Brain ischemia and reperfusion associated with cardiac arrest and resuscitation allows less than 10 percent of these patients to resume their normal lives: in addition, stroke is the leading cause of serious permanent disability. Our long-term goal is to gain sufficient understanding of the injury mechanisms to allow clinically effective therapy. The investigators have found that in vulnerable cortical and hippocampal neurons the marked inhibition of protein synthesis during reperfusion is associated with a rapid and large increase in alpha-subunit-phosphorylated eukaryotic initiation factor 2 [eIF2alpha(P)], which inhibits the delivery of the first amino-acyl tRNA. Initially, eIF2alpha(P) is only in the cytoplasm, but at four hours reperfusion vulnerable neurons also exhibit prominent nuclear eIF2alpha(P) and morphology suggesting early apoptosis. The researchers propose that activity of a specific eIF2alpha kinase and/or deglycosylation of p67 (binds eIF2 and blocks kinase access) are responsible for eIF2alpha phosphorylation and that eIF2alpha(P) binds nuclear DNA and may alter expression.
The specific aims are to (1) identify the brain kinase(s) responsible for eIF2alpha phosphorylation during reperfusion, (2) characterize the effects of ischemia and reperfusion on p67, and (3) identify the DNA sequence bound by eIF2alpha(P) and characterize the effects of this interaction. The investigators plan to isolate brain eIF2alpha kinases by affinity chromatography, microsequence them and obtain their cDNAs, utilize antibodies against them to map their brain localization, and utilize antisense oligonucleotides to test their individual responsiveness in cultured cell injury models and reperfused brains. The effects of ischemia and reperfusion on total p67, glycosylated p67, and the p67 deglycosylase will be examined by immunohistochemistry. They will utilize PCR to enrich the eIF2alpha(P)-bound fraction of DNA randomers, assess binding sequence specificity by competition titration, identify the consensus sequence in clones, identify genes containing this response element by consensus sequence-primed PCR, and produce response element-regulated reporter constructs to explore the effects of eIF2alpha(P) on expression.
