The Journal of Experimental Medicine
© 2008 Lange et al.
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 205 No. 5 1227-1242 www.jem.org/cgi/doi/
Free radicals and their reactive metabolites
(reactive oxygen species [ROS]) exist in neu-
ronal cells and tissues at low but measurable
concentrations ( 1 ). These tolerable equilib-
rium concentrations are the result of a tightly
controlled balance between the rates of pro-
duction and clearance, the latter being medi-
ated by a team of antioxidants including enzymes
and nonenzymatic compounds such as the tri-
Cells or tissues are in a stable oxidative
state if the rates of ROS production and scav-
enging capacity remain within a homeostatic
range. However, if this balance is disturbed,
either by an increase in ROS concentrations
or a decrease in antioxidant activities, the re-
sponse may not be suffi cient to keep the sys-
tem at a level compatible with survival. In
such cases, oxidants can modify cellular tar-
gets, leading to cell dysfunction or death ( 2 ).
Indeed, oxidative stress has been implicated in
virtually all of the major acute and chronic
neurodegenerative diseases ( 3 ).
In many cells, including cortical neurons,
the expression of genes with antioxidative ac-
tivity is precisely controlled by a synergistic
network of redox-sensing signaling cascades
( 4, 5 ). Specifi cally, aberrant levels of oxidants can
trigger the transcriptional induction of antioxi-
dative enzymes and other adaptive pathways ( 5 ).
Philipp S. Lange:
Rajiv R. Ratan:
Abbreviations used: ATF4,
activating transcription factor 4;
BHA, butylated hydroxyanisol;
BSO, buthionine sulfoximine;
DCF, 2 ? ,7 ? -dichlorofl uorescin;
? -GCS, ? -glutamylcysteine
synthetase; HCA, homocyste-
ate; MAP2, microtubule-associ-
ated protein 2; MCAo, middle
cerebral artery occlusion; MOI,
multiplicity of infection; ROS,
reactive oxygen species; TRB3,
tribbles homologue 3.
The online version of this article contains supplemental material.
ATF4 is an oxidative stress – inducible,
prodeath transcription factor in neurons
in vitro and in vivo
Philipp S. Lange , 1,2,9 Juan C. Chavez , 1,2,3 John T. Pinto , 4
Giovanni Coppola , 6 Chiao-Wang Sun , 8 Tim M. Townes , 8
Daniel H. Geschwind , 5,6,7 and Rajiv R. Ratan 1,2
1 Burke Medical Research Institute, White Plains, NY 10605
2 Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY 10021
3 Discovery Translational Medicine, Wyeth Research, Collegeville, PA 19426
4 Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595
5 Department of Human Genetics, 6 Program in Neurogenetics, Department of Neurology, and 7 Semel Institute,
David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095
8 Department of Biochemistry and Molecular Genetics, School of Medicine and School of Dentistry, University of Alabama
at Birmingham, Birmingham, AL 35294
9 Department of Anesthesiology and Intensive Care Medicine, University of Bonn, 53127 Bonn, Germany
Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of
oxidative stress – inducible transcription factors and their role in propagating the death
cascade are not well known. In an in vitro model of oxidative stress, the expression of the
bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathi-
one depletion and localized to the promoter of a putative death gene in neurons. Germline
deletion of ATF4 resulted in a profound reduction in oxidative stress – induced gene expres-
sion and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event
in the death pathway, as resistance to oxidative death by ATF4 deletion was associated
with decreased consumption of the antioxidant glutathione. Forced expression of ATF4 was
suffi cient to promote cell death and loss of glutathione. In ATF4 ? / ? neurons, restoration of
ATF4 protein expression reinstated sensitivity to oxidative death. In addition, ATF4 ? / ? mice
experienced signifi cantly smaller infarcts and improved behavioral recovery as compared
with wild-type mice subjected to the same reductions in blood fl ow in a rodent model of
ischemic stroke. Collectively, these fi ndings establish ATF4 as a redox-regulated, prodeath
transcriptional activator in the nervous system that propagates death responses to oxida-
tive stress in vitro and to stroke in vivo.
ATF4 IS A PRODEATH PROTEIN IN NEURONS | Lange et al.
dative stress alone was suffi cient to induce ATF4 protein
levels ( Fig. 1 C ).
Cortical neurons from ATF4 ? / ? brains are resistant
to oxidative stress – induced cell death
To verify that the protection by arginase and thapsigargin re-
quires ATF4, we cultured cortical neurons from brains of
embryonic ATF4 ? / ? mice (E15.5). Immunohistochemical
analysis ( Fig. 2 A ) using an antibody to microtubule-associ-
ated protein 2 (MAP2; which stains neural dendrites) showed
that basal viability and morphology of cell bodies and den-
drites did not display any diff erences between ATF4 +/+ and
ATF4 ? / ? neurons. We found that ATF4 mRNA was in-
duced in ATF4 +/+ neurons after oxidative stress and con-
fi rmed its absence in ATF4 ? / ? neurons ( Fig. 2 B ). We then
examined susceptibility to oxidative stress induced by the
glutamate analogue homocysteate (HCA) in ATF4 +/+ and
ATF4 ? / ? cortical neurons ( Fig. 2, C and D ). Contrary to our
hypothesis, we found that ATF4 ? / ? neurons were signifi -
cantly protected from oxidative stress – induced death. ATF4 ? / ?
neurons demonstrated enhanced resistance, as monitored by
MTT reduction ( Fig. 2 C ) and live/dead staining ( Fig. 2 D ).
Moreover, in ATF4 +/+ neurons, ATF4 induction by treat-
ment with HCA was a result of induction at the translational
level ( Fig. 2 E ). We confi rmed the nuclear accumulation of
ATF4 in ATF4 +/+ neurons exposed to oxidative stress ( Fig.
2 F ). Higher nuclear ATF4 activity after oxidative stress was
demonstrated by showing higher promoter activity of an
ATF4 target gene, tribbles homologue 3 (TRB3), that has
been recently associated with cell death. TRB3 mRNA is
induced upon HCA treatment in an ATF4-dependent man-
ner ( Fig. 2 G ). To characterize ATF4 binding on the pro-
moter of TRB3, we generated a 2-kb fragment of the mouse
TRB3 promoter ( Fig. 2 H ). This fragment contains a 33-bp
element whose human analogue has been characterized as a
DNA binding region of ATF4. Transfection of both ATF4 +/+
and ATF4 ? / ? neurons with the TRB3WT promoter con-
struct, along with a mutant version lacking the binding ele-
ment, revealed that this element plays an important but not
exclusive role in the induction of the TRB3 promoter after
oxidative stress. In the absence of ATF4, the TRB3 promoter
activity was not induced in response to oxidative stress.
Microarray analysis reveals that ATF4 regulates a subset
of genes that are induced in response to oxidative stress
In fi broblasts, previous studies ( 11 ) have established ATF4 as
an important prosurvival transcription factor in the context of
oxidative stress. Microarray analysis in that study ( 11 ) showed
that several ATF4-regulated genes in fi broblasts are involved
in amino acid import, glutathione biosynthesis, antioxidant
defense, and DNA repair. In contrast, in neurons, ATF4 has
classically been thought to act mainly as a transcriptional re-
pressor. In this context, ATF4 has been hypothesized to nega-
tively infl uence survival by repressing expression of genes
that are otherwise activated by prosurvival transcriptional ac-
tivators such as cAMP response element binding protein.
The cellular response to oxidative stress is tightly controlled
by a family of stress-responsive transcription factors ( 2, 6 ).
