Gary Krause - US grants

This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. More than 90% of the approximately 70,000 patients resuscitated from a cardiac arrest each year suffer permanent, often severe, brain injury as a sequela to the ischemia associated with the cardiac arrest. The broad, long-term objectives of our work are to understand the damage mechanisms operative in brain ischemia and reperfusion, and thereby identify clinically effective therapeutic interventions to forestall the frequent occurrence of severe brain injury. In those brain regions most vulnerable to damage by ischemia and reperfusion, neurons exhibit a substantial inhibition of post-ischemic protein synthesis. We propose that inhibition of protein synthesis (1) occurs at the level of formation of the initiation complex, (2) as a consequence of proteolysis and oxygen radical reactions, (3) which decrease levels or alters phosphorylation of specific translation initiation factors. We further propose that (4) translation competence during reperfusion can be protected or restored by either (a) blocking proteolysis, (b) providing the active initiation factor(s) involved (in vitro) or (c) by growth factor-mediated reversal of the reperfusion- induced phosphorylation alterations of initiation factors. The specific aims of this project are to (1) examine the effect of global brain ischemia and reperfusion on the rate of initiation of protein translation, (2) examine the levels, phosphorylation state, activity, and regional localization of key translation initiation factors before and after global brain ischemia and reperfusion, (3) examine the effects of calcium overload or radical damage to cultured neurons with respect to the above parameters, (4) examine the effects of adding purified phosphorylated/dephosphorylated initiation factor(s) on in vitro protein translation in brain homogenates obtained from normal and reperfused animals and on radical-damaged or calcium overloaded cultured neurons (5) and study the effects of an exogenous growth factor on initiation factor phosphorylation and translation competence in the cell culture model and in the reperfused brain. The experimental design (1) utilizes an animal model of cardiac arrest and resuscitation to characterize the effects of global brain ischemia and reperfusion on those initiation factors that control the initiation of protein synthesis (eIF-2, eIF-2B, eIF-4E, and EIF-4 gamma), and (2) utilizes a neuronal cell culture system (derived from induced differentiation of NB104 cells) to characterize the effects of a controlled radical or calcium overload insult on these same parameters. Because the growth factor insulin has been shown to improve neurologic outcome after brain ischemia and induce stimulation of the translation system through mechanisms including dephosphorylation of initiation factors, we will also study the effects of insulin administration on translation competence and initiation factors in both model systems.

DESCRIPTION (Adapted from applicant's abstract): 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.