R

N

D

PF

AhTiaueNedfidskstataP

Kf

INNMT

NCR

*5ESAdIv1scMPScpmw

Neuroscience 158 (2009) 995–1006

0d

EVIEW

F-�B SIGNALING IN CEREBRAL ISCHEMIA

Tfrutsbmait

AthtpfiolcirppttbecihrnpN

Tdpiom

. A. RIDDER AND M. SCHWANINGER*

harmacological Institute, University of Heidelberg, Im Neuenheimereld 366, 69120 Heidelberg, Germany

bstract—The transcription factor NF-�B is a key regulator ofundreds of genes involved in cell survival and inflammation.here is ample evidence that NF-�B is activated in cerebral

schemia, mainly in neurons. Despite its well known role asn antiapoptotic factor, in cerebral ischemia NF-�B contrib-tes to neuronal cell death, at least if the ischemia is severenough to lead to irreversible brain damage. In contrast,F-�B also seems to be responsible for the preconditioningffect of a transient and sublethal ischemia, perhaps byampening its own subsequent full activation. Among theve NF-�B subunits, RelA and p50 are responsible for theetrimental effect in cerebral ischemia. Activation of NF-�Bignaling is mediated by the upstream kinase inhibitor ofappaB kinase and is triggered by hypoxia, reactive oxygenpecies, and several inflammatory mediators. Interestingly,he complex NF-�B signaling pathway provides drug targetst several levels. Modulation of NF-�B signaling has the po-ential to interrupt multiple inflammatory and apoptotic mech-nisms through one specific molecular target. © 2009 IBRO.ublished by Elsevier Ltd. All rights reserved.

ey words: inflammation, apoptosis, stroke, transcriptionactor, cytokine, IKK.

Contentsnflammation and apoptosis in cerebral ischemia 995F-�B signaling 995F-�B in cerebral ischemia 997echanisms of NF-�B activation in cerebral ischemia 998he pro- and antiapoptotic function

of NF-�B 1001F-�B signaling as a drug target in cerebral ischemia 1002onclusions 1003eferences 1003

Corresponding author. Tel: �49-6221-548691; fax: �49-6221-48549.-mail address: markus.schwaninger@pharma.uni-heidelberg.de (M.chwaninger).bbreviations: Gpx1, glutathione peroxidase 1; HIF-1�, hypoxia-in-ucible transcription factor-1�; HMGB1, high-mobility group box 1;

CAM-1, intercellular adhesion molecule 1; ICE, interleukin-1�-con-erting enzyme; IKK, inhibitor of kappaB kinase; IL, interleukin; IL-RA, interleukin-1 receptor antagonist; iNOS, inducible nitric oxideynthase; LRP, lipoprotein receptor-related protein; MCAO, middleerebral artery occlusion; mGluR, metabotropic glutamate receptor;MP, matrix metallopeptidase; PDTC, pyrrolidine dithiocarbamate;PAR, proliferator-activated receptor; ROS, reactive oxygen species;OD, superoxide dismutase; TLR, toll-like receptor; TNF, tumor ne-rosis factor; TNFR, tumor necrosis factor receptor; tPA, tissue-typelasminogen activator; TUNEL, terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labeling; TWEAK, tumor necrosis factor–likeeak inducer of apoptosis.

306-4522/09 © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.oi:10.1016/j.neuroscience.2008.07.007

995

he brain responds very rapidly to ischemia. After only aew seconds cerebral function is impaired, though still in aeversible manner. Cerebral ischemia lasting several min-tes or longer may trigger a cascade of events that even-ually leads to irreversible deficits due to cell loss or othertructural changes. It takes at least several hours for full-lown ischemic damage to develop. However, this time lagay open a therapeutic window, in which irreversible dam-ge can be prevented. Therefore, delayed mechanisms of

schemic brain damage have received considerable atten-ion.

INFLAMMATION AND APOPTOSIS INCEREBRAL ISCHEMIA

fter onset of cerebral ischemia a multifaceted inflamma-ory reaction emerges over the course of the next fewours. Numerous inflammatory mediators are induced athe transcriptional level, including enzymes required forrostaglandin synthesis, cytokines of the tumor necrosisactor (TNF) family, and chemokines. The upregulation ofnflammatory genes is not restricted to glial cells but alsoccurs in neurons (Wang et al., 2008). Chemokines lure

eukocytes into the ischemic brain, mainly polymorphonu-lear leukocytes and monocytes, which release further

nflammatory mediators and contribute to the inflammatoryeaction. The inflammatory reaction becomes clinically ap-arent through a febrile response and the increase in acutehase protein levels in the peripheral blood of stroke pa-ients (Reith et al., 1996; Acalovschi et al., 2003). In addi-ion, inflammation contributes to the breakdown of thelood–brain barrier in cerebral ischemia (Candelario-Jalilt al., 2009). Disruption of the blood–brain barrier and theonsequent brain edema are major causes of acute lethal-

ty in stroke (Vahedi et al., 2007). Experimental studiesave firmly established that inflammation is closely inter-elated with neuronal cell death and thereby promotes theeurological deficit. Inflammation and apoptosis both de-end largely on gene expression and share key regulators,F-�B being a prominent example.

NF-�B SIGNALING

he transcription factor NF-�B consists of preformedimers. In mammals five different NF-�B subunits, p50,52, c-Rel, RelA, and RelB, form homo- and heterodimers

n various combinations. However, not all combinations doccur. For example, RelB does not form either ho-odimers or heterodimers with c-Rel nor RelA under nor-

al conditions (Ryseck et al., 1995). In neural extracts

d12

wIisRImsI1ae

ppp

acBrssttWiatRu

p(tIb

FSao

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–1006996

imers consisting of p50/RelA, p50/p50 (Schneider et al.,999), RelA/RelA, c-Rel/RelA, p50/c-Rel (Pizzi et al.,005) have been reported.

NF-�B dimers are retained in the cytosol by interactingith inhibitory I�B proteins. There are seven I�B proteins:

�B�, I�B�, I�B�, p100, p105, Bcl-3, and I�B�. Interest-ngly, the binding of I�B proteins to NF-�B dimers showsome specificity. I�B� and I�B� target predominantly p50/elA and p50/c-Rel heterodimers (Thompson et al., 1995);

�B� only interacts with RelA and c-Rel hetero- and ho-odimers (Whiteside et al., 1997); and Bcl-3 binds exclu-

ively to p50 or p52 homodimers (Nolan et al., 1993). I�B�,�B�, and I�B� are target genes of NF-�B (Brown et al.,993; Whiteside et al., 1997; Totzke et al., 2006) providingnegative feedback mechanism by which NF-�B activity isventually shut off.

In the canonical pathway of activation (Fig. 1) I�Broteins are phosphorylated (Ser32 and Ser36 of I�B�),olyubiquitinated by the SCF�TrCP ubiquitin ligase com-lex, and then degraded by the 26 S proteasome (Hacker

ig. 1. NF-�B signaling in cerebral ischemia. In the ischemic brain dier32 and Ser36. Upon phosphorylation, I�B� is degraded by the prot

nd initiate NF�B-dependent gene transcription. IKK also phosphorylates RelAf RelA. Phosphorylation of serine residues is labeled in yellow, phosphorylation

nd Karin, 2006). After degradation of I�B, NF-�B translo-ates into the nucleus and stimulates gene transcription.ecause phosphorylation of I�B proteins plays a pivotal

ole in the activation of NF-�B, identification of the respon-ible I�B kinase (inhibitor of kappaB kinase, IKK) repre-ents a major breakthrough. The IKK complex consists ofwo enzymatic subunits, IKK1 (IKK�) and IKK2 (IKK�), andhe regulatory subunit NF-�B essential modulator (NEMO).

hile IKK2 is essential for the canonical NF-�B pathwaynvolving phosphorylation of I�B�, I�B�, I�B� as outlinebove, IKK1 has been implicated in the so-called alterna-ive pathway of NF-�B activation leading to the formation ofelB/p52 dimers. The role of the latter in brain is largelynknown.

NF-�B is activated by a huge array of stimuli, includingroinflammatory cytokines such as TNF and interleukinIL)-1� that are recognized by specific membrane recep-ors such as tumor necrosis factor receptor (TNFR) andL-1R as well as microbial pathogens that are recognizedy members of the pattern recognition receptor family,

uli trigger activation of the IKK complex that phosphorylates I�B� atand p50/RelA heterodimers are released, translocate to the nucleus,

536

verse stimeasome,

at Ser . This phosphorylation increases the transcriptional activityof tyrosine residues in orange. NRs, NMDA-receptors; Glu, glutamate.

iaosicrtpNadiit

Tbv1edg1Nuhdcbs1Ics(entcn

fisstN(bp2eccfsw

oadeptltd–ascncsrts

eo(mwwebio

fmetamtjtnettss

dmprNtetetf

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–1006 997

ncluding toll-like receptors (TLRs). In addition to beingctivated by extracellular stimuli, some of the pattern rec-gnition receptors are also activated by intracellular stimuliuch as microbial components in the cytoplasm or in ves-cles. Other important inducers of NF-�B activity includeytotoxic agents, e.g. chemotherapeutic drugs, ionizingadiation, UV light, oxidative stress, and hypoxia. Most ofhese stimuli culminate in the activation of the IKK com-lex, but there are also alternative pathways of inducingF-�B activity. Several inducers of NF-�B signaling arelso known to play a role in cerebral ischemia. Here, weiscuss the mechanisms of NF-�B activation in cerebral

schemia and speculate on the complex signaling networknvolved in the pathophysiological events taking place inhe ischemic brain.

