Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2α is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine–nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E–eIF4G complexes; however, they did not induce eIF2α phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E–eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.
- eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)
- eukaryotic initiation factor 4G (eIF4G)
- nitric oxide (NO)
Brain ischaemia induces early and deep inhibition of protein synthesis that is persistent in vulnerable neurons such as CA1 hippocampal and striatal neurons [1–3]. In this regard, long-lasting inhibition of protein synthesis is one of the alterations that best correlates with neuronal injury after ischaemia. Indeed, translation recovery is associated with survival, whereas neurons unable to overcome protein synthesis blockade are destined to die . Translation is mainly inhibited via regulation of the initiation and elongation steps, by impairing the activity of certain eukaryotic initiation or elongation factors (eIFs or eEFs). Modulation of the phosphorylation status and/or degradation of these proteins accounts for their inactivation. For instance, changes in the phosphorylation status of eIF2α, 4E-BP1 (eIF4E-binding protein 1) or eEF2 are known to modulate the rate of protein synthesis . Phosphorylation of eIF2α by PERK [double-stranded RNA-activated protein kinase-like ER (endoplasmic reticulum) kinase] is sufficient to abrogate protein synthesis soon after reperfusion following an ischaemic episode [6–9]. In addition, a number of other factors are affected. For instance, depletion of ATP activates eEF2 kinase through AMPK (AMP-activated protein kinase) in cortical neurons and slows elongation . Furthermore, global cerebral ischaemia leads to eIF4G and eIF4E breakdown [11–13]. Ischaemia triggers the production of NO (nitric oxide) that is involved in inflammation and cerebral damage . In addition, NO decreases protein synthesis through eIF2α phosphorylation [15–17]; however, the contribution of NO to translation regulation under ischaemic conditions has not been investigated thoroughly.
Likewise, excitotoxicity is one of the main promoters of neuronal loss after an ischaemic episode, and is also known to inhibit protein synthesis [18–21]; however, the underlying mechanisms are not fully elucidated. The rise in intracellular Ca2+ induced by glutamate activates eEF2 kinase, which in turn phosphorylates eEF2 and results in the transient blockade of elongation . The increase in cytosolic Ca2+ concentration following NMDA (N-methyl-D-aspartate) exposure activates the cysteine protease calpain . eIF4G, the scaffolding protein that enables the assembly of eIF4E and eIF4A with the mRNA and the ribosomal 40 S subunit, is a substrate of calpain  and is thus a possible target of NMDA. However, most studies of eIF4G cleavage have reported that eIF4G is the target of caspase 3 under apoptotic conditions (reviewed in ). Caspase 3 cuts eIF4GI at two sites to generate three fragments, referred to as FAGs (fragments of apoptotic cleavage of eIF4G), N-FAG, M-FAG and C-FAG, corresponding to the N-terminal, middle and C-terminal parts of the protein respectively (reviewed in ). M-FAG533-1176 is the only fragment that remains able to promote some cap-dependent translation. Therefore proteolysis of eIF4G is presumably associated with the switch from cap-dependent to cap-independent translation and favours, for example, the translation of specific IRES (internal ribosome entry site)-containing mRNAs . Unlike with FAG, the role of eIF4G fragments resulting from calpain activity has not been explored.
4E-BP1 is another key regulator in protein synthesis because it associates with eIF4E . In cell cultures, insulin, amino acids and rapamycin modulate the phosphorylation status of 4E-BP1 through mTOR (mammalian target of rapamycin) signalling [25,26]. In the hypophosphorylated form, 4E-BP1 competes with the binding site of eIF4G and sequesters eIF4E, whereas the hyperphosphorylated γ form dissociates from eIF4E, enabling it to engage with other components of the eIF4F complex [24,27–31]. Therefore proteolysis of eIF4G, together with dephosphorylation of 4E-BP1, precludes the formation of eIF4F complexes  that may decrease the cap-mRNA translation.
