Neuroscience 158 (2009) 9951006

REVIEW
NF-B SIGNALING IN CEREBRAL ISCHEMIA
D. A. RIDDER AND M. SCHWANINGER*

The brain responds very rapidly to ischemia. After only a

few seconds cerebral function is impaired, though still in a
reversible manner. Cerebral ischemia lasting several minutes or longer may trigger a cascade of events that eventually leads to irreversible deficits due to cell loss or other
structural changes. It takes at least several hours for fullblown ischemic damage to develop. However, this time lag
may open a therapeutic window, in which irreversible damage can be prevented. Therefore, delayed mechanisms of
ischemic brain damage have received considerable attention.

Pharmacological Institute, University of Heidelberg, Im Neuenheimer

Feld 366, 69120 Heidelberg, Germany

AbstractThe transcription factor NF-B is a key regulator of

hundreds of genes involved in cell survival and inflammation.
There is ample evidence that NF-B is activated in cerebral
ischemia, mainly in neurons. Despite its well known role as
an antiapoptotic factor, in cerebral ischemia NF-B contributes to neuronal cell death, at least if the ischemia is severe
enough to lead to irreversible brain damage. In contrast,
NF-B also seems to be responsible for the preconditioning
effect of a transient and sublethal ischemia, perhaps by
dampening its own subsequent full activation. Among the
five NF-B subunits, RelA and p50 are responsible for the
detrimental effect in cerebral ischemia. Activation of NF-B
signaling is mediated by the upstream kinase inhibitor of
kappaB kinase and is triggered by hypoxia, reactive oxygen
species, and several inflammatory mediators. Interestingly,
the complex NF-B signaling pathway provides drug targets
at several levels. Modulation of NF-B signaling has the potential to interrupt multiple inflammatory and apoptotic mechanisms through one specific molecular target. 2009 IBRO.
Published by Elsevier Ltd. All rights reserved.

INFLAMMATION AND APOPTOSIS IN

CEREBRAL ISCHEMIA
After onset of cerebral ischemia a multifaceted inflammatory reaction emerges over the course of the next few
hours. Numerous inflammatory mediators are induced at
the transcriptional level, including enzymes required for
prostaglandin synthesis, cytokines of the tumor necrosis
factor (TNF) family, and chemokines. The upregulation of
inflammatory genes is not restricted to glial cells but also
occurs in neurons (Wang et al., 2008). Chemokines lure
leukocytes into the ischemic brain, mainly polymorphonuclear leukocytes and monocytes, which release further
inflammatory mediators and contribute to the inflammatory
reaction. The inflammatory reaction becomes clinically apparent through a febrile response and the increase in acute
phase protein levels in the peripheral blood of stroke patients (Reith et al., 1996; Acalovschi et al., 2003). In addition, inflammation contributes to the breakdown of the
blood brain barrier in cerebral ischemia (Candelario-Jalil
et al., 2009). Disruption of the blood brain barrier and the
consequent brain edema are major causes of acute lethality in stroke (Vahedi et al., 2007). Experimental studies
have firmly established that inflammation is closely interrelated with neuronal cell death and thereby promotes the
neurological deficit. Inflammation and apoptosis both depend largely on gene expression and share key regulators,
NF-B being a prominent example.

Key words: inflammation, apoptosis, stroke, transcription

factor, cytokine, IKK.
Contents
Inflammation and apoptosis in cerebral ischemia
NF-B signaling
NF-B in cerebral ischemia
Mechanisms of NF-B activation in cerebral ischemia
The pro- and antiapoptotic function
of NF-B
NF-B signaling as a drug target in cerebral ischemia
Conclusions
References

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*Corresponding author. Tel: 49-6221-548691; fax: 49-6221548549.

E-mail address: markus.schwaninger@pharma.uni-heidelberg.de (M.
Schwaninger).
Abbreviations: Gpx1, glutathione peroxidase 1; HIF-1, hypoxia-inducible transcription factor-1; HMGB1, high-mobility group box 1;
ICAM-1, intercellular adhesion molecule 1; ICE, interleukin-1-converting enzyme; IKK, inhibitor of kappaB kinase; IL, interleukin; IL1RA, interleukin-1 receptor antagonist; iNOS, inducible nitric oxide
synthase; LRP, lipoprotein receptor-related protein; MCAO, middle
cerebral artery occlusion; mGluR, metabotropic glutamate receptor;
MMP, matrix metallopeptidase; PDTC, pyrrolidine dithiocarbamate;
PPAR, proliferator-activated receptor; ROS, reactive oxygen species;
SOD, superoxide dismutase; TLR, toll-like receptor; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; tPA, tissue-type
plasminogen activator; TUNEL, terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling; TWEAK, tumor necrosis factorlike
weak inducer of apoptosis.

NF-B SIGNALING
The transcription factor NF-B consists of preformed
dimers. In mammals five different NF-B subunits, p50,
p52, c-Rel, RelA, and RelB, form homo- and heterodimers
in various combinations. However, not all combinations do
occur. For example, RelB does not form either homodimers or heterodimers with c-Rel nor RelA under normal conditions (Ryseck et al., 1995). In neural extracts

0306-4522/09 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuroscience.2008.07.007

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D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

dimers consisting of p50/RelA, p50/p50 (Schneider et al.,

1999), RelA/RelA, c-Rel/RelA, p50/c-Rel (Pizzi et al.,
2005) have been reported.
NF-B dimers are retained in the cytosol by interacting
with inhibitory IB proteins. There are seven IB proteins:
IB, IB, IB, p100, p105, Bcl-3, and IB. Interestingly, the binding of IB proteins to NF-B dimers shows
some specificity. IB and IB target predominantly p50/
RelA and p50/c-Rel heterodimers (Thompson et al., 1995);
IB only interacts with RelA and c-Rel hetero- and homodimers (Whiteside et al., 1997); and Bcl-3 binds exclusively to p50 or p52 homodimers (Nolan et al., 1993). IB,
IB, and IB are target genes of NF-B (Brown et al.,
1993; Whiteside et al., 1997; Totzke et al., 2006) providing
a negative feedback mechanism by which NF-B activity is
eventually shut off.
In the canonical pathway of activation (Fig. 1) IB
proteins are phosphorylated (Ser32 and Ser36 of IB),
polyubiquitinated by the SCFTrCP ubiquitin ligase complex, and then degraded by the 26 S proteasome (Hacker

and Karin, 2006). After degradation of IB, NF-B translocates into the nucleus and stimulates gene transcription.
Because phosphorylation of IB proteins plays a pivotal
role in the activation of NF-B, identification of the responsible IB kinase (inhibitor of kappaB kinase, IKK) represents a major breakthrough. The IKK complex consists of
two enzymatic subunits, IKK1 (IKK) and IKK2 (IKK), and
the regulatory subunit NF-B essential modulator (NEMO).
While IKK2 is essential for the canonical NF-B pathway
involving phosphorylation of IB, IB, IB as outline
above, IKK1 has been implicated in the so-called alternative pathway of NF-B activation leading to the formation of
RelB/p52 dimers. The role of the latter in brain is largely
unknown.
NF-B is activated by a huge array of stimuli, including
proinflammatory cytokines such as TNF and interleukin
(IL)-1 that are recognized by specific membrane receptors such as tumor necrosis factor receptor (TNFR) and
IL-1R as well as microbial pathogens that are recognized
by members of the pattern recognition receptor family,

Fig. 1. NF-B signaling in cerebral ischemia. In the ischemic brain diverse stimuli trigger activation of the IKK complex that phosphorylates IB at
Ser32 and Ser36. Upon phosphorylation, IB is degraded by the proteasome, and p50/RelA heterodimers are released, translocate to the nucleus,
and initiate NFB-dependent gene transcription. IKK also phosphorylates RelA at Ser536. This phosphorylation increases the transcriptional activity
of RelA. Phosphorylation of serine residues is labeled in yellow, phosphorylation of tyrosine residues in orange. NRs, NMDA-receptors; Glu, glutamate.

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

including toll-like receptors (TLRs). In addition to being

activated by extracellular stimuli, some of the pattern recognition receptors are also activated by intracellular stimuli
such as microbial components in the cytoplasm or in vesicles. Other important inducers of NF-B activity include
cytotoxic agents, e.g. chemotherapeutic drugs, ionizing
radiation, UV light, oxidative stress, and hypoxia. Most of
these stimuli culminate in the activation of the IKK complex, but there are also alternative pathways of inducing
NF-B activity. Several inducers of NF-B signaling are
also known to play a role in cerebral ischemia. Here, we
discuss the mechanisms of NF-B activation in cerebral
ischemia and speculate on the complex signaling network
involved in the pathophysiological events taking place in
the ischemic brain.

