Necrotic death of neurons following an excitotoxic insult isprevented by a peptide inhibitor of c-jun N-terminal kinase

Peter G. Arthur,* Graeme P. Matich,*,� Wei Wei Pang,* Dao-Yi Yu� and Marie A. Bogoyevitch*

*School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, Western Australia, Australia

�Centre for Ophthalmology and Visual Science, University of Western Australia, Crawley, Western Australia, Australia

Abstract

Peptide inhibitors of c-Jun N-terminal kinase (JNK) have been

shown to potently protect against cerebral ischemia. The

protective effect has been ascribed to prevention of apoptosis,

but cell death following cerebral ischemia is a consequence of

both apoptotic and necrotic cell death. We evaluated whether

a peptide inhibitor (TAT-TIJIP) of JNK could prevent necrotic

cell death in an in vitro model of excitotoxic neuronal death.

We find that TAT-TIJIP effectively prevented cell death by

interfering with several processes which have been identified

as leading to cell death by necrosis. In particular, reactive

oxygen species production was reduced, as indicated by an

88% decrease in the rate of dihydroethidium fluorescence in

the presence of TAT-TIJIP. Furthermore, TAT-TIJIP attenu-

ated the increase in cytosolic calcium following the excitotoxic

insult. The potent neuroprotective properties of JNK peptide

inhibitors likely reflects their abilities to prevent cell death by

necrosis as well as apoptosis.

Keywords: c-jun N-terminal kinase, excitotoxicity, necrosis,

neurons, peptide inhibitors, reactive oxygen species.

J. Neurochem. (2007) 102, 65–76.

Active c- jun N-terminal kinase (JNK) has been implicated inthe development of apoptosis (or programed cell death) ofneurons, initially in model culture systems such as PC12cells but also in vivo following JNK gene knockout studies(Xia et al. 1995; Yang et al. 1997). Recently, small peptideinhibitors directed against c- jun N-terminal kinases (JNKs)and derived from the scaffold protein JNK Interacting Protein(JIP1) have been developed. A 20 amino acid sequence (JNKBinding Domain; JBD20) and a shorter 11 amino acidsequence (Truncated Inhibitor of JNK Interacting Protein;TIJIP) block JNK access to substrates by a direct competitivemechanism (Barr et al. 2002, 2004; Borsello et al. 2003).These inhibitors are highly specific. When tested in assays of40 different protein kinases, only the JNKs and theirupstream activators mitogen activated protein kinase kinases4 and 7 were affected (Borsello et al. 2003). Cell penetratingpeptides capable of accessing intracellular JNKs have beenproduced by linking these peptide inhibitors to the 10 aminoacid Tat cell transporter sequence (TAT). The D-amino acidform of JBD20 linked to TAT has been termed D-JNKI-1(Borsello et al. 2003), and TIJIP linked to TAT is known asTAT-TIJIP (Kendrick et al. 2004).

D-JNKI-1 is a potent neuroprotectant against cerebralischemia, with a remarkably long therapeutic window of 6 h,

an important feature for any putative therapeutic agent(Gladstone et al. 2002). Borsello et al. (2003) suggested thatJNK inhibition was protective by preventing apoptosis.However, the protective effects of JNK peptide inhibitorsmay have extended beyond apoptosis, because considerablecell death by necrosis has been described in relatedexperimental models (Liu et al. 2004; Muller et al. 2004;

Received July 5, 2006; revised manuscript received December 21, 2006;accepted December 27, 2006.Address correspondence and reprint requests to Dr Peter G Arthur,

Biochemistry & Molecular Biology (M310), School of Biomedical,Biomolecular and Chemical Sciences, University of Western Australia,35 Stirling Highway, Crawley, WA 6009, Australia.E-mail: parthur@cyllene.uwa.edu.auAbbreviations used: au, arbitrary units; DHE, dihydroethidium; em,

emission; ex, excitation; FCCP, carbonyl cyanide p-(triflurometh-oxy)phenyl-hydrazone; GST, glutathione-S-transferase; Indo-1,1-{2-Amino-5-(6-carboxyindol-2-yl)phenoxy}-2-(2¢-amino-5¢-meth-ylphenoxy)ethane-N,N,N¢,N¢-tetraacetic acid pentaacetoxymethylester; JC-1, 5,5¢,6,6¢-Tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolyl-carbocyanine Iodide; JNK binding domain, JBD20; JNK, c-junN-terminal kinase; LDH, lactate dehydrogenase; NBS, neurobasalsaline; PI, propidium iodide; ROS, reactive oxygen species; TAT, Tatcell transporter sequence; TIJIP, Truncated Inhibitor of JNK Inter-acting Protein.

Journal of Neurochemistry, 2007, 102, 65–76 doi:10.1111/j.1471-4159.2007.04618.x

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 65–76 65

Unal-Cevik et al. 2004). There is also evidence indicatingthat inhibiting JNK activity protected livers from necroticcell death following ischemia reperfusion injury (Ueharaet al. 2004, 2005).

Neuronal death by necrosis is observed following a widerange of pathological insults to the brain, including cerebralischemia (Yamashima et al. 2003; Muller et al. 2004; Unal-Cevik et al. 2004; Rizk et al. 2005). The most prominentfeature of necrosis is swelling (oncosis) of the cell and itsorganelles followed by loss of cell membrane integrity.Necrosis is often considered irreversible once initiatedbecause the breakdown appears disorganized and lacks thecharacteristic patterns of the various forms of apoptosis.However, recent evidence suggests that there may be someconsistency in the early phases of necrosis, raising thepossibility that following an initiating event, it may bepossible to block downstream processes and prevent necroticcell death (Formigli et al. 2000).