There is compelling evidence of cross-talk between NO and NMDA. In pure neuron cultures, uncoupling between the NMDA receptor and nNOS (neuronal NO synthase) signalling pathways  and pharmacological inhibition of nNOS  strongly prevent neuronal death after an excitotoxic challenge. Reciprocally, production of NO by iNOS (inducible NO synthase) in activated glia leads to neuronal death, which is prevented by MK-801, an NMDA receptor antagonist [35–37], thus suggesting that NO leads to glutamate release  in a positive-feedback loop. Furthermore, NO challenge triggers excitotoxicity in cerebellar neurons . The mechanisms responsible for NO toxicity are not completely elucidated, but energy depletion is likely to be involved. NO induces a fast depolarization of the mitochondria, which leads to decreased ATP production . In addition, NO exposure triggers the activation of PARP [poly(ADP-ribose) polymerase], which leads to ATP consumption and may therefore account for energy depletion .
In the present study, we addressed the molecular mechanisms underlying NMDA-mediated long-term inhibition of protein synthesis in neuronal cultures, and considered whether NO is involved in this process.
MATERIALS AND METHODS
All products for culture, except for MEM (minimal essential medium), were purchased from Invitrogen. ATP bioluminescence assay kit, somatic cell ATP-releasing agent, chemicals and reagents were obtained from Sigma–Aldrich, unless stated otherwise. DEA/NO (diethylamine–NO sodium complex) was purchased from Tocris. The calpain inhibitors calpeptin and MDL28170, as well as rapamycin, were from Calbiochem. The non-specific NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and the nNOS inhibitor NPLA (NG-propyl-L-arginine) were purchased from Sigma–Aldrich. The mouse antibodies against eIF4E and STAT3 (signal transducer and activator of transcription 3) were from BD Transduction Laboratories. The goat antibodies against eIF4G (N-20) and anti-eIF2α were purchased from Santa Cruz Biotechnology. Rabbit anti-phospho-eIF2α (Ser51) was from Epitomics, Temecula, CA, U.S.A. and mouse anti-spectrin was from Chemicon, Burlingame, CA, U.S.A. Anti-4E-BP1 antibody was obtained from Cell Signaling Technology, Danvers, MA, U.S.A. Anti-mouse, anti-goat and anti-rabbit HRP (horseradish peroxidase)-conjugated antibodies were from GE Healthcare. Complete™ protease inhibitor cocktail and the antibody against β-tubulin were from Boehringer Mannheim. L-[4,5-3H]leucine (specific radioactivity=73 Ci/mmol) and m7GTP (7-methyl-GTP)–Sepharose were purchased from GE Healthcare.
Cell cultures and treatments
Mixed primary cortical cultures of neurons and glia were prepared from 18-day-old Sprague–Dawley rat embryos (Charles River Laboratories), as described previously . Animals were anaesthetized and killed by cervical dislocation. All procedures were approved by the Ethical Committee for Animal Use (CEEA) at the University of Barcelona. Neuron-enriched cultures were prepared as mixed cultures, but Ara-C (cytosine arabinoside) was added on DIV (day in vitro) 4 and then in the subsequent partial medium changes on DIV 7 and 10 to limit glial proliferation. Cells were seeded on 24-well plates at a density of 3680 cells/mm2. For the ATP content measurement, cells were seeded on six-well plates. All experiments were performed on 11–13 DIV cultures.
DEA/NO was prepared as a 20 mM stock in NaOH (pH 10) to avoid NO release and was stored at −20 °C until further use. A 2 mM DEA/NO solution in NaOH (pH 10) was prepared extemporaneously and added to the culture medium in order to achieve a final concentration of 4–40 μM. Cells were exposed for 30 min to DEA/NO and medium was thereafter replaced with MEM supplemented with B27 and gentamycin. Excitotoxic lesion was performed by treating cultures for 30 min with 35 μM NMDA. NOS inhibitors (L-NAME and NPLA) were added simultaneously with NMDA and also after medium change. Calpain inhibitors were added at 30 μM, unless stated otherwise, 1 h before NMDA or DEA/NO and also after medium change. Thapsigargin at 1 or 10 μM was added for 30 min and rapamycin at 1 or 2 μM for 3 h with no previous medium change. Calpeptin, MDL28170, thapsigargin and rapamycin were dissolved in DMSO.
