Molecular Pathways of Protein Synthesis Inhibition during Brain Reperfusion: Implications for Neuronal Survival or Death

Brain ischemia and reperfusion induce inhibition of translation initiation through molecular alterations in eIF2 and eIF4

The characteristics of postischemic PSI have been amply documented (Hossmann, 1993; Krause and Tiffany, 1993). Immediately on reperfusion after global ischemia, protein synthesis is inhibited in neurons throughout the entire brain. Dependent on the duration of ischemia, protein synthesis will gradually recover in brain regions more resistant to ischemic damage, but remains suppressed in selectively vulnerable neurons. Ischemic preconditioning can attenuate postischemic PSI (Kato et al., 1995), and the use of cycloheximide to block protein synthesis during preconditioning abolishes the subsequent neuroprotective effects (Barone et al., 1998). In the most vulnerable brain region, the CA1 of the hippocampus, protein synthesis never recovers fully during reperfusion even after relatively brief ischemia (Araki et al., 1990; Thilmann et al., 1986). Thus, a number of investigators have suggested that persistent PSI is a marker for neurons destined to undergo delayed neuronal death in the reperfused brain (Hossmann, 1993; Krause and Tiffany, 1993; White et al., 2000; Paschen, 2000). As we will see, our current understanding of the UPR and the regulation of translation initiation provide potential causal pathways for this inference.

In their seminal description of postischemic PSI, Kleihues and Hossmann (1971) observed polysome disaggregation and accumulation of free monosomes and implicated inhibition of translation initiation as the mechanism of PSI. It is now accepted that PSI during brain reperfusion is caused by inhibited translation initiation that involves modifications to eIF2 and eIF4 (Krause and Tiffany, 1993; DeGracia et al., 1996).

Roles of eIF2 and eIF4 in translation initiation.

In eukaryotes, translational control at initiation is coordinated by eIFs that orchestrate the assembly of ribosomal subunits, the messenger RNA (mRNA) to be translated, and the methionine-charged initiator transfer RNA (Met-tRNA_i) (Pain, 1996). A ternary complex comprising eIF2, Met-tRNA_i, and GTP delivers the peptide-initiating methionine to the 40S ribosomal subunit (Fig. 1), and this step of translation initiation controls the gross rate of protein synthesis (Pain, 1996). At a later stage of initiation, hydrolysis of this GTP generates an eIF2-GDP complex, and eIF2B (guanine nucleotide exchange factor) must catalyze the removal of this GDP for eIF2 to bind another GTP and participate in another round of translation initiation (Rowlands et al., 1988). The eIF2 containing an α-subunit phosphorylated on Ser-51 is a competitive inhibitor of eIF2B (Rowlands et al., 1988); thus, increased levels of eIF2α(P) downregulate the rate of global protein synthesis (Pain, 1996). Indeed, because the ratio of eIF2 to eIF2B is approximately 5:1 in the brain (Alcàzar et al., 1995), translation initiation becomes substantially inhibited when approximately 20% of eIF2 is phosphorylated (Pain, 1996). The inhibition of eIF2B by eIF2(αP) is an equilibrium process, so a small amount of ternary complex will remain available for residual translation even when eIF2(αP) levels are high. Phosphorylation of eIF2α occurs in response to diverse cellular stresses, including the UPR (Ron and Harding, 2000), viral infection (Kaufman, 2000), heme depletion in reticulocytes (Chen, 2000), and nutrient deprivation (Kimball and Jefferson, 2000), and occurs rapidly during postischemic brain reperfusion.

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FIG. 1.

Abbreviated steps in translation initiation showing the roles of eIF2 and eIF4. The GTP-bound eIF2 delivers the methionyl-charged initiator transfer RNA to the 40S ribosomal subunit as a ternary complex. At the end of initiation, the GTP on eIF2 is hydrolyzed, producing inactive eIF2-GDP. Active eIF2 is recycled by the guanine nucleotide exchange factor, eIF2B. Phosphorylation of the α subunit of eIF2 leads to sequestration of eIF2B, accumulation of eIF2-GDP, and inhibition of protein synthesis by lack of delivery of the first amino acid. eIF4 is a tetramer, eIF4E recognizes the m⁷-G cap of capped messenger RNAs, eIF4A and eIF4B possess RNA helicase activity and allow ATP-dependent scanning along the 5′ untranslated region of the messenger RNA to the AUG start codon, and eIF4G is a large scaffolding protein mediating interactions of the various translation initiation components. Proteolytic fragments of eIF4G can initiate translation in the absence of eIF4E by binding IRES in the 5′ untranslated region.

