Changes in the phosphorylation of initiation factor eIF‐2α, elongation factor eEF‐2 and p70 S6 kinase after transient focal cerebral ischaemia in mice

Abstract

Mice were subjected to 60 min occlusion of the left middle cerebral artery (MCA) followed by 1±6 h of reperfusion. Tissue samples were taken from the MCA territory of both hemispheres to analyse ischaemia-induced changes in the phosphorylation of the initiation factor eIF-2a, the elongation factor eEF-2 and p70 S6 kinase by western blot analysis. Tissue sections from additional animals were taken to evaluate ischaemia-induced changes in global protein synthesis by autoradiography and changes in eIF-2a phosphorylation by immunohistochemistry. Transient MCA occlusion induced a persistent suppression of protein synthesis. Phosphorylation of eIF-2a was slightly increased during ischaemia, it was markedly up-regulated after 1 h of reperfusion and it normalized after 6 h of recirculation despite ongoing suppression of protein synthesis. Similar changes in eIF-2a phosphorylation were induced in primary neuronal cell cultures by blocking of endoplasmic reticulum (ER) calcium pump, suggesting that disturbances of ER calcium homeostasis may play a role in ischaemia-induced changes in eIF-2a phosphorylation. Dephosphorylation of eIF-2a was not paralleled by a rise in levels of p67, a glycoprotein that protects eIF-2a from phosphorylation, even in the presence of active eIF-2a kinase. Phosporylation of eEF-2 rose moderately during ischaemia, but returned to control levels after 1 h of reperfusion and declined markedly below control levels after 3 and 6 h of recirculation. In contrast to the only shortlasting phosphorylation of eIF-2a and eEF-2, transient focal ischaemia induced a long-lasting dephosphorylation of p70 S6 kinase. The results suggest that blocking of elongation does not play a major role in suppression of protein synthesis induced by transient focal cerebral ischaemia. Investigating the factors involved in ischaemia-induced suppression of the initiation step of protein synthesis and identifying the underlying mechanisms may help to further elucidate those disturbances directly related to the pathological process triggered by transient cerebral ischaemia and leading to neuronal cell injury.

Damage to the Endoplasmic Reticulum and Activation of Apoptotic Machinery by Oxidative Stress in Ischemic Neurons

“…CHOP expression is a representative feature of ER stress-induced cell death (Harding et al, 2000;Oyadomari et al, 2001); thus, our results strongly indicate that oxidative ER stress is involved in ischemic neuronal cell death. This is a novel finding because it is known that the upper stream of the ER stress response occurs after ischemia, but whether this is actually relevant to neuronal cell death is uncertain (Althausen et al, 2001;Kumar et al, 2001;Hayashi et al, 2003a, b). In both the ischemia models used in this study, neurons with a prominent induction of ATF-4 and CHOP underwent apoptosis, but those without it Figure 7 Change in the level of CHOP expression in mouse brains after transient focal ischemia.…”

Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy

“…Previous reports have demonstrated that the ER is involved in ischemic neuronal cell death, protein synthesis inhibition [37,38] , phosphorylation of phospho-extracellular signal-regulated kinase (PERK) [39] and eukaryotic initiation factor 2 alpha (eIF2α) [40] , depletion of the ER calcium pool [41] , accumulation of unfolded proteins in the ER [42] , and induction of the ER molecular chaperone [43] . An increasing number of studies indicates that autophagy is induced by ER stress in organisms from yeast to mammals [44,45] .…”

Inhibition of Cap-initiation complexes linked to a novel mechanism of eIF4G depletion in acute myocardial ischemia

“…These data are consistent with previous reports that show no change in phosphorylation of eIF2a in cerebral ischemia. 10,13,34 As the 12 h ischemic samples are similar to the 6 h time points, this time point has been omitted from the remaining studies to be presented.…”

“…While initially surprising given reports of eIF2a phosphorylation in translational control of tissue culture cell hypoxia, 45 these results are consistent with previous reports in cerebral ischemia. [9][10][11]13 It is hypothesized that during ischemia, depletion of ATP is so severe that it may inactivate mechanisms that promote eIF2a phosphorylation. 11 It is widely accepted that eIF2a phosphorylation plays a significant role in translation inhibition during reperfusion, but this mechanism cannot account for suppression of protein synthesis during cerebral ischemia [9][10][11]13 or myocardial ischemia (as shown here).…”

“…[6][7][8] The mechanisms underlying protein synthesis inhibition during ischemia have been extensively studied in models of both transient focal and global cerebral ischemia. [9][10][11][12][13] In contrast, very little research has been conducted to understand the mechanisms of translation inhibition during myocardial ischemia. 6,14 Regulation of protein synthesis can occur through a number of different mechanisms involving both initiation and elongation.…”