We have therefore evaluated the effectiveness of a peptideinhibitor of JNK, in this case TAT-TIJIP, in preventingnecrotic cell death in an in vitro model of excitotoxicneuronal death. We found that TAT-TIJIP was effective inpreventing necrosis following exposure to glutamate. Fur-ther, TAT-TIJIP ameliorated the increase in superoxideproduction and cytosolic calcium which have been implica-ted in causing cell death by necrosis. These results implicateactive JNK in cell death by necrosis and indicate thatinhibiting JNK may be an effective strategy in preventingboth necrosis and apoptosis.

Materials and methods

Glutamate excitotoxicity and oxygen/glucose deprivation

experimental models

For the glutamate excitotoxicity model, primary cultures of rat

cortical neurons were established as described previously except

neurons were seeded onto polylysine-coated 35 mm plastic Petri

dishes (Munns et al. 2003). On day 12–13, media was removed,

replaced with 1 mL of neurobasal saline (NBS)-glucose (5.4 mmol/

L KCl, 0.8 mmol/L MgCl2, 90 mmol/L NaCl, 0.9 mmol/L

NaH2PO4, 20 mmol/L HEPES, 1.8 mmol/L CaCl2, 50 IU penicillin,

50 lg/mL streptomycin and 10 mmol/L gluocse at pH 7.4 and

235 mOsm/L) at 37�C for 15 min. After 15 min, 100 lmol/L

glutamate was added to experimental dishes and the equivalent

volume of vehicle added to control dishes. Upon removal of

medium, the dishes were washed twice with 1 mL of NBS-glucose

and incubated in a 5% CO2 incubator at 37�C in 1 mL of NB2

(Neurobasal medium; Invitrogen, Mt Waverley, Australia, supple-

mented with 2% B27 supplement; Invitrogen, 5 mmol/L creatine,

0.5 mmol/L glutamine, 25 IU penicillin and 25 lg/mL streptomy-

cin) and 1 mL of NBS-glucose for up to 24 h. In this model, cell

death was primarily necrotic. By 6 h after the excitotoxic insult

there was a significant increase (2–26%) in lactate dehydrogenase

(LDH) release relative to the initial measurement at 1 h (Fig. 1a). In

contrast, when neurons were treated with ceramide to induce

apoptosis (Movsesyan et al. 2002; Stoica et al. 2005), a significant

increase in LDH release was not evident until 24 h after glutamate

treatment (Fig. 1a). Furthermore, 24 h after transient exposure to

glutamate there was no change in the activity of caspase 3,

consistent with cell death by necrosis, whereas exposure to 50 lmol/

L ceramide produced a 4.5-fold increase in activity (Fig. 1b).

Propidium iodide (PI) uptake was also measured to provide an

alternative assessment of cell death. By 6 h after transient exposure

to 100 lmol/L glutamate, PI uptake was 2.8-fold higher than in

untreated controls (Fig. 1c). In contrast, by 6 h after treatment with

50 lmol/L ceramide there was no significant increase in PI uptake

(0.89-fold) relative to untreated neurons. Furthermore, few neurons

treated with glutamate stained positive for annexin V relative to

neurons treated with ceramide (Fig. 2c). Our observations are

consistent with previous work, in comparable experimental models,

showing exposure of neurons to 100 lmol/L glutamate causes cell

death by necrosis rather than apoptosis (Ankarcrona et al. 1995;Cheung et al. 1998; Yu et al. 2003).

Details of the oxygen/glucose deprivation neuronal death model

have been described (Arthur et al. 2004). On day 12, media was

0

10

20

30

40

50(a)

(b) (c)

1 2 6 24Time (h)

LDH

(%

rel

ease

)C

aspa

se 3

act

ivity

(re

lativ

e)

Pl u

ptak

e (r

elat

ive)

U G C

U G C U G C

U G C U G C U G C

0

100

200

300

400

500

0

100

200

300

400

Fig. 1 Cell death following transient glutamate insult. Panel (a) Glu-

tamate treatment caused LDH leakage 6 h after treatment. Petri dishes

were untreated (U) or treated with 100 lmol/L glutamate (G) for 15 min

or 50 lmol/L ceramide (C) for 24 h. Bars indicate significant difference

(p < 0.05) between times for the same treatment (n = 4). Panel (b)

Glutamate treatment did not increase caspase 3 activity at 24 h. Ca-

spase 3 activities are expressed relative to the caspase 3 activity of

untreated neurons (U = 100). Bar indicates significant difference

(p < 0.05) relative to untreated neurons (n = 3). Panel (c) Glutamate

treatment did increase PI uptake at 6 h. PI uptake is expressed relative

to uptake in untreated neurons (U = 100). Bar indicates significant dif-

ference (p < 0.05) relative to untreated neurons (n = 3).

66 P. G. Arthur et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 65–76� 2007 The Authors

removed and replaced with 2 mL NBS-lactate (as per NBS-glucose

with 2 mmol/L lactate replacing glucose) and gassed with humidified

100%nitrogen for 35–45 min at 37�C. Following gassing, 1.75 mLof

media was removed and 250 lL of NBN2 (Neurobasal medium;

Invitrogen, supplemented with 2% N2 supplement; Invitrogen,

5 mmol/L creatine, 0.5 mmol/L glutamine, 25 IU penicillin and

25 lg/mL streptomycin) was added with Petri dishes placed in a 5%

CO2 incubator at 37�C. In this model, cell death was primarily

apoptotic. Caspase 3 activity was elevated 2.7-fold, fragmented nuclei

were evident, neurons stained positive for annexin V without uptake

0

10

20

30

40

50

60(a)

(c)

(d) (e)

(b)

– + – +– – + +

)esaeler%(

HDL

GlutamateTAT−TIJIP

0

5

10

15

20

25

– + – +– – + +

Pl u

ptak

e (a

u)

GlutamateTAT−TIJIP

i

v

ii iii

iv vi vii

0

10

20

30

40

50

)esaeler%(

HDL

OGDTAT-TIJIP

––

+–

++

–+

0

50

100

150

200

250

OGDTAT-TIJIP

––

+–

++

–+

)evitaler(yt ivi tca

3e saps a

C

Fig. 2 TAT-TIJIP prevents necrotic cell death following transient glu-

tamate insult and apoptotic cell death after oxygen/glucose deprivation.