Among these transcription factors, the activating transcrip-
tion factor 4 (ATF4)/cAMP response element binding protein
2 may be a key player ( 7 – 9 ). ATF4 is expressed constitutively
only at low concentrations but becomes rapidly induced under
particular cell-stress conditions ( 10 ). ATF4 binds to the pro-
moter regions of an array of diff erent target genes, includ-
ing many involved in amino acid metabolism and redox
control ( 11 ). In fi broblasts, ATF4 coordinates the response to
amino acid depletion, oxidative stress, and endoplasmic retic-
ulum stress, and helps to balance redox homeostasis. Indeed,
ATF4-defi cient fi broblasts have been shown to be prone to
death after a host of stresses, including oxidative stress and
amino acid deprivation ( 11 ).
Interestingly, amino acid deprivation has been previously
reported to be neuroprotective in an in vitro model of oxida-
tive stress – induced cell death ( 12 ). This model employs im-
mature cortical neurons and takes advantage of the absence of
glutamate receptors at this stage of development to avoid ex-
citotoxicity. Instead, addition of glutamate analogues com-
petitively inhibits uptake of cyst(e)ine, the rate-limiting
precursor for the tripeptide glutathione. The resulting de-
cline in glutathione concentration is a primary event that
leads to neuronal cell death from oxidative stress ( 13 – 15 ), a
process that displays many features of apoptosis ( 14 – 16 ). This
glutathione depletion model facilitates the separation of bio-
chemical events that mediate death from those that are a con-
sequence of death, and it is highly relevant to pathological
conditions because an increase in cellular ROS production is
often observed in apoptotic processes triggered by diverse
stimuli associated with disease states. In this work, we defi ne
a novel prodeath role for ATF4 in neurons in vitro in re-
sponse to oxidative stress and in vivo in response to stroke, a
condition linked to oxidative stress.
Amino acid depletion and thapsigargin treatment
induce ATF4 and protect embryonic cortical neurons
from oxidative stress – induced cell death
Amino acid depletion via the arginine-degrading enzyme
arginase is a well-characterized strategy for protecting neu-
rons from oxidative stress – induced cell death ( 12, 17 ).
Studies in nonneural cells have shown that amino acid de-
privation activates cell-survival pathways by activating the
bZip transcription factor ATF4 ( 11 ). We initiated our stud-
ies with the hypothesis that arginase is a multipotent inhibi-
tor of apoptotic neuronal cell death by activating ATF4 and
its downstream gene targets. Because endoplasmic reticu-
lum stress by thapsigargin is also associated with increased
ATF4 expression ( 18 ), we examined the corollary hypoth-
esis that this agent could initiate tolerance to oxidative stress
in primary cortical neurons. As predicted, arginase and
thapsigargin led to nearly complete protection from neuro-
nal cell death ( Fig. 1, A and B ) and to accumulation of
ATF4 protein ( Fig. 1 C ). Interestingly, we found that oxi-
JEM VOL. 205, May 12, 2008
To begin to examine whether ATF4 functions primarily as a
repressor or activator in neurons and to determine the extent
to which these ATF4-regulated genes function similarly in
fi broblasts and neurons, we performed a global analysis of gene
expression using microarrays in ATF4 +/+ and ATF4 ? / ?
neurons ( Fig. 3 A ). More specifi cally, we assessed (a) the
physiological genomic eff ect of ATF4 knockout (comparing
ATF4 ? / ? with ATF4 +/+ neurons), and (b) the eff ect of ATF4
knockout on the response to oxidative stress (comparing
ATF4 ? / ? with ATF4 +/+ neurons after HCA treatment; Fig. 3
B and Table I ). At the chosen statistical threshold (5% false
discovery rate), 136 probes were down-regulated in ATF4 ? / ?
versus ATF4 +/+ neurons, compared with 53 that were up-
regulated, suggesting a role for ATF4 as a transcriptional acti-
vator. Functional gene ontology categories overrepresented in
this list include mitochondrion, oxidoreductase activity, and
amino acid metabolism. Several of the down-regulated genes
have been shown to be positively regulated by ATF4, includ-
ing the prodeath gene TRB3. Comparison of ATF4 ? / ? versus
ATF4 +/+ neurons after oxidative stress with HCA treatment
suggested a fundamental role of ATF4 in modulating oxida-
tive stress – induced gene expression. In fact, 119 probes are
dysregulated in response to oxidative stress in ATF4 +/+ neu-
rons, whereas only 3 change in ATF4 ? / ? neurons. Collec-
tively, these fi ndings suggest that ATF4 is a major upstream
regulator of oxidative stress – induced changes in gene ex-
pression. Additionally, for most genes in embryonic cortical
neurons, ATF4 functions as an activator and not a repressor.
Gene expression array data were obtained from ATF4 ? / ?
fi broblasts that display a higher susceptibility to oxidative stress
( 11 ). A signifi cant overlap could be observed between the array
data from ATF4 ? / ? fi broblasts and neurons (Table S1, available
indicating that ATF4 regulates at least a major part of target
genes in a cell type – independent manner. These similarities do
not explain the observation that ATF4 is prosurvival in fi bro-
blasts and prodeath in neurons, but they demonstrate the re-
liability in our analysis. In contrast, a subset of genes, including
the prodeath gene TRB3, were ATF4-regulated in neurons
but not in fi broblasts. Collectively, these fi ndings provide some
understanding of the tissue-specifi c gene regulation mediated
by ATF4 that could account for the divergent phenotypes in
dividing fi broblasts versus postmitotic neurons.
Dunn ’ s multiple comparisons test. (B) Live/dead assay of cortical neuronal
cultures (2 d in vitro). Live cells were detected by uptake and trapping of
calcein-AM (green fl uorescence). Dead cells failed to trap calcein but were
freely permeable to the highly charged DNA intercalating dye ethidium
homodimer (red fl uorescence). Bar, 50 μ m. (C) Treatment with 10 mM HCA
(shown as H) and 1 μ g/ml arginase (shown as A) or 1 μ M thapsigargin
(shown as T), alone or in combination with HCA, increases the expression of
ATF4 in cultured cortical neurons as compared with vehicle-treated control
(shown as C). Cells were harvested at the indicated time points, and nuclear
extracts were separated using gel electrophoresis and immunodetected
using an antibody against ATF4. YY1 was monitored as a loading control.
The immunoblot is a representative example of three experiments.
Figure 1. Amino acid depletion and thapsigargin treatment induce
ATF4 and protect embryonic cortical neurons from oxidative stress –
induced cell death. (A) Cortical neuronal cultures (1 d in vitro) were treated
with a vehicle control (shown as C), 10 mM of the glutamate analogue HCA,
1 μ g/ml arginase, 1 μ M thapsigargin, 1 μ g/ml arginase and 10 mM HCA, or
10 mM HCA and 1 μ M thapsigargin. 24 h later, cell viability was determined
using the MTT assay. The graph depicts mean (compared with control) ± SD
calculated from three separate experiments for each group ( n = 25). *, P <
0.05 from HCA-treated cultures by the Kruskal-Wallis test followed by
ATF4 IS A PRODEATH PROTEIN IN NEURONS | Lange et al.
Figure 2. Cortical neurons from ATF4 ? / ? brains are resistant to oxidative stress – induced cell death. (A) Immunocytochemistry of cultured ATF4 +/+ and
ATF4 ? / ? cortical neurons. Cells were stained with an antibody against MAP2 (which stains neural dendrites; red) and counterstained with Hoechst (blue). Bar, 50 μ m.