NF-B IN CEREBRAL ISCHEMIA

There is ample evidence that NF-B is activated in cerebral ischemia. Most investigators found NF-B to be activated in neurons (Clemens et al., 1997; Schneider et al.,
1999; Stephenson et al., 2000; Huang et al., 2001; Nurmi
et al., 2004). In addition, NF-B activation has also been
demonstrated in endothelial cells, astrocytes, and microglia (Terai et al., 1996; Howard et al., 1998; Gabriel et al.,
1999; Zhang et al., 2007a; Kaushal and Schlichter, 2008).
NF-B activation involves nuclear translocation of the subunits RelA and p50 and increased DNA binding of p50
homodimers and p50/RelA heterodimers (Fig. 1) (Schneider et al., 1999; Huang et al., 2001). In a transient middle
cerebral artery occlusion (MCAO) model increased DNA
binding could already be detected after 30 min of reperfusion following a 2-h period of MCAO (Schneider et al.,
1999). Increased DNA binding reflects activation of NF-B.
Indeed, in mouse models of both permanent and transient
cerebral ischemia a B-dependent -globin reporter gene
showed an increase in the transcriptional activity of NF-B
(Schneider et al., 1999; Nurmi et al., 2004). In these models nuclear translocation of RelA was detected mainly in
neurons. In postmortem human brain samples from patients who suffered a stroke, nuclear translocation of RelA
could also be observed in the brain areas surrounding the
necrotic infarct core (Nurmi et al., 2004).
To demonstrate the functional significance of these
findings, mice deficient in the p50 subunit of NF-B were
subjected to MCAO. The absence of p50 resulted in a
significant and comparable reduction in infarct size in both
transient and permanent stroke models, suggesting that
NF-B plays a detrimental role in cerebral ischemia
(Schneider et al., 1999; Nurmi et al., 2004). Because p50
both represses and transactivates gene transcription, depending on the dimerization partner (Hayden and Ghosh,
2008), it was difficult to interpret these results. Adenoviral
expression of a dominant-negative IB mutant gave
clearer evidence for the functional significance of NF-B in
cerebral ischemia. In this gene the phosphorylation sites
for IKK (Ser32 and Ser36) were mutated to alanine to
specifically inhibit NF-B activation. When this transgene
was expressed infarct size was reduced and neurological

997

outcome improved 24 h after a 2 h period of MCAO (Xu et

al., 2002). To determine what cell type is involved in the
detrimental function of NF-B activation in cerebral ischemia, transgenic mouse lines were generated that expressed the same dominant-negative IB mutant under
the control of neuron- or astrocyte-specific promoters. Selective inhibition of NF-B in neurons significantly reduced
the infarct size and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)
positive cells after 48 h of MCAO, whereas inhibition in
astrocytes had no statistically significant effect on infarct
size or neuronal survival (Zhang et al., 2005). Furthermore,
cultured primary cortical neurons expressing the dominantnegative IB were protected from cell death induced by
camptothecin, a DNA-damaging agent. Notably, expression of the dominant-negative IB did not completely
repress NF-B activity in these experiments. This is important as constitutive NF-B activity has been shown to
support neuronal survival (Bhakar et al., 2002).
Possibly, the five NF-B subunits also exert distinct
effects on cell survival. The unique phenotypes of knockout lines suggest that the subunits are functionally diverse
(Li and Verma, 2002; Gerondakis et al., 2006). Indeed, in
mice with a conditional deletion of RelA in the brain infarcts
were smaller than those in their control littermates,
whereas a germline deficiency of p52 and c-rel had no
effect on infarct size. Nevertheless, in wild-type mouse
brain all subunits showed both nuclear translocation and
increased DNA binding in an ELISA-based assay after 4 h
of ischemia (Inta et al., 2006).
Although NF-B is well known to have an antiapoptotic
function in diverse cell types, it plays a detrimental role in
most experimental settings of prolonged cerebral ischemia. Nevertheless, some controversial reports do not fit to
the concept of NF-B contributing to ischemic brain damage. One report claims that costaining with Fluoro-Jade, a
marker for neurodegeneration, does not colocalize with
transcriptional NF-B activity in a B-reporter mouse subjected to MCAO (Duckworth et al., 2006). According to
these findings, areas stained for active p50 did not show
neurodegeneration 4 days after transient cerebral ischemia. At this late stage it might be true that the antiapoptotic properties of NF-B have an impact. Possibly, only
the neurons in which NF-B activation is moderate have
survived up to that time, whereas others have already
succumbed to the ischemic insult.
Another controversial aspect, and one that at first sight
does not agree with the concept of NF-B playing a detrimental role in stroke, is the observation that ischemic
preconditioning relies on NF-B function. After a brief period of brain ischemia below the threshold of cell death,
NF-B is activated in neurons and confers neuroprotection
to a subsequent, prolonged period of ischemia (Blondeau
et al., 2001). This process seems to rely on gene transcription. Blocking NF-B activation pharmacologically with diethyldithiocarbamate or with B decoy DNA at the time of
the preconditioning stimulus abolished the protective effect. Interestingly, preconditioning the brain with sublethal
ischemia inhibited NF-B activation after a second, more

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D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

severe ischemic insult. This process was suggested to be

mediated through induction of IB (Fig. 1), which is a
well-known target gene of NF-B, and may account for the
neuroprotective effect of preconditioning. During a brief
period of nonlethal cerebral ischemia, the well-known antiapoptotic properties of NF-B seem to prevail. However,
the detrimental role of NF-B predominates during prolonged ischemia.
The mechanisms underlying the dual role of NF-Bactivation in cerebral ischemia have not yet been completely elucidated. NF-B interacts with several life/death
signaling pathways via expression and repression of dozens of pro- or antiapoptotic target genes (Barkett and
Gilmore, 1999; Dutta et al., 2006). In a model of permanent
cerebral ischemia, for example, we have found that expression of the proapoptotic Bcl-2-homology domain 3
(BH3) only genes Bim and Noxa depend on RelA and the
upstream kinase IKK (Inta et al., 2006). RelA stimulated
Bim and Noxa gene transcription in primary cortical neurons and bound to the promoter of both genes. As the ratio
of pro- and antiapoptotic Bcl-2 family members determines
cell fate, Bim and Noxa could be candidates that mediate
the detrimental effects of RelA activation in cerebral ischemia. Furthermore, NF-B is a central mediator of inflammatory processes. Several proinflammatory NF-B target
genes, including TNF, IL-1 and , IL-6, inducible nitric
oxide synthase (iNOS), intercellular adhesion molecule 1
(ICAM-1), and matrix metallopeptidase (MMP) 9, that are
known to be induced in cerebral ischemia could mediate
the deleterious effect (Wang et al., 2007; Gilmore, 2008).
Likewise, key genes of eicosanoid metabolism, which encode cytosolic phospholipase A2, cyclooxygenase 2, and
microsomal prostaglandin E synthase 1, are induced in the
ischemic brain, possibly contributing to ischemic damage
(Herrmann et al., 2005). Interestingly, it has recently been
shown that IKK2 also regulates the induction of hypoxiainducible transcription factor-1 (HIF-1) under hypoxic
conditions via activation of NF-B (Rius, 2008). Both detrimental and beneficial effects of HIF-1 have been described in cerebral ischemia depending on the cell type in
which HIF-1 was deleted (Helton et al., 2005; Baranova
et al., 2007).

MECHANISMS OF NF-B ACTIVATION IN

CEREBRAL ISCHEMIA
One of the very early and also pivotal steps in the pathophysiology of stroke seems to be the activation of the IKK
complex. In a mouse model of permanent cerebral ischemia, we could show increased IKK activity as early as half
an hour after the onset of ischemia using a kinase pulldown assay (Herrmann et al., 2005). The activation was
confined to the periphery of the ischemic territory, however. In the ischemic core, the lack of increased kinase
activity might be explained by degradation of IKK, as some
authors recently reported (Song et al., 2005). IKK activity
was increased for 5 h after the onset of MCAO. This was
also paralleled by a rather mild, but detectable increase in
IKK phosphorylation at Ser180/Ser181 in the periphery of