Measurement of LDH (lactate dehydrogenase) activity
Cell death was estimated 24 h after the lesion by measuring the activity of LDH released into the medium as described previously . Briefly, the decrease in 0.75 mM NADH absorbance at 340 nm was followed in a 50 mM phosphate buffer (pH 7.4) in the presence of 4.2 mM pyruvic acid as substrate. The kinetic assay of NADH consumption was monitored for 4 min, and the slope values of decreased absorbance at 340 nm (negative values) were converted into positive values and expressed in arbitrary units (AU).
PI (propidium iodide) nuclear staining
Cells were treated for 30 min with 35 μM NMDA and then fixed and stained with PI 24 h later as described previously . PI-positive nuclei were counted in three fields per well, and the sum of the three, corresponding to a total area of 0.4416 mm2, was calculated. The results were expressed as PI-positive cells/mm2.
Incorporation of [3H]leucine into proteins
Culture medium was withdrawn, and cells were incubated in 300 μl of MEM/B27 containing 396 μM unlabelled leucine and 4 μCi/ml [3H]leucine for 30 min at 37 °C. NMDA or DEA/NO was added immediately after the [3H]leucine to evaluate its effect on protein synthesis at 30 min. To assess protein synthesis 2 h after transient exposure to NMDA or DEA/NO, cells were incubated with NMDA or DEA/NO for 30 min, medium was changed to MEM/B27, and, 90 min later, cells were incubated with [3H]leucine for 30 min as described above. Medium was removed, proteins were precipitated with TCA (trichloroacetic acid), and lysates were processed as described previously  for the determination of [3H]leucine incorporation into proteins. Results are expressed as d.p.m. in the TCA fraction/mg of protein per min.
Measurement of ATP levels
After drug treatments, cells were washed with cold PBS and collected in a 5 mM EDTA and 0.1 M sodium phosphate (pH 7.5) buffer. After a 5 min spin at 590 g at 4 °C, pellets were resuspended in 200 μl of the same buffer, sonicated and centrifuged again at 10000 g for 15 min at 4 °C. ATP content was determined in the fresh supernatants using an ATP bioluminescent assay kit and following the manufacturer's instructions. Samples were run together with a 0.05–8 μM ATP standard curve, and luminescence was monitored in an Orion microplate luminometer (Berthold Detection Systems). RLU (relative light units) were converted into pmol of ATP, and results are expressed as nmol of ATP/mg of protein.
