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

Althausen, Sonja, Mengesdorf, Thorsten, Mies, Günter, Oláh, Laszlo, Nairn, Angus C., Proud, Christopher G. and Paschen, Wulf (2001) Changes in the phosphorylation of initiation factor eIF-2 alpha, elongation factor eEF-2 and p70 S6 kinase after transient focal cerebral ischaemia in mice. Journal of Neurochemistry, 78 (4), 779-787. (doi:10.1046/j.1471-4159.2001.00462.x).

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-2?, 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-2? phosphorylation by immunohistochemistry. Transient MCA occlusion induced a persistent suppression of protein synthesis. Phosphorylation of eIF-2? 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-2? 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-2? phosphorylation. Dephosphorylation of eIF-2? was not paralleled by a rise in levels of p67, a glycoprotein that protects eIF-2? from phosphorylation, even in the presence of active eIF-2? 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 short-lasting 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.

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