the infarcted tissue (Inta et al., 2006). The phosphorylation

of this site has been reported to be important for activation
of the kinase in several signaling pathways, supporting the
notion that IKK is activated (Perkins, 2006). It is known
that, in addition to the IB proteins, IKK also phosphorylates RelA at Ser536 (Fig. 1). Phosphorylation of RelA is
considered to increase its transcriptional activity (Perkins,
2006). Immunohistochemistry of mouse brain sections revealed a prominent increase in Ser536-phosphorylated
RelA after 1 h of MCAO adjacent to the ischemic core. The
staining colocalized with NeuN, a neuronal marker protein,
indicating that IKK phosphorylates RelA in neurons. In
addition, another recent publication also showed increased
RelA-phosphorylation in astrocytes (Zhang et al., 2007a).
To analyze the function of the IKK complex in cerebral
ischemia, Ikbkb, the gene encoding IKK2, was conditionally deleted in neurons or in neurons and glial cells. We
performed MCAO in these animals and observed a comparable reduction in infarct size in both mutant mouse
strains (Herrmann et al., 2005). In addition, the number of
TUNEL-positive cells was decreased in mice deficient for
IKK2 in neurons. These experiments did not address the
role of IKK1, though. As there is evidence that IKK1 and
IKK2 may have partially redundant or also opposing functions (Li et al., 2000; Lawrence et al., 2005), we were
interested in determining the effect of inhibiting both IKK1
and IKK2 in neurons. Therefore, a dominantnegative mutant of IKK2 (D145N) under the control of the tTA (tetracycline-controlled transactivator) was introduced in neurons. Both basal and inducible IKK activity in response to
cerebral ischemia was markedly diminished in this mouse
strain. These mice also showed infarct volumes that were
reduced by more than 50% (Herrmann et al., 2005). To
further test the validity of the concept of IKK contributing to
brain damage in cerebral ischemia, a transgenic mouse
line expressing a constitutively active IKK2 mutant (S177E,
S181E) in neurons was created. In accordance with the
previous results, these mice showed significantly larger
infarcts although no obvious signs of neurodegeneration
had been observed before the mice were subjected to
MCAO. Thus, IKK plays a major role in ischemic brain
damage but cannot induce neurodegeneration on its own.
The mechanisms underlying IKK activation in cerebral
ischemia have not been systematically investigated yet.
Several possible pathways come into question. IKK has
recently been shown to be activated by hypoxia and, in
turn, to mediate the activation of NF-B (Cummins et al.,
2006). These authors propose a model in which the prolyl
hydroxylases PHD-1, and to a lesser extent PHD-2, constitutively repress IKK by hydroxylation. As the PHDs have
an absolute requirement for molecular oxygen as cosubstrate, hypoxia then rapidly releases this repression of IKK
and activates NF-B. Cummins and colleagues (2006)
identified a potential hydroxylation site in IKK2 (Pro191),
which, when mutated, leads to a loss in hypoxic inducibility. Hypoxia also increased NF-B activity when cells were
stimulated with TNF. This enhanced susceptibility to cytokines under hypoxic conditions also underlines the central
role of IKK in the pathophysiology of stroke.

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

In addition to hypoxia, reactive oxygen species (ROS)

might contribute to NF-B activation in cerebral ischemia.
NF-B was shown to respond to oxidative stress induced
by ROS (Schreck et al., 1991). The formation of ROS in
cerebral ischemia is well known, especially in transient
ischemia models. Transgenic mice overexpressing the
ROS-scavenging enzymes superoxide dismutases (SOD)
1 or 2 and glutathione peroxidase 1 (Gpx1) are protected
against ischemic cell death (Sugawara et al., 2004). In
mice overexpressing SOD1, the activation of NF-B in the
ischemic brain is suppressed when compared with wildtype mice and therefore this could represent the mechanism underlying the neuroprotective effect (Huang et al.,
2001). The induction of the proapoptotic regulator c-myc, a
known NF-B-target, was also less pronounced in SOD1
transgenic mice. Moreover, infarct size is increased in
Gpx1-deficient mice and this is also associated with aggravated NF-B-activation (Crack et al., 2006). Furthermore, pharmacological data support the activation of
NF-B by ROS in cerebral ischemia (see below).
In addition to these rather direct mechanisms, NF-B
could also be activated through several receptor-mediated
signaling pathways. Recently, it was shown that the serine
protease tissue-type plasminogen activator (tPA) contributes to NF-B activation in cerebral ischemia via the lipoprotein receptor-related protein (LRP) (Zhang et al.,
2007a). This process is independent of the well-known
intravascular effect of tPA as a plasminogen activator and
suggests a cytokine-like function of tPA. LRP is present on
neurons and perivascular astrocytes. It is induced in the
ischemic hemisphere in a tPA-dependent manner. If LRPfunction was inhibited with anti-LRP antibodies or recombinant LRP-associated protein 1 (Lrpap1), infarct volumes
decreased, motor function recovered faster, and blood
brain barrier permeability following MCAO was reduced
(Polavarapu et al., 2007; Zhang et al., 2007a). In parallel,
NF-B activation was attenuated, as demonstrated by reduced DNA binding and less phosphorylation of RelA at
Ser536. Furthermore, a significant decrease in MCAO-induced nitric oxide production and iNOS expression accompanied these findings, which may reflect decreased NF-B
activation and account for the perceived neuroprotection
(Zhang et al., 2007a). Under ischemic conditions, the
ectodomain of LRP is shed from astrocytic end-feet, a
process dependent on tPA function and associated with
edema formation. As LRP has recently been shown to
regulate NF-B function (Yu et al., 2005), the authors of
these studies propose a model in which the shedding of
the ectodomain of LRP from perivascular astrocytes precedes the release of the intracellular domain of LRP into
the cytoplasm, which ultimately will activate the NF-B
pathway. This concept suggests that NF-B activation and
increased iNOS expression play a detrimental role in
astrocytes.
Besides tPA and LRP, the TLRs may function as upstream regulators of IKK activity in MCAO. The TLRs are a
family of at least 11 proteins that play a major role in innate
immunity, responding to microbial pathogens and also to
injury-induced endogenous ligands. By stimulating TLRs IKK

999

activity increases, mediating the activation of NF-B (Banerjee and Gerondakis, 2007). Recently published studies report
that mice deficient in either TLR2 or TLR4 show reduced
infarct sizes and improved neurological outcome when
compared with wild-type littermates (Caso et al., 2007;
Tang et al., 2007). Moreover, neurons deficient in TLR2 or
TLR4 showed increased resistance and less apoptotic cell
death when subjected to glucose deprivation as an in vitro
model of ischemic conditions. Immunohistochemistry demonstrated a rapid increase in TLR2 and TLR4 immunoreactivity in neurons and a delayed appearance of TLR2positive microglia (Tang et al., 2007), which is in line with
the concept that TLR signaling in the early phase activates
neuronal IKK, leading to neurodegeneration. Mice lacking
TLR4 also showed reduced expression of stroke-induced
cyclooxygenase 2 (COX2) and MMP9, known targets of
NF-B (Caso et al., 2007; Gilmore, 2008). Furthermore, in
a model of global cerebral ischemia, TLR4-deficient mice
showed less NF-B DNA-binding activity in the particularly
ischemia-sensitive hippocampal formation, less phosphorylated IB, and also increased neuronal survival than wildtype mice. The increase in several inflammatory mediators
(IL-6, TNF, Fas ligand (FasL) and high-mobility group box
1 (HMGB1)) was also clearly diminished (Hua et al., 2007).
The fact that the TLRs have also been linked to ischemic
preconditioning, an event also relying on NF-B function,
provides further evidence for a connection between the
TLRs, IKK, and NF-B in the pathophysiology of stroke
(Blondeau et al., 2001; Kariko et al., 2004; Stevens et al.,
2008). Ligands that possibly activate the TLRs in the ischemic brain could be molecules derived from injured tissue,
blood vessels, and necrotic cells. Fragments of extracellular matrix (hyaluronan, fibronectin, and heparan sulfate),
fibrin or fibrinogen and heat shock proteins activate TLR4.
RNA and chromatin-associated DNA activate TLR3 and
TLR9, respectively (Kariko et al., 2004). Another agonist of
TLR2 and 4, HMGB1 has also recently been shown to
have a proinflammatory and detrimental role in cerebral
ischemia (Kim et al., 2006).
Similar to the TLRs, another member of the patternrecognition receptor family, the type B scavenger receptor
CD36, was recently shown to participate in the inflammatory signaling pathways found in the ischemic brain and to
contribute to ischemic brain damage (Cho et al., 2005). In
sham-operated mice CD36 was observed in endothelial
cells. After 6 h of ischemia, CD36-positive microglia appeared in the infarcted brain and, after 3 days, astrocytes
in the periphery of the infarct also expressed CD36. In
CD36-deficient mice subjected to MCAO infarcts were
smaller and neurological deficits milder than in wild-type
controls. This was accompanied by an attenuated increase
in ischemia-induced ROS (Cho et al., 2005). The activation
of NF-B was alleviated in CD36-deficient mice after
stroke, although it could still be induced by i.c.v. administration of exogenous IL-1 (Kunz et al., 2008). This reduction in DNA-binding activity also went along with diminished infiltration of neutrophils, a suppressed glial reaction,
and curtailed expression of NF-B-dependent proinflammatory transcripts, including iNOS, ICAM-1, and endothe-

