Er Stress

The early damage to the ER and Golgi apparatus and the loss of ribosomes preceding mitochondrial damage indicate that the ER is a major target for oxidative damage during reperfusion (113,114). Folding of newly synthesized proteins requires high levels of Ca2+ in the ER lumen. Depletion of ER Ca2+ during reperfusion and oxidative damage to proteins and the ER lipid membrane result in accumulation of unfolded proteins (Fig. 2). The normal homeostatic response to accumulation of unfolded proteins is to decrease translation by regulation of eukaryotic initiation factor 2a (eIF2a) and to increase synthesis of chaperone proteins until unfolded proteins no longer accumulate (115). The chaperone protein glucose-regulated protein-78 (GRP78) binds to the unfolded proteins. With excess unfolded proteins, GRP78 dissociates from two kinases in the ER membrane: RNA-dependent protein kinase-like ER eIF2a kinase (PERK) and inositol-requiring enzyme (IRE1). As a result of GRP78 dissociation, PERK is activated and phosphorylates eIF2a,

Figure 2 ER stress after cerebral ischemia results in accumulation of unfolded/misfolded proteins in the ER lumen because of oxidative damage and loss of the high intralumen Ca2+. GRP78 in the ER membrane dissociates from PERK and IRE1 and binds to the excess of unfolded proteins in the lumen. Dissociated PERK forms dimers, which phosphorylate eIF2a and inhibit general protein synthesis. Restoration of protein synthesis requires dephosphorylation of eIF2a by GADD34, which, in turn, requires a minimal amount of intact translation machinery for expression of new protein after damage by ischemia. Dissociated IRE1 forms dimers that cleave X-box binding protein mRNA into a splice variant that acts as a transcription factor on chaperone genes. Proper folding of proteins requires expression of chaperone proteins, which, in turn, requires a minimal amount of intact translation machinery after damage by ischemia. Long-term survival of selective neurons depends on ability to express GADD34 and chaperone proteins. Abbreviations: ER, endoplasmic reticulum; PERK, protein kinase-like ER elF2a kinase; IRE1, Inositol-requiring enzyme; elF2a, eukaryotic initiation factor 2a; GRP78, glucose-regulated protein-78.

Figure 2 ER stress after cerebral ischemia results in accumulation of unfolded/misfolded proteins in the ER lumen because of oxidative damage and loss of the high intralumen Ca2+. GRP78 in the ER membrane dissociates from PERK and IRE1 and binds to the excess of unfolded proteins in the lumen. Dissociated PERK forms dimers, which phosphorylate eIF2a and inhibit general protein synthesis. Restoration of protein synthesis requires dephosphorylation of eIF2a by GADD34, which, in turn, requires a minimal amount of intact translation machinery for expression of new protein after damage by ischemia. Dissociated IRE1 forms dimers that cleave X-box binding protein mRNA into a splice variant that acts as a transcription factor on chaperone genes. Proper folding of proteins requires expression of chaperone proteins, which, in turn, requires a minimal amount of intact translation machinery after damage by ischemia. Long-term survival of selective neurons depends on ability to express GADD34 and chaperone proteins. Abbreviations: ER, endoplasmic reticulum; PERK, protein kinase-like ER elF2a kinase; IRE1, Inositol-requiring enzyme; elF2a, eukaryotic initiation factor 2a; GRP78, glucose-regulated protein-78.

which shuts down translation and leads to disaggregation of polyribosomes. In addition, IRE1 is activated and cuts X-box binding protein (xbp1) mRNA, which leads to synthesis of a variant protein that acts as a transcription factor for inducing ER chaperone proteins (115 ).

Evidence for ER stress after global cerebral ischemia includes increased accumulation of unfolded protein in the ER lumen, decreased binding of GRP78 to PERK, phosphorylation of eIF2a by PERK, and inhibition of protein synthesis (116,117). Markers of ER stress are reduced in animals with overexpression of Cu,Zn-SOD (118,119), consistent with a key role of oxygen radicals in initiating the stress response. However, depending on the severity and duration of ischemia, the homeostatic unfolded protein response may not be fully executed, and some neurons will eventually die. For example, the variant form of processed xbp1 may not be expressed because of damage to the ER machinery (120,121). Consequently, restitution of chaperone proteins may be impaired. Moreover, eIF2a is normally dephosphorylated by GADD34, and although GADD34 mRNA is increased after ischemia, protein expression of GADD34 remained suppressed in vulnerable regions (122). Thus, oxidative damage to the ER may limit its ability to synthesize the new proteins necessary to repair the dysfunctional ER. Persistent phosphorylation of eIF2a by the lack of GADD34 and activation of mitochondrial-dependent apoptosis occur in the same neurons that are destined to die (123). In addition to inhibiting translation, phosphorylation of eIF2a also leads to induction of activating transcription factor-4 (ATF-4) and proapoptotic C/EBP-homologous protein (CHOP). Induction of ATF-4 and CHOP was reduced after ischemia in animals that overexpressed Cu,Zn-SOD (119), again emphasizing the role of reactive oxygen species in ER stress. Although blocking synthesis of specific proteins during early reperfusion might be beneficial for cell survival, persistent ER stress and suppression of overall protein synthesis will lead to dysfunction of neurons in which proteins typically turnover every 1 to 2 days. Crosstalk between ER and mitochondria probably plays an important role in controlling cell death pathways after ischemia.

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