Neuron-enriched cultures were treated with NMDA, NMDA and L-NAME and NPLA, or DEA/NO for 30 min. After 90 min, cultures were washed with cold 10 mM PBS and were harvested in RIPA (radioimmunoprecipitation) lysis buffer (10 mM PBS, 1% Igepal AC-630, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with a protease inhibitor cocktail (Complete™) and 1 mM sodium orthovanadate. Protein content was determined using the Bradford assay (Bio-Rad Laboratories). Proteins (30 μg) were separated by electrophoresis on 7, 12 or 17% polyacrylamide gels (for spectrin-eIF4G, eIF2α and 4E-BP1 respectively) under denaturing conditions and transferred on to 0.2-μm-pore-size PVDF Immun-Blot® membranes (Bio-Rad Laboratories). Membranes were incubated overnight at 4 °C with the following primary antibodies: goat anti-eIF4G (diluted 1:500), goat anti-eIF2α (diluted 1:2000), rabbit anti-phospho-eIF2α (Ser51) (diluted 1:2000), rabbit anti-4E-BP1 (diluted 1:2000), mouse anti-spectrin (diluted 1:4000), mouse anti-STAT3 and mouse anti-eIF4E (each diluted 1:1000). After two washes in TBST (Tris-buffered saline containing Tween 20), membranes were then incubated for 1 h at room temperature (22 °C) with horseradish-peroxidase-conjugated anti-rabbit, anti-mouse or anti-goat antibodies. The reaction was visualized using a chemiluminescence detection system based on the luminol reaction. Autoradiograms were scanned with a GS-800 Densitometer scanner (Bio-Rad Laboratories), and band density was quantified with the Quantity One image analysis software (Bio-Rad Laboratories). For the analysis of eIF2α phosphorylation, the phosphorylated/total protein ratio was calculated for each sample. Since the molecular masses of spectrin and eIF4G are 240 and 220 kDa respectively, we decided to use the 90 kDa protein STAT3 of higher molecular mass than tubulin, to check for equal loading, since none of the treatments affected STAT3 expression (results not shown). The densities of the 145–150 kDa bands corresponding to cleaved spectrin were quantified together. The spectrin/STAT3 and eIF4G/STAT3 ratios were calculated for each sample. Data from all quantifications are expressed as a percentage of controls. In the case of 4E-BP1, the proportion of each phosphorylated form (α, β and γ) was calculated as a percentage of total 4E-BP1, as in .
Determination of the formation of eIF4E–eIF4G and eIF4E–eIF4E-B1 complexes
After the different treatments, cells were washed with buffer A containing 20 mM Tris/HCl (pH 7.6), 1 mM dithiothreitol, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 120 mM KCl, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, 10 μg/ml antipain, 50 mM sodium fluoride, 10 mM glycerophosphate and 10 mM sodium molybdate. They were then lysed with buffer A containing 0.5% Igepal AC-630 and 0.1% Triton X-100. Cell extracts were spun at 12000 g for 10 min at 4 °C, and the supernatants were collected and frozen at −80 °C until further use. Proteins (200 μg) were incubated with m7GTP–Sepharose for 45 min at 4 °C in buffer A containing 0.1 mM GTP. Proteins were eluted from m7GTP–Sepharose with SDS loading buffer and subjected to 7.5–17% SDS/PAGE and Western blot analysis. Membranes were incubated separately with antibodies against eIF4G, eIF4E and 4E-BP1.
Results are expressed as means±S.E.M. for n replicates, as indicated in the legend to each Figure. Unless indicated, one-way ANOVA with the Bonferroni post-hoc test was performed to evaluate significant differences between groups. Kruskal–Wallis analysis followed by Dunn's test for multiple comparisons was used to compare groups with non-homogenous variance.
NOS inhibitors protect against NMDA toxicity in mixed and neuron-enriched cultures
One of our objectives was to determine whether NO participates in NMDA-induced inhibition of protein synthesis in our primary cultures. First, we confirmed that NMDA toxicity was dependent on NO. When testing the effect of several NOS inhibitors, we found that the combination of the non-specific NOS inhibitor L-NAME with the specific nNOS inhibitor NPLA, at 2 mM and 100 μM respectively, provided almost complete protection against transient (30 min) exposure to 35 μM NMDA in mixed neuron/glia and neuron-enriched cultures, as assessed by LDH activity and PI staining assays (Figures 1A and 1B). Further experiments were therefore performed with the NAME+NPLA combination of NOS inhibitors (referred to as NOS inhibitors hereafter). Treatment with the above concentrations of NOS inhibitors did not affect neuronal viability (Figure 1A). To check for direct NO toxicity, i.e. that not induced by NMDA, we performed a dose–response study with DEA/NO, an NO donor. DEA/NO was applied for 30 min, and toxicity was assessed at 24 h. At 40 μM, DEA/NO induced toxicity similar to that induced upon treatment with 35 μM NMDA (Figure 1C), and this concentration of DEA/NO was therefore selected to perform further experiments.