NF-�B IN CEREBRAL ISCHEMIA

here is ample evidence that NF-�B is activated in cere-ral ischemia. Most investigators found NF-�B to be acti-ated in neurons (Clemens et al., 1997; Schneider et al.,999; Stephenson et al., 2000; Huang et al., 2001; Nurmit al., 2004). In addition, NF-�B activation has also beenemonstrated in endothelial cells, astrocytes, and micro-lia (Terai et al., 1996; Howard et al., 1998; Gabriel et al.,999; Zhang et al., 2007a; Kaushal and Schlichter, 2008).F-�B activation involves nuclear translocation of the sub-nits RelA and p50 and increased DNA binding of p50omodimers and p50/RelA heterodimers (Fig. 1) (Schnei-er et al., 1999; Huang et al., 2001). In a transient middleerebral artery occlusion (MCAO) model increased DNAinding could already be detected after 30 min of reperfu-ion following a 2-h period of MCAO (Schneider et al.,999). Increased DNA binding reflects activation of NF-�B.

ndeed, in mouse models of both permanent and transienterebral ischemia a �B-dependent �-globin reporter genehowed an increase in the transcriptional activity of NF-�BSchneider et al., 1999; Nurmi et al., 2004). In these mod-ls nuclear translocation of RelA was detected mainly ineurons. In postmortem human brain samples from pa-ients who suffered a stroke, nuclear translocation of RelAould also be observed in the brain areas surrounding theecrotic infarct core (Nurmi et al., 2004).

To demonstrate the functional significance of thesendings, mice deficient in the p50 subunit of NF-�B wereubjected to MCAO. The absence of p50 resulted in aignificant and comparable reduction in infarct size in bothransient and permanent stroke models, suggesting thatF-�B plays a detrimental role in cerebral ischemia

Schneider et al., 1999; Nurmi et al., 2004). Because p50oth represses and transactivates gene transcription, de-ending on the dimerization partner (Hayden and Ghosh,008), it was difficult to interpret these results. Adenoviralxpression of a dominant-negative I�B� mutant gavelearer evidence for the functional significance of NF-�B inerebral ischemia. In this gene the phosphorylation sitesor IKK (Ser32 and Ser36) were mutated to alanine topecifically inhibit NF-�B activation. When this transgene

as expressed infarct size was reduced and neurological i

utcome improved 24 h after a 2 h period of MCAO (Xu etl., 2002). To determine what cell type is involved in theetrimental function of NF-�B activation in cerebral isch-mia, transgenic mouse lines were generated that ex-ressed the same dominant-negative I�B� mutant under

he control of neuron- or astrocyte-specific promoters. Se-ective inhibition of NF-�B in neurons significantly reducedhe infarct size and the number of terminal deoxynucleoti-yl transferase-mediated dUTP nick-end labeling (TUNEL)positive cells after 48 h of MCAO, whereas inhibition instrocytes had no statistically significant effect on infarctize or neuronal survival (Zhang et al., 2005). Furthermore,ultured primary cortical neurons expressing the dominant-egative I�B� were protected from cell death induced byamptothecin, a DNA-damaging agent. Notably, expres-ion of the dominant-negative I�B� did not completelyepress NF-�B activity in these experiments. This is impor-ant as constitutive NF-�B activity has been shown toupport neuronal survival (Bhakar et al., 2002).

Possibly, the five NF-�B subunits also exert distinctffects on cell survival. The unique phenotypes of knock-ut lines suggest that the subunits are functionally diverseLi and Verma, 2002; Gerondakis et al., 2006). Indeed, inice with a conditional deletion of RelA in the brain infarctsere smaller than those in their control littermates,hereas a germline deficiency of p52 and c-rel had noffect on infarct size. Nevertheless, in wild-type mouserain all subunits showed both nuclear translocation and

ncreased DNA binding in an ELISA-based assay after 4 hf ischemia (Inta et al., 2006).

Although NF-�B is well known to have an antiapoptoticunction in diverse cell types, it plays a detrimental role inost experimental settings of prolonged cerebral isch-mia. Nevertheless, some controversial reports do not fit tohe concept of NF-�B contributing to ischemic brain dam-ge. One report claims that costaining with Fluoro-Jade, aarker for neurodegeneration, does not colocalize with

ranscriptional NF-�B activity in a �B-reporter mouse sub-ected to MCAO (Duckworth et al., 2006). According tohese findings, areas stained for active p50 did not showeurodegeneration 4 days after transient cerebral isch-mia. At this late stage it might be true that the antiapop-otic properties of NF-�B have an impact. Possibly, onlyhe neurons in which NF-�B activation is moderate haveurvived up to that time, whereas others have alreadyuccumbed to the ischemic insult.

Another controversial aspect, and one that at first sightoes not agree with the concept of NF-�B playing a detri-ental role in stroke, is the observation that ischemicreconditioning relies on NF-�B function. After a brief pe-iod of brain ischemia below the threshold of cell death,F-�B is activated in neurons and confers neuroprotection

o a subsequent, prolonged period of ischemia (Blondeaut al., 2001). This process seems to rely on gene transcrip-ion. Blocking NF-�B activation pharmacologically with di-thyldithiocarbamate or with �B decoy DNA at the time of

he preconditioning stimulus abolished the protective ef-ect. Interestingly, preconditioning the brain with sublethal

schemia inhibited NF-�B activation after a second, more

smwnpatl

apseGcp(uBroctemgo(ktLcmi(sicrswe

OpceadceaawaI

toontlc2vRsiaR

iappsTIrItiatcrcsrfblSpihMd

iSrt2hsasaiwisk

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–1006998

evere ischemic insult. This process was suggested to beediated through induction of I�B� (Fig. 1), which is aell-known target gene of NF-�B, and may account for theeuroprotective effect of preconditioning. During a brieferiod of nonlethal cerebral ischemia, the well-known anti-poptotic properties of NF-�B seem to prevail. However,he detrimental role of NF-�B predominates during pro-onged ischemia.

The mechanisms underlying the dual role of NF-�B-ctivation in cerebral ischemia have not yet been com-letely elucidated. NF-�B interacts with several life/deathignaling pathways via expression and repression of doz-ns of pro- or antiapoptotic target genes (Barkett andilmore, 1999; Dutta et al., 2006). In a model of permanenterebral ischemia, for example, we have found that ex-ression of the proapoptotic Bcl-2-homology domain 3BH3) –only genes Bim and Noxa depend on RelA and thepstream kinase IKK (Inta et al., 2006). RelA stimulatedim and Noxa gene transcription in primary cortical neu-

ons and bound to the promoter of both genes. As the ratiof pro- and antiapoptotic Bcl-2 family members determinesell fate, Bim and Noxa could be candidates that mediatehe detrimental effects of RelA activation in cerebral isch-mia. Furthermore, NF-�B is a central mediator of inflam-atory processes. Several proinflammatory NF-�B targetenes, including TNF, IL-1� and �, IL-6, inducible nitricxide synthase (iNOS), intercellular adhesion molecule 1ICAM-1), and matrix metallopeptidase (MMP) 9, that arenown to be induced in cerebral ischemia could mediatehe deleterious effect (Wang et al., 2007; Gilmore, 2008).ikewise, key genes of eicosanoid metabolism, which en-ode cytosolic phospholipase A2, cyclooxygenase 2, andicrosomal prostaglandin E synthase 1, are induced in the

schemic brain, possibly contributing to ischemic damageHerrmann et al., 2005). Interestingly, it has recently beenhown that IKK2 also regulates the induction of hypoxia-nducible transcription factor-1� (HIF-1�) under hypoxiconditions via activation of NF-�B (Rius, 2008). Both det-imental and beneficial effects of HIF-1� have been de-cribed in cerebral ischemia depending on the cell type inhich HIF-1 � was deleted (Helton et al., 2005; Baranovat al., 2007).

MECHANISMS OF NF-�B ACTIVATION INCEREBRAL ISCHEMIA

ne of the very early and also pivotal steps in the patho-hysiology of stroke seems to be the activation of the IKKomplex. In a mouse model of permanent cerebral isch-mia, we could show increased IKK activity as early as halfn hour after the onset of ischemia using a kinase pull-own assay (Herrmann et al., 2005). The activation wasonfined to the periphery of the ischemic territory, how-ver. In the ischemic core, the lack of increased kinasectivity might be explained by degradation of IKK, as someuthors recently reported (Song et al., 2005). IKK activityas increased for 5 h after the onset of MCAO. This waslso paralleled by a rather mild, but detectable increase in

KK phosphorylation at Ser180/Ser181 in the periphery of r

he infarcted tissue (Inta et al., 2006). The phosphorylationf this site has been reported to be important for activationf the kinase in several signaling pathways, supporting theotion that IKK is activated (Perkins, 2006). It is knownhat, in addition to the I�B proteins, IKK also phosphory-ates RelA at Ser536 (Fig. 1). Phosphorylation of RelA isonsidered to increase its transcriptional activity (Perkins,006). Immunohistochemistry of mouse brain sections re-ealed a prominent increase in Ser536-phosphorylatedelA after 1 h of MCAO adjacent to the ischemic core. Thetaining colocalized with NeuN, a neuronal marker protein,

ndicating that IKK phosphorylates RelA in neurons. Inddition, another recent publication also showed increasedelA-phosphorylation in astrocytes (Zhang et al., 2007a).