Panel (a) TAT-TIJIP prevents LDH leakage after glutamate treatment.

TAT-TIJIP (2 lmol/L) was incubated with neurons for 15 min prior to the

addition of glutamate to allow uptake by neurons. After treating neurons

with glutamate for 15 min, media in all dishes was replaced with media

free of TAT-TIJIP and glutamate. Cell death was assessed after 24 h by

LDH release (n = 4). Bar indicates significant difference (p < 0.05) re-

lative to neurons treated with glutamate. Panel (b) TAT-TIJIP prevents

PI uptake after glutamate treatment. The previous experiment was re-

peated except PI uptake was assessed (n = 4). Bar indicates significant

difference (p < 0.05) relative to neurons treated with glutamate. Panel

(c) Representative photographs showing phase contrast (i–iii) and

fluorescence microscopy (iv–vii) with staining for PI (red in the original

colour image) uptake and annexin v (green in the original colour image).

Neurons staining red are considered to be necrotic (examples indicated

by fi ), whereas neurons staining only green are considered to be

apoptotic (examples indicated by ‹ ). Neurons are untreated (i, iv),

treated with glutamate (ii, v), treated with glutamate in the presence of

TAT-TIJIP (iii, vi) or treated with ceramide (vii). Panel (d) Cell death by

apoptosis was prevented by TAT-TIJP. Petri dishes were assessed for

LDH leakage 24 h after being subjected to oxygen/glucose deprivation.

TAT-TIJIP (2 lmol/L) was added during and for 1 h post-oxygen/glu-

cose deprivation (OGD). Bar indicates significant difference (p < 0.05)

relative to oxygen/glucose deprivation treated neurons (n = 3). Panel

(e) TAT-TIJIP (2 lmol/L) application during and for 1 h post-oxygen/

glucose deprivation, prevented caspase 3 activation 24 h after being

subjected to oxygen/glucose deprivation (OGD). Caspase 3 activities

are expressed relative to untreated neurons (no oxygen/glucose depri-

vation, no TAT-TIJIP). Bar indicates significant difference (p < 0.05)

relative to oxygen/glucose deprivation treated neurons (n = 3).

A peptide JNK inhibitor prevents necrotic death 67

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 65–76

of the nuclear stain PI and the caspase inhibitors VAD-fmk and

IETD-fmk prevented cell death (Arthur et al. 2004).

Peptide JNK inhibitor

The inhibitor of JNK, TIJIP and the cell-permeable inhibitor of JNK,

TAT-TIJIP, were synthesized by Auspep (Parkville, Australia).

Peptide purity was at least 80% as determined by LC-mass

spectrometry. The sequence of TIJIP was RPKRPTTLNLF, and

that of TAT-TIJIP was GRKKRRQRRRPPRPKRPTTLNLF.

JNK activity assays

Assays to determine the kinetics of JNK isoform inhibition by TIJIP

were performed by assessing the activities of JNK1, JNK2 or JNK3

towards a glutathione-S-transferase (GST)-c-jun (1-135) substrate in

the presence of [c-32P]ATP and a range of TIJIP concentrations at

30�C. Each reaction was carried out in 30 lL volumes containing

the final concentrations of the following: 20 mmol/L HEPES pH

7.6, 20 mmol/L MgCl2, 20 mmol/L b-glycerophosphate, 500 lmol/

L dithiothreitol, 100 lmol/L sodium orthovanadate, 2.9 nmol/L

active JNK1, JNK2 or JNK3 (Upstate Biotechnology Inc.,

Charlottesville, VA, USA) and 0.7–31.5 lmol/L GST-c-jun (1-

135). The reactions were pre-treated with TIJIP inhibitor (0–4 lmol/

L) in the absence of ATP for 10 min at 30�C. Reactions were

initiated with the addition of 2 lCi [c-32P]ATP (3000 Ci/mmol) and

0.5–100 lmol/L ATP (final concentration) and incubated for 15 min

at 30�C. Where the concentration of GST-c-jun (1-135) was varied,

the ATP concentration was held constant at 50 lmol/L. Where the

ATP concentration was varied, the GST-c-jun(1-135) concentrationwas held constant at 8 lmol/L. Under these conditions, less than

10% of substrate was converted to product. Reactions were stopped

with 15 lL of 3X SDS Sample Buffer and then the phosphorylated

product was separated by SDS-PAGE and quantitated by Cerenkov

counting. Analysis of kinetic data for the inhibition of GST-c-jun(1-135) phosphorylation was performed using the non-linear least

squares regression analysis facility of the ScientistTM program as

previously described (Barr et al. 2004). From these fits, estimates of

Ki and Km and standard deviations of these estimates were

obtained.

JNK immunoblots

Prior to, or after treatment of neurons with oxygen/glucose

deprivation or glutamate, neurons were lysed in a cold stress lysis

buffer (20 mmol/L HEPES pH 7.7, 2.5 mmol/L MgCl2, 0.1 mmol/L

EDTA, 20 mmol/L b-glycerophosphate, 100 mmol/L NaCl,

0.5 mmol/L dithiothreitol, 0.1 mmol/L vanadate, 0.05% v/v Tri-

ton-X, 20 lg/mL leupeptin, 100 lg/ mL phenylmethylsulfonyl

fluoride and 20 lg/mL aprotinin). Total protein concentration in

the supernatant was measured and adjusted prior to being loaded

onto gels. The phosphorylation levels of JNKs were assayed by

immunoblotting cell lysates with anti-phospho-JNK antibodies. A

parallel immunoblot was also performed using anti-total JNK

antibodies to ensure equal loading and to assess the effect of the

treatment on total JNK.

