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Kumar 2001. 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. Wt. RIP. Wt. RIP.

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Owen in preparation

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


Wt

RIP

Wt

RIP

nic

I/R

Absorbance

Quantitation of immunoblot. After 20 minutes forebrain ischemia followed by 10 minutes reperfusion, there was a slight increase in eIF2a(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 eIF2a(P) levels after ischemia/reperfusion is due almost entirely to PERK.


Hayashi 2004
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 extensively

damaged in the wt animals. Massive neuronal degeneration was confirmed microscopically, and the infarcted area came very close to the ventricular wall. In the

tg 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 the

striatum were morphologically intact. Scale bar, 400 Am. (B) ATUNEL study showed that the positively labeled cells were distributed almost to the innermost

area 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 photomicrographs

and 20 Am for higher magnification photomicrographs.


Striatum

Cortex

Fig. 2. Change in phosphorylation status of PERK in the brain after ischemia. (A) Western blot analysis of samples from the striatum showed that the

phosphorylated 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 further

increased 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 < 0.01

compared with the wt animals at the same time point). (C) Immunohistochemistry for the phosphorylated form of PERK showed only weak immunoreactivity

in 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 h

but 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 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 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 and

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



Striatum

Cortex

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


Koumenis 2002
Koumenis 2002

FIG. 1. (A) Hypoxia, CoCl2, and thapsigargin (Thaps.) induce

phosphorylation of eIF2 on Ser51. Cells were exposed to 0.02% O2

for the indicated times or treated with 100 M CoCl2 for 2 h or 1 M

thapsigargin for 2 h. Shown at the top is an immunoblot with a rabbit

polyclonal antibody raised against eIF2 phosphorylated on Ser51.

The membrane was stripped and reprobed with a rabbit polyclonal

antibody that recognizes both phosphorylated and unphosphorylated

(total) eIF2 (bottom). The values between the two parts represent the

fold induction of phosphorylated eIF2 levels compared to control

levels after normalization to the total eIF2 levels, as determined by

densitometric analysis of the film and use of the ScionImage densitometric

analysis program (a commercial version of the NIH Image

shareware program). (B) Hypoxia, CoCl2, and thapsigargin reduce the

rates of protein synthesis. Cells were treated as described above and

labeled with [35S]methionine (50 Ci/ml) during the last 20 min of

treatment. TCA-precipitable counts were measured as described in

Materials and Methods. Concentrations of O2, CoCl2, and thapsigargin

were the same ones used in the experiment shown in Fig. 1A. Experiments

were performed in triplicate, and averages are reported along

with errors. Con, control; 35S-Meth. Inc., [35S]methionine incorporation.

Hypoxia only; no glucose depletion

A549 lung carcinoma cell line


FIG. 2. Kinetics and oxygen dependency of eIF2 phosphorylation

in normal human fibroblasts and HeLa cells. (A) AG1522 and HeLa

cells were exposed to hypoxia for the times indicated. Immunoblotting

was performed with the anti-total eIF2 antibody (left) or the anti-

Ser51-specific antibody (right). An anti-actin monoclonal antibody was

used as a control (C or Contr.). (B) HeLa cells were exposed to

different oxygen concentrations for the times indicated. The levels of

phosphorylation were analyzed by densitometry and are presented as

fold induction compared to that of untreated control cells (bottom

graph). Cells were also treated with 1 mM dithiothreitol (DTT) as a

positive control.


FIG. 3. Hypoxia-induced phosphorylation of eIF2 correlates with,

but is independent of, HIF-1 accumulation. (A) A549 cells were

treated with 2 or 4 h of hypoxia or with 4 h of hypoxia, followed by 30

min of reoxygenation (Reox.). Fifty micrograms of the protein extract

from the 4-h hypoxia treatment was pretreated with 10 U of calf

intestinal phosphatase (PPase) prior to gel electrophoresis. Membranes

were immunoblotted with the anti-phospho-specific eIF2 antibody

(top), anti-eIF2 antibody (middle), or a mouse monoclonal

antibody raised against human HIF-1 (bottom). (B) HIF-1 +/+ and

HIF-1 -/- cells were exposed to hypoxia for 1, 2, 4, and 8 h. Cell

lysates were probed by Western analysis for eIF2 phosphorylation as

described 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 in with,

PERK -/- , but not in PKR -/- , MEFs. PERK +/+ and PERK /-- MEFs

immortalized with the SV40 large-T antigen (A) and PKR +/+ and

PKR -/- MEFs (B) were exposed to moderate hypoxia (0.05% O2) for

the indicated times or to 1 M thapsigargin (Thaps.) for 2 h, and

protein extracts were subjected to immunoblotting with a goat antibody

raised against eIF2 phosphorylated on Ser51. The membrane

was stripped and reprobed with a rabbit polyclonal antibody that recognizes

both phosphorylated and total eIF2 . The values between the

two parts represent the fold induction of phosphorylated eIF2 levels

compared to control levels after normalization to total eIF2 levels.