To analyze the function of the IKK complex in cerebralschemia, Ikbkb, the gene encoding IKK2, was condition-lly deleted in neurons or in neurons and glial cells. Weerformed MCAO in these animals and observed a com-arable reduction in infarct size in both mutant mousetrains (Herrmann et al., 2005). In addition, the number ofUNEL-positive cells was decreased in mice deficient for

KK2 in neurons. These experiments did not address theole of IKK1, though. As there is evidence that IKK1 andKK2 may have partially redundant or also opposing func-ions (Li et al., 2000; Lawrence et al., 2005), we werenterested in determining the effect of inhibiting both IKK1nd IKK2 in neurons. Therefore, a dominant–negative mu-ant of IKK2 (D145N) under the control of the tTA (tetra-ycline-controlled transactivator) was introduced in neu-ons. Both basal and inducible IKK activity in response toerebral ischemia was markedly diminished in this mousetrain. These mice also showed infarct volumes that wereeduced by more than 50% (Herrmann et al., 2005). Tourther test the validity of the concept of IKK contributing torain damage in cerebral ischemia, a transgenic mouse

ine expressing a constitutively active IKK2 mutant (S177E,181E) in neurons was created. In accordance with therevious results, these mice showed significantly larger

nfarcts although no obvious signs of neurodegenerationad been observed before the mice were subjected toCAO. Thus, IKK plays a major role in ischemic brainamage but cannot induce neurodegeneration on its own.

The mechanisms underlying IKK activation in cerebralschemia have not been systematically investigated yet.everal possible pathways come into question. IKK has

ecently been shown to be activated by hypoxia and, inurn, to mediate the activation of NF-�B (Cummins et al.,006). These authors propose a model in which the prolylydroxylases PHD-1, and to a lesser extent PHD-2, con-titutively repress IKK by hydroxylation. As the PHDs haven absolute requirement for molecular oxygen as cosub-trate, hypoxia then rapidly releases this repression of IKKnd activates NF-�B. Cummins and colleagues (2006)

dentified a potential hydroxylation site in IKK2 (Pro191),hich, when mutated, leads to a loss in hypoxic inducibil-

ty. Hypoxia also increased NF-�B activity when cells weretimulated with TNF. This enhanced susceptibility to cyto-ines under hypoxic conditions also underlines the central

ole of IKK in the pathophysiology of stroke.

mNbciR1amitn2ktGgmN

cspup2isnifbdb(NdSdpa(eperttctpia

sfii

ajticTTdmoaptnTcNasiyt(1TppT(2eblfiRTThi

rCtcscpiCsciosttia

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–1006 999

In addition to hypoxia, reactive oxygen species (ROS)ight contribute to NF-�B activation in cerebral ischemia.F-�B was shown to respond to oxidative stress inducedy ROS (Schreck et al., 1991). The formation of ROS inerebral ischemia is well known, especially in transientschemia models. Transgenic mice overexpressing theOS-scavenging enzymes superoxide dismutases (SOD)or 2 and glutathione peroxidase 1 (Gpx1) are protected

gainst ischemic cell death (Sugawara et al., 2004). Inice overexpressing SOD1, the activation of NF-�B in the

schemic brain is suppressed when compared with wild-ype mice and therefore this could represent the mecha-ism underlying the neuroprotective effect (Huang et al.,001). The induction of the proapoptotic regulator c-myc, anown NF-�B-target, was also less pronounced in SOD1ransgenic mice. Moreover, infarct size is increased inpx1-deficient mice and this is also associated with ag-ravated NF-�B-activation (Crack et al., 2006). Further-ore, pharmacological data support the activation ofF-�B by ROS in cerebral ischemia (see below).

In addition to these rather direct mechanisms, NF-�Bould also be activated through several receptor-mediatedignaling pathways. Recently, it was shown that the serinerotease tissue-type plasminogen activator (tPA) contrib-tes to NF-�B activation in cerebral ischemia via the li-oprotein receptor-related protein (LRP) (Zhang et al.,007a). This process is independent of the well-known

ntravascular effect of tPA as a plasminogen activator anduggests a cytokine-like function of tPA. LRP is present oneurons and perivascular astrocytes. It is induced in the

schemic hemisphere in a tPA-dependent manner. If LRP-unction was inhibited with anti-LRP antibodies or recom-inant LRP-associated protein 1 (Lrpap1), infarct volumesecreased, motor function recovered faster, and blood–rain barrier permeability following MCAO was reducedPolavarapu et al., 2007; Zhang et al., 2007a). In parallel,F-�B activation was attenuated, as demonstrated by re-uced DNA binding and less phosphorylation of RelA ater536. Furthermore, a significant decrease in MCAO-in-uced nitric oxide production and iNOS expression accom-anied these findings, which may reflect decreased NF-�Bctivation and account for the perceived neuroprotectionZhang et al., 2007a). Under ischemic conditions, thectodomain of LRP is shed from astrocytic end-feet, arocess dependent on tPA function and associated withdema formation. As LRP has recently been shown toegulate NF-�B function (Yu et al., 2005), the authors ofhese studies propose a model in which the shedding ofhe ectodomain of LRP from perivascular astrocytes pre-edes the release of the intracellular domain of LRP intohe cytoplasm, which ultimately will activate the NF-�Bathway. This concept suggests that NF-�B activation and

ncreased iNOS expression play a detrimental role instrocytes.

Besides tPA and LRP, the TLRs may function as up-tream regulators of IKK activity in MCAO. The TLRs are aamily of at least 11 proteins that play a major role in innatemmunity, responding to microbial pathogens and also to

njury-induced endogenous ligands. By stimulating TLRs IKK m

ctivity increases, mediating the activation of NF-�B (Baner-ee and Gerondakis, 2007). Recently published studies reporthat mice deficient in either TLR2 or TLR4 show reducednfarct sizes and improved neurological outcome whenompared with wild-type littermates (Caso et al., 2007;ang et al., 2007). Moreover, neurons deficient in TLR2 orLR4 showed increased resistance and less apoptotic celleath when subjected to glucose deprivation as an in vitroodel of ischemic conditions. Immunohistochemistry dem-nstrated a rapid increase in TLR2 and TLR4 immunore-ctivity in neurons and a delayed appearance of TLR2-ositive microglia (Tang et al., 2007), which is in line withhe concept that TLR signaling in the early phase activateseuronal IKK, leading to neurodegeneration. Mice lackingLR4 also showed reduced expression of stroke-inducedyclooxygenase 2 (COX2) and MMP9, known targets ofF-�B (Caso et al., 2007; Gilmore, 2008). Furthermore, inmodel of global cerebral ischemia, TLR4-deficient mice

howed less NF-�B DNA-binding activity in the particularlyschemia-sensitive hippocampal formation, less phosphor-lated I�B, and also increased neuronal survival than wild-ype mice. The increase in several inflammatory mediatorsIL-6, TNF, Fas ligand (FasL) and high-mobility group box(HMGB1)) was also clearly diminished (Hua et al., 2007).he fact that the TLRs have also been linked to ischemicreconditioning, an event also relying on NF-�B function,rovides further evidence for a connection between theLRs, IKK, and NF-�B in the pathophysiology of stroke

Blondeau et al., 2001; Kariko et al., 2004; Stevens et al.,008). Ligands that possibly activate the TLRs in the isch-mic brain could be molecules derived from injured tissue,lood vessels, and necrotic cells. Fragments of extracel-

ular matrix (hyaluronan, fibronectin, and heparan sulfate),brin or fibrinogen and heat shock proteins activate TLR4.NA and chromatin-associated DNA activate TLR3 andLR9, respectively (Kariko et al., 2004). Another agonist ofLR2 and 4, HMGB1 has also recently been shown toave a proinflammatory and detrimental role in cerebral

schemia (Kim et al., 2006).Similar to the TLRs, another member of the pattern-

ecognition receptor family, the type B scavenger receptorD36, was recently shown to participate in the inflamma-

ory signaling pathways found in the ischemic brain and toontribute to ischemic brain damage (Cho et al., 2005). Inham-operated mice CD36 was observed in endothelialells. After 6 h of ischemia, CD36-positive microglia ap-eared in the infarcted brain and, after 3 days, astrocytes

n the periphery of the infarct also expressed CD36. InD36-deficient mice subjected to MCAO infarcts weremaller and neurological deficits milder than in wild-typeontrols. This was accompanied by an attenuated increase

n ischemia-induced ROS (Cho et al., 2005). The activationf NF-�B was alleviated in CD36-deficient mice aftertroke, although it could still be induced by i.c.v. adminis-ration of exogenous IL-1� (Kunz et al., 2008). This reduc-ion in DNA-binding activity also went along with dimin-shed infiltration of neutrophils, a suppressed glial reaction,nd curtailed expression of NF-�B-dependent proinflam-

atory transcripts, including iNOS, ICAM-1, and endothe-

loRTspv2fNba

oVua2feinbo1istm(bsamptnabNctfpt

utinDtdoaplio

ogmssAiii(eiTiocsa1azNtabi2lIwauvpDbaac

nhcpkwmTAs2iavpsra

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–10061000

ial adhesion molecule 1 (ELAM-1). Notably, the activationf NF-�B was independent of the formation of ROS as aOS scavenger did not attenuate it (Kunz et al., 2008).he ligands that possibly activate CD36-dependent NF-�Bignaling comprise a wide variety of molecules, for exam-le, modified lipids, amyloid-�, thrombospondin-1, and ad-anced glycation end products (Febbraio and Silverstein,007). Interestingly, it has also been suggested that CD36orms a signaling complex with TLR2/6 that activatesF-�B in response to bacterial cell wall components (Feb-raio and Silverstein, 2007), possibly indicating a cooper-tive role in cerebral ischemia as well.