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D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

lial adhesion molecule 1 (ELAM-1). Notably, the activation

of NF-B was independent of the formation of ROS as a
ROS scavenger did not attenuate it (Kunz et al., 2008).
The ligands that possibly activate CD36-dependent NF-B
signaling comprise a wide variety of molecules, for example, modified lipids, amyloid-, thrombospondin-1, and advanced glycation end products (Febbraio and Silverstein,
2007). Interestingly, it has also been suggested that CD36
forms a signaling complex with TLR2/6 that activates
NF-B in response to bacterial cell wall components (Febbraio and Silverstein, 2007), possibly indicating a cooperative role in cerebral ischemia as well.
As already mentioned, IL-1 is another possible inducer
of NF-B activity in the ischemic brain (Kunz et al., 2008).
Via activation of IKK, IL-1 and IL-1 are well-known stimuli of the NF-B signaling pathway. Both IL-1 and IL-1
are rapidly induced in cerebral ischemia (Allan et al.,
2005). Deleting both cytokines markedly reduces the infarct size in mice subjected to MCAO, whereas deleting
either gene alone, perhaps owing to compensatory effects,
is ineffective (Boutin et al., 2001). Administering exogenous IL-1 intracerebroventricularly exacerbated ischemic
brain damage, whereas administration or overexpression
of the endogenous interleukin-1 receptor antagonist (IL1RA) greatly reduces ischemic brain injury. Mice deficient
in IL-1RA show increased ischemic brain damage, further
supporting the notion that IL-1 plays a detrimental role in
the pathophysiology of stroke (Allan et al., 2005). Furthermore, mice lacking interleukin-1-converting enzyme
(ICE), also known as caspase-1, showed less ischemic
brain injury, edema formation, and inflammatory responses (Huang et al., 2003). Interestingly, this was paralleled by reduced activation of NF-B in electrophoretic
mobility shift assays and a significant reduction in the
phosphorylation of the RelA subunit. Immunohistochemistry revealed that the phospho-RelA positive cells were
neurons, which is consistent with the concept that IKK
activates NF-B in neurons. These data provide a link
between the detrimental functions of IL-1 and ICE to
NF-B and its proinflammatory and proapoptotic role in
cerebral ischemia. The fact that IL-1 and IL-1 are also
target genes of NF-B (Gilmore, 2008) suggests a positive
feedback loop by which NF-B activation is sustained,
perpetuating the inflammatory response and worsening
the outcome.
In addition to IL-1, TNF might also be a stimulus regulating NF-B activity in the ischemic brain. TNF is known
to induce NF-B activity in a wide range of cell types,
including neurons (Herrmann et al., 2005), astrocytes (Ginis et al., 2002), and endothelial cells (Trickler et al., 2005).
Depending on the stimulus, receptor context, and the activation state of the cells, TNF can be associated with cell
death or survival, induction of inflammation, or with control
or resolution of inflammation, features that have also been
attributed to NF-B. Like NF-B, TNF was shown to play a
protective role in ischemic preconditioning, but during prolonged ischemia it is considered to exacerbate neuronal
injury (Hallenbeck, 2002). Preconditioning with low doses
of LPS conferred neuroprotection in both a mouse model

of stroke and in cultured neurons when subjected to oxygen glucose deprivation (Rosenzweig et al., 2007). Remarkably, this effect seems to rely on TNF induction and
secretion after stimulation with LPS. Notably, TNF expression is known to be regulated by NF-B (Gilmore, 2008).
As TNF expression increases at the mRNA level within 1 h
in the ischemic zone in cerebral ischemia, followed by
increases in protein levels within 2 6 h of the onset of
ischemia, it may contribute to the activation of NF-B
(Hallenbeck, 2002). Administering exogenous TNF intracerebroventricularly after the onset of MCAO increased the
infarct size in mice (Rosenzweig et al., 2007). Antibodies to
TNF and pharmacological inhibition of TNF-alpha converting enzyme (TACE) (Wang et al., 2004) have been demonstrated to confer neuroprotection in brain ischemia. In
contrast, mice lacking the TNFRs TNFR1 and TNFR2
showed significantly larger infarcts than wild-type controls
and oxidative stress was also increased (Bruce et al.,
1996). This has been attributed to the inability of these
animals to induce the neuroprotective and antioxidant enzyme SOD2. Interestingly, SOD2 is also a target gene of
NF-B (Gilmore, 2008). When stimulated with TNF cultured astrocytes showed increased expression of SOD2
and ICAM-1 (Ginis et al., 2002), an adhesion molecule
believed to increase the inflammatory response in the
ischemic brain and worsen the outcome (Wang et al.,
2007). Preconditioning with TNF or ceramide, a sphingolipid messenger in TNF signaling, curtailed the increase in
ICAM-1 when the cells were re-challenged with TNF,
whereas SOD2 induction remained unweakened (Ginis et
al., 2002). In this paradigm, ICAM-1 transcription was regulated by RelA associated with the transcriptional coactivator p300. In preconditioned cells RelA remained unphosphorylated and did not associate with p300 although RelA
DNA-binding activity did not change. This illustrates a link
between NF-B and TNF in ischemic preconditioning and
also demonstrates the double-edged impact of both TNF
and NF-B on the outcome with respect to the molecular
context.
Another member of the TNF family of cytokines, tumor
necrosis factorlike weak inducer of apoptosis (TWEAK),
has also been implicated in the activation of NF-B in
cerebral ischemia. It has proangiogenic and proapoptotic
properties and induces cell death in tumor cell lines (Winkles, 2008). By massively parallel signature sequencing,
we detected an increase in TWEAK at the mRNA level in a
mouse model of cerebral ischemia (Potrovita et al., 2004).
TWEAK acts through Fn14, a member of the TNFR family.
Although Fn14 does not contain a death domain, it was
shown to mediate TWEAK-induced cell death (Aggarwal,
2003). Interestingly, Fn14 was also induced by cerebral
ischemia, predominantly in the periphery of the infarcted
area. A neutralizing anti-TWEAK antibody increased survival in cortical neurons subjected to oxygen glucose deprivation and reduced infarct size in a mouse model of
stroke, demonstrating a detrimental role of TWEAK in cerebral ischemia (Potrovita et al., 2004). This finding was
also confirmed by others using a soluble form of Fn14 and
by subjecting Fn14-deficient mice to MCAO (Zhang et al.,

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

2007b). In these mice both induction of NF-B DNA-binding activity and the increases in IKK and RelA phosphorylation were lower after MCAO. In addition to promoting
infarct size, TWEAK-Fn14 signaling also increased the
permeability of the blood brain barrier in cerebral ischemia. This effect is probably mediated by the induction of
MMP9 activity via NF-B and the subsequent basement
membrane laminin degradation (Zhang et al., 2007b).
Fn14 is expressed in cortical neurons. Stimulation with recombinant TWEAK activates NF-B through the IKK complex
and triggered apoptosis in a moderate number of cortical
neurons in vitro. Inhibition of NF-B in neurons reduced
TWEAK-induced apoptosis, suggesting that NF-B is playing
a proapoptotic role in the context of TWEAK stimulation
(Potrovita et al., 2004).
Excitotoxicity is a key event in ischemic brain damage
(Dirnagl et al., 1999). Energy depletion leads to a loss of
the membrane potential, and neurons depolarize. Voltagegated Ca2 channels become activated, whereby excitatory neurotransmitters, including glutamate, accumulate in
the extracellular space. Glutamate might also induce
NF-B activity in cerebral ischemia as it has been shown to
activate NF-B in neurons in vitro via NMDA-receptor activation and an increase in intracellular Ca2 (Guerrini et
al., 1995; Grilli et al., 1996). In addition, metabotropic
glutamate receptors (mGluRs) are responsible for activating the subunit c-Rel in cortical neurons (Pizzi et al., 2005).
Recently, it was shown in an in vitro model of the ischemic
penumbra that neuronal glutamate release led to increased NF-B activity in microglia via their group II
mGluRs. The activated microglia exerted neurotoxic effects and killed nave neurons through an apoptotic mechanism that was mediated by TNF and involved activation of
both caspase-3 and caspase-8 (Kaushal and Schlichter,
2008). This illustrates the functional significance and the
detrimental effects of NF-B activation in microglial cells.
Most of the listed stimuli seem to activate NF-B via
activation of the IKK complex. However, there might be
also IKK-independent mechanisms of NF-B activation in
cerebral ischemia. Several post-translational modifications
of the IBs and the NF-B subunits are known to control
NF-B activity (Perkins, 2006). One example for IKK-independent NF-B activation in cerebral ischemia might be
tyrosine phosphorylation of IB. After hypoxia and reoxygenation Src phosphorylates IB at Tyr42 and thereby
induces NF-B activity (Fan et al., 2003). Src was also
shown to play a detrimental role in glutamate-induced
excitotoxicity in neurons (Khanna et al., 2007). Furthermore, in a liver model of ischemia and reperfusion Srcdependent phosphorylation of IB at Tyr42 was found to
be important for NF-B activation (Fan et al., 2004). The
activation of NF-B also exerted a detrimental effect in this
model. Interestingly, in a stroke model both pharmacological inhibition of Src and genetic deficiency in Src conferred
neuroprotection (Paul et al., 2001). We hypothesize that
NF-B might be a downstream effector of Src that accounts for its deleterious function in cerebral ischemia and
that tyrosine phosphorylation of IB might also play a role
in the pathophysiology of stroke.

