Brain ischemia and reperfusion injury prevents greater than 90% of the 70,000 patients per year resuscitated from cardiac arrest from resuming their normal lives. Our long-term goal is sufficient understanding of the injury mechanisms to formulate clinically effective therapy. Inhibition of protein synthesis during brain reperfusion correlates with regional selective vulnerability and neuronal death, and is due to modification of two translation initiation factors: the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), and the proteolytic fragmentation of eukaryotic initiation factor 4G (eIF4G). eIF2 phosphorylation and eIF4G fragmentation affect not only the overall protein synthesis rate, but also which peptides are synthesized from the available mRNAs. Moreover, the kinase that phosphorylates eIF2alpha immediately after brain ischemia and reperfusion, PERK, is known to be activated only by the endoplasmic reticulum stress signaling system termed the unfolded protein response (UPR). The UPR can signal either an adaptive pro-survival response, or it can trigger cell death. Thus suppression of protein translation is likely to be part of a more comprehensive cellular response that determines the ultimate fate of reperfused neurons. We hypothesize: (1) the UPR is activated during early brain reperfusion, (2) vulnerable, but not resistant, neurons fail to resolve the UPR, and (3) there is synthesis of only a limited number of proteins during early reperfusion, as a consequence of eIF2alpha phosphorylation and eIF4G fragmentation, that may determine the outcome of neuronal recovery or death.
Our Specific Aims are the following.
Aim I will compare in ischemia and reperfusion vulnerable and resistant brain regions the activation of the UPR by characterizing activation of its three effectors ATF6, IRE1alpha, and PERK.
Aim 2 will examine in ischemia and reperfusion vulnerable and resistant brain regions whether the UPR is resolved (by determining if synthesis of the pro-survival proteins GRP78, XBP-1, GADD34 and SERCA2b occurs), or if the UPR fails to resolve (by determining if synthesis of the pro-cell death proteins ATF4 and CHOP occurs).
Aim 3 will identify those proteins being synthesized by residual translation during the early hours of reperfusion and compare them between ischemia and reperfusion vulnerable and resistant brain regions. This approach provides an integrated examination during brain ischemia and reperfusion of: (1) the occurrence and the consequences of UPR activation, (2) the consequences of translation initiation factor alterations on residual protein synthesis, and (3) the relationship of these two events to the selective vulnerability of the brain to ischemia and reperfusion injury.