The eIF4F complex that delivers the mRNA to the 40S ribosomal subunit (Fig. 1) also regulates translation initiation (Pain, 1996). The eIF4F consists of the following three subunits: (1) eIF4E, which binds the m⁷G-cap of the mRNA, (2) eIF4A, an mRNA helicase and ATPase, and (3) eIF4G, a scaffolding protein with binding sites for eIF4E, eIF3, and polyA binding protein. After binding the eIF4F complex with eIF4B, a cofactor of eIF4A, the small ribosomal subunit uses ATP to scan the 5′ untranslated region of the mRNA until it encounters the AUG start codon; the factors affecting recognition of the start codon are complex and can involve cis and trans factors at the 5′ and 3′ ends of the mRNA (Gray and Wickens, 1998). Phosphorylation of eIF4E on Ser-53 increases its binding to eIF4G (Lamphear and Panniers, 1990), and eIF4E activity is also controlled by the phosphorylation state of the eIF4E-binding proteins, which sequester eIF4E when unphosphorylated (Gingras et al., 1999). The eIF4G is also phosphorylated at multiple sites, but the function of this phosphorylation is unknown (Yan et al., 1992). Increasing the quantity or activity of eIF4F enhances the translation of mRNAs that are in low abundance (e.g., transcription factors, growth factor receptors) or have complex secondary structures (Gray and Wickens, 1998).

I/R-induced alterations of eIF2 and eIF4 are expected to cause major changes in the repertoire of new peptides synthesized by residual translation in vulnerable neurons

In simultaneous immunohistochemical examination of eIF2α(P) and quantitative autoradiographic assessment of protein synthesis in CA1 neurons at 90-minute reperfusion, approximately 15% of the control level of protein synthesis and intense eIF2α(P) immunostaining were observed (Sullivan et al., 1999). There has been tremendous focus on altered gene expression during I/R, and it is clearly important to determine if these transcripts are being translated (Read et al., 2001). However, protein expression levels generally cannot be determined from quantitative mRNA data (Gygi et al., 1999). Moreover, during cerebral I/R, residual protein synthesis occurs in an environment of altered translation initiation, which poses significant constraints on the repertoire of proteins synthesized.

The levels of eIF2α(P) seen during the first hours of reperfusion not only greatly reduce the overall rate of translation, but can also cause paradoxically enhanced translation of some proteins by the process of “bypass-scanning” (Dever et al., 1995) (Fig. 2). In yeast, the mRNA for the transcription factor GCN4 is polycistronic with a long 5′ leader containing three short ORFs followed by the actual code for GCN4 in the last ORF. Normally, the ready availability of eIF2-GTP–Met-tRNA_i complexes allows the translation initiation at the ORF nearest the 5′ terminal followed by the disassembly of the translation complex at the stop codon of this ORF, thereby precluding the synthesis of GCN4. However, with increased levels of eIF2(αP), the ribosome is less likely to find a competent ternary complex in time to initiate translation at the upstream ORFs (Dever et al., 1995). The probability of a ternary complex binding to the small ribosomal subunit is enhanced over longer scanning times; thus, GCN4 is translated because initiation does not occur on the ORFs nearer the 5′ end of the message (Dever et al., 1995). An important mammalian transcript translated by eIF2α(P)-mediated bypass scanning is ATF4 (Harding et al., 2000a), the transcription factor for apoptosis-inducing CHOP (GADD153) (Zinszner et al., 1998).

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FIG. 2.

Schematic representation showing how high levels of eIF2α(P) mediate bypass scanning on messenger RNAs with 5′ uORFs. (A) When eIF2α(P) levels are low, ternary complex (TC) availability is high, allowing a scanning ribosome (yellow) to initiate at an uORF (blue), synthesize a short ORF peptide, and terminate translation, thereby precluding translation of the actual protein-coding sequence (green). (B) When eIF2α(P) levels are high and TC levels are greatly reduced, the ribosome without a TC scans through uORFs, and prolonged scanning of the rest of the 5′ leader increases the probability of the ribosome binding a TC before reaching the authentic AUG start codon (green). Examples of such messenger RNAs include yeast GCN4 and mammalian ATF4.