1001

THE PRO- AND ANTIAPOPTOTIC FUNCTION

OF NF-B
NF-B is well known for its antiapoptotic function (Barkett
and Gilmore, 1999; Dutta et al., 2006). Indeed, the protective effect of ischemic preconditioning has been shown to
be mediated by NF-B (Blondeau et al., 2001). Preconditioning induces the expression of erythropoietin in astrocytes (Ruscher et al., 2002; Prass et al., 2003). Upon
release, erythropoietin protects adjacent neurons through
a NF-B-dependent mechanism (Digicaylioglu and Lipton,
2001). Other preconditioning stimuli, such as LPS, are
likely to operate through NF-B as well (Rosenzweig et al.,
2007).
However, findings in manifest cerebral ischemia seem
to be in conflict with the concept that NF-B is an antiapoptotic factor. Once severe ischemia occurs, NF-B activation contributes to ischemic brain damage. Although unusual, this proapoptotic function of NF-B is not without
precedence (Qin et al., 1999; Pizzi et al., 2002; Luedde et
al., 2005). What could be the basis for this surprising
double life of NF-B? In the following we offer four possible explanations for this enigma.
1. Cell type-specific effects. NF-B is a key regulator of
innate immunity and inflammation. Activation of microglia by cerebral ischemia depends on NF-B activation
(Kaushal and Schlichter, 2008). Thus, a strong inflammatory reaction controlled by NF-B activation in microglia and other inflammatory cells may override a
potential antiapoptotic effect of NF-B in neurons.
Whereas this hypothesis seems attractive, it does not
explain why selective inhibition of NF-B signaling in
neurons reduced the infarct size (Herrmann et al.,
2005; Zhang et al., 2005). Furthermore, activation
of NF-B in neurons may either induce apoptosis
or protect against it, depending on the stimulus
(Kaltschmidt et al., 2002; Pizzi et al., 2002; Tarabin
and Schwaninger, 2004).
2. The kinetics of NF-B activation have profound effects
on the pattern of induced genes. Some genes require
persistent NF-B activity whereas others are already
stimulated by transient activation (Hoffmann et al.,
2002). Thus, transient NF-B activation may upregulate antiapoptotic genes and protect against ischemia,
whereas sustained activation may induce a proapoptotic set of genes and promote apoptosis. Indeed, the
neuroprotective effect of an antioxidant drug was associated with a reduction of the sustained, but not the
transient activation of NF-B (Clemens et al., 1998).
This mechanism would also explain the opposing role
of NF-B in transient preconditioning ischemia and in
manifest ischemia (Schneider et al., 1999; Blondeau et
al., 2001).
3. As previously outlined, NF-B is a heterogeneous factor that is formed by five subunits in various combinations. Pizzi and Spano (2006) have provided convincing evidence that RelA and c-Rel have opposing effects on the survival of neurons. Pizzi et al. (2002)
showed that the toxic effect of glutamate in cerebellar

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D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

granule cells is mediated by the NF-B subunits p50

and RelA. In contrast, IL-1 and activation of the
mGluR type 5 increased neuronal survival through cRel (Pizzi et al., 2002, 2005). Interestingly, both RelA
and c-Rel are activated in cerebral ischemia (Schneider et al., 1999; Inta et al., 2006). In accordance with
the notion that the two subunits have a different function, neural deficiency of RelA reduced the infarct volume in MCAO but c-Rel deficiency had no effect. In
these experiments compensatory mechanisms in response to the germline knockout of c-Rel may have
masked its neuroprotective action. A preconditioning
paradigm may be better suited to reveal the protective
action of c-Rel in vivo. So far, little is known about how
these two subunits are activated differentially.
4. The effect of NF-B signaling on cell survival is shaped
by the interaction with numerous other signaling cascades and transcription factors (for review see Perkins,
2007). Interestingly, several of these interaction partners are also activated in cerebral ischemia and may
determine the transcriptional response and the effect
of NF-B on cell survival. In accordance with this concept, we found no increased cell loss in mice in which
the IKK was constitutively active in neurons (Herrmann
et al., 2005). However, these mice were more susceptible to cerebral ischemia. Thus, NF-B signaling cannot induce neuronal cell death by itself but seems to
synergize with other pathways. A prominent example is
the JNK signaling pathway that promotes cell death in
cerebral ischemia (Borsello et al., 2003). NF-B inhibits the JNK pathway at several levels but may also
stimulate it through its target gene PKC (Perkins,
2007). PKC is induced in cerebral ischemia, suggesting that a positive interaction between the two pathways may be possible (Koponen et al., 2000). Likewise, p53 interacts with NF-B in a complex manner
involving mutual inhibition and induction. It is clear that
p53 is activated in cerebral ischemia (Culmsee and
Mattson, 2005). However, the functional consequences of the interaction between NF-B and p53 for
neuronal survival are still disputed (Aleyasin et al.,
2004; Culmsee and Mattson, 2005). The specific interactions of NF-B with many other neuronal signaling
pathways are largely unknown and remain to be
determined.

NF-B SIGNALING AS A DRUG TARGET IN

CEREBRAL ISCHEMIA
The detrimental effect of NF-B in cerebral ischemia suggests that inhibition of the signaling pathway may represent a treatment strategy in stroke. Because of the pivotal
role of NF-B in several inflammatory disorders, considerable efforts have been undertaken to identify inhibitors of
the NF-B signaling cascade. Numerous compounds were
shown to interfere with NF-B activation although the molecular mechanism was not identified in each case. The
best characterized drug target in NF-B signaling is the
kinase IKK. Already in 1996 Spanos group provided evi-

dence that aspirin is neuroprotective via inhibition of NF-B

signaling (Grilli et al., 1996). Later work identified the kinase IKK as the molecular target of aspirin in NF-B signaling (Yin et al., 1998). Because aspirin has numerous
other effects, it is questionable whether NF-B inhibition
contributes to the established efficacy of aspirin in the
treatment of acute stroke. In recent years, highly specific
inhibitors of IKK were developed (Karin et al., 2004). The
selective IKK inhibitor BMS-345541 blocked IKK activation
in a mouse model of stroke and reduced the infarct volume
in a dose-dependent manner (Herrmann et al., 2005).
BMS-345541 was still effective when administered up to
4.5 h after onset of ischemia suggesting that IKK inhibition
may have a therapeutic time window suitable for clinical
use. Another compound, MLN1145, was found to be neuroprotective in a rat model of stroke (Tortella and Williams,
2005). In contrast, a peptide inhibitor of IKK worsened
ischemic brain damage in neonatal rats (van den Tweel et
al., 2006). Whether this is an age-specific effect is still not
known.
For several structurally diverse compounds, correlative
evidence has been presented suggesting that NF-B inhibition is the cause of their neuroprotective action in cerebral ischemia. Antioxidants such as N-acetyl-cysteine,
LY231617, LY341122, caffeic acid phenethyl ester, and
taxifolin reduce ischemic brain damage and inhibit NF-B
activation (Carroll et al., 1998; Clemens et al., 1998; Stephenson et al., 2000; Wang et al., 2005; Khan et al., 2007).
Pyrrolidine dithiocarbamate (PDTC) is another antioxidant
drug that is both neuroprotective and an inhibitor of NF-B
(Nurmi et al., 2004). Initially, it was thought to interfere with
NF-B signaling through its antioxidant properties but later
data showed that PDTC inhibits ubiquitin ligase activity,
which is essential for NF-B activation (Hayakawa et al.,
2003). Other ubiquitin ligase inhibitors have not yet been
tested in cerebral ischemia. Further data on proteasome
inhibitors, such as MLN519 and CVT-634, indicate that
these compounds ameliorate ischemic brain damage and
inhibit NF-B activity in parallel (Buchan et al., 2000; Williams et al., 2003). NF-B is inhibited by nuclear receptors
such as the estrogen receptor and the peroxisome proliferator-activated receptor (PPAR) (De Bosscher et al.,
2006). The inhibitory effect on NF-B may contribute to the
well-established neuroprotection afforded by estrogens
and PPAR activators (Wen et al., 2004; Pereira et al.,
2006).
For the clinical development of NF-B inhibitors safety
issues will play an important role. Potential untoward effects might include an increased susceptibility to infections, interferences in lymphopoiesis, hepatotoxicity, and
teratogenicity (Strnad and Burke, 2007). As genetic defects in the NF-kB pathway in humans lead to increased
susceptibility to infection, pharmacological blockers of IKK
may have a similar effect. Moreover, administration of the
IKK inhibitor ML120B to mice resulted in an increased
number of apoptotic cells in thymus, spleen and bone
marrow and in a reduction of viable pre-B-cells (Nagashima et al., 2006). Because IKK2-deficient mice die
due to liver apoptosis during embryonic development, pos-

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

sible untoward effects of IKK inhibitors may include teratogenicity. In adults, genetic deletion of the IKK subunit
NEMO in hepatocytes caused steatohepatitis and hepatocellular carcinoma (Luedde et al., 2007).
These untoward effects may be relevant if IKK inhibitors are used for the treatment of chronic inflammatory
diseases. In the treatment of stroke, a short-term treatment
might be sufficient and is less likely to cause untoward
effects than long-term treatment. A couple of IKK inhibitors
have been discovered. Preclinical evaluation of these substances in animal stroke models and phase I studies are
now needed to evaluate their possible usage in the treatment of human stroke.

CONCLUSIONS
Studies in cerebral ischemia have revealed an unusual
role of the transcription factor NF-B. While it has antiapoptotic functions in many other paradigms, its main action in the ischemic brain seems to contribute to acute
neurodegeneration. The specific causes of this unusual
role of NF-B are still rather hypothetical. Also, many
details of the signaling pathways that lead to the activation
of NF-B in cerebral ischemia remain to be defined. However, with present knowledge it is already apparent that
NF-B signaling offers many targets for therapeutic intervention in cerebral ischemia.

