No abstract

Protein synthesis inhibition occurs in neurons immediately on reperfusion after ischemia and involves at least alterations in eukaryotic initiation factors 2 (eIF2) and 4 (eIF4). Phosphorylation of the alpha subunit of eIF2 [eIF2(alphaP)] by the endoplasmic reticulum transmembrane eIF2alpha kinase PERK occurs immediately on reperfusion and inhibits translation initiation. PERK activation, along with depletion of endoplasmic reticulum Ca2+ and inhibition of the endoplasmic reticulum Ca2+ -ATPase, SERCA2b, indicate that an endoplasmic reticulum unfolded protein response occurs as a consequence of brain ischemia and reperfusion. In mammals, the upstream unfolded protein response components PERK, IRE1, and ATF6 activate prosurvivial mechanisms (e.g., transcription of GRP78, PDI, SERCA2b ) and proapoptotic mechanisms (i.e., activation of Jun N-terminal kinases, caspase-12, and CHOP transcription). Sustained eIF2(alphaP) is proapoptotic by inducing the synthesis of ATF4, the CHOP transcription factor, through "bypass scanning" of 5' upstream open-reading frames in ATF4 messenger RNA; these upstream open-reading frames normally inhibit access to the ATF4 coding sequence. Brain ischemia and reperfusion also induce mu-calpain-mediated or caspase-3-mediated proteolysis of eIF4G, which shifts message selection to m 7 G-cap-independent translation initiation of messenger RNAs containing internal ribosome entry sites. This internal ribosome entry site-mediated translation initiation (i.e., for apoptosis-activating factor-1 and death-associated protein-5) can also promote apoptosis. Thus, alterations in eIF2 and eIF4 have major implications for which messenger RNAs are translated by residual protein synthesis in neurons during brain reperfusion, in turn constraining protein expression of changes in gene transcription induced by ischemia and reperfusion. Therefore, our current understanding shifts the focus from protein synthesis inhibition to the molecular pathways that underlie this inhibition, and the role that these pathways play in prosurvival and proapoptotic processes that may be differentially expressed in vulnerable and resistant regions of the reperfused brain.

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons, which persists in vulnerable neurons, that is caused by the inhibition of translation initiation as a result of the phosphorylation of the a-subunit of eukaryotic initiation factor 2 (eIF2a). To identify kinases responsible for eIF2a phosphorylation [eIF2a(P)] during brain reperfusion, we induced ischemia by bilateral carotid artery occlusion followed by post-ischemic assessment of brain eIF2a(P) in mice with homozygous functional knockouts in the genes encoding the heme-regulated eIF2a kinase (HRI), or the amino acidregulated eIF2a kinase (GCN2). A 10-fold increase in eIF2a(P) was observed in reperfused wild-type mice and in the HRI±/± or GCN2±/± mice. However, in all reperfused groups, the RNA-dependent protein kinase (PKR)-like endoplasmic reticulum eIF2a kinase (PERK) exhibited an isoform mobility shift on SDS±PAGE, consistent with the activation of the kinase. These data indicate that neither HRI nor GCN2 are required for the large increase in post-ischemic brain eIF2a(P), and in conjunction with our previous report that eIF2a(P) is produced in the brain of reperfused PKR±/± mice, provides evidence that PERK is the kinase responsible for eIF2a phosphorylation in the early post-ischemic brain.

Postischemic brain reperfusion is associated with a substantial and long-lasting reduction of protein synthesis in selectively vulnerable neurons. Because the overall translation initiation rate is typically regulated by altering the phosphorylation of serine 51 on the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha), we used an antibody specific to phosphorylated eIF-2 alpha [eIF-2(alpha P)] to study the regional and cellular distribution of eIF-2(alpha P) in normal, ischemic, and reperfused rat brains. Western blots of brain postmitochondrial supernatants revealed that approximately 1% of all eIF-2 alpha is phosphorylated in controls, eIF-2(alpha P) is not reduced by up to 30 minutes of ischemia, and eIF-2(alpha P) is increased approximately 20-fold after 10 and 90 minutes of reperfusion. Immunohistochemistry shows localization of eIF-2(alpha P) to astrocytes in normal brains, a massive increase in eIF-2(alpha P) in the cytoplasm of neurons within the first 10 minutes of reperfusion, accumulation of eIF-2(alpha P) in the nuclei of selectively vulnerable neurons after 1 hour of reperfusion, and morphology suggesting pyknosis or apoptosis in neuronal nuclei that continue to display eIF-2(alpha P) after 4 hours of reperfusion. These observations, together with the fact that eIF-2(alpha P) inhibits translation initiation, make a compelling case that eIF-2(alpha P) is responsible for reperfusion-induced inhibition of protein synthesis in vulnerable neurons.

We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors elF-4E, elF-4G, and elF-2a to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 mm of cardiac arrest followed by resuscitation and 90 mm of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was -~0.4 fmol of leucine/min/ 1.tg of protein and was unaffected by 10 mm of cardiac arrest, but 90 mm of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 mm of global brain ischemia nor 90 mm of reperfusion induced significant alterations in the quantity or serine phosphorylation of elF-4E. However, we observed in all 90-min-reperfused samples elF-4G fragments that also bound elF-4E. The amount of elF-2a was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated elF-2a in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated elF-2a was uniformly present after 90 mm of reperfusion and represented 24 ± 3% of the elF-2a in these samples. The serine phosphorylation of elF-2a and partial fragmentation of elF-4G observed after 90 mm of reperfusion offer an explanation for the inhibition of protein synthesis.