Fragmentation of eIF4G can also induce large changes in which mRNAs are translated. Loss of intact eIF4G can cause failed docking of eIF4E-bound m⁷G-capped mRNAs to the small ribosomal subunit during translation initiation. This extensively alters mRNA selection by favoring only those messages that contain an IRES (Gingras et al., 1999), which do not require the cap-binding mechanism to load onto ribosomes. The issue of IRES-sequence characteristics is complex and few of the known IRES sequences exhibit consensus; furthermore, there is an unpredictable involvement of idiosyncratic cofactors that affect IRES function (Hellen and Sarnow, 2001). For example, the ER chaperone GRP78 is vital for UPR resolution. Although its transcript has an IRES, a polypyrimidine-binding protein known to upregulate IRES-dependent translation of several other transcripts downregulates GRP78 translation (Hellen and Sarnow, 2001). Nevertheless, poliovirus research has shown the truly massive effect of eIF4G degradation on mRNA selection. The poliovirus uses proteolysis of eIF4G to shut off translation of most constitutive host mRNAs while IRES-containing viral mRNAs are translated (Johannes et al., 1999). According to complimentary DNA microarray assays, there is little change in the total levels of host mRNAs when eIF4G is fragmented, but approximately 97% of host mRNAs no longer appear on ribosomes whereas there is a substantial enrichment of approximately 3% of host mRNAs (Johannes et al., 1999).

Involvement in IRES-mediated translation by truncated eIF4G isoforms lacking the eIF4E binding domain may be greater than just the exclusion of capped messages (Henis-Korenblit et al., 2000). Caspase-3 cleaves eIF4G at multiple sites but leaves a stable 76-kd “middle fragment” that may continue to play a significant role in message selection during apoptosis (Hellen and Sarnow, 2001). Truncated eIF4G isoforms can also be generated by transcription of separate genes. For example, death-associated protein-5 (DAP5) is an eIF4G isoform that lacks the eIF4E binding domain, and on caspase-mediated removal of 11 kd of its C-terminal, DAP5 is activated to facilitate IRES-initiated translation of mRNAs, including its own (Henis-Korenblit et al., 2000). This mechanism maintains a high level of DAP5 during caspase-mediated cell death. Another important example of mammalian IRES-mediated translation is the synthesis of apoptosis-activating factor-1, which occurs only by an IRES-mediated mechanism, again serving to maintain sufficient levels of this protein even when cap-dependent translation initiation mechanisms are compromised (Coldwell et al., 2000). Indeed, PSI involving both initial eIF2α phosphorylation and proteolytic fragmentation of eIF4G now appears to be stereotypical for apoptosis induced by a variety of mechanisms (Marissen and Lloyd, 1998; Morley et al., 2000).

Although there have been numerous studies of transcripts induced by I/R, the predicted large shift in the repertoire of peptides synthesized by residual translation in reperfused vulnerable neurons necessitates substantial additional proteomic investigation because the real message is indeed in the translation (Richter and Theurkauf, 2001). In this regard, some experiments have examined the whole hippocampus (Kiessling et al., 1986; Wengenack et al., 1998). The work of Kiessling et al. (1986) found enhanced synthesis of proteins with mobilities of 110, 70, 65, 50, and 27 kd at 3-hour reperfusion after 30-minute 4VO. Although these studies asked prescient questions, inclusion of the entire hippocampus introduced resistant regions (DG, CA2, and much of CA3). Compared with the CA1, these regions (1) have initially less eIF2α(P) and substantially more clearance of this isoform by 90-minute to 4-hour reperfusion (DeGracia et al., 1997), (2) display less evidence of activation of μ-calpain (Roberts-Lewis et al., 1994) and caspase-3 (Chen et al., 1998) and are thus likely to have more limited or even absent eIF4G degradation, and (3) have better preservation of global protein synthesis (Thilman et al., 1986). Currently available tools are adequate for the identification of pulse-labeled proteins from a single brain ventral median nucleus (Mobbs et al., 1989), and the use of this technology to identify the repertoire of proteins produced in the reperfused CA1 by residual translation is now urgent.

Phosphorylation of eIF2α during initial reperfusion is caused by the UPR elicited by stress to the ER

With recognition of the rapidity and extent of I/R-induced eIF2α phosphorylation, it became important to understand the mechanism leading to eIF2α(P) formation because this would likely provide insight into a stress pathway leading to a major component of PSI. The eIF2α can become phosphorylated by the following three mechanisms: (1) deglycosylation of the eIF2-binding protein p67 that is permissive for eIF2α phosphorylation (Gupta et al., 1997) (2) inhibition of an eIF2α(P) phosphatase, or (3) activation of an eIF2α kinase.