REFERENCES
Acalovschi D, Wiest T, Hartmann M, Farahmi M, Mansmann U, Auffarth GU, Grau AJ, Green FR, Grond-Ginsbach C, Schwaninger M
(2003) Multiple levels of regulation of the interleukin-6 system in
stroke. Stroke 34:1864 1869.
Aggarwal BB (2003) Signalling pathways of the TNF superfamily: a
double-edged sword. Nat Rev Immunol 3:745756.
Aleyasin H, Cregan SP, Iyirhiaro G, OHare MJ, Callaghan SM, Slack
RS, Park DS (2004) Nuclear factor-(kappa)B modulates the p53
response in neurons exposed to DNA damage. J Neurosci
24:29632973.
Allan SM, Tyrrell PJ, Rothwell NJ (2005) Interleukin-1 and neuronal
injury. Nat Rev Immunol 5:629 640.
Banerjee A, Gerondakis S (2007) Coordinating TLR-activated signaling pathways in cells of the immune system. Immunol Cell Biol
85:420 424.
Baranova O, Miranda LF, Pichiule P, Dragatsis I, Johnson RS, Chavez
JC (2007) Neuron-specific inactivation of the hypoxia inducible
factor 1{alpha} increases brain injury in a mouse model of transient
focal cerebral ischemia. J Neurosci 27:6320 6332.
Barkett M, Gilmore TD (1999) Control of apoptosis by Rel/NF-kappaB
transcription factors. Oncogene 18:6910 6924.
Bhakar AL, Tannis L-L, Zeindler C, Russo MP, Jobin C, Park DS,
MacPherson S, Barker PA (2002) Constitutive nuclear factorkappa B activity is required for central neuron survival. J Neurosci
22:8466 8475.
Blondeau N, Widmann C, Lazdunski M, Heurteaux C (2001) Activation
of the nuclear factor-kappaB is a key event in brain tolerance.
J Neurosci 21:4668 4677.
Borsello T, Clarke PG, Hirt L, Vercelli A, Repici M, Schorderet DF,
Bogousslavsky J, Bonny C (2003) A peptide inhibitor of c-Jun
N-terminal kinase protects against excitotoxicity and cerebral ischemia. Nat Med 9:1180 1186.
Boutin H, LeFeuvre RA, Horai R, Asano M, Iwakura Y, Rothwell NJ
(2001) Role of IL-1alpha and IL-1beta in ischemic brain damage.
J Neurosci 21:5528 5534.

1003

Brown K, Park S, Kanno T, Franzoso G, Siebenlist U (1993) Mutual

regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. Proc Natl Acad Sci U S A 90:25322536.
Bruce AJ, Boling W, Kindy MS, Peschon J, Kraemer PJ, Carpenter
MK, Holtsberg FW, Mattson MP (1996) Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice
lacking TNF receptors. Nat Med 2:788 794.
Buchan AM, Li H, Blackburn B (2000) Neuroprotection achieved with
a novel proteasome inhibitor which blocks NF-kappaB activation.
Neuroreport 11:427 430.
Candelario-Jalil E, Yang Y, Rosenberg G (2009) Diverse roles of
matrix metalloproteinases and tissue inhibitors of metalloproteinases in neuroinflammation and cerebral ischemia. Neuroscience,
158:983994.
Carroll JE, Howard EF, Hess DC, Wakade CG, Chen Q, Cheng C
(1998) Nuclear factor-kappa B activation during cerebral reperfusion: effect of attenuation with N-acetylcysteine treatment. Brain
Res Mol Brain Res 56:186 191.
Caso JR, Pradillo JM, Hurtado O, Lorenzo P, Moro MA, Lizasoain I
(2007) Toll-like receptor 4 is involved in brain damage and inflammation after experimental stroke. Circulation 115:1599 1608.
Cho S, Park EM, Febbraio M, Anrather J, Park L, Racchumi G,
Silverstein RL, Iadecola C (2005) The class B scavenger receptor
CD36 mediates free radical production and tissue injury in cerebral
ischemia. J Neurosci 25:2504 2512.
Clemens JA, Stephenson DT, Yin T, Smalstig EB, Panetta JA, Little
SP (1998) Drug-induced neuroprotection from global ischemia is
associated with prevention of persistent but not transient activation
of nuclear factor-kappaB in rats. Stroke 29:677 682.
Clemens JA, Stephenson DT, Smalstig EB, Dixon EP, Little SP (1997)
Global ischemia activates nuclear factor-B in forebrain neurons of
rats. Stroke 28:10731081.
Crack PJ, Taylor JM, Ali U, Mansell A, Hertzog PJ (2006) Potential
contribution of NF-kappaB in neuronal cell death in the glutathione
peroxidase-1 knockout mouse in response to ischemia-reperfusion
injury. Stroke 37:15331538.
Culmsee C, Mattson MP (2005) p53 In neuronal apoptosis. Biochem
Biophys Res Commun 331:761777.
Cummins EP, Berra E, Comerford KM, Ginouves A, Fitzgerald KT,
Seeballuck F, Godson C, Nielsen JE, Moynagh P, Pouyssegur J,
Taylor CT (2006) Prolyl hydroxylase-1 negatively regulates IkappaB kinase-beta, giving insight into hypoxia-induced NFkappaB
activity. Proc Natl Acad Sci U S A 103:18154 18159.
De Bosscher K, Vanden Berghe W, Haegeman G (2006) Cross-talk
between nuclear receptors and nuclear factor kappaB. Oncogene
25:6868 6886.
Digicaylioglu M, Lipton SA (2001) Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling
cascades. Nature 412:641 647.
Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22:391397.
Duckworth EA, Butler T, Collier L, Collier S, Pennypacker KR (2006)
NF-kappaB protects neurons from ischemic injury after middle
cerebral artery occlusion in mice. Brain Res 1088:167175.
Dutta J, Fan Y, Gupta N, Fan G, Gelinas C (2006) Current insights into
the regulation of programmed cell death by NF-kappaB. Oncogene
25:6800 6816.
Fan C, Li Q, Ross D, Engelhardt JF (2003) Tyrosine phosphorylation
of I kappa B alpha activates NF kappa B through a redox-regulated
and c-Src-dependent mechanism following hypoxia/reoxygenation. J Biol Chem 278:20722080.
Fan C, Li Q, Zhang Y, Liu X, Luo M, Abbott D, Zhou W, Engelhardt JF
(2004) IkappaBalpha and IkappaBbeta possess injury contextspecific functions that uniquely influence hepatic NF-kappaB induction and inflammation. J Clin Invest 113:746 755.
Febbraio M, Silverstein RL (2007) CD36: implications in cardiovascular disease. Int J Biochem Cell Biol 39:20122030.

1004

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

Gabriel C, Justicia C, Camins A, Planas AM (1999) Activation of

nuclear factor-kappaB in the rat brain after transient focal ischemia.
Brain Res Mol Brain Res 65:61 69.
Gerondakis S, Grumont R, Gugasyan R, Wong L, Isomura I, Ho W,
Banerjee A (2006) Unravelling the complexities of the NF-kappaB
signalling pathway using mouse knockout and transgenic models.
Oncogene 25:6781 6799.
Gilmore TD (2008) Rel/NF-B transcription factors. www.nf-kb.org.
Ginis I, Jaiswal R, Klimanis D, Liu J, Greenspon J, Hallenbeck JM
(2002) TNF-alpha-induced tolerance to ischemic injury involves
differential control of NF-kappaB transactivation: the role of NFkappaB association with p300 adaptor. J Cereb Blood Flow Metab
22:142152.
Grilli M, Pizzi M, Memo M, Spano P (1996) Neuroprotection by aspirin
and sodium salicylate through blockade of NF-B activation. Science 274:13831385.
Guerrini L, Blasi F, Denis-Donini S (1995) Synaptic activation of NFkappa B by glutamate in cerebellar granule neurons in vitro. Proc
Natl Acad Sci U S A 92:90779081.
Hacker H, Karin M (2006) Regulation and function of IKK and IKKrelated kinases. Sci STKE 2006:re13.
Hallenbeck JM (2002) The many faces of tumor necrosis factor in
stroke. Nat Med 8:13631368.
Hayakawa M, Miyashita H, Sakamoto I, Kitagawa M, Tanaka H, Yasuda H, Karin M, Kikugawa K (2003) Evidence that reactive oxygen
species do not mediate NF-kappaB activation. EMBO J 22:
3356 3366.
Hayden MS, Ghosh S (2008) Shared principles in NF-kappaB signaling. Cell 132:344 362.
Helton R, Cui J, Scheel JR, Ellison JA, Ames C, Gibson C, Blouw B,
Ouyang L, Dragatsis I, Zeitlin S, Johnson RS, Lipton SA, Barlow C
(2005) Brain-specific knock-out of hypoxia-inducible factor-1{alpha} reduces rather than increases hypoxic-ischemic damage.
J Neurosci 25:4099 4107.
Herrmann O, Baumann B, de Lorenzi R, Muhammad S, Zhang W,
Kleesiek J, Malfertheiner M, Kohrmann M, Potrovita I, Maegele I,
Beyer C, Burke JR, Hasan MT, Bujard H, Wirth T, Pasparakis M,
Schwaninger M (2005) IKK mediates ischemia-induced neuronal
death. Nat Med 11:13221329.
Hoffmann A, Levchenko A, Scott ML, Baltimore D (2002) The IkappaBNF-kappaB signaling module: temporal control and selective gene
activation. Science 298:12411245.
Howard EF, Chen Q, Cheng C, Carroll JE, Hess D (1998) NF-kappa B
is activated and ICAM-1 gene expression is upregulated during
reoxygenation of human brain endothelial cells. Neurosci Lett
248:199 203.
Hua F, Ma J, Ha T, Xia Y, Kelley J, Williams DL, Kao RL, Browder IW,
Schweitzer JB, Kalbfleisch JH, Li C (2007) Activation of Toll-like
receptor 4 signaling contributes to hippocampal neuronal death
following global cerebral ischemia/reperfusion. J Neuroimmunol
190:101111.
Huang CY, Fujimura M, Noshita N, Chang YY, Chan PH (2001) SOD1
down-regulates NF-kappaB and c-Myc expression in mice after
transient focal cerebral ischemia. J Cereb Blood Flow Metab
21:163173.
Huang FP, Wang ZQ, Wu DC, Schielke GP, Sun Y, Yang GY (2003)
Early NFkappaB activation is inhibited during focal cerebral ischemia in interleukin-1beta-converting enzyme deficient mice. J Neurosci Res 73:698 707.
Inta I, Paxian S, Maegele I, Zhang W, Pizzi M, Spano P, Sarnico I,
Muhammad S, Herrmann O, Inta D, Baumann B, Liou H-C, Schmid
RM, Schwaninger M (2006) Bim and noxa are candidates to mediate the deleterious effect of the NF-{kappa}B subunit RelA in
cerebral ischemia. J Neurosci 26:12896 12903.
Kaltschmidt B, Heinrich M, Kaltschmidt C (2002) Stimulus-dependent
activation of NF-kappaB specifies apoptosis or neuroprotection in
cerebellar granule cells. Neuromol Med 2:299 309.