(B) Real-time PCR of ATF4 mRNA expression in ATF4 +/+ and ATF4 ? / ? neurons in response to treatment with 10 mM HCA. The value obtained from the ATF4 +/+
control was arbitrarily defi ned as 1. Mean ± SD was calculated from three separate experiments. Each data point was performed in duplicate. (C) Cortical neuro-
nal cultures (1 d in vitro) prepared from brains from ATF4 +/+ and ATF4 ? / ? embryos were treated with a vehicle control (shown as C) or 10 mM HCA. 24 h later, cell
viability was determined using the MTT assay. The graph depicts mean (compared with control) ± SD calculated from data from fi ve separate experiments ( n = 27
ATF4 +/+ and 73 ATF4 ? / ? ). *, P < 0.05 from ATF4 +/+ untreated cultures by the Kruskal-Wallis test followed by Dunn ’ s multiple comparisons test. The difference be-
tween treated and untreated ATF4 ? / ? neurons was not signifi cant (n.s.). (D) Representative live/dead assay displaying untreated and HCA-treated ATF4 +/+ and
ATF4 ? / ? neurons. Bar, 50 μ m. (E) ATF4 +/+ neurons were transfected with a reporter plasmid (pGL3 backbone) containing the mouse ATF4 5 ? UTR and AUG fused to
luciferase. Cortical neurons were cotransfected with a plasmid expressing Renilla to allow normalization for transfection effi ciency. 24 h after transfection, neu-
rons were treated with vehicle control (shown as C) or 10 mM HCA. Cells were harvested in luciferase assay buffer 12 h after the onset of treatment. Values were
JEM VOL. 205, May 12, 2008
ATF4 has a negative impact on neuronal glutathione
The loss of glutathione is central to the in vitro model of
oxidative stress used in this study. This model has been suc-
cessfully used to describe signaling pathways that prevent
cell death downstream of glutathione depletion. However,
in fi broblasts, ATF4 has been described as a positive regula-
tor of glutathione metabolism ( 11 ). Therefore, we performed
glutathione measurements to determine whether ATF4 per-
mits cell death in response to HCA by direct infl uence of the
neuronal glutathione metabolism or by acting downstream
of glutathione depletion. In untreated neurons, glutathione
concentrations in ATF4 +/+ and ATF4 ? / ? neurons did not
diff er ( Fig. 6 A ). As anticipated, HCA treatment caused a
progressive loss of glutathione in ATF4 +/+ neurons. How-
ever, ATF4 ? / ? neurons displayed a markedly slower decline
in glutathione concentration. To rule out the possibility that
an increased rate of glutathione synthesis is responsible for
decreased sensitivity to cystine deprivation – induced gluta-
thione depletion ( 20 ), we used buthionine sulfoximine (BSO)
to directly inhibit the rate-limiting enzyme of glutathione
synthesis, ? -glutamylcysteine synthetase ( ? -GCS; Fig. 6 B ).
BSO resulted in cell death in ATF4 +/+ neurons, whereas
ATF4 ? / ? neurons were essentially resistant ( Fig. 6, C and D ).
As expected from these results, ? -GCS expression did not
diff er between ATF4 +/+ and ATF4 ? / ? neurons at the pro-
tein level and did not appear in the gene array ( Fig. 6 E ).
Similar to HCA, BSO treatment led to a decline in the con-
centration of glutathione in ATF4 +/+ neurons, whereas
ATF4 ? / ? neurons displayed a distinctly slower reduction of
glutathione ( Fig. 6 F ). These fi ndings are consistent with the
notion that the diff erences in reduction of glutathione after
HCA or BSO is not attributable to changes in synthesis. To
determine whether ATF4 directly regulates the neuronal
glutathione content, we measured glutathione in neurons
with forced expression of ATF4WT, ATF4 ? RK, and GFP
( Fig. 6 G ). Consistent with the fi nding that forced expression
of ATF4WT can reduce neuronal viability, we found that
this viability reduction correlates with a reduction in the
neuronal glutathione content. Because cell death attribut-
able to glutathione depletion can be completely blocked by
a whole host of classical antioxidants, we combined forced
expression of ATF4WT with butylated hydroxyanisol
(BHA) treatment ( Fig. 6, H and I ). BHA is a well characterized
Overexpression of ATF4 is suffi cient to restore sensitivity
to glutathione depletion – induced cell death and is capable
of inducing cell death by itself
Because we used germline knockout animals ( 19 ) in this study,
we could not exclude the possibility that the decreased sus-
ceptibility to oxidative stress was a consequence of a compen-
satory eff ect distantly related to ATF4 defi ciency. To address
this question, we used adenoviral overexpression of a mouse
ATF4WT construct. As a control, we used a dominant-
negative ATF4 construct harboring a mutation in its DNA
binding domain ( 292 RYRQKKR 298 to 292 GYLEAAA 298 ). To
confi rm the diff erent DNA binding properties of these con-
structs, we analyzed the 33-bp binding region of the TRB3
promoter by gel-shift ( Fig. 4 A ) and chromatin immuno-
precipitation studies ( Fig. 4 B ). In fact, only nuclear extracts
from cells overexpressing ATF4WT in combination with
WT oligonucleotide formed a band specifi c for ATF4. Con-
sistently, only TRB3 promoter chromatin from cells over-
expressing ATF4WT could be PCR amplifi ed using primers
fl anking the ATF4 binding site. Cotransfection of WT and
mutant ATF4 constructs with reporter constructs containing
the WT and the mutant binding site confi rmed the activating
eff ect ATF4 has on transcription ( Fig. 4 C ).
To determine whether forced expression of ATF4 in
ATF4 ? / ? neuronal cultures can restore sensitivity to oxidative
stress and/or has an eff ect itself, we overexpressed both constructs
along with a GFP control in both ATF4 +/+ and ATF4 ? / ? cor-
tical neuronal cultures, followed by treatment with HCA ( Fig.
5 ). We confi rmed the effi cient expression of both ATF4 con-
structs in cortical neurons by immunohistochemistry ( Fig. 5 A )
and Western blotting ( Fig. 5 B ). Specifi cally, ? 50% of neurons
and a small number of glia are infected with the adenoviruses.
Infection with ATF4 ? RK was able to protect ATF4 +/+ corti-
cal neuronal cultures from HCA-induced toxicity ( Fig. 5, C
and D ), whereas infection with ATF4WT was capable of ren-
dering ATF4 ? / ? neurons sensitive to HCA ( Fig. 5, E and F ).
Moreover, infection with ATF4WT itself signifi cantly reduced
the viability in both WT and ATF4 ? / ? neurons. Combination
of forced expression of ATF4WT and treatment with HCA re-
sulted in a higher loss of viability than infection with ATF4WT
alone, suggesting that at least one more pathway in addition to
ATF4 is involved in HCA toxicity. Collectively, these results
are consistent with the notion that ATF4 indeed plays a key
role in oxidative stress – mediated cell death.
calculated from three separate experiments and are given as the ratio of luciferase and Renilla activities (mean ± SD; n = 3). The value for treatment with vehicle
control was arbitrarily defi ned as 1. (F) Oxidative stress results in nuclear accumulation of ATF4 in cultured cortical neurons. 60 μ g of nuclear extracts from corti-
cal neurons treated with 10 mM HCA or vehicle control (shown as C) were separated using gel electrophoresis and immunodetected using an antibody against
ATF4. YY1 was monitored as a loading control. (G) Real-time PCR of TRB3 mRNA expression in ATF4 +/+ and ATF4 ? / ? neurons in response to treatment with 10
mM HCA. The value obtained from the ATF4 +/+ control was arbitrarily defi ned as 1. Mean ± SD was calculated from three separate experiments. Each data point
was performed in duplicate. (H) ATF4 +/+ and ATF4 ? / ? neurons were transfected with a luciferase reporter plasmid (pGL3 backbone) containing a 2-kb fragment of
the mouse TRB3 promoter (TRB3WT), with a mutant version of this promoter lacking the 33-bp ATF4 binding site (TRB3 ? 33bp) or with the empty vector (pGL3
basic). Cortical neurons were cotransfected with a plasmid expressing Renilla to allow normalization for transfection effi ciency. 24 h after transfection, neurons
were treated with vehicle control (shown as C) or 10 mM HCA. Cells were harvested in luciferase assay buffer 12 h after the onset of treatment. Values are given
as the ratio of luciferase and Renilla activities (mean ± SD) and were calculated from three separate experiments. Each data point was performed in duplicate.