Using Western blot analysis, we did not observe the loss or deglycosylation of p67 associated with global brain I/R, and using immunohistochemistry we also found no loss of glycosylated p67 from vulnerable neurons (Owen et al., 2001). Althausen et al. (2001) recently confirmed no change in p67 glycosylation in the focal ischemia model.

The in vivo eIF2α(P) phosphatase is protein phosphatase 1 (Redpath and Proud, 1990). We found in our global ischemia model no difference (based on multipoint time courses) in the rate of in vitro dephosphorylation of exogenous eIF2(αP) by rat forebrain homogenates from control, ischemic, and reperfused animals (DeGracia et al., 1998). In a reperfusion time course obtained after 30-minute 4VO, Martin de la Vega et al. (2001b) reported a modest and transient I/R-induced reduction of exogenous eIF2α(P) dephosphorylation during in vitro reactions assayed at a single 15-minute reaction time point. This latter study failed to show a correlation between eIF2α(P) phosphatase activity and endogenous levels of eIF2α(P). Our more detailed kinetic study suggests that loss of eIF2α(P) phosphatase activity may not have a major causal role in the accumulation of eIF2α(P) during reperfusion.

There is, however, a fundamental methodologic concern regarding these studies that may account for their inconsistent results. The protein phosphatase 1 catalytic subunit activity against any given substrate is regulated primarily through targeting subunits specific to the substrates (Lee et al., 1999). The aforementioned studies necessarily used brain homogenates with the attendant disruption of subcellular molecular localization in all of the different neuronal, glial, and vascular components of the tissue. This fact, taken together with the targeting subunit issue, argues that without confirmation of distinct I/R-induced posttranslational modification of an eIF2α(P) phosphatase or a targeting subunit in neurons displaying eIF2α(P), the results of gross activity assays must be viewed with substantial caution. Indeed, the in vivo activities of phosphatases or kinases could easily be masked during in vitro activity assays in gross homogenates.

Assessment of the effect of I/R on gross eIF2α kinase activity in brain homogenates has not been more compelling than that of the eIF2α(P) phosphatase assays. In general, the effect of I/R has been seen as a marginal increase (Burda et al., 1998) or an actual decrease (DeGracia et al., 1998; Martin de la Vega et al., 2001b) in in vitro eIF2α kinase activity of gross brain homogenates at reperfusion times when eIF2α(P) levels are increasing severalfold in vivo. Therefore, it is essential to directly inspect the individual eIF2α kinases.

Genetic and biochemical analyses indicate that there are only four homologous mammalian eIF2α kinases: GCN2 (Kimball and Jefferson, 2000), HRI (Chen, 2000), PKR (Kaufman, 2000), and PERK (Ron and Harding, 2000). The amino acid-regulated eIF2α kinase GCN2 is expressed in mammalian brain (Sood et al., 2000), and potential accumulation of uncharged transfer RNAs consequent to lowered ATP levels in ischemic neurons might activate GCN2. HRI coordinates globin synthesis with heme availability in erythroid cells. Although there appears to be little HRI in the brain (Pal et al., 1991), its activation by nitric oxide (Uma et al., 2001), heavy metals such as iron (Hurst et al., 1987), oxidized glutathione, and polyunsaturated fatty acids and lipoperoxides (De Herreros et al., 1985) rendered HRI an inviting candidate for mediating I/R-induced eIF2α phosphorylation. PKR is activated not only by viral nucleotides but also by heparin and is widely distributed in various tissues, including the brain (Haines et al., 1993). PERK (or PEK-pancreatic eIF2α kinase) spans the ER membrane, and recognition of unfolded luminal peptides by GRP78 dissociation from the PERK N-terminal domain causes activation of the eIF2α kinase in the cytoplasmic C-terminal domain (Bertolotti et al., 2000). PERK is most highly expressed in pancreas, but is also expressed in brain (Shi et al., 1998).