Kariko K, Weissman D, Welsh FA (2004) Inhibition of toll-like receptor

and cytokine signaling-a unifying theme in ischemic tolerance.
J Cereb Blood Flow Metab 24:1288 1304.
Karin M, Yamamoto Y, Wang QM (2004) The IKK NF-kappa B system:
a treasure trove for drug development. Nat Rev Drug Discov
3:1726.
Kaushal V, Schlichter LC (2008) Mechanisms of microglia-mediated
neurotoxicity in a new model of the stroke penumbra. J Neurosci
28:22212230.
Khan M, Elango C, Ansari MA, Singh I, Singh AK (2007) Caffeic acid
phenethyl ester reduces neurovascular inflammation and protects
rat brain following transient focal cerebral ischemia. J Neurochem
102:365377.
Khanna S, Roy S, Park HA, Sen CK (2007) Regulation of c-Src activity
in glutamate-induced neurodegeneration. J Biol Chem 282:23482
23490.
Kim JB, Sig Choi J, Yu YM, Nam K, Piao CS, Kim SW, Lee MH, Han
PL, Park JS, Lee JK (2006) HMGB1, a novel cytokine-like mediator
linking acute neuronal death and delayed neuroinflammation in the
postischemic brain. J Neurosci 26:6413 6421.
Koponen S, Goldsteins G, Keinanen R, Koistinaho J (2000) Induction
of protein kinase C[delta] subspecies in neurons and microglia
after transient global brain ischemia. J Cereb Blood Flow Metab
20:93102.
Kunz A, Abe T, Hochrainer K, Shimamura M, Anrather J, Racchumi G,
Zhou P, Iadecola C (2008) Nuclear factor-kappaB activation and
postischemic inflammation are suppressed in CD36-null mice after
middle cerebral artery occlusion. J Neurosci 28:1649 1658.
Lawrence T, Bebien M, Liu GY, Nizet V, Karin M (2005) IKKalpha limits
macrophage NF-kappaB activation and contributes to the resolution of inflammation. Nature 434:1138 1143.
Li Q, Estepa G, Memet S, Israel A, Verma IM (2000) Complete lack of
NF-kappaB activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation. Genes Dev 14:1729 1733.
Li Q, Verma IM (2002) NF-kappaB regulation in the immune system.
Nat Rev Immunol 2:725734.
Luedde T, Assmus U, Wuestefeld T, Zu Vilsendorf A, Roskams T,
Schmidt-Supprian M, Rajewsky K, Brenner DA, Manns MP,
Pasparakis M, Trautwein C (2005) Deletion of IKK2 in hepatocytes
does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion-injury. J Clin Invest 115:849 859.
Luedde T, Beraza N, Kotsikoris V, van Loo G, Nenci A, De Vos R,
Roskams T, Trautwein C, Pasparakis M (2007) Deletion of NEMO/
IKKgamma in liver parenchymal cells causes steatohepatitis and
hepatocellular carcinoma. Cancer Cell 11:119 132.
Nagashima K, Sasseville VG, Wen D, Bielecki A, Yang H, Simpson C,
Grant E, Hepperle M, Harriman G, Jaffee B, Ocain T, Xu Y, Fraser
CC (2006) Rapid TNFR1-dependent lymphocyte depletion in vivo
with a selective chemical inhibitor of IKKbeta. Blood 107:
4266 4273.
Nolan GP, Fujita T, Bhatia K, Huppi C, Liou HC, Scott ML, Baltimore
D (1993) The bcl-3 proto-oncogene encodes a nuclear I kappa
B-like molecule that preferentially interacts with NF-kappa B p50
and p52 in a phosphorylation-dependent manner. Mol Cell Biol
13:35573566.
Nurmi A, Lindsberg PJ, Koistinaho M, Zhang W, Juettler E, Karjalainen-Lindsberg ML, Weih F, Frank N, Schwaninger M, Koistinaho J (2004) Nuclear factor-kappaB contributes to infarction after
permanent focal ischemia. Stroke 35:987991.
Paul R, Zhang ZG, Eliceiri BP, Jiang Q, Boccia AD, Zhang RL, Chopp
M, Cheresh DA (2001) Src deficiency or blockade of Src activity in
mice provides cerebral protection following stroke. Nat Med
7:222227.
Pereira MP, Hurtado O, Cardenas A, Bosca L, Castillo J, Davalos A,
Vivancos J, Serena J, Lorenzo P, Lizasoain I, Moro MA (2006)
Rosiglitazone and 15-deoxy-Delta12,14-prostaglandin J2 cause
potent neuroprotection after experimental stroke through noncom-

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

pletely overlapping mechanisms. J Cereb Blood Flow Metab
26:218 229.
Perkins ND (2006) Post-translational modifications regulating the activity and function of the nuclear factor kappa B pathway. Oncogene 25:6717 6730.
Perkins ND (2007) Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 8:49 62.
Pizzi M, Goffi F, Boroni F, Benarese M, Perkins SE, Liou HC, Spano
P (2002) Opposing roles for NF-kappa B/Rel factors p65 and c-Rel
in the modulation of neuron survival elicited by glutamate and
interleukin-1beta. J Biol Chem 277:2071720723.
Pizzi M, Sarnico I, Boroni F, Benarese M, Steimberg N, Mazzoleni G,
Dietz GP, Bahr M, Liou HC, Spano PF (2005) NF-kappaB factor
c-Rel mediates neuroprotection elicited by mGlu5 receptor agonists against amyloid beta-peptide toxicity. Cell Death Differ
12:761772.
Pizzi M, Spano P (2006) Distinct roles of diverse nuclear factorkappaB complexes in neuropathological mechanisms. Eur J Pharmacol 545:2228.
Polavarapu R, Gongora MC, Yi H, Ranganthan S, Lawrence DA,
Strickland D, Yepes M (2007) Tissue-type plasminogen activatormediated shedding of astrocytic low-density lipoprotein receptorrelated protein increases the permeability of the neurovascular
unit. Blood 109:3270 3278.
Potrovita I, Zhang W, Burkly L, Hahm K, Lincecum J, Wang MZ,
Maurer MH, Rossner M, Schneider A, Schwaninger M (2004)
Tumor necrosis factor-like weak inducer of apoptosis-induced neurodegeneration. J Neurosci 24:8237 8244.
Prass K, Scharff A, Ruscher K, Lowl D, Muselmann C, Victorov I,
Kapinya K, Dirnagl U, Meisel A (2003) Hypoxia-induced stroke
tolerance in the mouse is mediated by erythropoietin. Stroke
34:19811986.
Qin ZH, Chen RW, Wang Y, Nakai M, Chuang DM, Chase TN (1999)
Nuclear factor kappaB nuclear translocation upregulates c-Myc
and p53 expression during NMDA receptor-mediated apoptosis in
rat striatum. J Neurosci 19:4023 4033.
Reith J, Jorgensen H, Pedersen P, Nakayama H, Raaschou H,
Jeppesen L, Olsen T (1996) Body temperature in acute stroke:
relation to stroke severity, infarct size, mortality, and outcome.
Lancet 347:422 425.
Rius J (2008) NF-B links innate immunity to the hypoxic response
through transcriptional regulation of HIF-1. Nature, 453:807 811.
Rosenzweig HL, Minami M, Lessov NS, Coste SC, Stevens SL, Henshall DC, Meller R, Simon RP, Stenzel-Poore MP (2007) Endotoxin
preconditioning protects against the cytotoxic effects of TNFalpha
after stroke: a novel role for TNFalpha in LPS-ischemic tolerance.
J Cereb Blood Flow Metab 27:16631674.
Ruscher K, Freyer D, Karsch M, Isaev N, Megow D, Sawitzki B, Priller
J, Dirnagl U, Meisel A (2002) Erythropoietin is a paracrine mediator
of ischemic tolerance in the brain: evidence from an in vitro model.
J Neurosci 22:1029110301.
Ryseck RP, Novotny J, Bravo R (1995) Characterization of elements
determining the dimerization properties of RelB and p50. Mol Cell
Biol 15:3100 3109.
Schneider A, Martin-Villalba A, Weih F, Vogel J, Wirth T, Schwaninger
M (1999) NF-B is activated and promotes cell death in focal
cerebral ischemia. Nat Med 5:554 559.
Schreck R, Rieber P, Baeuerle PA (1991) Reactive oxygen intermediates as apparently widely used messengers in the activation of
the NF-kappa B transcription factor and HIV-1. EMBO J 10:
22472258.
Song YS, Lee YS, Chan PH (2005) Oxidative stress transiently decreases the IKK complex (IKKalpha, beta, and gamma), an upstream component of NF-kappaB signaling, after transient focal
cerebral ischemia in mice. J Cereb Blood Flow Metab 25:
13011311.
Stephenson D, Yin T, Smalstig EB, Hsu MA, Panetta J, Little S,
Clemens J (2000) Transcription factor nuclear factor-kappa B is