NMDA and NO induce protein synthesis failure
We have demonstrated previously that NMDA decreases the protein synthesis rate after 1 h of exposure . In the present study, we explored whether transient exposure to NMDA caused long-lasting inhibition of protein synthesis. A time-course study showed that NMDA inhibited the incorporation of [3H]leucine into proteins after 30 min of exposure in mixed (Figure 2A) and neuron-enriched (Figure 2B) cultures by 52 and 43% respectively. After removal of NMDA, protein synthesis recovered partially, but remained significantly lower than in controls 2 h later (i.e. 90 min after NMDA removal) in both mixed (Figure 2A) and neuron-enriched (Figure 2B) cultures. NOS inhibitors were ineffective at 30 min (during NMDA exposure), but did promote complete recovery of protein synthesis at 2 h. Treatment with NOS inhibitors alone had no effect on protein synthesis (Figure 2B). DEA/NO also led to a slight decrease in protein synthesis (less than 20%) at the end of a 30 min exposure, and the effect was more pronounced 2 h later (33% decrease) in neuron-enriched cultures (Figure 2C). Altogether, these data suggest that NO impairs translation and contributes to the late phase of protein synthesis inhibition induced by NMDA.
NMDA and NO induce a moderate decrease in ATP content and do not regulate the phosphorylation of eIF2α
ATP depletion can induce ER stress, which leads to phosphorylation of eIF2α and subsequent inhibition of protein synthesis [15,42]. In the present study, we evaluated ATP content after the treatments to determine whether ATP depletion was involved in the observed inhibition of protein synthesis. NMDA caused a 26% decrease in ATP levels at the end of the 30 min exposure, and this effect was maintained 2 h later (Figure 3A). Although DEA/NO decreased ATP content to 53% after 30 min of exposure, this effect was not significantly different from controls 2 h later (Figure 3B), thus indicating that, in contrast with NMDA, the action of NO on ATP is not long-lasting. NOS inhibitors did not prevent the ATP decrease 2 h after NMDA exposure (Figure 3C), and this illustrates that NO was not involved in the persistent ATP decrease after NMDA. Our data suggest that the late protein synthesis inhibition caused by NMDA is not mediated by the corresponding decreased ATP levels.
Phosphorylated eIF2α binds to the GDP/GTP exchanger eIF2B and inhibits its function (reviewed in ). The inability to restore the binding of GTP to eIF2 prevents the formation of new eIF2·GTP·Met-tRNAi (initiator methionine tRNA) ternary complexes and consequently protein synthesis is inhibited. NMDA and NO did not induce eIF2α phosphorylation on Ser51 (Figures 3D and 3E), indicating that eIF2 activity was not inhibited. In order to determine whether our cultures might show increased phosphorylation of eIF2α, neurons were treated for 30 min with thapsigargin. This ER stress inducer is known to cause eIF2α phosphorylation through PERK activation. Figure 3(F) shows that thapsigargin triggers eIF2α phosphorylation when compared with control cells.
NMDA and NO lead to calpain activation and eIF4G proteolysis
NMDA is known to induce a cytosolic Ca2+ increase [10,20] during the period of exposure. As expected, NMDA activated calpain, a Ca2+-dependent cysteine protease, which was assessed by the proteolysis of α-spectrin (Figures 4A and 4C). The 240 kDa full-length protein is processed into 150 and 145 kDa fragments by calpain and to 150 kDa and 120 kDa fragments by caspase 3 . NMDA generated the formation of the 145–150 kDa fragments only (Figure 4A), this being indicative of calpain-dependent hydrolysis. Furthermore, eIF4G was cleaved 2 h after NMDA exposure (39% decrease, P<0.05; Figures 4A and 4C). Proteolysis of both spectrin and eIF4G was blocked completely in the presence of NOS inhibitors (Figures 4A and 4C), thus indicating an NO-dependent mechanism. In agreement with a previous report , NO dramatically stimulated calpain and decreased eIF4G levels by 52% (Figures 4B and 4D). Several calpain inhibitors were then used to determine the contribution of calpain to eIF4G processing, and we found that two of them, calpeptin and MDL28170, completely inhibited calpain activation and blocked the eIF4G degradation induced by NMDA (Figures 5A and 5C) and DEA/NO (Figures 5B and 5D). Therefore eIF4G degradation was totally dependent on calpain activation.