When the effect of homozygous eIF2α kinase functional knockouts on I/R-induced eIF2α(P) was studied, PKR-, HRI-, and GCN2-knockout mice showed no reduction of reperfusion-induced eIF2α(P) (DeGracia et al., 1998; Kumar et al., 2001). It was not possible to analyze PERK knockouts because these mice die from type I-like diabetes within 6 to 8 postnatal weeks due to apoptosis of pancreatic β and acinar cells (Harding et al., 2001). However, PERK activation by autophosphorylation induces a characteristic sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shift (Harding et al., 1999). We found that brain I/R always induced a shift in PERK mobility from the inactive to the activated isoform (Kumar et al., 2001). Martin de la Vega et al. (2001b) reported no change in the eIF2α kinase activity of PERK immunoprecipitated from control and reperfused samples; however, Western blot analysis was not used to confirm that PERK had been immunoprecipitated, or to examine the effect of I/R on its electrophoretic mobility. In fact, assays of immunoprecipitated PERK activity are unlikely to be a useful approach to its in vivo activation state because the in vitro kinase reaction conditions will themselves activate PERK, even if it is immunoprecipitated in the inactive state (Bertolotti et al., 2000).

Brain I/R does not affect p67 glycosylation or levels, has little or no effect on eIF2α(P) phosphatase activity, and PKR, HRI, and GCN2 are not needed for phosphorylation of eIF2α during reperfusion. However, brain I/R consistently induces transformation of PERK to its active isoform, and PERK activation together with the immunolocalization of eIF2α(P) provide direct molecular evidence that an ER UPR is occurring in neurons as a consequence of brain I/R.

Current understanding of the UPR

The ER is the site of lipid synthesis and of maturation and processing of secreted and transmembrane proteins (Stevens and Argon, 1999). The UPR occurs when cells are subjected to ER stress, and can be thought of as a stimulus-effector system. The stimuli triggering the UPR are those that upset the functions occurring in the ER lumen related to protein folding, maturation, posttran-scriptional processing, or lipid synthesis (Kaufman, 1999). The effectors of the UPR include both immediate and long-term transcriptional, translational, and posttranslational modification components that operate in parallel. Even in yeast, the overall UPR transcriptional response is complex and involves approximately 5% of the genome, including genes for proteins involved at all stages of secreted and membrane-bound protein processing (Travers et al., 2000). It is expected that a similarly large change in gene transcription occurs during the mammalian UPR. The biologic objective of the UPR is to reduce the requirement for ER protein processing by temporary PSI while simultaneously upregulating the transcription of genes or processes that will abate the effects of the ER insult or push the cell into apoptosis if ER dysfunction persists (Patil and Walter, 2001). The primary effectors of the mammalian UPR are three ER-transmembrane proteins: PERK, IRE1, and ATF6.

Several disruptions of ER biochemistry activate the UPR. These changes include depletion of ER Ca²⁺ stores (e.g., by the ER Ca²⁺-ATPase inhibitor thapsigargin), shift of the lumen from an oxidizing to a reducing environment (e.g., by dithiothreitol or β-mercaptoethanol), inhibition of protein glycosylation (e.g., with tunicamy-cin), glucose or oxygen deprivation (Kaufman, 1999; Ron and Harding, 2000), and disruption of ER-associated protein degradation (Patil and Walter, 2001).

Both PERK (Shi et al., 1998; Harding et al., 2001) and IRE1(Miyoshi et al., 2000) are present in the brain and expressed in neurons. Activation of PERK and IRE1α is mediated by their homologous ER-luminal N-terminal sequences (Fig. 3). Normally, the luminal domains of the inactive forms of PERK and IRE1α form 1:1 complexes with the chaperone GRP78 (also know as BiP or KAR2) (Bertolotti et al., 2000). In the presence of unfolded ER-luminal proteins, GRP78 dissociates from PERK and IRE1α to bind the unfolded proteins to prevent their aggregation (Ron and Harding, 2001). We have found that immunoprecipitation of PERK from homogenates of nonischemic brains coimmunoprecipitates GRP78, but PERK coimmunoprecipitation of GRP78 is lost after a 10-minute cardiac arrest and 10-minute reperfusion (DeGracia et al, 2000). Once dissociated from GRP78, IRE1α and PERK homooligomerize and activate by transautophosphorylation (Bertolotti et al., 2000; Welihinda and Kaufman, 1996).

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FIG. 3.