Values are given as the ratio of luciferase and Renilla activities (mean ± SD; n = 3). The value for empty pGL3 was arbitrarily defi ned as 1.
ATF4 IS A PRODEATH PROTEIN IN NEURONS | Lange et al.
ing the redox-sensitive probe 2 ? ,7 ? -dichlorofluorescin
(DCF; Fig. S1, available at http://www.jem.org/cgi/content/
full/jem.20071460/DC1). Indeed, ATF4 ? / ? neurons dis-
played lower levels of ROS in response to HCA treatment
than ATF4 +/+ neurons. These fi ndings are consistent with
antioxidant known to block HCA-induced cell death in
cortical neurons ( 6 ). In fact, in neurons overexpressing
ATF4WT, BHA signifi cantly increased neuronal viability
and protected against the additional toxic eff ect of HCA
treatment. Finally, we measured production of ROS by us-
Figure 3. Gene expression array analysis of ATF4 +/+ and ATF4 ? / ? neurons before and after treatment with HCA. (A) Heatmap with cluster
dendrogram of the differentially expressed genes (log2 fold change vs. control) at a false discovery rate of 5%. Unsupervised clustering groups samples
by genotype and by treatment. Genes are in rows and samples are in columns. Column color coding is as follows: red, ATF4 ? / ? versus ATF4 +/+ untreated
neurons; blue, HCA-treated ATF4 ? / ? versus untreated ATF4 ? / ? neurons; and green, HCA-treated ATF4 +/+ versus untreated ATF4 +/+ neurons. (B) The num-
ber of genes that are up- (red) and down-regulated (green). Shown are three different contrasts: ATF4 ? / ? versus ATF4 +/+ untreated neurons, HCA-treated
ATF4 +/+ versus untreated ATF4 +/+ neurons, and HCA-treated ATF4 ? / ? versus untreated ATF4 ? / ? neurons. The complete list of differentially expressed
genes is available in Table S3 (available at http://www.jem.org/cgi/content/full/jem.20071460/DC1). ANOVA FDR, analysis of variance false discovery
rate; C, control.
JEM VOL. 205, May 12, 2008
been associated with stroke pathology and repletion of anti-
oxidants has reduced neuronal damage ( 22 ). Therefore, we
hypothesized that ATF4 defi ciency might positively infl u-
ence the antioxidant status in the adult brain and thereby im-
prove the outcome after ischemia-reperfusion injury. To test
the hypothesis that ATF4 germline knockout animals have
less neuronal loss after stroke, we used a transient middle ce-
rebral artery occlusion (MCAo) model for ischemia-reperfu-
sion injury, as previously described ( 23 ).
the notion that ATF4 positively infl uences ROS levels and
leads to a higher consumption of glutathione independent of
ATF4 ? / ? animals are less susceptible to ischemic brain damage
Oxidative stress is an established mediator of neuronal loss in
cerebral ischemia ( 21 ). It is generally accepted that glutathi-
one acts as a major endogenous cerebral antioxidant in the
adult brain. In fact, depletion of intracellular antioxidants has
Table I. Differentially expressed genes in ATF4 -/- neurons versus ATF4 +/+ neurons before and after HCA treatment
From each of the three contrasts, the top 30 genes with the highest fold change were selected. Ratios are log2 transformed. Up-regulated genes (fold change > 0.2) are
highlighted in red, and down-regulated genes (fold change < ? 0.2) are highlighted in green.
ATF4 IS A PRODEATH PROTEIN IN NEURONS | Lange et al.
cerebral blood vessels of the circle of Willis ( Fig. 7 A , middle)
( 23 ). Besides a higher degree of tortuosity in the MCA, no
major abnormalities were detected in the brains obtained from
ATF4 ? / ? mice. Finally, using immunofl uorescent staining for
the endothelial-specifi c marker CD31/PECAM, we did not
observe signifi cant diff erences in the density or morphology
of the microvessels ( Fig. 7 A , bottom). In addition, important
First, we performed morphological studies to rule out that
germline ATF4 defi ciency causes a fundamental abnormality
in the adult brain that would bias stroke outcome ( 19, 24 – 26 ).
Examining sagittal sections of brains from ATF4 +/+ and
ATF4 ? / ? animals ( Fig. 7 A , top), we did not detect any sig-
nifi cant structural diff erences in the cerebellum, hippocam-
pus, or cortex. We then assessed the morphology of major
Figure 4. ATF4 binds to a 33-bp binding element within the TRB3 promoter. (A) EMSA performed with 10 μ g of dialyzed nuclear extracts
from HT22 cells transfected with ATF4WT, mutant ATF4 (ATF4 ? RK), or GFP, respectively. Extracts were incubated with a radioactively labeled WT
oligonucleotide containing the TRB3 promoter binding site or with a mutant oligonucleotide. Binding of ATF4WT to the TRB3WT promoter binding
site was confi rmed by supershift analysis (arrow) performed with an antibody (Ab) directed against ATF4. (B) Overexpressed ATF4WT protein occupies
its putative binding site within the TRB3 promoter in HT22 cells, as shown by chromatin immunoprecipitation assay. HT22 cells were transfected with
ATF4WT, mutant ATF4 (ATF4 ? RK), or GFP. An anti-myc antibody was used to precipitate the proteins in nuclear extracts of cross-linked HT22 cells.
Coprecipitated DNA fragments were detected using PCR with a set of primers specifi c for the ATF4 binding site in the TRB3 promoter, yielding a
190-bp product. A representative example of three experiments is shown. (C) HT22 cells were transfected with the expression plasmids for ATF4WT,
mutant ATF4 (ATF4 ? RK), or GFP. The cells were cotransfected with either a luciferase reporter vector containing the 33-bp ATF4WT binding site (33 bp
WT), a reporter vector containing a mutant form of this binding site (33 bp MUT), or empty vector (pGL3 basic). In parallel, the transfection mix con-
tained a plasmid expressing Renilla to allow normalization for transfection effi ciency. The value for empty pGL3 cotransfected with GFP was arbi-
trarily defi ned as 1. Shown are ratios of luciferase and Renilla activities (mean ± SD for three independent experiments; each data point was
performed in duplicate).
