Kumar 2001

Wt RIP

Total protein. Equal amounts of protein from non-ischemic brain homogenates were electrophoresed and proteins were detected by staining the gel with Sypro Ruby. There is no difference in total protein between the groups (p=0.50).

Owen in preparation

eIF2 α ( )P

2Total eIF α

Wt nic RIP nic /Wt I R /RIP I R

Immunoblot for phosphorylated eIF2α and total eIF2α in forebrainhomogenates. Wt nic, non-ischemic wild type; RIP nic, non-ischemicRIP; Wt I/R, reperfused wild type; RIP I/R, reperfused RIP

Quantitation of immunoblot. After 20 minutes forebrain ischemia followed by 10 minutes reperfusion, there was a slight increase in eIF2α(P) in the RIP mice that did not reach statistical significance (p=0.4). There were no significant changes in total eIF2 (p=0.22) This indicates that the large elevation in eIF2α(P) levels after ischemia/reperfusion is due almost entirely to PERK.

nicWt WtRIP RIP

I/R

Abs

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QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Hayashi 2004

Fig. 1. Histological analysis of the brain 1 day after reperfusion. (A) Cresyl violet staining showed that the striatum on the ischemic side was extensivelydamaged in the wt animals. Massive neuronal degeneration was confirmed microscopically, and the infarcted area came very close to the ventricular wall. In thetg animals, the size of the infarct area was substantially smaller than in the wt animals. The photomicrograph showed that neurons on the medial side of thestriatum were morphologically intact. Scale bar, 400 Am. (B) ATUNEL study showed that the positively labeled cells were distributed almost to the innermostarea of the striatum in the wt animals, although they did not appear in that area in the tg animals. Scale bar is 400 Am for lower magnification photomicrographsand 20 Am for higher magnification photomicrographs.

Fig. 2. Change in phosphorylation status of PERK in the brain after ischemia. (A) Western blot analysis of samples from the striatum showed that thephosphorylated form of PERK was barely detectable in the sham-operated (c) wt and tg animals. In the wt animals, it was markedly increased at 1 h and furtherincreased at 4 h, but decreased at 1 day. In the tg animals, it was increased at 1 h to a milder degree than in the wt animals, but was decreased at 4 h and 1 day.(B) Quantitative analysis of Western blot showed that tg overexpression of SOD1 significantly prevented accumulation of phospho-PERK at 4 h (**P < 0.01compared with the wt animals at the same time point). (C) Immunohistochemistry for the phosphorylated form of PERK showed only weak immunoreactivityin the sham-operated (c) medial part of the striatum of the wt and tg animals. In the wt animals, the immunoreactivity became strong at 1 h and stronger at 4 h,but was substantially decreased at 1 day. Note that many neurons were degenerated at this time point. In the tg animals, the immunoreactivity was increased at 1 hbut the degree was milder than in the wt animals. At 4 h and 1 day, the immunoreactivity gradually decreased. Higher magnification clearly confirmed that SOD1 overexpression reduced the degree of PERK phosphorylation at 4 h. (D) Western blot analysis of samples from the cortex, showed that phospho-PERK wasbarely detectable in the sham-operated (c) wt and tg animals. In both the wt and tg animals, it was increased at 1 h and gradually decreased thereafter.(E) Quantitative analysis showed that there was no difference in PERK phosphorylation in the cortex between the wt and tg animals. (F) In both the wt and tg animals, immunohistochemical analysis showed that phospho-PERK was barely detectable in the sham-operated (c) cortices, was dramatically increased at 1 h,and then gradually decreased. Even with higher magnification, the differences in immunoreactivity for phospho-PERK were not ascertained between the wt andtg animals at 4 h. Scale bars are 50 Am for large panels and 20 Am for insets.