Schematic representation showing how PERK and IRE1 are activated by the dissociation of GRP78, homooligomerization, and autophosphorylation. (Left) In unstressed cells, PERK and IRE1 are ER transmembrane proteins maintained in an inactive state by binding GRP78 on their ER-lumenal domains. (Right) On ER stress, GRP78 dissociates and binds unfolded or misfolded proteins, allowing homooligomerization of PERK or IRE1 or both, leading to autophosphorylation of these enzymes and activation toward their substrates.

Activated PERK phosphorylates eIF2α, thereby inhibiting most protein synthesis. However, David Ron's group has shown that at least the mammalian mRNA for ATF4 undergoes translation initiation during the UPR by “bypass-scanning” similar to that seen for yeast GCN4 mRNA (Harding et al., 2000a) (Fig. 2). Like the mRNA for GCN2, ATF4 mRNA has a long 5′ leader with multiple uORFs. This mRNA is constitutively present in mammalian cells, including neurons (Yukawa et al., 1999), but is not efficiently translated until there is increased eIF2α phosphorylation (Harding et al., 2000a). ATF4 is a transcription factor, originally identified as CREB-2 in the family of cyclic AMP-responsive genes (Hai et al., 1989). ATF4 is responsible for CHOP transcription (Fawcett et al., 1999). Thus, PERK activation, by way of increased eIF2α(P), is responsible not only for general PSI but also for at least one important change in gene expression (i.e., the upregulation of CHOP) through bypass scanning-mediated enhanced translation of ATF4 during the UPR (Fig. 2).

The functions of mammalian IRE1 are more complex and less completely understood. The cytoplasmic domains of all IRE1 isoforms also include an endonucleolytic RNAse domain (Sidrauski and Walter, 1997) that is activated by IRE1 transautophosphorylation. In yeast, the activated IRE1 RNAse removes a 252-nucleotide translation-attenuator sequence from the constitutively expressed but inefficiently translated HAC1 mRNA (Kawahara et al., 1997), which is then ligated by transfer RNA ligase (Sidrauski and Walter, 1997) and translated efficiently (Kawahara et al., 1997). HAC1 protein is a member of the basic leucine zipper (bZIP) family of transcription factors; in yeast it binds to the UPR element (Kohno et al., 1993) in the promoter of UPR-responsive yeast genes (including that for GRP78) and thereby up-regulates their transcription. Although a sequence analogue of yeast HAC1 does not appear to exist in mammalian genomes, the mammalian UPR induces accurate intron splicing of vector-introduced HAC1 mRNA by IRE1 isoforms (Niwa et al., 1999), and expression of the yeast HAC1 protein in mammalian cells induces the promoter activity of the GRP78 gene on UPR induction (Foti et al., 1999). Cross-linking experiments have recently shown that UPR induction in mammalian cells is associated with complex formation between IRE1α and an unidentified constitutively expressed RNA (Bertolotti and Ron, 2001), and several investigators have shown that overexpression of mammalian IRE1 isoforms induces enhanced GRP78 transcription. It is not yet clear whether this UPR-responsive transcriptional effect of mammalian IRE1 involves its processing of an mRNA (as in yeast) or a role in upstream activation of ATF6 (Wang et al., 2000), but dominant negative mutants of IRE1 inhibit UPR-induced transcription dependent on an ATF6-binding minimum consensus sequence TGACGT (Wang et al., 2000).

As PERK can modulate apoptosis via CHOP expression, both IRE1α and IRE1β (expressed mainly in the gut) are also connected to mammalian apoptotic mechanisms (Fig. 4). Recently, Iwawaki et al. (2001) showed that overexpression of IRE1β can induce apoptosis by a mechanism involving cleavage of the 28S ribosomal RNA by the IRE1 β RNAse, and that a chimeric protein with the ER luminal domain of IRE1β and the cytoplasmic domain of IRE1α also cleaves the 28S ribosomal RNA. Although it is not yet known whether native IRE1α can process ribosomal RNA, such a mechanism has the potential to explain persistent PSI in an unresolved UPR, even after levels of eIF2α(P) are reduced. Moreover, activation of IRE1α leads to activation of the apoptosis-associated stress-induced protein Ser/Thr kinases known as SAPKs (stress-activated protein kinases) or JNKs via interaction of the IRE1α cytoplasmic domain with the TRAF2 (tumor necrosis factor receptor-associated factor-2) adaptor protein (Urano et al., 2000) and JIK (Jun inhibitor kinase) (Yoneda et al., 2001). The same complex of activated IRE1α with JIK and TRAF2 is also implicated in inducing activation of ER-localized procaspase-12 (Yoneda et al., 2001).