Enzyme assays for lactate dehydrogenase and caspase 3

For enzyme assays, media was removed and the dishes were washed

once with 1 mL of NBS-glucose. Extraction buffer (400 lL of

50 mmol/L Tris–HCl, 0.5 mmol/L EDTA, 20% glycerol, pH 7) was

added to the dish and neurons were scraped into a 2 mL microfuge

tube, sonicated twice and then centrifuged at 1000 g for 5 min.

Supernatant and media samples were assayed for LDH activity or

for caspase 3 activity after minimal storage at 4�C or )80�Crespectively. LDH activity was assessed in media samples and cell

extracts by measuring the rate of change in absorbance at 340 nm

after mixing 50 lL of assay reagent (80 mmol/L Imidazole pH 7.1,

500 lmol/L NADH, 1.5 mmol/L pyruvate, 0.07% bovine serum

albumin) with 100 lL of sample (diluted as required). LDH release

was expressed relative to total LDH activity (LDH activity of the

supernatant(s) plus LDH activity of the cell extract). Caspase 3

activity and protein content was assessed as previously described

with caspase 3 activity expressed relative to protein (Arthur et al.2004).

Fluorescent assays

The fluorescence of neuronal cultures containing dihydroethidium

(DHE), 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolylcarbo-

cyanine Iodide (JC-1), PI or 1-{2-Amino-5-(6-carboxyindol-2-yl)

phenoxy}-2-(2¢-amino-5¢-methylphenoxy)ethane-N,N,N¢,N¢-tetra-acetic acid pentaacetoxymethyl ester (Indo-1) were read at 1 min

intervals on a fluorescent plate reader (Fluostar Optima; BMG

LABTECH, Offenburg, Germany) at 37�C. The fluorescent signal

was corrected for background by including a neuronal culture without

the relevant dye. DHE (5 lmol/L) fluorescence was read (ex 544 nm,

bandwidth 15 nm; em 590 nm, bandwidth 15 nm) after pre-incuba-

tion for 35 min at 37�C. JC-1 (640 nmol/L) fluorescence (ex 485 nm,

bandwidth 20 nm; em 1, 520 nm, bandwidth 15 nm; em 2, 590 nm,

bandwidth 15 nm) was read after pre-incubation for 105 min at 37�C.PI (5 lmol/L) fluorescence was read (ex 344 nm, bandwidth 20 nm;

em 590 nm, bandwidth 15 nm) for 25 min in NBS-glucose, 6 h after

glutamate exposure. Dishes pre-incubated with Indo-1 (2 lmol/L) for

45 min at 37�C were washed twice with 1 mL NBS-glucose and then

fluorescence (em355 nm, bandwidth 20 nm; ex 1 405 nm, bandwidth

15 nm; ex 2 460 nm, bandwidth 25 nm) was read following the

addition of 1 mL NBS-glucose. Fluorescence results were calculated

as five-point moving averages after background subtraction for DHE

andPI and as five-pointmoving averages after background subtraction

of the 590 nm/520 nm ratio for JC-1 and 405 nm/460 nm ratio for

Indo-1.

Oxygen consumption

Neurons were seeded onto polylysine-coated 25 mm diameter glass

coverslips, placed in 35 mm plastic dishes at a density of 1.5 million

neurons per dish and cultured as described earlier. To measure

oxygen consumption, coverslips were transferred to a Bioptechs

FCS2 perfusion chamber (Bioptechs Inc., Butler, PA, USA) filled

with NBS-glucose and the decrease in oxygen concentration after

passage through the chamber was measured. Neurons were grown

on glass to prevent underestimating oxygen consumption caused by

oxygen leakage from plastic Petri dishes (Munns and Arthur 2002).

Oxygen consumption was calculated from the change in oxygen

concentration after passage through the chamber multiplied by the

flow rate.

Statistics

All data points are expressed as mean ± SEM from measurements

made on neuronal cultures taken from (n) culture preparations

68 P. G. Arthur et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 65–76� 2007 The Authors

prepared from different animals. Experiments were independent in

that separate culture preparations were not from the same primary

preparation. Statistical difference was determined using Student’s

t-test or ANOVA with Tukey post hoc testing. Data were considered

significantly different when p < 0.05.

Results

JNK isoforms are inhibited by TIJIP and JNK is

phosphorylated in the cultured neuronal models

We performed kinetic assays using activated JNK1, JNK2 orJNK3 where either GST-c-jun(1-135) or ATP concentrationswere varied at a fixed concentration of the other substrate in thepresence or absence of TIJIP. Data obtained from experimentsperformed at fixed ATP concentration, where GST-c-jun (1-135) concentrations were varied in the presence of increasingconcentrations of TIJIP, were indicative of competitiveinhibition for all JNK isoforms. All three JNK isoforms wereinhibited by TIJIP, with the following calculated kineticparameters: JNK1, Km (jun) 3.8 ± 0.6 lmol/L, Ki (TIJIP)0.73 ± 0.12 lmol/L; JNK2,Km (jun) 0.97 ± 0.33 lmol/L, Ki(TIJIP) 0.50 ± 0.18 lmol/L; JNK3,Km (jun) 2.4 ± 0.6 lmol/L, Ki (TIJIP) 0.9 ± 0.2 lmol/L (n = 3).

A representative autoradiograph showing the inhibition ofthe activity of JNK3 towards c-jun is shown in Fig. 3. Thisshows that TAT-TIJIP when included at a final concentrationof 2 lmol/L is an effective inhibitor of JNK3 activity.Therefore, TIJIP effectively inhibits all 3 JNK isoforms,including JNK3 that has been previously implicated as acritical mediator of cell death following stroke (Kuan et al.2003).

To assess JNK activity in cultured neurons, immunoblotsof the active phosphorylated forms of JNK were per-formed. JNK phosphorylation was evident following aninsult with oxygen/glucose deprivation or an excitotoxicinsult with glutamate (Fig. 3). Unstressed cells also showhigh basal levels of JNK activity which is consistent withobservations showing that neuronal tissue is characterizedby high levels of constitutively active JNK (Bjorkblomet al. 2005).