As already mentioned, IL-1 is another possible inducerf NF-�B activity in the ischemic brain (Kunz et al., 2008).ia activation of IKK, IL-1� and IL-1� are well-known stim-li of the NF-�B signaling pathway. Both IL-1� and IL-1�re rapidly induced in cerebral ischemia (Allan et al.,005). Deleting both cytokines markedly reduces the in-arct size in mice subjected to MCAO, whereas deletingither gene alone, perhaps owing to compensatory effects,

s ineffective (Boutin et al., 2001). Administering exoge-ous IL-1� intracerebroventricularly exacerbated ischemicrain damage, whereas administration or overexpressionf the endogenous interleukin-1 receptor antagonist (IL-RA) greatly reduces ischemic brain injury. Mice deficient

n IL-1RA show increased ischemic brain damage, furtherupporting the notion that IL-1 plays a detrimental role inhe pathophysiology of stroke (Allan et al., 2005). Further-ore, mice lacking interleukin-1�-converting enzyme

ICE), also known as caspase-1, showed less ischemicrain injury, edema formation, and inflammatory re-ponses (Huang et al., 2003). Interestingly, this was par-lleled by reduced activation of NF-�B in electrophoreticobility shift assays and a significant reduction in thehosphorylation of the RelA subunit. Immunohistochemis-ry revealed that the phospho-RelA positive cells wereeurons, which is consistent with the concept that IKKctivates NF-�B in neurons. These data provide a linketween the detrimental functions of IL-1 and ICE toF-�B and its proinflammatory and proapoptotic role inerebral ischemia. The fact that IL-1� and IL-1� are alsoarget genes of NF-�B (Gilmore, 2008) suggests a positiveeedback loop by which NF-�B activation is sustained,erpetuating the inflammatory response and worseninghe outcome.

In addition to IL-1, TNF might also be a stimulus reg-lating NF-�B activity in the ischemic brain. TNF is knowno induce NF-�B activity in a wide range of cell types,ncluding neurons (Herrmann et al., 2005), astrocytes (Gi-is et al., 2002), and endothelial cells (Trickler et al., 2005).epending on the stimulus, receptor context, and the ac-

ivation state of the cells, TNF can be associated with celleath or survival, induction of inflammation, or with controlr resolution of inflammation, features that have also beenttributed to NF-�B. Like NF-�B, TNF was shown to play arotective role in ischemic preconditioning, but during pro-

onged ischemia it is considered to exacerbate neuronalnjury (Hallenbeck, 2002). Preconditioning with low doses

f LPS conferred neuroprotection in both a mouse model b

f stroke and in cultured neurons when subjected to oxy-en glucose deprivation (Rosenzweig et al., 2007). Re-arkably, this effect seems to rely on TNF induction and

ecretion after stimulation with LPS. Notably, TNF expres-ion is known to be regulated by NF-�B (Gilmore, 2008).s TNF expression increases at the mRNA level within 1 h

n the ischemic zone in cerebral ischemia, followed byncreases in protein levels within 2–6 h of the onset ofschemia, it may contribute to the activation of NF-�BHallenbeck, 2002). Administering exogenous TNF intrac-rebroventricularly after the onset of MCAO increased the

nfarct size in mice (Rosenzweig et al., 2007). Antibodies toNF and pharmacological inhibition of TNF-alpha convert-

ng enzyme (TACE) (Wang et al., 2004) have been dem-nstrated to confer neuroprotection in brain ischemia. Inontrast, mice lacking the TNFRs TNFR1 and TNFR2howed significantly larger infarcts than wild-type controlsnd oxidative stress was also increased (Bruce et al.,996). This has been attributed to the inability of thesenimals to induce the neuroprotective and antioxidant en-yme SOD2. Interestingly, SOD2 is also a target gene ofF-�B (Gilmore, 2008). When stimulated with TNF cul-

ured astrocytes showed increased expression of SOD2nd ICAM-1 (Ginis et al., 2002), an adhesion moleculeelieved to increase the inflammatory response in the

schemic brain and worsen the outcome (Wang et al.,007). Preconditioning with TNF or ceramide, a sphingo-

ipid messenger in TNF signaling, curtailed the increase inCAM-1 when the cells were re-challenged with TNF,hereas SOD2 induction remained unweakened (Ginis etl., 2002). In this paradigm, ICAM-1 transcription was reg-lated by RelA associated with the transcriptional coacti-ator p300. In preconditioned cells RelA remained unphos-horylated and did not associate with p300 although RelANA-binding activity did not change. This illustrates a linketween NF-�B and TNF in ischemic preconditioning andlso demonstrates the double-edged impact of both TNFnd NF-�B on the outcome with respect to the molecularontext.

Another member of the TNF family of cytokines, tumorecrosis factor–like weak inducer of apoptosis (TWEAK),as also been implicated in the activation of NF-�B inerebral ischemia. It has proangiogenic and proapoptoticroperties and induces cell death in tumor cell lines (Win-les, 2008). By massively parallel signature sequencing,e detected an increase in TWEAK at the mRNA level in aouse model of cerebral ischemia (Potrovita et al., 2004).WEAK acts through Fn14, a member of the TNFR family.lthough Fn14 does not contain a death domain, it washown to mediate TWEAK-induced cell death (Aggarwal,003). Interestingly, Fn14 was also induced by cerebral

schemia, predominantly in the periphery of the infarctedrea. A neutralizing anti-TWEAK antibody increased sur-ival in cortical neurons subjected to oxygen glucose de-rivation and reduced infarct size in a mouse model oftroke, demonstrating a detrimental role of TWEAK in ce-ebral ischemia (Potrovita et al., 2004). This finding waslso confirmed by others using a soluble form of Fn14 and

y subjecting Fn14-deficient mice to MCAO (Zhang et al.,

2iyipeMmFcanTa(

(tgttNatagiRpcmfab2d

aacoNptgisemdbaminNcti

Natbtcra2l2

tttupa“s

1

2

3

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–1006 1001

007b). In these mice both induction of NF-�B DNA-bind-ng activity and the increases in IKK and RelA phosphor-lation were lower after MCAO. In addition to promotingnfarct size, TWEAK-Fn14 signaling also increased theermeability of the blood–brain barrier in cerebral isch-mia. This effect is probably mediated by the induction ofMP9 activity via NF-�B and the subsequent basementembrane laminin degradation (Zhang et al., 2007b).n14 is expressed in cortical neurons. Stimulation with re-ombinant TWEAK activates NF-�B through the IKK complexnd triggered apoptosis in a moderate number of corticaleurons in vitro. Inhibition of NF-�B in neurons reducedWEAK-induced apoptosis, suggesting that NF-�B is playingproapoptotic 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 ofhe membrane potential, and neurons depolarize. Voltage-ated Ca2� channels become activated, whereby excita-

ory neurotransmitters, including glutamate, accumulate inhe extracellular space. Glutamate might also induceF-�B activity in cerebral ischemia as it has been shown toctivate NF-�B in neurons in vitro via NMDA-receptor ac-ivation and an increase in intracellular Ca2� (Guerrini etl., 1995; Grilli et al., 1996). In addition, metabotropiclutamate receptors (mGluRs) are responsible for activat-

ng the subunit c-Rel in cortical neurons (Pizzi et al., 2005).ecently, it was shown in an in vitro model of the ischemicenumbra that neuronal glutamate release led to in-reased NF-�B activity in microglia via their group IIGluRs. The activated microglia exerted neurotoxic ef-

ects and killed naïve neurons through an apoptotic mech-nism that was mediated by TNF and involved activation ofoth caspase-3 and caspase-8 (Kaushal and Schlichter,008). This illustrates the functional significance and theetrimental effects of NF-�B activation in microglial cells.