NMDA and DEA/NO induce the dephosphorylation of 4E-BP1 and abrogate the formation of eIF4F complexes
The formation of eIF4F complexes plays a key role in the regulation of translation initiation. Since eIF4E is a component of the eIF4F complex and it is a rate-limiting translation factor for cap-mRNA, we measured its level of expression, observing that none of the treatments altered its cell content (Figure 6A).
The phosphorylation status of 4E-BP1 also governs the formation of eIF4F complexes. 4E-BP1 resolves into three electrophoretic forms, γ, β and α, of increasing migration velocity. Only the hyperphosphorylated γ form of 4E-BP1 does not bind eIF4E  and it therefore increases the availability of eIF4E to interact with eIF4G. We studied the phosphorylation of 4E-BP1 and performed a semi-quantitative analysis of the proportion of γ, β and α forms with respect to total 4E-BP1 (Figure 6B and Table 1). NMDA was observed to promote a shift from the γ form to the α form (Table 1), an effect that was abrogated by NOS inhibitors (Figure 6B and Table 1). Furthermore, DEA/NO induced the dephosphorylation of 4E-BP1, illustrated by a decrease in the γ form and an increase in the α form, which is the band of lowest mobility (Figure 6B and Table 1). In contrast, none of the treatments affected the β form.
Since eIF4G and 4E-BP1 compete for the same binding site in eIF4E, we evaluated the proteins bound to eIF4E after its purification by m7GTP–Sepharose affinity chromatography. DEA/NO and NMDA almost completely abolished the interaction between eIF4G and eIF4E, and the NMDA effect was prevented by NOS inhibitors (Figure 6C).
We also studied whether calpain inhibitors influenced the phosphorylation status of 4E-BP1. Calpeptin and MDL28170 did not reverse the dephosphorylation of 4E-BP1 induced by NMDA and DEA/NO respectively (Figure 6B and Table 1). Moreover, calpeptin decreased the γ form and enhanced the formation of the α form compared with NMDA alone (Figure 6B and Table 1). When the rate of protein synthesis was measured, we observed that none of the calpain inhibitors prevented the blockade of protein synthesis induced by NMDA or DEA/NO (Figure 6D), thus suggesting that the prevention of eIF4G cleavage alone is not sufficient to abrogate the inhibition of protein synthesis. In contrast, NOS inhibitors that blocked eIF4G processing and dephosphorylation of 4E-BP1 did promote the complete recovery of translation (Figures 2B, 4C and 6B). In addition, we observed that none of the calpain inhibitors rescued neurons from death (Figure 6E), suggesting that survival is compromised when the normal rate of protein synthesis is not achieved.
Rapamycin decreases the formation of eIF4F complexes and inhibits protein synthesis in a similar way to NMDA
In order to determine whether hypophosphorylation of 4E-BP1 was by itself sufficient to decrease the formation of eIF4F complexes in the absence of eIF4G degradation, we incubated neurons with rapamycin, an inhibitor of the 4E-BP1 kinase mTOR (reviewed in ). At 3 h after addition of rapamycin, we observed a dephosphorylation of 4E-BP1, but no loss of eIF4G (Figure 7A). When we looked at the amount of eIF4G bound to eIF4E after m7GTP–Sepharose affinity chromatography, we found that rapamycin induced a dose-dependent inhibition of eIF4G binding to eIF4E (Figure 7B) that correlated with a decrease in protein synthesis (Figure 7C) of similar proportions to that achieved with NMDA (Figure 6D).
