3-

4 per time point) of results expressed as dpm/mg proteins corresponding to the ratio between the

radioactivity in the TCA soluble fraction and the amount of proteins. Steady state of [3H]leucine

in the medium and the cells is reached after 20 min. B, NMDA causes a concentration-dependent

reduction of amino acid incorporation into proteins. Cultures were incubated for 30 min with

increasing concentrations of NMDA. Medium was then removed and cells were incubated for 30

min with 300 µl of MEM/B27 containing [3H]leucine. Samples were processed as indicated in

Experimental Procedures. Data are mean ± SEM (n=4 cultures). C, NMDA at 35 µM (N) induced

a reduction of amino acid incorporation into proteins that was prevented by 100 ng/ml TGF-α

(N+T). Reduced incorporation was also found in the presence of 40 µM PD 98059 (N+T+PD and

PD alone). Data are mean ± SEM (n=12-16 cultures from 4 independent experiments) of results

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expressed as TCA precipitable dpm/mg of protein/min. ***P<0.001, *P<0.05 vs. control,

$$$P<0.001 vs. NMDA and &&P<0.01 vs NMDA+TGF-α.

FIG. 4: NMDA decreases eIF4E phosphorylation and TGF-αα stimulates it through activation

of Erk1/2. eIF2αα phosphorylation is not affected by any treatment. A, Cells were treated with

NMDA for 30 min, changed to a fresh medium and harvested 30 min later. TGF-α was added

simultaneously to NMDA (60) or after the lesion (30). Proteins from duplicate samples were

resolved on 12% SDS-PAGE. Immunoblots were incubated first with anti-eIF2α-P, then with

anti-eIF2-α and finally with anti-β-tubulin. B, Blot A was incubated with anti-eIF4E-P and then

with anti-eIF4E. C, Cultures treated for 5 days with TGF-α were exposed to NMDA. PD 98059

was added as described in Experimental Procedures. Cells were harvested 30 min after medium

removal and processed for eIF4E-P, eIF4E and β-tubulin Western blot.

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TABLE I

Effect of treatments on intracellular free leucine and leucyl-tRNA.

Treatment TCA soluble [3H]

(dpm/mg prot)

Intracellular leucine

(nmol leucine/mg prot)

Leucyl-tRNA SA

(dpm/nmol)

Control 268567 ± 13263 (15) 27.00 ± 1.77 (6) 1010.43 TGF-α 100 ng/ml 206910 ± 8665 (12)** 23.20 ± 1.65 (6) 1057.59 NMDA 35 µM 215380 ± 8589 (16)* 22.98 ± 2.29 (6) 1112.95 PD 98059 40 µM 240149 ± 14146 (16) 26.45 ± 0.67 (6) n.d. NMDA + TGF-α 172546 ± 10550 (11)*** 24.27 ± 0.70 (6) n.d. NMDA + TGF-α + PD 166760 ± 8186 (14)*** 24.26 ± 1.17 (6) n.d.

Intracellular TCA soluble radioactivity, concentration of leucine and leucyl-tRNA SA were determined

as described in Experimental Procedures. Data are mean ± SEM (n), n.d.: not determined. One-way

ANOVA was performed for statistical analysis. ***P<0.001, **P<0.01 and *P<0.05 indicate a

significant difference with control.

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Valérie Petegnief, Bibiana Friguls, Coral Sanfeliu, Cristina Suñol and Anna M. Planaspreventing protein synthesis inhibition through an Erk1/2-dependent mechanism

attenuates NMDA toxicity in cortical cultures byαTransforming growth factor-

published online May 27, 2003J. Biol. Chem. 

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