Most of the listed stimuli seem to activate NF-�B viactivation of the IKK complex. However, there might belso IKK-independent mechanisms of NF-�B activation inerebral ischemia. Several post-translational modificationsf the I�Bs and the NF-�B subunits are known to controlF-�B activity (Perkins, 2006). One example for IKK-inde-endent NF-�B activation in cerebral ischemia might beyrosine phosphorylation of I�B�. After hypoxia and reoxy-enation Src phosphorylates I�B� at Tyr42 and thereby

nduces NF-�B activity (Fan et al., 2003). Src was alsohown to play a detrimental role in glutamate-inducedxcitotoxicity in neurons (Khanna et al., 2007). Further-ore, in a liver model of ischemia and reperfusion Src-ependent phosphorylation of I�B� at Tyr42 was found toe important for NF-�B activation (Fan et al., 2004). Thectivation of NF-�B also exerted a detrimental effect in thisodel. Interestingly, in a stroke model both pharmacolog-

cal inhibition of Src and genetic deficiency in Src conferredeuroprotection (Paul et al., 2001). We hypothesize thatF-�B might be a downstream effector of Src that ac-ounts for its deleterious function in cerebral ischemia andhat tyrosine phosphorylation of I�B� might also play a role

n the pathophysiology of stroke.

THE PRO- AND ANTIAPOPTOTIC FUNCTIONOF NF-�B

F-�B is well known for its antiapoptotic function (Barkettnd Gilmore, 1999; Dutta et al., 2006). Indeed, the protec-ive effect of ischemic preconditioning has been shown toe mediated by NF-�B (Blondeau et al., 2001). Precondi-ioning induces the expression of erythropoietin in astro-ytes (Ruscher et al., 2002; Prass et al., 2003). Uponelease, erythropoietin protects adjacent neurons throughNF-�B-dependent mechanism (Digicaylioglu and Lipton,

001). Other preconditioning stimuli, such as LPS, areikely to operate through NF-�B as well (Rosenzweig et al.,007).

However, findings in manifest cerebral ischemia seemo be in conflict with the concept that NF-�B is an antiapop-otic factor. Once severe ischemia occurs, NF-�B activa-ion contributes to ischemic brain damage. Although un-sual, this proapoptotic function of NF-�B is not withoutrecedence (Qin et al., 1999; Pizzi et al., 2002; Luedde etl., 2005). What could be the basis for this surprisingdouble life” of NF-�B? In the following we offer four pos-ible explanations for this enigma.

. Cell type-specific effects. NF-�B is a key regulator ofinnate immunity and inflammation. Activation of micro-glia by cerebral ischemia depends on NF-�B activation(Kaushal and Schlichter, 2008). Thus, a strong inflam-matory reaction controlled by NF-�B activation in mi-croglia and other inflammatory cells may override apotential antiapoptotic effect of NF-�B in neurons.Whereas this hypothesis seems attractive, it does notexplain why selective inhibition of NF-�B signaling inneurons reduced the infarct size (Herrmann et al.,2005; Zhang et al., 2005). Furthermore, activationof NF-�B in neurons may either induce apoptosisor protect against it, depending on the stimulus(Kaltschmidt et al., 2002; Pizzi et al., 2002; Tarabinand Schwaninger, 2004).

. The kinetics of NF-�B activation have profound effectson the pattern of induced genes. Some genes requirepersistent NF-�B activity whereas others are alreadystimulated by transient activation (Hoffmann et al.,2002). Thus, transient NF-�B activation may upregu-late antiapoptotic genes and protect against ischemia,whereas sustained activation may induce a proapop-totic set of genes and promote apoptosis. Indeed, theneuroprotective effect of an antioxidant drug was as-sociated with a reduction of the sustained, but not thetransient activation of NF-�B (Clemens et al., 1998).This mechanism would also explain the opposing roleof NF-�B in transient preconditioning ischemia and inmanifest ischemia (Schneider et al., 1999; Blondeau etal., 2001).

. As previously outlined, NF-�B is a heterogeneous fac-tor that is formed by five subunits in various combina-tions. Pizzi and Spano (2006) have provided convinc-ing evidence that RelA and c-Rel have opposing ef-fects on the survival of neurons. Pizzi et al. (2002)

showed that the toxic effect of glutamate in cerebellar

4

Tgsratslbk

dsnnoctisiiB4mur2iak

ebbLtapPd(Ndw2titilse2wa2

ifttfsmInmg

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–10061002

granule cells is mediated by the NF-�B subunits p50and RelA. In contrast, IL-1� and activation of themGluR type 5 increased neuronal survival through c-Rel (Pizzi et al., 2002, 2005). Interestingly, both RelAand c-Rel are activated in cerebral ischemia (Schnei-der et al., 1999; Inta et al., 2006). In accordance withthe notion that the two subunits have a different func-tion, neural deficiency of RelA reduced the infarct vol-ume in MCAO but c-Rel deficiency had no effect. Inthese experiments compensatory mechanisms in re-sponse to the germline knockout of c-Rel may havemasked its neuroprotective action. A preconditioningparadigm may be better suited to reveal the protectiveaction of c-Rel in vivo. So far, little is known about howthese two subunits are activated differentially.

. The effect of NF-�B signaling on cell survival is shapedby the interaction with numerous other signaling cas-cades and transcription factors (for review see Perkins,2007). Interestingly, several of these interaction part-ners are also activated in cerebral ischemia and maydetermine the transcriptional response and the effectof NF-�B on cell survival. In accordance with this con-cept, we found no increased cell loss in mice in whichthe IKK was constitutively active in neurons (Herrmannet al., 2005). However, these mice were more suscep-tible to cerebral ischemia. Thus, NF-�B signaling can-not induce neuronal cell death by itself but seems tosynergize with other pathways. A prominent example isthe JNK signaling pathway that promotes cell death incerebral ischemia (Borsello et al., 2003). NF-�B inhib-its the JNK pathway at several levels but may alsostimulate it through its target gene PKC� (Perkins,2007). PKC� is induced in cerebral ischemia, suggest-ing that a positive interaction between the two path-ways may be possible (Koponen et al., 2000). Like-wise, p53 interacts with NF-�B in a complex mannerinvolving mutual inhibition and induction. It is clear thatp53 is activated in cerebral ischemia (Culmsee andMattson, 2005). However, the functional conse-quences of the interaction between NF-�B and p53 forneuronal survival are still disputed (Aleyasin et al.,2004; Culmsee and Mattson, 2005). The specific inter-actions of NF-�B with many other neuronal signalingpathways are largely unknown and remain to bedetermined.

NF-�B SIGNALING AS A DRUG TARGET INCEREBRAL ISCHEMIA

he detrimental effect of NF-�B in cerebral ischemia sug-ests that inhibition of the signaling pathway may repre-ent a treatment strategy in stroke. Because of the pivotalole of NF-�B in several inflammatory disorders, consider-ble efforts have been undertaken to identify inhibitors of

he NF-�B signaling cascade. Numerous compounds werehown to interfere with NF-�B activation although the mo-

ecular mechanism was not identified in each case. Theest characterized drug target in NF-�B signaling is the

inase IKK. Already in 1996 Spano’s group provided evi- d

ence that aspirin is neuroprotective via inhibition of NF-�Bignaling (Grilli et al., 1996). Later work identified the ki-ase IKK as the molecular target of aspirin in NF-�B sig-aling (Yin et al., 1998). Because aspirin has numerousther effects, it is questionable whether NF-�B inhibitionontributes to the established efficacy of aspirin in thereatment of acute stroke. In recent years, highly specificnhibitors of IKK were developed (Karin et al., 2004). Theelective IKK inhibitor BMS-345541 blocked IKK activation

n a mouse model of stroke and reduced the infarct volumen a dose-dependent manner (Herrmann et al., 2005).MS-345541 was still effective when administered up to.5 h after onset of ischemia suggesting that IKK inhibitionay have a therapeutic time window suitable for clinicalse. Another compound, MLN1145, was found to be neu-oprotective in a rat model of stroke (Tortella and Williams,005). In contrast, a peptide inhibitor of IKK worsened

schemic brain damage in neonatal rats (van den Tweel etl., 2006). Whether this is an age-specific effect is still notnown.

For several structurally diverse compounds, correlativevidence has been presented suggesting that NF-�B inhi-ition is the cause of their neuroprotective action in cere-ral ischemia. Antioxidants such as N-acetyl-cysteine,Y231617, LY341122, caffeic acid phenethyl ester, andaxifolin reduce ischemic brain damage and inhibit NF-�Bctivation (Carroll et al., 1998; Clemens et al., 1998; Ste-henson et al., 2000; Wang et al., 2005; Khan et al., 2007).yrrolidine dithiocarbamate (PDTC) is another antioxidantrug that is both neuroprotective and an inhibitor of NF-�BNurmi et al., 2004). Initially, it was thought to interfere withF-�B signaling through its antioxidant properties but laterata showed that PDTC inhibits ubiquitin ligase activity,hich is essential for NF-�B activation (Hayakawa et al.,003). Other ubiquitin ligase inhibitors have not yet beenested in cerebral ischemia. Further data on proteasomenhibitors, such as MLN519 and CVT-634, indicate thathese compounds ameliorate ischemic brain damage andnhibit NF-�B activity in parallel (Buchan et al., 2000; Wil-iams et al., 2003). NF-�B is inhibited by nuclear receptorsuch as the estrogen receptor and the peroxisome prolif-rator-activated receptor (PPAR) � (De Bosscher et al.,006). The inhibitory effect on NF-�B may contribute to theell-established neuroprotection afforded by estrogensnd PPAR� activators (Wen et al., 2004; Pereira et al.,006).