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FIG. 4.

The mammalian UPR is a stimulus-effector system. Several mechanisms, including depletion of ER Ca²⁺, can lead to misfolding of proteins being processed in the ER lumen. Malfolded lumenal proteins result in activation of three known effectors (PERK, IRE1, and ATF6). These effectors cause reduction of the processing-requiring peptide load and induce transcription for resolution of the ER stress, but two of these effectors (PERK and IRE1) also initiate apoptotic pathways that will result in the death of the cell if ER stress resolution is unsuccessful. PERK-mediated eIF2α phosphorylation generally inhibits protein synthesis, but also paradoxically increases bypass scanning-mediated translation of at least proapoptotic ATF4. IRE1 may be involved in induction of transcription for ER chaperones through mechanisms involving RNA processing or the activation of ATF6 or both; however, IRE1 also induces proapoptotic activation of JNKs and caspase-12, and its cytoplasmic RNAse domain can degrade 28S ribosomal RNA. After site-2 metalloprotease releases ATF6 from the ER membrane, it is translocated into the nucleus and promotes transcription for ER-resident proteins needed for resolution of the UPR; ATF6 has no known proapoptotic effects.

ATF6 is a key transcription factor in the resolution of the mammalian UPR (Patil and Walter, 2001), and unlike the situation with IRE1 and PERK, there is no evidence that ATF6 is also involved in proapoptotic mechanisms (Fig. 4). Normally resident as an ER transmembrane protein, on UPR induction ATF6 is proteolyzed by the ER-localized site-2 metalloproteinase (S2P) (Ye et al., 2000). As noted previously, there is no consensus whether IRE1 activation is upstream of ATF6 proteolysis (Patil and Walter, 2001). The liberated cytoplasmic domain of ATF6 translocates to the nucleus and collaborates with other transcription factors (NF-Y and YY1) (Li et al., 2000) to induce transcription of genes containing the mammalian ER stress-response element (Roy and Lee, 1999).

Overexpression of dominant negative mutants of the mammalian IRE1s (Wang et al., 1998), S2P (Ye et al., 2000), or ATF6 (Wang et al., 2000) attenuate the mammalian UPR, and PERK-knockout cells exhibit enhanced susceptibility to UPR-induced death (Harding et al., 2000b). Therefore, at least in the initial stage of UPR induction, all three ER-transmembrane upstream effectors are important for an effective UPR response.

The ER UPR is induced in vulnerable neurons by brain ischemia and reperfusion

Paschen has been a seminal advocate of the concept that ER stress represents an important fundamental mechanism of I/R-induced brain injury (Paschen, 1996, 2000). His insight grew in part from the foundation developed by Brostrom and colleagues, who showed that depletion of ER Ca²⁺ induced PSI through inhibited translation initiation (Brostrom and Brostrom, 1998). It is now clear that low levels of ER [Ca²⁺] inhibit ER-luminal protein-disulfide isomerases, causing malfolding in the ER lumen of newly synthesized peptides (Corbett et al., 1995; Oliver et al., 1999).

Depletion of ER Ca²⁺ is caused by brain I/R. Kohno et al. (1997) have shown that after 5-minute ischemia in the gerbil, ER Ca²⁺ in vulnerable neurons is lost by 15-minute reperfusion and is not recovered after 3 hours of reperfusion. Free arachidonic acid, which is produced during ischemia (Avaldano and Bazan, 1975), induces depletion of ER Ca²⁺ stores, phosphorylation of eIF2α, and inhibition of protein synthesis (Fleming and Mellow, 1995; O'Neil et al., 1999). Furthermore, microsomes from postischemic brains display decreased Ca²⁺ uptake (Parsons et al., 1997; Racay et al, 2000) that may be related to inactivation of SERCA2b by a nitric oxide-mediated mechanism (Doutheil et al., 2000) and prolonged uncoupling of Ca²⁺ transport and ATPase activity (Parsons et al., 1999). In this regard, Paschen's group has shown that in primary neuronal cultures, the nitric oxide donor SNAP induces inhibition of SERCA2b and depletion of ER Ca²⁺ stores (Doutheil et al., 2000). We have recently found that in eNOS- and nNOS-knockout mice, a greater duration of ischemia is needed to induce PERK activation (Kumar et al., 2001) and eIF2α phosphorylation (Kumar et al., 2000) during reperfusion, suggesting that nitric oxide production exacerbates ER stress.

