Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

Abstract

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) 2alpha 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 eIF2alpha 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.

SIRT1 Regulation Modulates Stroke Outcome

“…Indeed, ischemia causes the transient inhibition of translation, partly due to changes in the phosphorylation of several translation factors (eIF2α and 4E-BP). The recovery of protein synthesis during the reperfusion is essential to neuron survival whereas the persistent blockage of translation is lethal [76][77][78]. SIRT1 inhibition with NAM or sirtinol, as well as SIRT1 silencing, prevented cell death in cultured neurons subjected to oxidative stress [79] through reducing the IGF-1/Erk1/2 signaling pathway.…”

Calpain-Mediated Signaling Mechanisms in Neuronal Injury and Neurodegeneration

“…Decreases in eIF4G levels were demonstrated to be calpain-mediated following cardiac arrest, as the pharmacological calpain inhibitor, MDL-28,170, abrogated diminished protein levels [61]. Calpain-mediated decreases in eIF4G were also shown following NMDA toxicity in primary neuronal culture as the calpain inhibitor calpeptin also blocked reductions in eIF4G protein levels [62]. Reduced eIF4G levels were correlated with inhibition of protein synthesis; however, in this model, although calpain inhibition was neuroprotective, it did not restore protein synthesis, leaving the significance of eIF4G reduction for neuronal survival in excitotoxicity unknown.…”

Estimation of the effective orientation of the SHG source in primary cortical neurons

“…Primary cortical neuron cultures were prepared from 18-day-old Sprague-Dawley rat embryos (Charles River Laboratories) as described previously [24]. Briefly, animals were anaesthetized and sacrificed by cervical dislocation.…”