For the clinical development of NF-�B inhibitors safetyssues will play an important role. Potential untoward ef-ects might include an increased susceptibility to infec-ions, interferences in lymphopoiesis, hepatotoxicity, anderatogenicity (Strnad and Burke, 2007). As genetic de-ects in the NF-kB pathway in humans lead to increasedusceptibility to infection, pharmacological blockers of IKKay have a similar effect. Moreover, administration of the

KK inhibitor ML120B to mice resulted in an increasedumber of apoptotic cells in thymus, spleen and bonearrow and in a reduction of viable pre-B-cells (Na-ashima et al., 2006). Because IKK2-deficient mice die

ue to liver apoptosis during embryonic development, pos-

sgNc

tdmehsnm

SratnrdoeNv

A

A

A

A

B

B

B

B

B

B

B

B

B

B

C

C

C

C

C

C

C

C

C

D

D

D

D

D

F

F

F

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–1006 1003

ible untoward effects of IKK inhibitors may include terato-enicity. In adults, genetic deletion of the IKK subunitEMO in hepatocytes caused steatohepatitis and hepato-ellular carcinoma (Luedde et al., 2007).

These untoward effects may be relevant if IKK inhibi-ors are used for the treatment of chronic inflammatoryiseases. In the treatment of stroke, a short-term treatmentight be sufficient and is less likely to cause untowardffects than long-term treatment. A couple of IKK inhibitorsave been discovered. Preclinical evaluation of these sub-tances in animal stroke models and phase I studies areow needed to evaluate their possible usage in the treat-ent of human stroke.

CONCLUSIONS

tudies in cerebral ischemia have revealed an unusualole of the transcription factor NF-�B. While it has anti-poptotic functions in many other paradigms, its main ac-ion in the ischemic brain seems to contribute to acuteeurodegeneration. The specific causes of this unusualole of NF-�B are still rather hypothetical. Also, manyetails of the signaling pathways that lead to the activationf NF-�B in cerebral ischemia remain to be defined. How-ver, with present knowledge it is already apparent thatF-�B signaling offers many targets for therapeutic inter-ention in cerebral ischemia.

REFERENCES

calovschi D, Wiest T, Hartmann M, Farahmi M, Mansmann U, Auf-farth GU, Grau AJ, Green FR, Grond-Ginsbach C, Schwaninger M(2003) Multiple levels of regulation of the interleukin-6 system instroke. Stroke 34:1864–1869.

ggarwal BB (2003) Signalling pathways of the TNF superfamily: adouble-edged sword. Nat Rev Immunol 3:745–756.

leyasin H, Cregan SP, Iyirhiaro G, O’Hare MJ, Callaghan SM, SlackRS, Park DS (2004) Nuclear factor-(kappa)B modulates the p53response in neurons exposed to DNA damage. J Neurosci24:2963–2973.

llan SM, Tyrrell PJ, Rothwell NJ (2005) Interleukin-1 and neuronalinjury. Nat Rev Immunol 5:629–640.

anerjee A, Gerondakis S (2007) Coordinating TLR-activated signal-ing pathways in cells of the immune system. Immunol Cell Biol85:420–424.

aranova O, Miranda LF, Pichiule P, Dragatsis I, Johnson RS, ChavezJC (2007) Neuron-specific inactivation of the hypoxia induciblefactor 1{alpha} increases brain injury in a mouse model of transientfocal cerebral ischemia. J Neurosci 27:6320–6332.

arkett M, Gilmore TD (1999) Control of apoptosis by Rel/NF-kappaBtranscription factors. Oncogene 18:6910–6924.

hakar AL, Tannis L-L, Zeindler C, Russo MP, Jobin C, Park DS,MacPherson S, Barker PA (2002) Constitutive nuclear factor-kappa B activity is required for central neuron survival. J Neurosci22:8466–8475.

londeau N, Widmann C, Lazdunski M, Heurteaux C (2001) Activationof the nuclear factor-kappaB is a key event in brain tolerance.J Neurosci 21:4668–4677.

orsello T, Clarke PG, Hirt L, Vercelli A, Repici M, Schorderet DF,Bogousslavsky J, Bonny C (2003) A peptide inhibitor of c-JunN-terminal kinase protects against excitotoxicity and cerebral isch-emia. Nat Med 9:1180–1186.

outin 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.

rown K, Park S, Kanno T, Franzoso G, Siebenlist U (1993) Mutualregulation of the transcriptional activator NF-kappa B and its inhib-itor, I kappa B-alpha. Proc Natl Acad Sci U S A 90:2532–2536.

ruce AJ, Boling W, Kindy MS, Peschon J, Kraemer PJ, CarpenterMK, Holtsberg FW, Mattson MP (1996) Altered neuronal and mi-croglial responses to excitotoxic and ischemic brain injury in micelacking TNF receptors. Nat Med 2:788–794.

uchan AM, Li H, Blackburn B (2000) Neuroprotection achieved witha novel proteasome inhibitor which blocks NF-kappaB activation.Neuroreport 11:427–430.

andelario-Jalil E, Yang Y, Rosenberg G (2009) Diverse roles ofmatrix metalloproteinases and tissue inhibitors of metalloprotein-ases in neuroinflammation and cerebral ischemia. Neuroscience,158:983–994.

arroll JE, Howard EF, Hess DC, Wakade CG, Chen Q, Cheng C(1998) Nuclear factor-kappa B activation during cerebral reperfu-sion: effect of attenuation with N-acetylcysteine treatment. BrainRes Mol Brain Res 56:186–191.

aso JR, Pradillo JM, Hurtado O, Lorenzo P, Moro MA, Lizasoain I(2007) Toll-like receptor 4 is involved in brain damage and inflam-mation after experimental stroke. Circulation 115:1599–1608.

ho S, Park EM, Febbraio M, Anrather J, Park L, Racchumi G,Silverstein RL, Iadecola C (2005) The class B scavenger receptorCD36 mediates free radical production and tissue injury in cerebralischemia. J Neurosci 25:2504–2512.

lemens JA, Stephenson DT, Yin T, Smalstig EB, Panetta JA, LittleSP (1998) Drug-induced neuroprotection from global ischemia isassociated with prevention of persistent but not transient activationof nuclear factor-kappaB in rats. Stroke 29:677–682.

lemens JA, Stephenson DT, Smalstig EB, Dixon EP, Little SP (1997)Global ischemia activates nuclear factor-�B in forebrain neurons ofrats. Stroke 28:1073–1081.

rack PJ, Taylor JM, Ali U, Mansell A, Hertzog PJ (2006) Potentialcontribution of NF-kappaB in neuronal cell death in the glutathioneperoxidase-1 knockout mouse in response to ischemia-reperfusioninjury. Stroke 37:1533–1538.

ulmsee C, Mattson MP (2005) p53 In neuronal apoptosis. BiochemBiophys Res Commun 331:761–777.

ummins 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 Ikap-paB kinase-beta, giving insight into hypoxia-induced NFkappaBactivity. Proc Natl Acad Sci U S A 103:18154–18159.

e Bosscher K, Vanden Berghe W, Haegeman G (2006) Cross-talkbetween nuclear receptors and nuclear factor kappaB. Oncogene25:6868–6886.

igicaylioglu M, Lipton SA (2001) Erythropoietin-mediated neuropro-tection involves cross-talk between Jak2 and NF-kappaB signallingcascades. Nature 412:641–647.

irnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology of isch-aemic stroke: an integrated view. Trends Neurosci 22:391–397.

uckworth EA, Butler T, Collier L, Collier S, Pennypacker KR (2006)NF-kappaB protects neurons from ischemic injury after middlecerebral artery occlusion in mice. Brain Res 1088:167–175.

utta J, Fan Y, Gupta N, Fan G, Gelinas C (2006) Current insights intothe regulation of programmed cell death by NF-kappaB. Oncogene25:6800–6816.

an C, Li Q, Ross D, Engelhardt JF (2003) Tyrosine phosphorylationof I kappa B alpha activates NF kappa B through a redox-regulatedand c-Src-dependent mechanism following hypoxia/reoxygen-ation. J Biol Chem 278:2072–2080.

an C, Li Q, Zhang Y, Liu X, Luo M, Abbott D, Zhou W, Engelhardt JF(2004) IkappaBalpha and IkappaBbeta possess injury context-specific functions that uniquely influence hepatic NF-kappaB in-duction and inflammation. J Clin Invest 113:746–755.

ebbraio M, Silverstein RL (2007) CD36: implications in cardiovascu-

lar disease. Int J Biochem Cell Biol 39:2012–2030.