1005

activated in neurons after focal cerebral ischemia. J Cereb Blood

Flow Metab 20:592 603.
Stevens SL, Ciesielski TM, Marsh BJ, Yang T, Homen DS, Boule JL,
Lessov NS, Simon RP, Stenzel-Poore MP (2008) Toll-like receptor
9: a new target of ischemic preconditioning in the brain. J Cereb
Blood Flow Metab 28:1040 1047.
Strnad J, Burke JR (2007) IkappaB kinase inhibitors for treating autoimmune and inflammatory disorders: potential and challenges.
Trends Pharmacol Sci 28:142148.
Sugawara T, Fujimura M, Noshita N, Kim GW, Saito A, Hayashi T,
Narasimhan P, Maier CM, Chan PH (2004) Neuronal death/survival signaling pathways in cerebral ischemia. NeuroRx 1:1725.
Tang SC, Arumugam TV, Xu X, Cheng A, Mughal MR, Jo DG, Lathia
JD, Siler DA, Chigurupati S, Ouyang X, Magnus T, Camandola S,
Mattson MP (2007) Pivotal role for neuronal toll-like receptors in
ischemic brain injury and functional deficits. Proc Natl Acad Sci
U S A 104:13798 13803.
Tarabin V, Schwaninger M (2004) The role of NF-kappaB in 6-hydroxydopamine- and TNFalpha-induced apoptosis of PC12 cells.
Naunyn Schmiedebergs Arch Pharmacol 369:563569.
Terai K, Matsuo A, McGeer EG, McGeer PL (1996) Enhancement of
immunoreactivity for NF-B in human cerebral infarction. Brain Res
739:343349.
Thompson JE, Phillips RJ, Erdjument-Bromage H, Tempst P, Ghosh S
(1995) I[kappa]B-[beta] regulates the persistent response in a biphasic activation of NF-[kappa]B. Cell 80:573582.
Tortella FC, Williams AJ (2005) A novel inhibitory kinase (IKK)
inhibitor, MLN1145, reduces brain infarction and improves outcome following focal ischemic brain injury in rats. vol. Program No.
589.6. 2005 Abstract Viewer/Itinerary Planner Washington, DC:
Society for Neuroscience.
Totzke G, Essmann F, Pohlmann S, Lindenblatt C, Janicke RU,
Schulze-Osthoff K (2006) A novel member of the I{kappa}B family,
human I{kappa}B-{zeta}, inhibits transactivation of p65 and its DNA
binding. J Biol Chem 281:1264512654.
Trickler WJ, Mayhan WG, Miller DW (2005) Brain microvessel endothelial cell responses to tumor necrosis factor-alpha involve a
nuclear factor kappa B (NF-kappaB) signal transduction pathway.
Brain Res 1048:24 31.
Vahedi K, Hofmeijer J, Juettler E, Vicaut E, George B, Algra A,
Amelink GJ, Schmiedeck P, Schwab S, Rothwell PM, Bousser MG,
van der Worp HB, Hacke W (2007) Early decompressive surgery in
malignant infarction of the middle cerebral artery: a pooled analysis
of three randomised controlled trials. Lancet Neurol 6:215222.
van den Tweel ER, Kavelaars A, Lombardi MS, Groenendaal F, May
M, Heijnen CJ, van Bel F (2006) Selective inhibition of nuclear
factor-kappaB activation after hypoxia/ischemia in neonatal rats is
not neuroprotective. Pediatr Res 59:232236.
Wang HK, Park UJ, Kim SY, Lee JH, Kim SU, Gwag BJ, Lee YB (2008)
Free radical production in CA1 neurons induces MIP-1{alpha} expression, microglia recruitment, and delayed neuronal death after
transient forebrain ischemia. J Neurosci 28:17211727.
Wang Q, Tang XN, Yenari MA (2007) The inflammatory response in
stroke. J Neuroimmunol 184:53 68.
Wang X, Feuerstein GZ, Xu L, Wang H, Schumacher WA, Ogletree
ML, Taub R, Duan JJ, Decicco CP, Liu RQ (2004) Inhibition of
tumor necrosis factor-alpha-converting enzyme by a selective antagonist protects brain from focal ischemic injury in rats. Mol Pharmacol 65:890 896.
Wang YH, Wang WY, Chang CC, Liou KT, Sung YJ, Liao JF, Chen CF,
Chang S, Hou YC, Chou YC, Shen YC (2005) Taxifolin ameliorates
cerebral ischemia-reperfusion injury in rats through its anti-oxidative effect and modulation of NF-kappa B activation. J Biomed Sci
115.
Wen Y, Yang S, Liu R, Perez E, Yi KD, Koulen P, Simpkins JW (2004)
Estrogen attenuates nuclear factor-kappa B activation induced by
transient cerebral ischemia. Brain Res 1008:147154.

1006

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 9951006

Whiteside ST, Epinat JC, Rice NR, Israel A (1997) I kappa B epsilon,
a novel member of the I kappa B family, controls RelA and cRel
NF-kappa B activity. EMBO J 16:14131426.
Williams AJ, Hale SL, Moffett JR, Dave JR, Elliott PJ, Adams J,
Tortella FC (2003) Delayed treatment with MLN519 reduces infarction and associated neurologic deficit caused by focal ischemic
brain injury in rats via antiinflammatory mechanisms involving nuclear factor-kappaB activation, gliosis, and leukocyte infiltration.
J Cereb Blood Flow Metab 23:75 87.
Winkles JA (2008) The TWEAK-Fn14 cytokine-receptor axis: discovery,
biology and therapeutic targeting. Nat Rev Drug Discov 7:411425.
Xu L, Zhan Y, Wang Y, Feuerstein GZ, Wang X (2002) Recombinant
adenoviral expression of dominant negative IkappaBalpha protects
brain from cerebral ischemic injury. Biochem Biophys Res Commun 299:14 17.
Yin MJ, Yamamoto Y, Gaynor RB (1998) The anti-inflammatory agents
aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta.
Nature 396:77 80.

Yu G, Rux AH, Ma P, Bdeir K, Sachais BS (2005) Endothelial expression of E-selectin is induced by the platelet-specific chemokine
platelet factor 4 through LRP in an NF-kappaB-dependent manner.
Blood 105:35453551.
Zhang W, Potrovita I, Tarabin V, Herrmann O, Beer V, Weih F,
Schneider A, Schwaninger M (2005) Neuronal activation of NF-B
contributes to cell death in cerebral ischemia. J Cereb Blood Flow
Metab 25:30 40.
Zhang X, Polavarapu R, She H, Mao Z, Yepes M (2007a) Tissuetype plasminogen activator and the low-density lipoprotein receptor-related protein mediate cerebral ischemia-induced nuclear factor-kappaB pathway activation. Am J Pathol 171:
12811290.
Zhang X, Winkles JA, Gongora MC, Polavarapu R, Michaelson JS,
Hahm K, Burkly L, Friedman M, Li XJ, Yepes M (2007b) TWEAKFn14 pathway inhibition protects the integrity of the neurovascular
unit during cerebral ischemia. J Cereb Blood Flow Metab 27:
534 544.

(Accepted 3 July 2008)

(Available online 10 July 2008)