Striatum Cortex

Ethidium = SOD expression

Fig. 4. Change in phosphorylation status of eIF2a in the brain after ischemia. (A) Western blot analysis of samples from the striatum showed that phosphoeIF2a was barely detectable in the sham-operated (c) wt and tg animals. In the wt animals, it was markedly increased at 1 h and further increased at 4 h, but decreased at 1 day. In the tg animals, it was only mildly increased at 1 h, but gradually decreased thereafter. (B) Quantitative analysis of Western blot showed that the tg overexpression of SOD1 significantly prevented accumulation of phospho-eIF2a at 1 and 4 h (*P < 0.05, **P < 0.01 compared with the wt animals at the same time point). (C) Immunohistochemistry for phospho-eIF2a showed very weak immunoreactivity in the sham-operated (c) medial part of the striatum in the wt and tg animals. In the wt animals, the immunoreactivity became strong at 1 h and stronger at 4 h, but was substantially decreased at 1 day. Note that many neurons were degenerated at this time point. In the tg animals, the immunoreactivity was mildly increased at 1 h, and decreased thereafter. Higher magnification clearly confirmed that SOD1 overexpression reduced the degree of eIF2a phosphorylation at 4 h. (D) Western blot analysis of cortical samples showed that phospho-eIF2a was barely detectable in the sham-operated (c) wt and tg animals. In both the wt and tg animals, it was increased at 1 h and gradually decreased thereafter. (E) Quantitative analysis showed that there was no difference in eIF2a phosphorylation in the cortex between the wt and tg animals. (F) In both the wt and tg animals, immunohistochemical analysis showed that phospho-eIF2a was barely detectable in the sham-operated (c) cortices, was dramatically increased at 1 h, and then gradually decreased. Even with higher magnification, differences in immunoreactivity for phospho-PERK were no ascertained between the wt and tg

cortices at 4 h. Scale bars are 50 Am for large panels and 20 Am for insets.

Striatum Cortex

Striatum Cortex

FIG. 1. (A) Hypoxia, CoCl2, and thapsigargin (Thaps.) inducephosphorylation of eIF2 on Ser51. Cells were exposed to 0.02% O2for the indicated times or treated with 100 M CoCl2 for 2 h or 1 Mthapsigargin for 2 h. Shown at the top is an immunoblot with a rabbitpolyclonal antibody raised against eIF2 phosphorylated on Ser51.The membrane was stripped and reprobed with a rabbit polyclonalantibody that recognizes both phosphorylated and unphosphorylated(total) eIF2 (bottom). The values between the two parts represent thefold induction of phosphorylated eIF2 levels compared to controllevels after normalization to the total eIF2 levels, as determined bydensitometric analysis of the film and use of the ScionImage densitometricanalysis program (a commercial version of the NIH Imageshareware program). (B) Hypoxia, CoCl2, and thapsigargin reduce therates of protein synthesis. Cells were treated as described above andlabeled with [35S]methionine (50 Ci/ml) during the last 20 min oftreatment. TCA-precipitable counts were measured as described inMaterials and Methods. Concentrations of O2, CoCl2, and thapsigarginwere the same ones used in the experiment shown in Fig. 1A. Experimentswere performed in triplicate, and averages are reported alongwith errors. Con, control; 35S-Meth. Inc., [35S]methionine incorporation.

Koumenis 2002

Hypoxia only; no glucose depletion

A549 lung carcinoma cell line

FIG. 2. Kinetics and oxygen dependency of eIF2 phosphorylationin normal human fibroblasts and HeLa cells. (A) AG1522 and HeLacells were exposed to hypoxia for the times indicated. Immunoblottingwas performed with the anti-total eIF2 antibody (left) or the anti-Ser51-specific antibody (right). An anti-actin monoclonal antibody wasused as a control (C or Contr.). (B) HeLa cells were exposed todifferent oxygen concentrations for the times indicated. The levels ofphosphorylation were analyzed by densitometry and are presented asfold induction compared to that of untreated control cells (bottomgraph). Cells were also treated with 1 mM dithiothreitol (DTT) as apositive control.

FIG. 3. Hypoxia-induced phosphorylation of eIF2 correlates with,but is independent of, HIF-1 accumulation. (A) A549 cells weretreated with 2 or 4 h of hypoxia or with 4 h of hypoxia, followed by 30min of reoxygenation (Reox.). Fifty micrograms of the protein extractfrom the 4-h hypoxia treatment was pretreated with 10 U of calfintestinal phosphatase (PPase) prior to gel electrophoresis. Membraneswere immunoblotted with the anti-phospho-specific eIF2 antibody(top), anti-eIF2 antibody (middle), or a mouse monoclonalantibody raised against human HIF-1 (bottom). (B) HIF-1 +/+ andHIF-1 -/- cells were exposed to hypoxia for 1, 2, 4, and 8 h. Celllysates were probed by Western analysis for eIF2 phosphorylation asdescribed in the legend to Fig. 1. DTT, dithiothreitol.