G

G

GG

G

G

H

H

H

H

H

H

H

H

H

H

H

I

K

K

K

K

K

K

K

K

K

L

L

L

L

L

N

N

N

P

P

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–10061004

abriel C, Justicia C, Camins A, Planas AM (1999) Activation ofnuclear factor-kappaB in the rat brain after transient focal ischemia.Brain Res Mol Brain Res 65:61–69.

erondakis S, Grumont R, Gugasyan R, Wong L, Isomura I, Ho W,Banerjee A (2006) Unravelling the complexities of the NF-kappaBsignalling pathway using mouse knockout and transgenic models.Oncogene 25:6781–6799.

ilmore TD (2008) Rel/NF-�B transcription factors. www.nf-kb.org.inis I, Jaiswal R, Klimanis D, Liu J, Greenspon J, Hallenbeck JM

(2002) TNF-alpha-induced tolerance to ischemic injury involvesdifferential control of NF-kappaB transactivation: the role of NF-kappaB association with p300 adaptor. J Cereb Blood Flow Metab22:142–152.

rilli M, Pizzi M, Memo M, Spano P (1996) Neuroprotection by aspirinand sodium salicylate through blockade of NF-�B activation. Sci-ence 274:1383–1385.

uerrini L, Blasi F, Denis-Donini S (1995) Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro. ProcNatl Acad Sci U S A 92:9077–9081.

acker H, Karin M (2006) Regulation and function of IKK and IKK-related kinases. Sci STKE 2006:re13.

allenbeck JM (2002) The many faces of tumor necrosis factor instroke. Nat Med 8:1363–1368.

ayakawa M, Miyashita H, Sakamoto I, Kitagawa M, Tanaka H, Ya-suda H, Karin M, Kikugawa K (2003) Evidence that reactive oxygenspecies do not mediate NF-kappaB activation. EMBO J 22:3356–3366.

ayden MS, Ghosh S (2008) Shared principles in NF-kappaB signal-ing. Cell 132:344–362.

elton 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{al-pha} reduces rather than increases hypoxic-ischemic damage.J Neurosci 25:4099–4107.

errmann 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 neuronaldeath. Nat Med 11:1322–1329.

offmann A, Levchenko A, Scott ML, Baltimore D (2002) The IkappaB-NF-kappaB signaling module: temporal control and selective geneactivation. Science 298:1241–1245.

oward EF, Chen Q, Cheng C, Carroll JE, Hess D (1998) NF-kappa Bis activated and ICAM-1 gene expression is upregulated duringreoxygenation of human brain endothelial cells. Neurosci Lett248:199–203.

ua 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-likereceptor 4 signaling contributes to hippocampal neuronal deathfollowing global cerebral ischemia/reperfusion. J Neuroimmunol190:101–111.

uang CY, Fujimura M, Noshita N, Chang YY, Chan PH (2001) SOD1down-regulates NF-kappaB and c-Myc expression in mice aftertransient focal cerebral ischemia. J Cereb Blood Flow Metab21:163–173.

uang FP, Wang ZQ, Wu DC, Schielke GP, Sun Y, Yang GY (2003)Early NFkappaB activation is inhibited during focal cerebral isch-emia in interleukin-1beta-converting enzyme deficient mice. J Neu-rosci Res 73:698–707.

nta I, Paxian S, Maegele I, Zhang W, Pizzi M, Spano P, Sarnico I,Muhammad S, Herrmann O, Inta D, Baumann B, Liou H-C, SchmidRM, Schwaninger M (2006) Bim and noxa are candidates to me-diate the deleterious effect of the NF-{kappa}B subunit RelA incerebral ischemia. J Neurosci 26:12896–12903.

altschmidt B, Heinrich M, Kaltschmidt C (2002) Stimulus-dependentactivation of NF-kappaB specifies apoptosis or neuroprotection in

cerebellar granule cells. Neuromol Med 2:299–309.

ariko K, Weissman D, Welsh FA (2004) Inhibition of toll-like receptorand cytokine signaling-a unifying theme in ischemic tolerance.J Cereb Blood Flow Metab 24:1288–1304.

arin M, Yamamoto Y, Wang QM (2004) The IKK NF-kappa B system:a treasure trove for drug development. Nat Rev Drug Discov3:17–26.

aushal V, Schlichter LC (2008) Mechanisms of microglia-mediatedneurotoxicity in a new model of the stroke penumbra. J Neurosci28:2221–2230.

han M, Elango C, Ansari MA, Singh I, Singh AK (2007) Caffeic acidphenethyl ester reduces neurovascular inflammation and protectsrat brain following transient focal cerebral ischemia. J Neurochem102:365–377.

hanna S, Roy S, Park HA, Sen CK (2007) Regulation of c-Src activityin glutamate-induced neurodegeneration. J Biol Chem 282:23482–23490.

im JB, Sig Choi J, Yu YM, Nam K, Piao CS, Kim SW, Lee MH, HanPL, Park JS, Lee JK (2006) HMGB1, a novel cytokine-like mediatorlinking acute neuronal death and delayed neuroinflammation in thepostischemic brain. J Neurosci 26:6413–6421.

oponen S, Goldsteins G, Keinanen R, Koistinaho J (2000) Inductionof protein kinase C[delta] subspecies in neurons and microgliaafter transient global brain ischemia. J Cereb Blood Flow Metab20:93–102.

unz A, Abe T, Hochrainer K, Shimamura M, Anrather J, Racchumi G,Zhou P, Iadecola C (2008) Nuclear factor-kappaB activation andpostischemic inflammation are suppressed in CD36-null mice aftermiddle cerebral artery occlusion. J Neurosci 28:1649–1658.

awrence T, Bebien M, Liu GY, Nizet V, Karin M (2005) IKKalpha limitsmacrophage NF-kappaB activation and contributes to the resolu-tion of inflammation. Nature 434:1138–1143.

i Q, Estepa G, Memet S, Israel A, Verma IM (2000) Complete lack ofNF-kappaB activity in IKK1 and IKK2 double-deficient mice: addi-tional defect in neurulation. Genes Dev 14:1729–1733.

i Q, Verma IM (2002) NF-kappaB regulation in the immune system.Nat Rev Immunol 2:725–734.

uedde 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 hepatocytesdoes not sensitize these cells to TNF-induced apoptosis but pro-tects from ischemia/reperfusion-injury. J Clin Invest 115:849–859.

uedde 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 andhepatocellular carcinoma. Cancer Cell 11:119–132.

agashima K, Sasseville VG, Wen D, Bielecki A, Yang H, Simpson C,Grant E, Hepperle M, Harriman G, Jaffee B, Ocain T, Xu Y, FraserCC (2006) Rapid TNFR1-dependent lymphocyte depletion in vivowith a selective chemical inhibitor of IKKbeta. Blood 107:4266–4273.

olan GP, Fujita T, Bhatia K, Huppi C, Liou HC, Scott ML, BaltimoreD (1993) The bcl-3 proto-oncogene encodes a nuclear I kappaB-like molecule that preferentially interacts with NF-kappa B p50and p52 in a phosphorylation-dependent manner. Mol Cell Biol13:3557–3566.

urmi A, Lindsberg PJ, Koistinaho M, Zhang W, Juettler E, Kar-jalainen-Lindsberg ML, Weih F, Frank N, Schwaninger M, Koisti-naho J (2004) Nuclear factor-kappaB contributes to infarction afterpermanent focal ischemia. Stroke 35:987–991.

aul R, Zhang ZG, Eliceiri BP, Jiang Q, Boccia AD, Zhang RL, ChoppM, Cheresh DA (2001) Src deficiency or blockade of Src activity inmice provides cerebral protection following stroke. Nat Med7:222–227.

ereira 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-

P

P

P

P

P

P

P

P

Q

R

R

R

R

R

S

S

S

S

S

S

S

T

T

T

T

T

T

T

V

v

W

W

W

W

W

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–1006 1005

pletely overlapping mechanisms. J Cereb Blood Flow Metab26:218–229.

erkins ND (2006) Post-translational modifications regulating the ac-tivity and function of the nuclear factor kappa B pathway. Onco-gene 25:6717–6730.

erkins ND (2007) Integrating cell-signalling pathways with NF-kap-paB and IKK function. Nat Rev Mol Cell Biol 8:49–62.

izzi M, Goffi F, Boroni F, Benarese M, Perkins SE, Liou HC, SpanoP (2002) Opposing roles for NF-kappa B/Rel factors p65 and c-Relin the modulation of neuron survival elicited by glutamate andinterleukin-1beta. J Biol Chem 277:20717–20723.

izzi M, Sarnico I, Boroni F, Benarese M, Steimberg N, Mazzoleni G,Dietz GP, Bahr M, Liou HC, Spano PF (2005) NF-kappaB factorc-Rel mediates neuroprotection elicited by mGlu5 receptor ago-nists against amyloid beta-peptide toxicity. Cell Death Differ12:761–772.

izzi M, Spano P (2006) Distinct roles of diverse nuclear factor-kappaB complexes in neuropathological mechanisms. Eur J Phar-macol 545:22–28.

olavarapu R, Gongora MC, Yi H, Ranganthan S, Lawrence DA,Strickland D, Yepes M (2007) Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascularunit. Blood 109:3270–3278.

