Characterization of the eIF2-associated protein p67 during brain ischemia and reperfusion.

OBJECTIVES: Within the first few minutes of reperfusion after global brain ischemia, there is a severe depression of protein translation owing to phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2). There is a 67 kDa peptide (p67) that, in its glycosylated form, binds to eIF2 and protects eIF2alpha from phosphorylation. Moreover, cells with high p67 content exhibit enhanced resistance to eIF2alpha phosphorylation. To examine the possibilities that deglycosylation of brain p67 occurs during ischemia and/or early reperfusion or that p67 deglycosylation may be more extensive in the vulnerable neurons, these experiments were undertaken to characterize the localization and activation state of p67 during early brain reperfusion METHODS: Western blots using antibodies that recognize total p67, glycosylated p67 and phosphorylated eIF2alpha were used to characterize total p67 and glycosylated p67 during reperfusion-induced phosphorylation of eIF2alpha. We also characterized the immunohistochemical distribution of glycosylated p67 before and after brain ischemia and reperfusion. RESULTS: There was a large increase in phosphorylated eIF2alpha, but there was no decrease in the levels of total or glycosylated p67 from those observed in controls following 10 minutes complete brain ischemia and 10 or 60 minutes subsequent reperfusion. Furthermore, there was no reduction in localized immunostaining for glycosylated p67 in vulnerable neurons during ischemia and reperfusion. DISCUSSION: It does not appear that p67 plays a significant role in regulating the phosphorylation of eIF2alpha following transient brain ischemia.

Persistent eIF2alpha(P) is colocalized with cytoplasmic cytochrome c in vulnerable hippocampal neurons after 4 hours of reperfusion following 10-minute complete brain ischemia.

Upon brain reperfusion following ischemia, there is widespread inhibition of neuronal protein synthesis that is due to phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha), which persists in selectively vulnerable neurons (SVNs) destined to die. Other investigators have shown that expression of mutant eIF2alpha (S51D) mimicking phosphorylated eIF2alpha induces apoptosis, and expression of non-phosphorylatable eIF2alpha (S51A) blocks induction of apoptosis. An early event in initiating apoptosis is the release of cytochrome c from mitochondria, and cytochrome c release corresponds to the selective vulnerability of hippocampal CA1 neurons in rats after transient global cerebral ischemia. At present the signaling pathways leading to this are not well defined. We hypothesized that persistent eIF2alpha(P) reflects injury mechanisms that are causally upstream of release of cytochrome c and induction of apoptosis. At 4 h of reperfusion following 10-min cardiac arrest, vulnerable neurons in the striatum, hippocampal hilus and CA1 showed colocalized intense immunostaining for both persistent eIF2alpha(P) and cytoplasmic cytochrome c, while resistant neurons in the dentate gyrus and elsewhere did not immunostain for either. A lower intensity of persistent eIF2alpha(P) immunostaining was present in cortical layer V pyramidal neurons without cytoplasmic cytochrome c, possibly reflecting the lesser vulnerability of this area to ischemia. We did not observe cytoplasmic cytochrome c in any neurons that did not also display persistent eIF2alpha(P) immunostaining. Because phosphorylation of eIF2alpha during early brain reperfusion is carried out by PERK, these findings suggest that there is prolonged activation of the unfolded protein response in the reperfused brain.

Molecular pathways of protein synthesis inhibition during brain reperfusion: implications for neuronal survival or death.

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.