The neurological complications following cerebral ischemic events (i.e. stroke) produce severe clinical disease. Our long term goal is thus to elucidate mechanisms following ischemia that produce clinical disease to potentially identify novel therapeutic strategies. Ischemia and reperfusion cause immediate and global arrest of protein synthesis in all ischemia-affected neurons. Initial protein synthesis inhibition (PSI) is either followed by near complete restoration of protein synthesis or there is a persistence of PSI. It is this persistent PSI that is highly correlated with neurodegeneration and eventual neuronal death. Thus our working hypothesis is that persistent PSI mediates cell death following cerebral ischemia. Our specific hypothesis is that proteolytic cleavage of eukaryotic initiation factor 4G (elF4G) by proteases such as calpain may mediate persistent PSI, and prevention of elF4G cleavage may block persistent PSI and thus provide neuroprotection against ischemic injury. This hypothesis is based on the following data: 1) elF4G cleavage begins prior to decreases in other protein synthesis initiation factors in vitro and in vivo, 2) the time course of cleavage is correlated with that of persistent PSI in vitro, 3) in vitro ischemia results in the production of a novel size-specific cleavage product, and 4) inhibition of calpain activity by overexpression of its endogenous inhibitor calpastatin partially restores protein synthesis following in vitro ischemia. As a result of these data, our specific focus is to determine the role of calpain in elF4G cleavage and to identify the role of elF4G cleavage in persistent PSI and cell death.
Our specific aims are: 1) to test the hypothesis that elF4G cleavage and persistent PSI after in vitro ischemia is mediated by calpain activation. We will i) determine if elF4G cleavage is prevented following in vitro ischemia (i) by overexpression the endogenous calpain inhibitor calpastatin and (ii) using neurons derived from calpain knock out mice. We will also (iii) determine if prevention of elF4G cleavage via calpain inhibition can rescue protein synthesis following in vitro ischemia. 2) To test the hypothesis that prevention of elF4G cleavage alleviates persistent PSI and confers neuroprotection against in vitro ischemia. We will (i) overexpress elF4G to attempt to restore baseline elF4G levels, (ii) determine if overexpression of elF4G will confer neuroprotection following in vitro ischemia and (iii) identify the cleavage sites of elF4G by calpain using recombinant proteins in vitro.
Ultimately, the delineation of the mechanism governing persistent PSI may lead to a novel therapeutic strategy for cerebral ischemia.
| Stetler, R Anne; Gao, Yanqin; Zhang, Lili et al. (2012) Phosphorylation of HSP27 by protein kinase D is essential for mediating neuroprotection against ischemic neuronal injury. J Neurosci 32:2667-82 |
| Vosler, Peter S; Gao, Yanqin; Brennan, Christopher S et al. (2011) Ischemia-induced calpain activation causes eukaryotic (translation) initiation factor 4G1 (eIF4GI) degradation, protein synthesis inhibition, and neuronal death. Proc Natl Acad Sci U S A 108:18102-7 |
| Vosler, Peter S; Graham, Steven H; Wechsler, Lawrence R et al. (2009) Mitochondrial targets for stroke: focusing basic science research toward development of clinically translatable therapeutics. Stroke 40:3149-55 |
| Vosler, Peter S; Sun, Dandan; Wang, Suping et al. (2009) Calcium dysregulation induces apoptosis-inducing factor release: cross-talk between PARP-1- and calpain-signaling pathways. Exp Neurol 218:213-20 |