The peptide inhibitor TAT-TIJIP prevents cell death

We examined the effectiveness of TAT-TIJIP (2 lmol/L) inpreventing necrotic cell death caused by transient exposure to100 lmol/L glutamate for 15 min. TAT-TIJIP prevented90 ± 1% (n = 4) of the release of LDH measured in theabsence of TAT-TIJIP (Fig. 2a). The protective effect of TAT-TIJIP was confirmed in a separate series of experimentswhere TAT-TIJIP prevented 93 ± 3% (n = 4) of the uptake ofPI measured in the absence of TAT-TIJIP (Fig. 2b). Theprotective effect of TAT-TIJIP was corroborated by phasecontrast (at 24 h) and fluorescence microscopy (at 6 h) wherenumerous intact neurons with processes were evident after

treatment with glutamate (Fig. 2c-iii and c-vi), in contrast toneurons treated with glutamate only (Fig. 2c-ii, c-v). Overall,these observations indicate TAT-TIJIP prevents necrotic celldeath caused by glutamate exposure.

Cell death by necrosis is not considered to involvetranscriptionally mediated processes (Niquet et al. 2003).However, JNK is known to phosphorylate transcriptionfactors, so alterations in the synthesis of various proteinscould account for the protective effect of TAT-TIJIP. To testthis possibility we used actinomycin D to block RNAsynthesis and then tested for the protective effect of TAT-TIJIP. Actinomycin D did not affect cell viability of controlcells or the magnitude of cell death following glutamateaddition, yet TAT-TIJIP continued to be protective in thepresence of actinomycin D (Fig. 4).

The JNK inhibitor peptide D-JNKI-1 has previously beenshown to prevent apoptosis in cultured neurons following anexcitotoxic insult (Levraut et al. 2003). As described in theintroduction, the amino acid sequence of TAT-TIJIP is

Control TAT-TIJIP

P-cJun

1 2 3 4 5 6

Control A Control B Glutamate

0 min 30 min 60 min

54 kDa

46 kDa

54 kDa

46 kDa

(a)

(b)

(c)

Fig. 3 JNK inhibition by TIJIP and JNK phosphorylation in neuronal

cultures. Panel (a) Example of JNK inhibition by TIJIP. JNK3 activity

towards the transcription protein c-jun (lanes 1–3) was inhibited in the

presence of 2 lmol/L TIJIP (lanes 4–6). JNK activity was measured by

incorporation of [c-32P]ATP and replicate experiments are shown in

lanes 1–3 and lanes 4–6. Panel (b) JNK phosphorylation, as assessed

by immunoblots, was evident prior to (lane 1, 0 min) and following

(lane 2, 30 min and lane 3, 60 min) oxygen/glucose deprivation. The

immunoblot is representative of three separate experiments. Total

JNK, as assessed by immunoblot, was stable (data not shown). Panel

(c) JNK phosphorylation, as assessed by immunoblots, was evident in

the absence (lane 1, Control A at 0 min; lane 2, Control B at 15 min)

and presence of 100 lmol/L glutamate (lane 3, 15 min after glutamate

addition). The immunoblot is representative of three separate experi-

ments. The level of total JNK, as assessed by immunoblot, was con-

stant (data not shown).

A peptide JNK inhibitor prevents necrotic death 69

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 65–76

comparable to the longer D-amino acid peptide D-JNKI-1. Totest if TAT-TIJIP was also capable of preventing apoptosiswe used an experimental model in which oxygen/glucosedeprivation causes cell death predominantly by apoptosis(Arthur et al. 2004). The presence of 2 lmol/L TAT-TIJIPprevented 76% (±1%, n = 3) of the release of LDH measuredin the absence of TAT-TIJIP (Fig. 2d). Additionally, activa-tion of caspase 3 activity in this model was prevented by thepresence of TAT-TIJIP (Fig. 2e). These observations areconsistent with previous work showing that the inhibition ofJNK prevents apoptotic cell death (Putcha et al. 2003;Okuno et al. 2004; Guan et al. 2005).

TAT-TIJIP decreases ROS generation by mitochondria

TAT-TIJIP was ineffective in preventing necrotic death if itwas added after glutamate exposure (data not shown)indicating TAT-TIJIP could be acting to protect neurons byblocking the events directly associated with glutamateexposure. There is considerable evidence for an involvementof elevated intracellular calcium and ROS in neuronal celldeath following an excitotoxic insult (Cazevieille et al. 1993;Dutrait et al. 1995; Reynolds and Hastings 1995). Therefore,TAT-TIJIP may have prevented necrotic death by attenuatingthe increase in calcium and/or ROS associated with glutam-ate exposure.

Before testing this concept, we first confirmed that therewas a significant increase in ROS levels as measured by theincrease in fluorescence caused by the oxidation of DHEfollowing treatment of neurons with glutamate. DHE isoxidized by superoxide to produce a fluorescent compound

and has previously been used to indirectly monitor increasesin ROS in neuronal cultures (Kim-Han et al. 2001; Okunoet al. 2004; Tezel and Yang 2004). Following the addition ofglutamate, the rate of increase in DHE fluorescence was 7.2-fold higher (Fig. 5). This may underestimate the increase inROS because DHE does not respond proportionately toincreases in ROS (Benov et al. 1998). In the presence of2 lmol/L TAT-TIJIP, the rate of increase in DHE fluores-cence following the addition of glutamate was significantlyattenuated (1.7-fold higher, Fig. 5b).

