Jonathon M. Sullivan - US grants

Research Training Program: Applicant: Dr. Jonathon M. Sullivan is committed to academic medicine and research in the area of brain ischemia and reperfusion. He completed his Emergency Medicine Research Fellowship at Wayne State University, and should complete his Physiology doctoral dissertation within 12 months. Institution: Research training at Wayne State University is strong. It has the nation's largest public graduate school, the third largest medical school, and is ranked in the top 2.5% of all U.S. colleges and universities by the Carnegie Classification "Research University I." The sponsors of the this application are experienced in studying the pathophysiology of brain ischemia and reperfusion and will provide Dr. Sullivan with extensive training in this area and assure that he has additional training in academic processes such as manuscript preparation, journal manuscript review, grant review for the Emergency Medicine Foundation, didactic presentations in both basic science and clinical disciplines, and clinical teaching. Research Investigation Program: It has been known since 1973 that protein synthesis is inhibited during reperfusion in selectively vulnerable neurons; however, until very recently the mechanisms involved in this were unknown. Our laboratory has found during ischemia calpain-mediated proteolysis of eIF-4G (crucial for cap- binding-dependent translation) and during reperfusion an about20-fold increase in ser-51(P)-eIF-2alpha, which inhibits the generation of eIF-2-GTP that is required for all translation initiation. Our Preliminary Data shows that we have developed an antibody specific for ser-51(P)-eIF-2alpha and that Dr. Sullivan has utilized this antibody to map after 1 hour reperfusion the accumulation of ser-51(P)-eIF-2alpha to vulnerable neurons, found evidence that insulin reduces the accumulation of ser-51(P)-eIF-2alpha in vulnerable hippocampal neurons, and developed a microautoradiographic technique to closely assess new protein synthesis in vulnerable neurons. The hypotheses proposed are that insulin administration during brain reperfusion (1) is associated with dephosphorylation of ser-51 on eIF- 2alpha and (2) will induce restoration of cap-independent translation in selectively vulnerable neurons. Dr. Sullivan will utilize an in vivo model of complete brain ischemia and reperfusion by cardiac arrest and resuscitation for time course histopathologic studies during reperfusion of (I) the location and progression of ser-51(P)-eIF- 2alpha, (ii) the location and progression of general protein synthesis, (iii) the location and progression of synthesis of HSP-70, and (iv) the effects of insulin administered at the onset of reperfusion on these parameters. Examination of the effects of insulin during reperfusion on general synthesis, cap-binding-independent synthesis of HSP-70, and neuronal levels of ser-51(P)-eIF-2alpha could connect the known neuroprotective effects of insulin directly to signal transduction mechanisms affecting translation initiation and message selection.

DESCRIPTION (Adapted from applicant's abstract): Brain ischemia/reperfusion prevents more than 90 percent of patients resuscitated from cardiac arrest from resuming their normal lives. The long-term goal is sufficient understanding of the injury mechanisms to formulate clinically effective therapy. The inhibition of protein synthesis during brain reperfusion is associated with a rapid increase in alpha-subunit phosphorylated eukaryotic initiation factor 2 [eIF2alpha(P)], which inhibits translation initiation. Insulin administration at resuscitation from cardiac arrest induces dephosphorylation of eIF2alpha(P) and rescue of protein synthesis in vulnerable neurons. Growth factors such as insulin mediate cell survival through the kinase Akt, a downstream affector of the phosphatidylinositol-3 kinase (PI3K). Akt inhibits apoptosis by phosphorylating protein substrates such as the pro-apoptotic protein Bad. Akt is also involved in the regulation of translation initiation, in part through its deactivation of glycogen synthase kinase 3beta (GSK3beta). It is proposed that therapeutic growth factor signaling through the PI3K/Akt signaling system during reperfusion results in both dephosphorylation of eIF2alpha(P) and also activation of cell survival pathways. Specific Aims are to 1) characterize the effects of 10 min cardiac arrest and various durations of reperfusion on phosphorylation of Akt, GSK3beta, Bad, eIF2alpha and the regulatory subunit of PI3K, 2) investigate the effects of insulin administration at resuscitation after 10 min cardiac arrest on phosphorylation of these proteins during reperfusion, and 3) investigate whether insulin-induced changes in eIF2alpha (P), Akt, Bad and GSK3beta are all PI3K-dependent. A rat model of cardiac arrest and resuscitation will be used to prepare brain tissue for analysis by immunoblotting and immunohistochemistry. Utilizing intracerebroventricular injection of the PI3K inhibitor, wortmannin, followed by cardiac arrest and resuscitation, it will be investigated whether the insulin-induced changes in phosphorylation states of Akt, Bad, GSK3beta and eIF2alpha(P) are all PI3K-dependent. This approach will permit integrated examination of a signaling system that may link growth factor mediated survival and translational competence in the setting of brain reperfusion.