The present study investigated the mechanisms underlying excitotoxicity-mediated translation repression. In particular, we explored the contribution of eIF4G proteolysis and 4E-BP1 dephosphorylation to NMDA- and DEA/NO-induced protein synthesis inhibition. To our knowledge, this is the first study to report molecular changes in translation factors that might be involved in the long-lasting inhibition of protein synthesis after transient exposure to NMDA. We showed that neurons can withstand a transient blockade of translation with limited neuronal loss, and also provided evidence that protein synthesis recovery closely correlates with neuronal survival, as is observed in animal models of stroke . Indeed, the inactivation of NOS enabled full recovery of global protein synthesis, along with an almost complete rescue from NMDA-induced neurotoxicity; this latter finding has been reported previously by other groups [44,45]. Interestingly, NOS inhibitors were able to prevent the delayed, but not the initial, inhibition of protein synthesis following NMDA exposure. Similarly, ischaemic pre-conditioning suppresses persistent, but not acute, inhibition of translation (reviewed in ).
NOS inhibitors did not impede NMDA-induced decreased ATP levels, despite the fact that they promoted a complete recovery of protein synthesis. Therefore the moderate decrease in ATP after NMDA and DEA/NO treatments is unlikely to be the cause of protein synthesis inhibition. This result is in agreement with previously published data showing that severe protein synthesis inhibition precedes the ATP decrease after treatment with an NO donor . Furthermore, suppression of protein synthesis occurs in the penumbra where ATP levels are maintained in transient focal ischaemia . Interestingly, our data are in accordance with studies suggesting that transient inhibition of protein synthesis is not lethal, whereas the persistent phase of translation suppression is related to neurodegeneration [18,46–48].
Release of Ca2+ from ER stores through blockade of Ca2+/ATPase with thapsigargin  was shown to induce protein synthesis alterations due to phosphorylation of eIF2α. Glutamate also mobilizes Ca2+ from ER reservoirs, but depresses protein synthesis in an eIF2α-phosphorylation-independent manner , in agreement with our data. We did not detect phosphorylation of eIF2α after exposure to DEA/NO, a fast NO-releasing complex, in contrast with previous observations in neuroepithelial cells . SNAP (S-nitroso-N-acetyl-DL-penicillamine), a slow NO-releasing agent, has been shown to mobilize thapsigargin-sensitive Ca2+ ER stores in neurons  and to phosphorylate eIF2α similarly to thapsigargin in neuroblastoma . The shorter exposure time, faster NO release and lower concentration of NO donor used in the present study may explain the discrepancies with those studies. Therefore, under our experimental conditions, DEA/NO inhibited translation by a mechanism that was independent of eIF2α phosphorylation, at least in the late phase. Our data suggest that NO does not contribute to the initial inhibition of protein synthesis by NMDA, but rather takes part in delayed translation deterioration.
Calpain activation and subsequent proteolysis of its substrates is known to occur after excitotoxicity in neurons [22,39,43]. Although some studies have indicated an attenuation of NMDA toxicity with pharmacological inhibitors of calpain , other studies, including this present one, indicated no protection . Nevertheless, we found that calpain was responsible for eIF4G breakdown after exposure to NMDA or DEA/NO, in accordance with previous observations in the ischaemic brain . Proteolysis of several translation factors has been reported, mainly under apoptotic conditions. Research has shown that eIF2α and 4E-BP1 are the substrates for caspase 3 (reviewed in ), whereas eIF4E and 4E-BP1 are the substrates for calpain in vitro [12,49]. However, to the best of our knowledge, there is no evidence that eIF4E and 4E-BP1 are cleaved by calpain in vivo [12,49]. In the present study, we did not observe any degradation of eIF2α, eIF4E or 4E-BP1, suggesting that calpain specifically processed eIF4G in our experimental set-up. The eIF4G fragment generated by calpain cleavage does not bind to eIF4E, thus precluding the formation of eIF4F complexes, as shown in both the present study and a model of global cerebral ischaemia . It is therefore likely that the strong proteolysis of eIF4G contributed to impaired translation in our experimental conditions.