(C) Effects of hypoxia and thapsigargin on protein synthesis rates in

PERK +/+ and PERK -/- MEFs. MEFs were exposed to hypoxia

(0.05%) for the indicated times or treated with thapsigargin (1 M) for

2 h. During the last 20 min of the treatments, cells were labeled with

[35S]methionine and TCA-precipitable counts were measured as described

in Materials and Methods. The results shown are averages of

three independent experiments the standard errors. Incorp., incorporation;

Contr., control.


FIG. 8. Loss of PERK reduces cell survival following hypoxic with,

stress. (A) Clonogenic survival of PERK /+ + and PERK -/- MEFs after

a 24-h exposure to hypoxia (Hypox.). Following hypoxic stress, 300

cells 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. Colonies

representing 50 cells were counted, and cell survival fractions were

calculated for control (untreated) and treated (exposed to hypoxia)

cells. Average cell survival fractions (hypoxia/control [Hyp/Con]) from

three independent experiments are reported for each cell line, along

with the standard deviations. (C) Proliferation of PERK +/+ and

PERK -/- MEFs under normoxic conditions. Five thousand cells were

plated in 24-well plates, and the viable cells (cells excluding trypan

blue) were counted with a hemocytometer every 24 h. WT, wild type;

KO, knockout.


Munoz 2000
Munoz 2000 with,

FIG. 1. PC12 cell morphology. A:

PC12 cells differentiated with

NGF (100 ng/ml) for 5 days. B and

C: Differentiated control cells

maintained in LSM without NGF

for 4 (B) or 6 (C) h. D: Differentiated

cells subjected to experimental

ischemia for 4 h. E and F:

Differentiated cells subjected to

experimental ischemia for 4 h followed

by 2 h of recovery in the

absence (E) or presence (F) of

NGF. Phase contrast optics. Bar

5 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 with,

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 described

in Materials and Methods. Data are means 6 SEM of three or

four different cultures (each sample run in triplicate). C, control PC12

cells; I, 4-h ischemia; I/R30 and I/R2, 4-h ischemia followed by 30-min

and 2-h reperfusion, respectively. ND, not determined.

a p , 0.05, bp , 0.01, cp , 0.001, significant differences between

control and ischemic cells.


FIG. 2. Protein synthesis rate after ischemia/recovery. Protein

synthesis was measured in PC12 cells after ischemia and recovery,

as described in Materials and Methods. Results are expressed

as the percentage of control values and represent

means 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 bands

corresponding to eIF2a and eIF2(aP) were analyzed by protein

immunoblot as described in Materials and Methods. Results are

expressed as the percentage of eIF2(aP) over the total eIF2 and

represent means 6 SEM of three to five different cultures. Inset:

A typical experiment is shown. Lane 1, control (C); lane 2, 4-h

ischemia (I); lane 3, 4-h ischemia/15-min recovery (I/R15); lane 4,

4-h ischemia/30-min recovery (I/R30). Statistical significance is

as in Fig. 2.


TABLE 2. Effect of ischemia and glucose or oxygen ischemia/recovery.

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 cells

that were maintained under glucose or oxygen deprivation or subjected

to ischemia for 4 h as described in Materials and Methods. Data are

means 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 the

dephosphorylation of eIF2(a32P) (0.5 mg) by concentrated lysates

as described in Materials and Methods. Bands corresponding

to eIF2(a32P) were quantitated, and data represent the

means 6 SEM of three or four different cultures. Data are expressed

as 32P released in arbitrary units (A.U.). C, control; I, 4-h

ischemia; I/R30, 4-h ischemia/30-min recovery. Inset: A typical

autoradiograph is shown. Lane 1, eIF2(a32P) incubated for 30

min in the absence of extracts; lanes 2 and 3, control cells; lanes

4 and 5, ischemic cells. The arrow indicates the position of

eIF2(a32P).

FIG. 7. eIF2a phosphatase activity after ischemia/recovery.

eIF2a phosphatase activity was determined by measuring the

dephosphorylation of eIF2(aP) (3 mg) by concentrated lysates as

described in Materials and Methods. eIF2(aP) bands in western

blots were quantified, and the phosphate released was calculated

as the difference between eIF2(aP) band in the absence of

extracts (lane 1) and bands corresponding to the different extracts

(lanes 2–5). Data are expressed in arbitrary units (A.U.) and

represent the means 6 SEM of four to eight different cultures.

Inset: A typical western blot corresponding to the analysis of the

corresponding reaction mixtures (2 ml) is shown. Lane 1,

eIF2(aP) incubated for 30 min in the absence of lysate, showing

that the factor used as substrate is highly phosphorylated (80–

90%); lanes 2–5, eIF2(aP) incubated for 30 min in the presence

of lysates from control (C; lanes 2 and 3), ischemic (I; lane 4), or

4-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 detectable

eIF2(aP). Statistical significance is as in Fig. 2.


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