Hu et al. (2000, 2001) recently provided ultrastructural evidence that brain I/R induces abnormal protein aggregates in several neuronal subcellular compartments, including the ER. These aggregates clear from injury-resistant neurons but persist in vulnerable CA1 neurons that progress to cell death. The persistence of these aggregates is associated with increased ubiquination of detergent-insoluble proteins, suggesting that they are being targeted for degradation but not in fact being cleared.

Further support for the I/R-induced UPR in vulnerable neurons derives from studies of individual UPR pathways. As discussed previously, PERK activation and greatly increased levels of eIF2α(P) are evident within 5 minutes of reperfusion. Furthermore, N-methyl-d-aspartate neurotoxicity (Laabich et al., 2001) and brain I/R (Jin et al., 2001; Paschen et al., 1998a) induce CHOP transcription, and GADD34 is transcribed by neurons in response to thapsigargin-induced ER Ca²⁺ depletion (Mengesdorf et al., 2001) and brain I/R (Doutheil et al., 1999; Imai et al., 2001). Although direct studies of I/R-induced IRE1α activation have yet to be performed, JNK activation during early reperfusion is consistent with the consequences of IRE1α activation. Herdegen et al. (1998) showed markedly enhanced phosphorylation of neuronal Jun after 3 hours of reperfusion (the earliest time examined) after 90-minute middle cerebral artery occlusion. Subsequently, Takagi et al. (2000) used 20-minute bilateral carotid artery occlusion in mice to show, by Western blot analysis, activation of JNK1 within 10 minutes of reperfusion, and by immunohistochemical analysis showed a large increase in activated JNK in vulnerable cortical and CA1 neurons after 30-minute reperfusion. Thus, there is JNK activation at the same early reperfusion time in which PERK activation occurs, consistent with UPR-induced pathways. Similarly, although ATF6 proteolysis has not yet been examined in brain I/R, transcription of the ER-resident protein disulfide isomerase ERP72 (Paschen et al., 1998b) and the ER chaperones GRP78 and GRP94 (Lowenstein et al., 1994; Wang et al., 1993) is upregulated. Together with our current understanding of the UPR, these findings strongly suggest that further examination of IRE1α activation, ATF6 proteolysis and nuclear translocation, caspase-12 activation, and translation of ATF4, CHOP, and other UPR-expressed genes is needed.

A general pathophysiologic view of I/R-induced PSI

As Lipton (1999) has recently noted, it is surprisingly difficult to answer to the seemingly straightforward question of whether PSI during brain reperfusion is protective or pathologic. Given the complexity of the relations between the system components reviewed here, this question may not in fact be helpful. Indeed, the data reviewed suggest the need to shift the emphasis from PSI to the underlying pathways that mediate PSI and to the issue of residual translation products. Such a transformation of viewpoint suggests that reperfusion-induced PSI may be both protective and pathologic.

During initial reperfusion, eIF2α(P)-mediated PSI may be a protective response to ischemia-induced ER stress. Downregulation of the UPR through the expression of dominant negative IREα (Miyoshi et al., 2000) or PERK (Harding et al., 2000b) enhances cell death. However, persistence of eIF2α(P) coupled with eIF4G degradation may result in a pathologic transition involving major alterations of message selection that lead to disproportionate expression of proapoptotic genes by residual translation. In fact, the molecular evidence we have reviewed shows that UPR activation (essentially immediately on reperfusion) includes components that may be the earliest proapoptotic pathway activated by brain I/R. This finding suggests that inducing early resolution of the UPR and reducing eIF4G degradation are likely to be important targets for therapeutic interventions for the survival of selectively vulnerable neurons. A proteomic comparison of vulnerable and resistant neurons regarding their translation products during the first several hours of reperfusion should provide more insight into the validity of this view.

The evidence we have reviewed strongly supports the arguments of Richter and Theurkauf (2001) that “the message is in the translation,” of Read et al. (2001) that translation assessment is essential to understanding brain ischemia-induced gene expression, and of Niwa and Walter (2000) that it is at the level of translational control that cells decide their fate when they “pause” in an UPR. Therefore, the issue of injury-resistant versus vulnerable neurons now seems to involve which messages are on the ribosomes, and whether during early reperfusion residual translation produces proteins involved in the resolution of the UPR and other cell stresses or those mediating apoptosis.