otrovita 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 neu-rodegeneration. J Neurosci 24:8237–8244.

rass K, Scharff A, Ruscher K, Lowl D, Muselmann C, Victorov I,Kapinya K, Dirnagl U, Meisel A (2003) Hypoxia-induced stroketolerance in the mouse is mediated by erythropoietin. Stroke34:1981–1986.

in ZH, Chen RW, Wang Y, Nakai M, Chuang DM, Chase TN (1999)Nuclear factor kappaB nuclear translocation upregulates c-Mycand p53 expression during NMDA receptor-mediated apoptosis inrat striatum. J Neurosci 19:4023–4033.

eith 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.

ius J (2008) NF-�B links innate immunity to the hypoxic responsethrough transcriptional regulation of HIF-1�. Nature, 453:807–811.

osenzweig HL, Minami M, Lessov NS, Coste SC, Stevens SL, Hen-shall DC, Meller R, Simon RP, Stenzel-Poore MP (2007) Endotoxinpreconditioning protects against the cytotoxic effects of TNFalphaafter stroke: a novel role for TNFalpha in LPS-ischemic tolerance.J Cereb Blood Flow Metab 27:1663–1674.

uscher K, Freyer D, Karsch M, Isaev N, Megow D, Sawitzki B, PrillerJ, Dirnagl U, Meisel A (2002) Erythropoietin is a paracrine mediatorof ischemic tolerance in the brain: evidence from an in vitro model.J Neurosci 22:10291–10301.

yseck RP, Novotny J, Bravo R (1995) Characterization of elementsdetermining the dimerization properties of RelB and p50. Mol CellBiol 15:3100–3109.

chneider A, Martin-Villalba A, Weih F, Vogel J, Wirth T, SchwaningerM (1999) NF-�B is activated and promotes cell death in focalcerebral ischemia. Nat Med 5:554–559.

chreck R, Rieber P, Baeuerle PA (1991) Reactive oxygen interme-diates as apparently widely used messengers in the activation ofthe NF-kappa B transcription factor and HIV-1. EMBO J 10:2247–2258.

ong YS, Lee YS, Chan PH (2005) Oxidative stress transiently de-creases the IKK complex (IKKalpha, beta, and gamma), an up-stream component of NF-kappaB signaling, after transient focalcerebral ischemia in mice. J Cereb Blood Flow Metab 25:1301–1311.

tephenson D, Yin T, Smalstig EB, Hsu MA, Panetta J, Little S,

Clemens J (2000) Transcription factor nuclear factor-kappa B is

activated in neurons after focal cerebral ischemia. J Cereb BloodFlow Metab 20:592–603.

tevens SL, Ciesielski TM, Marsh BJ, Yang T, Homen DS, Boule JL,Lessov NS, Simon RP, Stenzel-Poore MP (2008) Toll-like receptor9: a new target of ischemic preconditioning in the brain. J CerebBlood Flow Metab 28:1040–1047.

trnad J, Burke JR (2007) IkappaB kinase inhibitors for treating auto-immune and inflammatory disorders: potential and challenges.Trends Pharmacol Sci 28:142–148.

ugawara T, Fujimura M, Noshita N, Kim GW, Saito A, Hayashi T,Narasimhan P, Maier CM, Chan PH (2004) Neuronal death/sur-vival signaling pathways in cerebral ischemia. NeuroRx 1:17–25.

ang SC, Arumugam TV, Xu X, Cheng A, Mughal MR, Jo DG, LathiaJD, Siler DA, Chigurupati S, Ouyang X, Magnus T, Camandola S,Mattson MP (2007) Pivotal role for neuronal toll-like receptors inischemic brain injury and functional deficits. Proc Natl Acad SciU S A 104:13798–13803.

arabin V, Schwaninger M (2004) The role of NF-kappaB in 6-hy-droxydopamine- and TNFalpha-induced apoptosis of PC12 cells.Naunyn Schmiedebergs Arch Pharmacol 369:563–569.

erai K, Matsuo A, McGeer EG, McGeer PL (1996) Enhancement ofimmunoreactivity for NF-�B in human cerebral infarction. Brain Res739:343–349.

hompson JE, Phillips RJ, Erdjument-Bromage H, Tempst P, Ghosh S(1995) I[kappa]B-[beta] regulates the persistent response in a bi-phasic activation of NF-[kappa]B. Cell 80:573–582.

ortella FC, Williams AJ (2005) A novel inhibitory � kinase (IKK)inhibitor, MLN1145, reduces brain infarction and improves out-come following focal ischemic brain injury in rats. vol. Program No.589.6. 2005 Abstract Viewer/Itinerary Planner Washington, DC:Society for Neuroscience.

otzke 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 DNAbinding. J Biol Chem 281:12645–12654.

rickler WJ, Mayhan WG, Miller DW (2005) Brain microvessel endo-thelial cell responses to tumor necrosis factor-alpha involve anuclear factor kappa B (NF-kappaB) signal transduction pathway.Brain Res 1048:24–31.

ahedi 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 inmalignant infarction of the middle cerebral artery: a pooled analysisof three randomised controlled trials. Lancet Neurol 6:215–222.

an den Tweel ER, Kavelaars A, Lombardi MS, Groenendaal F, MayM, Heijnen CJ, van Bel F (2006) Selective inhibition of nuclearfactor-kappaB activation after hypoxia/ischemia in neonatal rats isnot neuroprotective. Pediatr Res 59:232–236.

ang HK, Park UJ, Kim SY, Lee JH, Kim SU, Gwag BJ, Lee YB (2008)Free radical production in CA1 neurons induces MIP-1{alpha} ex-pression, microglia recruitment, and delayed neuronal death aftertransient forebrain ischemia. J Neurosci 28:1721–1727.

ang Q, Tang XN, Yenari MA (2007) The inflammatory response instroke. J Neuroimmunol 184:53–68.

ang X, Feuerstein GZ, Xu L, Wang H, Schumacher WA, OgletreeML, Taub R, Duan JJ, Decicco CP, Liu RQ (2004) Inhibition oftumor necrosis factor-alpha-converting enzyme by a selective an-tagonist protects brain from focal ischemic injury in rats. Mol Phar-macol 65:890–896.

ang YH, Wang WY, Chang CC, Liou KT, Sung YJ, Liao JF, Chen CF,Chang S, Hou YC, Chou YC, Shen YC (2005) Taxifolin amelioratescerebral ischemia-reperfusion injury in rats through its anti-oxida-tive effect and modulation of NF-kappa B activation. J Biomed Sci1–15.

en 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 1008:147–154.

W

W

W

X

Y

Y

Z

Z

Z

D. A. Ridder and M. Schwaninger / Neuroscience 158 (2009) 995–10061006

hiteside ST, Epinat JC, Rice NR, Israel A (1997) I kappa B epsilon,a novel member of the I kappa B family, controls RelA and cRelNF-kappa B activity. EMBO J 16:1413–1426.

illiams AJ, Hale SL, Moffett JR, Dave JR, Elliott PJ, Adams J,Tortella FC (2003) Delayed treatment with MLN519 reduces infarc-tion and associated neurologic deficit caused by focal ischemicbrain injury in rats via antiinflammatory mechanisms involving nu-clear factor-kappaB activation, gliosis, and leukocyte infiltration.J Cereb Blood Flow Metab 23:75–87.

inkles JA (2008) The TWEAK-Fn14 cytokine-receptor axis: discovery,biology and therapeutic targeting. Nat Rev Drug Discov 7:411–425.

u L, Zhan Y, Wang Y, Feuerstein GZ, Wang X (2002) Recombinantadenoviral expression of dominant negative IkappaBalpha protectsbrain from cerebral ischemic injury. Biochem Biophys Res Com-mun 299:14–17.

in MJ, Yamamoto Y, Gaynor RB (1998) The anti-inflammatory agentsaspirin and salicylate inhibit the activity of I(kappa)B kinase-beta.

Nature 396:77–80.

u G, Rux AH, Ma P, Bdeir K, Sachais BS (2005) Endothelial expres-sion of E-selectin is induced by the platelet-specific chemokineplatelet factor 4 through LRP in an NF-kappaB-dependent manner.Blood 105:3545–3551.

hang W, Potrovita I, Tarabin V, Herrmann O, Beer V, Weih F,Schneider A, Schwaninger M (2005) Neuronal activation of NF-�Bcontributes to cell death in cerebral ischemia. J Cereb Blood FlowMetab 25:30–40.

hang X, Polavarapu R, She H, Mao Z, Yepes M (2007a) Tissue-type plasminogen activator and the low-density lipoprotein re-ceptor-related protein mediate cerebral ischemia-induced nu-clear factor-kappaB pathway activation. Am J Pathol 171:1281–1290.

hang X, Winkles JA, Gongora MC, Polavarapu R, Michaelson JS,Hahm K, Burkly L, Friedman M, Li XJ, Yepes M (2007b) TWEAK-Fn14 pathway inhibition protects the integrity of the neurovascularunit during cerebral ischemia. J Cereb Blood Flow Metab 27:

534–544.

(Accepted 3 July 2008)(Available online 10 July 2008)