JEM VOL. 205, May 12, 2008
The procedure used to occlude the MCA (occlusion time =
45 min) led to reproducible infarcts in WT animals involving
both the cerebral cortex and the striatum, with sparing of
physiological parameters did not diff er between ATF4 +/+ and
ATF4 ? / ? animals (Fig. S2 and Table S2, available at http://
Figure 5. Overexpression of ATF4WT restores sensitivity to oxidative stress and reduces neuronal viability itself. (A) Representative immuno-
cytochemistry of ATF4 +/+ and ATF4 ? / ? cortical neurons infected with the adenoviral constructs ATF4WT and ATF4 ? RK. Neurons were stained with anti-
bodies against myc (green) and MAP2 (red), and were counterstained with Hoechst dye (blue). Bar, 50 μ m. (B) Whole-cell extracts obtained from both
ATF4 +/+ and ATF4 ? / ? neurons infected with GFP, ATF4WT, and ATF4 ? RK were separated using gel electrophoresis and immunodetected using an antibody
directed against the myc tag. Total eIF2 ? was monitored as a loading control. (C) ATF4 +/+ cortical neurons were infected with GFP, ATF4WT, and ATF4 ? RK
adenoviruses at an MOI of 100. 24 h after infection, neurons were treated with vehicle control (shown as C) or 10 mM HCA. 24 h later, cell viability was
determined using the MTT assay. The graph depicts mean (compared with control) ± SD calculated from four separate experiments for each group ( n =
45). P < 0.05 by the Kruskal-Wallis test followed by Dunn ’ s multiple comparisons test from untreated ATF4WT-overexpressing neurons (*) and from HCA-
treated neurons overexpressing GFP ( § ). (D) Live/dead assay. Bar, 50 μ m. (E) ATF4 ? / ? cortical neurons were infected with GFP, ATF4WT, and ATF4 ? RK ad-
enoviruses at an MOI of 100. 24 h after infection, neurons were treated with vehicle control (shown as C) or 10 mM HCA. 24 h later, cell viability was
determined using the MTT assay. The graph depicts mean (compared with control) ± SD calculated from four separate experiments for each group ( n =
28). P < 0.05 by the Kruskal-Wallis test followed by Dunn ’ s multiple comparisons test from untreated ATF4WT-overexpressing neurons (*) and from HCA-
treated neurons overexpressing GFP ( § ). (F) Live/dead assay. Bar, 50 μ m.
ATF4 IS A PRODEATH PROTEIN IN NEURONS | Lange et al.
Figure 6. ATF4 has a negative impact on the neuronal glutathione metabolism. (A) ATF4 +/+ ( ? ) and ATF4 ? / ? ( ? ) cortical neurons were treated with
10 mM HCA. At the indicated time points, cells were trypsinized, washed, and pelleted. Reduced glutathione (GSH) was determined in the cell pellets using HPLC
electrochemical detection. Data are from three separate cultures, and each data point was measured in duplicate. Graph depicts mean ± SD. (B) Schematic
overview over cysteine uptake and glutathione synthesis and their inhibition. (C) ATF4 +/+ and ATF4 ? / ? cortical neurons were treated with a vehicle control
(shown as C) or 200 μ M BSO. 24 h later, cell viability was determined using the MTT assay. The graph depicts mean (compared with control) ± SD calculated
from fi ve separate experiments for each group ( n = 46 ATF4 +/+ and 58 ATF4 ? / ? ). *, P < 0.05 from untreated ATF4 +/+ cultures by the Kruskal-Wallis test followed
by Dunn ’ s multiple comparisons test. The difference between treated and untreated ATF4 ? / ? neurons was not signifi cant (n.s.). (D) Live/dead assay displaying
untreated and BSO-treated ATF4 +/+ and ATF4 ? / ? neurons. Bar, 50 μ m. (E) Protein expression of ? -GCS does not differ between ATF4 +/+ and ATF4 ? / ? cortical
JEM VOL. 205, May 12, 2008
pression reinstated sensitivity to oxidative death. We establish
the relevance of protection by ATF4 defi ciency by demon-
strating that the ATF4 homozygous knockout is capable of
protecting the adult mouse brain from stroke-induced injury
and disability. Indeed, we show that ATF4 ? / ? animals recover
more easily and maintain proper motor function more effi -
ciently. Although known to be a stress-responsive protein,
these results for the fi rst time establish ATF4 as a redox-regu-
lated protein that can function to lower the threshold for oxi-
dative stress – induced death in neurons.
ATF4 is a redox-regulated transcription factor
Classically, stress-mediated enhancement of ATF4 levels is
known to occur via enhanced effi ciency of translation of
constant levels of ATF4 mRNA ( 29, 30 ). Consistent with
this model, we found an up-regulation of ATF4 mRNA
translational effi ciency after glutathione depletion. How-
ever, we also observed an increase in ATF4 mRNA levels
after oxidative stress, thereby confi rming a recent report
describing a role for transcriptional regulation in ATF4 in-
duction by cell stress ( 31 ). The distinct increase in ATF4
protein levels in the nucleus of neurons undergoing oxida-
tive stress corresponded to an ATF4-dependent induction
of a TRB3 promoter-luciferase reporter. Collectively, these
observations argue that oxidative stress – induced changes in
ATF4 mRNA levels and translational effi ciency lead to in-
creased ATF4 activity in neurons. Because ATF4 overex-
pression is suffi cient to induce death, limiting the amount
of ATF4, at least under basal conditions, seems to be neces-
sary for neuronal survival. Mechanisms that control and
limit basal ATF4 activity ( 32 – 34 ) appear to be important
determinants of neuronal fate. Although this low level of
ATF4 appears to be suffi cient to regulate a host of genes, its
absence does not grossly infl uence neuronal morphology or
ATF4 has a direct impact on glutathione metabolism
Several converging lines of inquiry demonstrate a central
role for glutathione metabolism in degeneration and aging
in the nervous system. Our fi ndings confi rm the primary
role that glutathione can play in neuronal death. However,
the mechanisms by which a global depletion of glutathione
in neurons is transduced into apoptotic events remained
elusive. The data presented in this study favor a model in
the hippocampus. However, a signifi cantly smaller infarct
was observed when the procedure was performed in ATF4 ? / ?
animals ( Fig. 7, C and D ). The smaller infarct area could be
observed in all sections of the brain. Thus, the higher resis-
tance to ischemia-reperfusion injury was not limited to a spe-
cifi c brain area. In fact, the smaller infarct volume was
associated with a faster recovery in ATF4 ? / ? animals, as mea-
sured by a simple neurological score ( Fig. 7 E ). In addition,
two simple behavioral tests ( Fig. 7, F and G ) complemented
the morphological data. In consideration of the eye lens mal-
formation regularly observed in ATF4 ? / ? animals ( 25 ), these
tests are not dependent on the visual sense. Consistent with
the morphological data, ATF4 ? / ? animals performed signifi -
cantly better than the ATF4 +/+ control group, suggesting that
ATF4 defi ciency facilitates recovery after stroke.
ATF4 is a prodeath transcription factor
Solid evidence supports the hypothesis that oxidative stress is
an initiator or propagator of neuronal dysfunction or death in
prevalent neurological diseases, including stroke, spinal cord
injury, Alzheimer ’ s disease, and Parkinson ’ s disease ( 3, 27 ).
These studies have led to the somewhat surprising conclusion
that oxidants can trigger neuronal death via highly regulated
signaling pathways and subsequent activation of prodeath tran-
scription factors, leading to the controlled demise known as
apoptosis ( 28 ). The current study adds to our understanding of
the cellular transcription factors that regulate neuronal viability
and function after oxidative stress in primary cortical neurons.