HIF-1a is a hypoxia-inducible protein. It has a very short half life, but rapidly accumulates under hypoxic conditions

FIG. 7. Hypoxia fails to induce phosphorylation of eIF2 inPERK -/- , but not in PKR -/- , MEFs. PERK +/+ and PERK /-- MEFsimmortalized with the SV40 large-T antigen (A) and PKR +/+ andPKR -/- MEFs (B) were exposed to moderate hypoxia (0.05% O2) forthe indicated times or to 1 M thapsigargin (Thaps.) for 2 h, andprotein extracts were subjected to immunoblotting with a goat antibodyraised against eIF2 phosphorylated on Ser51. The membranewas stripped and reprobed with a rabbit polyclonal antibody that recognizesboth phosphorylated and total eIF2 . The values between thetwo parts represent the fold induction of phosphorylated eIF2 levelscompared to control levels after normalization to total eIF2 levels.(C) Effects of hypoxia and thapsigargin on protein synthesis rates inPERK +/+ and PERK -/- MEFs. MEFs were exposed to hypoxia(0.05%) for the indicated times or treated with thapsigargin (1 M) for2 h. During the last 20 min of the treatments, cells were labeled with[35S]methionine and TCA-precipitable counts were measured as describedin Materials and Methods. The results shown are averages ofthree independent experiments Contr., control.

the standard errors. Incorp., incorporation;

FIG. 8. Loss of PERK reduces cell survival following hypoxicstress. (A) Clonogenic survival of PERK /+ + and PERK -/- MEFs aftera 24-h exposure to hypoxia (Hypox.). Following hypoxic stress, 300cells were plated into dishes and allowed to grow under normoxia(Normox.) for 12 days. Experiments were performed in triplicate.(B) Quantitation of cell survival following hypoxic stress. Coloniesrepresenting 50 cells were counted, and cell survival fractions werecalculated for control (untreated) and treated (exposed to hypoxia)cells. Average cell survival fractions (hypoxia/control [Hyp/Con]) fromthree independent experiments are reported for each cell line, alongwith the standard deviations. (C) Proliferation of PERK +/+ andPERK -/- MEFs under normoxic conditions. Five thousand cells wereplated in 24-well plates, and the viable cells (cells excluding trypanblue) were counted with a hemocytometer every 24 h. WT, wild type;KO, knockout.

Munoz 2000

FIG. 1. PC12 cell morphology. A:PC12 cells differentiated withNGF (100 ng/ml) for 5 days. B andC: Differentiated control cellsmaintained in LSM without NGFfor 4 (B) or 6 (C) h. D: Differentiatedcells subjected to experimentalischemia for 4 h. E and F:Differentiated cells subjected toexperimental ischemia for 4 h followedby 2 h of recovery in theabsence (E) or presence (F) ofNGF. Phase contrast optics. Bar5 15 mm.

This is hypoxia AND glucose depletion (OGD)

Cell viability declined to 59% at 4hr of ischemia. No cell death during recovery was observed. Cell viability parameters were the same for both NGF treated and non-treated cells

TABLE 1. ATP and GTP levels and glutamate release after ischemia/recovery Glutamate (mM ) ATP (nmol/mg) GTP (nmol/mg) C 17.1 +/- 6.8 30.5 +/- 2.1 12.6 +/- 1.2 I 73.2 +/- 16.5a 10.8 +/- 1.7c 6.7 +/- 0.5b I/R30 7.9 +/- 3.6 11.4 +/- 1.0c 7.8 +/- 1.1a I/R2 ND 16.9 +/- 1.5a 6.4 +/- 0.9a

ATP, GTP, and glutamate concentrations were determined as describedin Materials and Methods. Data are means 6 SEM of three orfour different cultures (each sample run in triplicate). C, control PC12cells; I, 4-h ischemia; I/R30 and I/R2, 4-h ischemia followed by 30-minand 2-h reperfusion, respectively. ND, not determined.a p , 0.05, bp , 0.01, cp , 0.001, significant differences betweencontrol and ischemic cells.