Mitochondria are a possible source of the increase in ROSwhen neurons are exposed to glutamate. Increased mito-chondrial calcium initiated by glutamate exposure has beenlinked to the increase in ROS generation by mitochondria(Dugan et al. 1995; Reynolds and Hastings 1995; Votyakova

Glutamate TAT–TIJIP

Actinomycin

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+––

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)esaeler%(

HDL

Fig. 4 TAT-TIJIP protected neuronal viability in the presence of ac-

tinomycin D. Neurons were exposed to 100 lmol/L glutamate for

15 min in the presence and absence of 1 lmol/L actinomycin D and

cell death was assessed by LDH release after 24 h (n = 3). Bars

indicate significant difference (p < 0.05) relative to neurons treated

with glutamate without TAT-TIJIP.

0

2

4

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8

evitaler(ecnecseroulf

EH

D)

GlutamateTAT-TIJIP

––

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++

-1000

0

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2000

3000

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6000

5 10 15 20 25 30Time (min)

)ua(ecnecseroulf

EH

D

(a)

(b)

Fig. 5 TAT-TIJIP prevents an increase in the rate of DHE fluores-

cence following the addition of glutamate. Panel (a) Representative

example showing DHE fluorescence of neuronal cultures following

addition of 100 lmol/L glutamate (arrow) at 15 min to cultures in the

absence (d) and presence of TAT-TIJIP (s). Glutamate free neuronal

cultures served as controls in the absence (+) and presence of TAT-

TIJIP (·). Where TAT-TIJIP was added, neuronal cultures were pre-

incubated with 2 lmol/L TAT-TIJIP for 15 min (time = 0). Linear

regression analysis over 20–30 min was used to calculate rate of in-

crease in DHE signal (shaded region). Panel (b) Rates of increase in

DHE signal are expressed relative (=1) to rates of increase for neur-

onal cultures not treated with glutamate or TAT-TIJIP. Bar indicates

significant difference relative (p < 0.05) to neurons treated with glu-

tamate (n = 4).

70 P. G. Arthur et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 65–76� 2007 The Authors

and Reynolds 2005). Since the uptake of calcium by themitochondria is dependent on mitochondrial membranepotential, it is possible to reduce mitochondrial calciumuptake by dissipating the membrane potential. Mitochondrialmembrane potential was dissipated with carbonyl cyanidep-(trifluromethoxy)phenyl-hydrazone (FCCP), which uncou-ples mitochondria, and myxothiazol, which inhibits complexI and complex III of the electron transport chain. We alsoincluded oligomycin in these experiments to prevent dissi-pation of cytosolic ATP by reversal of the ATP synthasepump (Budd and Nicholls 1996a,b). In the presence of FCCP,or myxothiazol, the rate of increase in DHE fluoresence

following glutamate addition was significantly attenuated(Fig. 6). Calcium uptake by mitochondria can also beinhibited with ruthenium red (Reed and Bygrave 1974;Trollinger et al. 2000). In the presence of ruthenium red therate of increase in DHE fluorescence following glutamateaddition was also significantly attenuated (Fig. 6).

Taken together, our observations indicate TAT-TIJIP likelyprevents an increase in ROS generation by mitochondriafollowing the addition of glutamate. Preventing ROSgeneration is likely to prevent necrotic cell death. Consistentwith the concept that ROS were contributors to cell death inthis model the antioxidants glutathione and tocopherolreduced LDH release by 36% and 62% relative to neuronstreated with glutamate (Fig. 7). These observations areconsistent with previous work implicating ROS in necroticcell death (Ali et al. 2001; Yaglom et al. 2003).

The peptide inhibitor TAT-TIJIP attenuates the change

in cytosolic calcium following glutamate addition

Increased intracellular calcium following glutamate exposurehas also been implicated in neuronal cell death, so TAT-TIJIPcould exert its protective effect by attenuating the increase incalcium (Castilho et al. 1998; Nicholls et al. 1999). Toexamine this possibility, we first confirmed there was anincrease in intracellular calcium levels, as measured byIndo1, following treatment of neurons with glutamate in our

0

2

4

6

8

10

12

Glutamate – + +Rot

+FCCP

+Ruth

)evitaler(ecnecseroulf

EH

D

-1000

0

1000

2000

3000

4000

5000(a)

(b)

5 10 15 20 25 30Time (min)

ua(ecnecseroulf

EH

D)

Fig. 6 The increase in superoxide following glutamate addition is

likely mitochondrial in origin. Panel (a) shows a representative

example of the increase in DHE fluorescence in arbitrary units (au)

following the addition of glutamate (arrow). Traces show neurons

untreated (+) or treated with glutamate (d) at 15 min (arrow). At 0 min

the mitochondrial specific inhibitor rotenone (5 lmol/L, h) or the mit-

ochondrial uncoupler FCCP (1.25 lmol/L, s) were used to disrupt

mitochondrial function in the presence of 2.5 lmol/L oligomycin. At

0 min, 10 lmol/L Ruthenium Red (·) was used to prevent calcium

uptake into the mitochondria. Linear regression analysis over 20–

30 min was used to calculate the rate of increase in DHE signal

(shaded region). Panel (b) shows rates of increase in DHE signal

expressed relative (=1) to rates of increase for neuronal cultures not

treated with glutamate. Neurons were treated with rotenone (Rot),

FCCP and Ruthenium Red (Ruth) as described. Bars indicate signi-

ficant difference (p < 0.05) from neurons treated with glutamate

(n = 4).

0

25

50

75

Glutamate – + +GSH

+Toc.

)esaeler%(

HDL

Fig. 7 Glutathione and tocopherol attenuate LDH release in neurons

treated with glutamate. Neurons treated with glutathione (GSH) were

pre-incubated with 5 mmol/L oxidized glutathione overnight, and then

pre-incubated with 5 mmol/L GSH for 15 min prior to 100 lmol/L

glutamate exposure. After 15 min, glutamate was removed and the

GSH concentration was maintained. Neurons treated with tocopherol

(Toc.) were pre-incubated with 50 lmol/L tocopherol for 3 h prior to

100 lmol/L glutamate exposure. After 15 min, glutamate was re-

moved and the tocopherol concentration was maintained. LDH release

was measured 24 h post-glutamate insult. Bars indicate significant

difference (p < 0.05) relative to neurons treated with glutamate only

(n = 4).