Specifi cally, we show that the transcription factor ATF4 is in-
duced by oxidative stress caused by depletion of the major an-
tioxidant tripeptide, glutathione. We further demonstrate that
transgenic deletion of ATF4 renders neurons resistant to neu-
ronal cell death. Prevention of cell death by germline knock-
out of ATF4 is associated with a preservation of glutathione
levels, the primary mediator of death in our oxidative stress
model. Consistent with ATF4 ’ s role in regulating an early,
upstream aspect of the oxidative neuronal death pathway, we
found that ATF4 defi ciency causes global down-regulation of
gene expression and blocks the up-regulation of many genes
that are induced by oxidative stress in WT neurons. Accord-
ingly, forced expression of ATF4 was suffi cient to promote
cell death and loss of glutathione. In addition, we demonstrate
that in ATF4 ? / ? neurons, restoration of ATF4 protein ex-
neurons. Cytoplasmic extracts were separated using gel electrophoresis and immunodetected using an antibody against ? -GCS. Total eIF2 ? was monitored
as a loading control. (F) ATF4 +/+ ( ? ) and ATF4 ? / ? ( ? ) cortical neurons were treated with 200 μ M BSO. At the indicated time points, cells were trypsinized,
washed, and pelleted. GSH was determined in the cell pellets using HPLC electrochemical detection. Data are from three separate cultures, and each data point
was measured in duplicate. Graph depicts mean ± SD. (G) ATF4 +/+ cortical neurons were infected with GFP ( ? ), ATF4WT ( ? ), and ATF4 ? RK ( ? ) adenoviruses
at an MOI of 100. At the indicated time points after infection, cells were trypsinized, washed, and pelleted. GSH was determined in the cell pellets using HPLC
electrochemical detection. The graph depicts mean ± SD calculated from three separate experiments for each group, and each data point was measured in
duplicate. The value obtained from noninfected neurons was arbitrarily defi ned as 100%. (H) ATF4 +/+ cortical neurons were infected with GFP and ATF4WT ad-
enoviruses at an MOI of 100. 24 h after infection, neurons were treated with vehicle control (shown as C), 10 mM HCA, 10 μ M BHA, or a combination of both.
The graph depicts mean (compared with control) ± SD calculated from fi ve separate experiments for each group ( n = 29). *, P < 0.05 from untreated neurons
overexpressing ATF4WT by the Kruskal-Wallis test followed by Dunn ’ s multiple comparisons test. The difference between neurons overexpressing ATF4WT
treated with BHA alone and neurons overexpressing ATF4WT treated with both BHA and HCA was not signifi cant (n.s.). (I) Live/dead assay. Bar, 50 μ m.
ATF4 IS A PRODEATH PROTEIN IN NEURONS | Lange et al.
vation includes the increased production of ROS and turn-
over of glutathione. Although our data clearly establish that
ATF4 induction does not infl uence glutathione synthesis,
we cannot determine whether the increased turnover of
which glutathione depletion causes an ATF4-dependent
up-regulation of a coordinated set of genes that orchestrate
the timely and irreversible demise of the cell. Part of the
orchestrated sequence of events downstream of ATF4 acti-
Figure 7. Role of ATF4 in brain ischemia. (A, top) Sagittal sections of ATF4 +/+ (left) and ATF4 ? / ? (right) brains stained with cresyl violet. Bar, 3,000 μ m.
(A, middle) Ventral view of large cerebral blood vessels of representative ATF4 +/+ (left) and ATF4 ? / ? (right) mice that were perfused with India ink.
Note the higher degree of tortuosity of the MCA in the brain from the ATF4 ? / ? mouse. Bar, 3,000 μ m. (A, bottom) Representative microscopic views of
brain sections from ATF4 +/+ and ATF4 ? / ? mice that were immunostained for the endothelial cell – specifi c marker CD31 (green). Bar, 100 μ m. (B) Represen-
tative brain sections at 4 d after MCAo from ATF4 +/+ ( n = 5) and ATF4 ? / ? ( n = 6) mice from rostral to caudal stained with cresyl violet to determine the
infarct area. Bar, 1,000 μ m. (C) The infarct area in ATF4 +/+ ( ? ) and ATF4 ? / ? ( ? ) brains was measured in 12 sequential sections taken from ATF4 +/+ ( n = 5)
and ATF4 ? / ? ( n = 6) mice at rostral to caudal regular intervals. Graph depicts mean ± SD. (D) The infarct volume was assessed by adding the infarct vol-
umes based on the infarct area in each section. Graph depicts mean ± SD. *, P < 0.0001 by the t test. (E) Scoring of neurological defi cit was assessed at
different time points of recovery in ATF4 +/+ ( n = 5) and ATF4 ? / ? ( n = 4) mice. Graph depicts mean ± SD. *, P < 0.05 by the Mann-Whitney test. (F) Inclined
plane test at different time points after stroke in ATF4 +/+ ( n = 5) and ATF4 ? / ? ( n = 4) mice. The test measured the time a mouse managed to hold itself on
an inclined glass plate angled at 50 ° before sliding down. Graph depicts mean ± SD. *, P < 0.05 by the t test. (G) Hanging wire test at different time points
after stroke in ATF4 +/+ ( n = 5) and ATF4 ? / ? ( n = 4) mice. The hanging wire test determined the time it took an animal to cross a distance of 45 cm on a
freely hanging narrow metal bar. Graph depicts mean ± SD. *, P < 0.05 by the t test.
JEM VOL. 205, May 12, 2008
addition to ATF4 induction ( 2, 45 ). Which of these signaling
events are capable of preventing the deleterious consequences
of ATF4 activation requires further investigation. In general,
the similarity of data obtained from fi broblasts and from cor-
tical neurons is limited. However, despite the opposite eff ects
on glutathione metabolism and cell survival, a comparison
between the array data from fi broblasts with our gene ex-
pression data indicates a highly signifi cant overlap. Even
though the role of each of the many target genes of ATF4
warrant further investigation, the diff erent metabolic de-
mands of postmitotic neurons and actively dividing fi bro-
blasts might help to explain the distinct role of ATF4 in
distinct cell types ( 46 ). Additionally, diff erently expressed di-
merization partners of ATF4 might also signifi cantly modify
ATF4 ’ s function. Indeed, several diff erent cancer cell lines
show an elevated level of ATF4 expression ( 10, 47 – 50 ).
Moreover, ATF4 is already known to have functions that are
limited to a specifi c cell type such as osteoblasts ( 40, 51, 52 ).
The precise characterization of these additional signaling
events warrants further investigation, and might help to ex-
plain why and how these signaling events are capable of pre-
venting the deleterious consequences of ATF4 induction.
ATF4 is a prodeath transcription factor in vivo
We have established that ATF4 also plays an important pro-
death role in an in vivo model of stroke. However, the use of
conventional ATF4 ? / ? animals poses a risk of bias on the
outcome in a complex paradigm such as stroke ( 53 ). There-
fore, the results need to be interpreted with caution. We can-
not rule out the possibility that ATF4 defi ciency in all cell
types of the brain and the body has an impact on stroke out-
come. However, no overt diff erence was observed when
comparing the brain morphology between WT and ATF4 ? / ?
brains, including the vascular system. In addition, the smaller
infarct size in the ATF4 ? / ? mice could not be attributed to
changes in systemic physiological parameters, including body
temperature, or changes in cerebral blood fl ow under basal
conditions or after MCAo. However, an issue that was not
addressed in this study is whether the ATF4 ? / ? mice dis-
played an altered infl ammatory response after ischemia that
could explain the smaller infarct size. Although a higher pro-
pensity to infections or other immune-related alterations has
not yet been observed in ATF4 ? / ? mice, we cannot rule out
the possibility that an altered infl ammatory response ( 54 ) is
one reason for the smaller infarct volume in the ATF4 ? / ?
mice. In summary, the strong correlation in the outcome be-
tween in vitro and in vivo studies lets us conclude that our
results, even when reviewed carefully, suggest an important
role for ATF4 in the induction of neuronal cell death. Thus,
ATF4 might be an important target for therapeutic interven-
tion against stroke and other neurodegenerative diseases.