FIG. 2. Protein synthesis rate after ischemia/recovery. Proteinsynthesis was measured in PC12 cells after ischemia and recovery,as described in Materials and Methods. Results are expressedas the percentage of control values and representmeans 6 SEM of three to six different cultures. Average of[3H]methionine incorporated in controls: 49,000 cpm 3 106 cells.C, control PC12 cells; I, 4-h ischemia; I/R15, I/R30, and I/R2h,4 h of ischemia followed by 15 and 30 min and 2 h of recovery,respectively. Statistical significance between control and ischemia/recovery: *p , 0.05, **p , 0.01, ***p , 0.001.

+NGF = -NGF

All subsequent experiments done without NGF

FIG. 3. eIF2a phosphorylation status after ischemia/recovery.Cell lysates were subjected to IEF electrophoresis, and bandscorresponding to eIF2a and eIF2(aP) were analyzed by proteinimmunoblot as described in Materials and Methods. Results areexpressed as the percentage of eIF2(aP) over the total eIF2 andrepresent means 6 SEM of three to five different cultures. Inset:A typical experiment is shown. Lane 1, control (C); lane 2, 4-hischemia (I); lane 3, 4-h ischemia/15-min recovery (I/R15); lane 4,4-h ischemia/30-min recovery (I/R30). Statistical significance isas in Fig. 2.

TABLE 2. Effect of ischemia and glucose or oxygen deprivation on cell viability, protein synthesis rate, and eIF2(aP) levels Protein Cytotoxicity synthesis rate eIF2(aP) (% of dead cells) (% of control) (%) Control 0.0 100 13.3 +/- 0.95 Anoxia 10.1+/- 4.1 79.2 +/-2.2 12.0 +/- 2.5 Glucose Deprivation 22.7 +/- 7.5 88.2 +/- 4.3 19.2 +/- 0.51a Ischemia 59.8 +/- 1.8 20.5 +/- 2.2b 41.7 +/- 1.91b

All three parameters were determined in differentiated PC12 cellsthat were maintained under glucose or oxygen deprivation or subjectedto ischemia for 4 h as described in Materials and Methods. Data aremeans 6 SEM of two to five different cultures.a p , 0.01, bp , 0.001, significant differences between control,ischemic, or deprived (glucose or oxygen) cells.

FIG. 6. eIF2(a32P) dephosphorylation after ischemia/recovery.eIF2a phosphatase activity was determined by measuring thedephosphorylation of eIF2(a32P) (0.5 mg) by concentrated lysatesas described in Materials and Methods. Bands correspondingto eIF2(a32P) were quantitated, and data represent themeans 6 SEM of three or four different cultures. Data are expressedas 32P released in arbitrary units (A.U.). C, control; I, 4-hischemia; I/R30, 4-h ischemia/30-min recovery. Inset: A typicalautoradiograph is shown. Lane 1, eIF2(a32P) incubated for 30min in the absence of extracts; lanes 2 and 3, control cells; lanes4 and 5, ischemic cells. The arrow indicates the position ofeIF2(a32P).

FIG. 7. eIF2a phosphatase activity after ischemia/recovery.eIF2a phosphatase activity was determined by measuring thedephosphorylation of eIF2(aP) (3 mg) by concentrated lysates asdescribed in Materials and Methods. eIF2(aP) bands in westernblots were quantified, and the phosphate released was calculatedas the difference between eIF2(aP) band in the absence ofextracts (lane 1) and bands corresponding to the different extracts(lanes 2–5). Data are expressed in arbitrary units (A.U.) andrepresent the means 6 SEM of four to eight different cultures.Inset: A typical western blot corresponding to the analysis of thecorresponding reaction mixtures (2 ml) is shown. Lane 1,eIF2(aP) incubated for 30 min in the absence of lysate, showingthat the factor used as substrate is highly phosphorylated (80–90%); lanes 2–5, eIF2(aP) incubated for 30 min in the presenceof lysates from control (C; lanes 2 and 3), ischemic (I; lane 4), or4-h ischemia/30-min recovery (I/R30; lane 5) cells; lanes 6–8,lysates incubated in the absence of added eIF2(aP) from C, I,and I/R30, respectively, to show that they do not contain detectableeIF2(aP). Statistical significance is as in Fig. 2.