The binding of 4E-BP1 to eIF4E also compromises the association of eIF4F components . We observed that NMDA and DEA/NO insults led to the dephosphorylation of 4E-BP1, which is expected to facilitate its binding to eIF4E. A similar process has been described after global ischaemia and was shown to cause a deficit in eIF4E–eIF4G complexes and therefore to contribute to translation inhibition . NOS inhibitors enabled the complete recovery of protein synthesis by preventing eIF4G proteolysis and by precluding the dephosphorylation of 4E-BP1. The lack of protein synthesis recovery in the presence of a normal amount of eIF4G (in the presence of calpain inhibitors) suggests that the hypophosphorylation of 4E-BP1 is sufficient to decrease the protein synthesis rate. This result is supported by the data obtained with rapamycin. Rapamycin, which dephosphorylated 4E-BP1, but maintained eIF4G integrity, decreased the rate of protein synthesis. The rapamycin-induced inhibition of translation correlated with the decreased formation of eIF4E–eIF4G complexes. eIF4E is crucial for cap-dependent translation initiation and is a mandatory element of the eIF4F complex since it functions as the cap-binding protein. Decreased availability of eIF4E resulting from protease-mediated degradation or trapping by 4E-BP1 would severely compromise cap-mRNA translation. Therefore eIF4E is a rate-limiting factor, and its sequestration by 4E-BP1 might slow down the process of translation. The lower amount of full-length eIF4G may worsen the process by decreasing the probability of eIF4E–eIF4G binding. Our data suggest that preventing eIF4G degradation and 4E-BP1 dephosphorylation are necessary to unlock translation repression. The maintenance of eIF4G levels while 4E-BP1 is hypophosphorylated is not sufficient to prevent protein synthesis inhibition. Our results are in agreement with research showing that ischaemic tolerance, which promotes translation recovery, tends to prevent eIF4G processing and to favour the phosphorylation of 4E-BP1 [47,50].
Since eIF4G and eIF4E play a key role in the translation of cap-mRNAs, we propose that limited availability of both proteins accounts for long-lasting translation failure (summarized in Figure 8) and contributes to neuronal loss after NMDA and NO challenges. Further studies are required to unravel the mechanisms controlling 4E-BP1 dephosphorylation.
We thank Araceli Salinas and Elisabet Gomez for their excellent technical assistance and the Language Advice Service from the University of Barcelona for revising the English. V. P. is a recipient of a Ramon y Cajal grant, and M. F.-N. is a recipient of an FPI (Formación de Personal Investigador) grant from the Spanish Ministry of Education and Science (MEC). This study was supported by grants FIS05/0271 from the Spanish Ministry of Health and SAF2005-05793-C01 from the Ministry of Education and Science.
Abbreviations: AU, arbitrary units; DEA/NO, diethylamine–nitric oxide sodium complex; DIV, day in vitro; eEF, eukaryotic elongation factor; eIF, eukaryotic initiation factor; 4E-BP1, eIF4E-binding protein 1, ER, endoplasmic reticulum; FAG, fragment of apoptotic cleavage of eIF4G; LDH, lactate dehydrogenase; m7GTP, 7-methyl-GTP; MEM, minimal essential medium; mTOR, mammalian target of rapamycin; L-NAME, NG-nitro-L-arginine methyl ester; NMDA, N-methyl-D-aspartate; NOS, nitric oxide synthase; nNOS, neuronal NOS; NPLA, NG-propyl-L-arginine; PERK, double-stranded RNA-activated protein kinase-like ER kinase; PI, propidium iodide; STAT3, signal transducer and activator of transcription 3; TCA, trichloroacetic acid
- © The Authors Journal compilation © 2008 Biochemical Society