A peptide JNK inhibitor prevents necrotic death 71

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 65–76

experimental model (Fig. 8). When glutamate was added inthe presence of TAT-TIJIP there was a significant attenuationin intracellular calcium concentrations (Fig. 8).

The significant attenuation of the increase in intracellularcalcium caused by TAT-TIJIP could be a result of reducedcalcium influx into the cytosol, increased calcium removalor calcium buffering by the mitochondria. To evaluate the roleof mitochondria in this process we used ruthenium red toblock calcium uptake into the mitochondria (Fig. 9). In thepresence of ruthenium red there was still a marked increase incysotolic calcium concentration following the addition ofglutamate, which was not affected by the presence of TAT-TIJIP (Fig. 9). Since TAT-TIJIP was no longer effective, thisobservation indicates mitochondria are a likely target for TAT-TIJIP. Ruthenium red has also been shown to inhibit calciumrelease from the endoplasmic reticulum (Kessel et al. 2005).Consequently, increased calcium accumulation by endoplas-

mic reticulum could have been responsible for the attenuatedincrease in calcium caused by TAT-TIJIP. This was tested byblocking calcium uptake into the endoplasmic reticulum withthapsigargin (Paschen et al. 1996). In the presence ofthapsigargin, the increase in cytosolic calcium was partiallyattenuated following the addition of glutamate (Fig. 9).Nevertheless, TAT-TIJIP was still effective in substantiallyattenuating the increase in cytosolic calcium following theaddition of glutamate in the presence of thapsigargin. Takentogether these observations are consistent with TAT-TIJIPacting to maintain calcium buffering by the mitochondria.

TAT-TIJIP maintains mitochondria membrane potential

and oxygen consumption

A combination of oxidative stress and calcium overload hasbeen implicated in causing mitochondrial dysfunction (Nich-olls 2004). Mitochondrial dysfunction, in turn, has been

-0.1

0

0.1

0.2

0.3

0.4

0.5(a)

(b)

5 10 15 20 25 30

Time (min)

)oitar(1-

OD

NI

0

0.1

0.2

0.3

0.4

0.5

GlutamateTAT-TIJIP

––

+–

++

)oitar(1-

OD

NI

Fig. 8 TAT-TIJIP attenuates the increase in calcium caused by

treatment with glutamate. Panel (a) shows a representative example

of Indo-1 fluorescence in neuronal cultures following addition of

100 lmol/L glutamate (arrow) at 15 min to cultures in the absence (d)

and presence of TAT-TIJIP (s). Glutamate free neuronal cultures

served as controls (+). Where TAT-TIJIP was added, neuronal cul-

tures were pre-incubated with 2 lmol/L TAT-TIJIP for 15 min (added

at time = 0). Panel (b) shows the change in Indo-1 fluoresence ratio

after 15 min of glutamate exposure for the above experiment ex-

pressed relative to the value immediately prior to glutamate addition.

Bar indicates significant difference (p < 0.05) relative to glutamate

treated neurons (n = 3).

0

0.1

0.2

0.3

0.4

)oitar(1-

OD

N I

GlutamateTAT-TIJIP

+–

++

+–

—— Ruth—— —— Thaps ——

++

-0.1

0

0.1

0.2

0.3

0.4

5 10 15 20 25 30Time (min)

)oitar(1-

OD

NI

(a)

(b)

Fig. 9 Ruthenium Red, but not Thapsigargin, blocks the attenuation

caused by TAT-TIJIP. Panel (a) shows a representative example of

Indo-1 fluorescence in neuronal cultures, in the presence of 10 lmol/L

ruthenium red (s,d) or 10 lmol/L thapsigargin (+, ·), following addi-

tion of 100 lmol/L glutamate (arrow) at 15 min to cultures in the ab-

sence (d, ·) and presence of 2 lmol/L TAT-TIJIP (+,s). Ruthenium

red (Ruth.) or thapsigargin (Thaps.) were added at 0 min. Panel (b)

shows the change in Indo-1 fluoresence ratio after 15 min of glutamate

exposure for the above experiment expressed relative to value

immediately prior to glutamate addition. Bar indicates significant dif-

ference (p < 0.05) relative to glutamate treated neurons (n = 4).

72 P. G. Arthur et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 65–76� 2007 The Authors

implicated in causing cell death by necrosis (Almeida et al.2002). We therefore examined if TAT-TIJIP protectedmitochondria from excitotoxic stress by assessing mito-chondrial membrane potential with JC-1, and by measuringoxygen consumption following excitotoxic stress.

Membrane potential in cells not treated with glutamate didnot change significantly over 15 min (Fig. 10). Followingthe addition of glutamate there was a rapid decline inmembrane potential which had essentially recovered by15 min. In the presence of TAT-TIJIP the change inmitochondrial membrane potential following glutamateaddition was only 37% of the change seen with glutamateonly (Fig. 10). TAT-TIJIP did not significantly affect mem-brane potential over 15 min in the absence of glutamate.

Damage to the mitochondria would impair the ability ofthe mitochondria to synthesize ATP and consume oxygen.We monitored the activity of the electron transport chain bymeasuring the oxygen consumption of neurons in thepresence of the uncoupler FCCP. Oxygen consumptionmeasured 2 h after the addition of 100 lmol/L glutamate for15 min, was significantly depressed relative to a control(Fig. 10). In contrast, the oxygen consumption of neuronstreated with glutamate in the presence of 2 lmol/L TAT-TIJIP were significantly higher than that treated withglutamate only. The decline in uncoupled oxygen consump-tion was not likely caused by cell death because whole celloxygen consumption was unaffected and there was littleevidence of cell death by 2 h.