MATERIALS AND METHODS
Animals. All animal procedures were performed according to protocols
approved by the Institutional Animal Care and Use Committee of the Weill
Medical College of Cornell University. Germline ATF4 ? / ? mice have been
described previously ( 19 ). For the culture of mouse embryonic cortical
glutathione is primarily attributable to a change in a gluta-
thione-degrading enzyme with a subsequent increase of ROS,
or a secondary consequence of ATF4-mediated ROS pro-
duction. Increased ROS production would be expected to
decrease glutathione levels by enzyme-mediated consump-
tion of the thiol tripeptide. Of note, many mitochondrial
genes that could facilitate production of deleterious ROS
during respiration are down-regulated in the ATF4 ? / ?
neurons. Future studies will clarify whether ATF4 mediates
death primarily by altering glutathione turnover or by dial-
ing up the production of free radicals.
ATF4 induces global transcriptional changes
ATF4 was originally described as a transcriptional repressor
( 35, 36 ). However, our gene array data suggest that ATF4
acts at least in part as a transcriptional activator. This fi nding
is in line with several more recent publications that describe
the activating eff ect of ATF4 on several target genes, such as
heme oxygenase 1 ( 37, 38 ), stanniocalcin 2 ( 39 ), osteocalcin
( 40 ), gadd153/CHOP ( 41 ), and TRB3 ( 42 ). Some of these
target genes have been shown to play an important role in
cell death and cell survival in the brain. Pharmacological in-
duction of heme oxygenase 1 was demonstrated to protect
neurons from oxidative insults both in vitro and in vivo ( 43 ).
Indeed, heme oxygenase 1 is induced in WT neurons by oxi-
dative stress; however, it was down-regulated in ATF4 ? / ?
neurons. Likewise, stanniocalcin 2 is up-regulated in response
to oxidative stress in WT neurons but was down-regulated in
ATF4 ? / ? neurons. In a recent work ( 39 ), stanniocalcin 2 was
identifi ed as an oxidative stress – responsive gene with cyto-
protective properties. A prodeath ATF4 target gene, TRB3,
was strongly dependent on ATF4 and up-regulated in re-
sponse to oxidative stress. TRB3 ’ s role in neuronal apoptosis
was described fi rst in a neuronal cell line ( 44 ) and has been
recently confi rmed ( 42 ).
The prodeath role of ATF4 might be context
and cell type specifi c
The data presented in this work are in support of a strong
prodeath role of ATF4. However, previous fi ndings regard-
ing the roles of ATF4 have yielded partly diff erent results.
ATF4 ? / ? fi broblasts are impaired in expressing genes in-
volved in amino acid import, glutathione biosynthesis, and
resistance to oxidative stress ( 11 ). Therefore, ATF4 was pro-
posed to play a key role in rendering the cell more resistant
against the metabolic consequences of endoplasmic reticulum
oxidation. Therefore, we initially hypothesized that ATF4
induction by endoplasmic reticulum stress or amino acid de-
pletion is the key event in the protection against oxidative
stress mediated by HCA treatment ( Fig. 1 ). The fi nding that
ATF4 activation can be associated with protection from oxi-
dative stress – induced cell death, although it clearly acts as a
prodeath transcription factor, points to a complex and con-
text-specifi c role of ATF4 in the propagation of neuronal cell
death. However, both endoplasmic reticulum stress or amino
acid depletion initiate several diff erent signaling events in
ATF4 IS A PRODEATH PROTEIN IN NEURONS | Lange et al.
http://www.bioconductor.org; reference 57 ). Raw data were log2 trans-
formed and normalized using quantile normalization. Analysis of diff erential
expression was performed using a linear model fi tting (LIMMA package; ref-
erence 58 ). The obtained p-values were corrected for multiple testing using
the false discovery rate method, and a threshold of 0.05 was applied. Micro-
array data have been deposited in the National Center for Biotechnology
Information Gene Expression Omnibus under accession no. GSE10470 .
DNA EMSA. Crude nuclear extracts were purifi ed by dialysis using Slide-
A-Lyzer MINI Dialysis Units (Thermo Fisher Scientifi c). The following
oligonucleotide probes (Invitrogen) corresponding to the ATF4 binding site
of the TRB3 gene were used: TRB3WT, 5 ? -GATTAGCTCAGGTTTA-
CATCAGCCGGGCGGGGA-3 ? ; and TRB3MUT, 5 ? -GATTAGCT-
CAGTCTAAACCTATAGGGGCGGGGA-3 ? . The oligonucleotides were
annealed with complementary DNA and radiolabeled with ? -[ 32 P]ATP us-
ing T4 polynucleotide kinase. After incubation with nuclear extracts, the
DNA – protein complexes were resolved in 5% polyacrylamide gels, and the
signal was visualized using a PhosphorImager (Fujifi lm).
Chromatin immunoprecipitation assay and PCR amplifi ca-
tion. Formaldehyde cross-linking and chromatin immunoprecipitation
were performed as described previously ( 59 ). The cross-linked chromatin
suspension was sonicated using a Sonicator 3000 (Mosonix). Immuno-
precipitation was performed with an anti-myc antibody. DNA – protein
cross-linking was reversed, followed by an overnight incubation at 65 ° C.
DNA was isolated by phenol/chloroform extraction and subjected to PCR
analysis using the primers 5 ? -GGTCACAGATGGTGCAATCC-3 ? and
5 ? -AACTGAGCAGCTCTCGGAGTC-3 ? .
Glutathione determination using HPLC electrochemical detection
and ROS detection using DCF fl uorescence. Concentrations of reduced
glutathione were measured using HPLC (PerkinElmer) equipped with an
eight-channel coulometric array detector (ESA, Inc.). Cells were lysed in 5%
(wt/vol) metaphosphoric acid and centrifuged at 10,000 g for 10 min to sedi-
ment protein. Cell-pellet precipitates were saved for protein determinations.
Glutathione concentrations of supernatant fractions were determined by in-
jecting 5- μ l aliquots onto an Ultrasphere 5 u, 4.6 × 250 mm, C18 column
(Beckman Coulter), and eluting with a mobile phase of 50 mM NaH 2 PO 4 ,
0.05 mM octane sulfonic acid, 1.5% acetonitrile (pH 2.62) at a fl ow rate of
1 ml/min. Peak areas were analyzed using software from ESA, Inc. Intracellular
generation of ROS was determined by DCF. Cortical neurons were seeded in
96-well fl uorescence plates. After the treatments indicated in the fi gures, the
cultures were incubated for 45 min with 1.25 μ M DCF-H 2 . Cells were washed
with HBSS and read on a plate reader (Fluorometer; MDS Analytical Tech-
nologies) at 485-nm excitation and 530-nm emission wavelengths.
Cerebral ischemia. Transient focal cerebral ischemia was induced by
MCAo using the intraluminal fi lament method, as previously described ( 23 ).
Male mice were anesthetized. A small incision was performed in the skin
covering the scalp, and a fi ber optic probe was glued to the parietal bone and
connected to a laser-Doppler fl owmeter (Perifl ux System 5010; Perimed) for
continuous monitoring of cerebral blood fl ow. For MCAo, a heat-blunted
black monofi lament surgical suture (6-0) was inserted into the exposed ex-
ternal carotid artery, advanced into the internal carotid artery, and wedged
into the circle of Willis to obstruct the origin of the MCA. The fi lament was
left in place for 45 min and withdrawn.
Physiological parameters. Animal physiology was assessed in ATF4 +/+
and ATF4 ? / ? mice ( n = 3 each). For this purpose, the left femoral artery was
cannulated using a polyethylene tube (PE-10; BD Biosciences) to record
mean arterial blood pressure. Blood samples were taken for chemical analysis
using a hand-held blood analyzer (iSTAT; Abbot Laboratories).
Quantifi cation of infarct volume. Coronal brain sections were serially
cut in a cryostat (Leica) and stained with cresyl violet to identify viable tissue.
neurons, embryos (15.5 days post conceptionem) were obtained from the
mating of ATF4 heterozygous mice.
Primary cortical neurons and cell culture. Primary neuronal cultures
were prepared from the cerebral cortices of mouse embryos, as described
previously ( 55 ), with minor modifi cations. The mouse hippocampal cell line
HT22 was maintained according to standard procedures ( 56 ). Transfection of
both cortical neurons and HT22 cells was performed using Lipofectamine
2000 (Invitrogen) according to the manufacturer ’ s instructions. Recombinant
arginase was provided by D. Ash (Temple University, Philadelphia, PA).
Genotyping. Genomic DNA for PCR was prepared using the DNeasy
genomic DNA isolation kit (QIAGEN). The WT alleles were detected us-
ing the primer 5 ? -AGCAAAACAAGACAGCAGCCACTA-3 ? . The ATF4 ? / ?
alleles (neomycin) were detected using the primer 5 ? -ATATTGCTG-
A A G A G CTTGGCGGC-3 ? . As a common reverse primer, we used
5 ? - G T T T C T A CAGCTTCCTCCTCCACTCTT -3 ? .
Cell viability/cell death assays. Cell viability was assessed by propidium
iodide uptake and retention of calcein (Invitrogen) using an inverted epi-
fl uorescence microscope (Axiovert 200M; Carl Zeiss, Inc.). MTT assay was
performed according to the manufacturer ’ s instructions (Promega). In brief,
absorbance was measured at 570 nm. From each data point, the reference
absorbance measured at 690 nm was subtracted. Finally, values were calcu-
lated as the percentage of untreated control cells.
Plasmids. The expression plasmids for ATF4WT and dominant-negative
ATF4 ? RK (provided by J. Alam, Ochsner Foundation, New Orleans, LA)
( 37 ), and the mouse ATF4 5 ? UTR luciferase construct (D. Ron, New York
University, New York, NY) have been previously described. The promoter
construct pTRB3 was generated by amplifi cation of genomic mouse DNA
using the primers 5 ? -CTCACTCAGGTGCCTGTAGTGCTCG-3 ? and 5 ? -
TCAGCAGAAGCAGCCAGAGGTGTAG-3 ? , followed by a second round
of PCR with the primers 5 ? -ACACTCGAGAGAGAAACAAATGTGT-
CATG-3 ? and 5 ? -ACAAAGCTTCTAGAGAGCAAGGAAGAAAG-3 ? be-
fore cloning into the vector pGL3basic (Promega). The mutant form lacking
the 33-bp ATF4 binding site was generated using a site-directed mutagenesis
kit (QuikChange; Stratagene). To generate the reporter plasmids with the
33-bp ATF4 binding sequence, the following oligonucleotides were annealed
and cloned into pGL3 basic: p33WT, 5 ? -TCGAGGCAGATTAGCT-
CAGGTTTACATCAGCCGGGCGGGGATCCA-3 ? and 5 ? -AGCTTG-
GATCCCCGCCCGGCTGATGTAAACCTGAGCTAATCTGCC-3 ? ;
and p33MUT (mutant form), 5 ? -TCGAGGCAGATTAGCTCAGTCTA-
AACCTATAGGGGCGGGGATCCA-3 ? and 5 ? -AGCTTGGATCCCC-
GCCCCTATAGGTTTAGACTGAGCTAATCTGCC-3 ? . All sequences
were verifi ed by automatic DNA sequencing.
Luciferase assay. Primary neurons and HT22 cells were transfected with
the respective reporter construct along with the pTK-Renilla control
(Promega). A dual luciferase assay (Promega) was performed using a biolu-
minometer (MDS Analytical Technologies), according to the manufacturer ’ s
Adenoviruses. To generate the adenoviruses ATF4WT and ATF4 ? RK,
the expression cassettes of each construct were cloned into the shuttle vector
ad5 pVQ-K-NpA. The correct sequence was confi rmed by automatic DNA
sequencing. Virus generation and amplifi cation were performed by Vira-
Quest. Infection with adenoviruses was performed at a multiplicity of infec-
tion (MOI) of 100.
Gene array analysis. RNA quantity was assessed with a spectrophotometer
(Nanodrop; Thermo Fisher Scientifi c), and quality was assessed with nano-
chips (Bioanalyzer; Agilent Technologies). Total RNA was amplifi ed,
labeled, and hybridized on arrays (MouseRef-8 Expression BeadChip; Illu-
mina). Data analysis was performed using Bioconductor packages (available at
JEM VOL. 205, May 12, 2008 Download full-text
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To correct for the eff ect of edema, the infarcted area was determined indi-
rectly by subtracting the area of the healthy tissue in the ipsilateral hemi-
sphere from the area of the contralateral hemisphere on each section.
Infarction volume was calculated by integration of infarct areas measured in
20 equidistant brain sections that encompassed the whole lesion. Volumes
from all sections were summed to calculate total infarct volume.
Cerebral macrovascular morphology. To assess the morphology of ma-
jor cerebral blood vessels of the circle of Willis, deeply anesthetized mice
were perfused transcardially with a prewarm (37 ° C) saline solution contain-
ing gelatin (20% wt/vol) and India ink (0.25% vol/vol).
Neurological evaluation. Neurological scores were assigned the following
values: 0, normal motor function; 1, fl exion of torso and contralateral fore-
limb when the mouse was lifted by the tail; 2, circling to the contralateral
side when the mouse was held by the tail on a fl at surface, but normal pos-
ture at rest; 3, leaning to the contralateral side at rest; and 4, no spontaneous
motor activity. The inclined plane test measured the time a mouse managed
to hold itself on an inclined glass plate angled at 50 ° before sliding down.
The hanging wire test determined the time it took an animal to cross a dis-
tance of 45 cm on a freely hanging narrow metal bar.
Online supplemental material. Table S1 compares the array data from fi -
broblasts ( 11 ) with the microarray data obtained from neurons. Table S2
summarizes physiological data in ATF4 +/+ and ATF4 ? / ? mice. Table S3
shows a complete list of all genes diff erentially expressed after statistical anal-
ysis, including p-values and gene ontology information, and a list with the
complete gene ontology analysis. Fig. S1 shows measurements of DCF fl uo-
rescence in ATF4 +/+ and ATF4 ? / ? neurons in response to HCA. Fig. S2
shows measurements of cerebral blood fl ow, body temperature, and blood
pressure during MCAo. Online supplemental material is available at http://
The authors wish to thank Jawed Alam for providing us with the expression
plasmids for ATF4. The ATF4 5 ? UTR reporter plasmid was a gift from David Ron.
David Ash provided us with recombinant arginase. We thank Brett Langley,
Ambreena Siddiq, JoAnn Gensert, Alvaro G. Estevez, Jin Son, Bruce Volpe, and
Gary Gibson for sharing various materials and for their invaluable advice and
encouragement throughout this study. This work was possible because of the
outstanding technical support from Hsin-Hwa Lee, Chu-Hui Peng, Lucretia Batten,
and Elizabeth Newman.
This study was supported by the W.M. Burke Foundation, by a grant from the
National Institutes of Health to Rajiv R. Ratan (NS40591), and by the Dr. Miriam and
Sheldon G. Adelson Medical Research Foundation. Philipp S. Lange is a recipient of a
postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (LA 1483/1-1)
and the Koeln Fortune Program/Faculty of Medicine, University of Cologne
(125/2003). Juan C. Chavez is the recipient of a Scientist Development Grant from
the Northeast Affi liate of the American Heart Association (0635556T).
The authors have no confl icting fi nancial interests.
Submitted: 16 July 2007
Accepted: 11 April 2008
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