Discussion

Previous work has shown that the inhibition of JNK preventsapoptotic cell death (Xia et al. 1995; Yang et al. 1997;Levraut et al. 2003; Putcha et al. 2003; Okuno et al. 2004;Guan et al. 2005). Consistent with these earlier results wedemonstrated that the JNK peptide inhibitor, TAT-TIJIP, wasalso capable of preventing neuronal cell death by apoptosis.We expand on earlier studies by showing that the protectiveeffect of TAT-TIJIP extends to preventing cell death bynecrosis. Overall, TAT-TIJIP effectively interfered withseveral processes which have been identified as leading tocell death by necrosis. A key finding was that the inhibitorwas particularly effective in preventing the increase in ROS(DHE signal) following the addition of glutamate. This issignificant because the increase in ROS generation followingan excitotoxic insult have been implicated in causing celldeath by necrosis (Reynolds and Hastings 1995; Yaglomet al. 2003). In addition, TAT-TIJIP attenuated the increase incytosolic calcium caused by the excitotoxic insult. Elevatedcytosolic calcium has been proposed to cause necrotic celldeath through destabilization of lysosomes, activation ofphospholipases and damage to mitochondria (Almeida et al.2002; Yamashima et al. 2003; Liu et al. 2006). Finally, therewas evidence TAT-TIJIP prevented mitochondrial dysfunc-tion which has also been implicated in necrotic cell death(Almeida et al. 2002).

Necrosis is often regarded as a disorganized and irrevers-ible process once initiated. The effectiveness of TAT-TIJIP inpreventing necrosis indicates some consistency, in at least the

-0.4

-0.2

0

0.2

5

(a)

(b)

(c)

15 25 35Time (min)

Glutamate FCCP

85

90

95

100

105

110

115)oitar(ecnecseroulf

1-CJ

)oitar(ecnecseroulf

1-CJ

GlutamateTAT-TIJIP

Time

––

–+

++

——— 7 min ———

––

+–

–+

++

+–

——— 15 min ———

0

5

10

15

20

25

noitpmusnoc

n egyxO

)ni etor pg

m/nim/lo

mn(

GlutamateTAT-TIJIP

––

+–

++

Fig. 10 TAT-TIJIP attenuates disruption to mitochondrial function

caused by treatment with glutamate. Panel (a) Shows a representative

example of changes in mitochondrial membrane potential, as assessed

by JC-1 fluorescence, following addition of 100 lmol/L glutamate

(arrow) at 15 min to neuronal cultures in the absence (d) and presence

of TAT-TIJIP (s). Glutamate free neuronal cultures served as controls in

the absence (+) and presence of TAT-TIJIP (·). Where TAT-TIJIP was

added, neuronal cultures were pre-incubated with 2 lmol/L TAT-TIJIP

for 15 min (added at time = 0). Panel (b) shows membrane potential at 7

and 15 min after the addition of glutamate and is expressed relative to

membrane potential immediately prior to the addition of glutamate. Bars

indicate significant difference (p < 0.05) between conditions (n = 3).

Panel (c) shows oxygen consumption was significantly depressed in

neurons treated with glutamate and then uncoupled with FCCP to

assess maximal mitochondrial function. In contrast, the oxygen con-

sumption of neurons treated with glutamate in the presence of TAT-

TIJIP were significantly higher. Oxygen consumption was measured 2 h

after the addition of glutamate. Bars indicate significant difference

(p < 0.05) between conditions (n = 3).

A peptide JNK inhibitor prevents necrotic death 73

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 65–76

early phases of necrosis. A complete explanation of howTAT-TIJIP prevents cell death will require a better under-standing of the progression of molecular events leading tonecrosis. This will require considerable effort and mayrequire identifying additional protein substrates for JNKs.This was beyond the scope of this paper, but our observationsindicate several possible ways in which TAT-TIJIP could bepreventing necrosis. A number of protein substrates for JNKshave already been identified, and it is noteworthy that severalmitochondrial proteins have been identified, in addition toseveral transcription factors (Court et al. 2004; Okuno et al.2004).

It is unlikely the protective effect of TAT-TJIIP is a resultof inhibiting transcriptional processes. Necrotic cell deathfollowing an excitotoxic insult is not considered to involvetranscriptional components as has been demonstrated withsome forms of apoptosis (Hou et al. 2001; Desagher et al.2005). Our observations support this contention. First, thetranscriptional inhibitor actinomycin D did not block theprotective effect of TAT-TIJIP. Second, TAT-TIJIP waseffective in preventing a substantial increase in ROS andintracellular calcium. The rapid effects of TAT-TIJIP are notconsistent with the longer time-scales normally associatedwith transcriptionally controlled processes. Furthermore,TAT-TIJIP was ineffective when added after the exposureof neurons to glutamate (data not shown). This wouldindicate the critical protective effect of TAT-TIJIP is exertedduring the exposure of neurons to glutamate. Consequently,TAT-TIJIP was likely effective because it interfered withcritical post-translational phosphorylation(s) by JNKs.

One explanation for the protective effect of TAT-TIJIPthat is consistent with our observations is that blocking JNKactivity prevents an increase in cytosolic calcium which inturn prevents ROS generation. Reduced cytosolic calciumunder these circumstances could be a result of reducedinflux through ion channels or increased removal bycalcium ATPase pumps (Osborn et al. 2004). An alternativepossibility is that by blocking JNK activity, the JNKinhibitor decreases mitochondrial sensitivity to increasedcalcium following glutamate addition. Under these circum-stances, the detrimental effects of mitochondrial ROS oncalcium ATPase pumps would be prevented (Nicholls andBudd 2000).

Neuronal tissue is characterized by high levels of consti-tutively active JNK and in our experimental models therewas evidence of high basal JNK phosphorylation (Bjorkblomet al. 2005). The significance of high basal JNK phosphory-lation with the respect to cell death by necrosis is uncertain.There is evidence that JNK1 is the predominant form of basalJNK activity, so it is possible that acute inhibition of JNK1was preventing necrotic cell death (Shackelford and Yeh2006). However, there is also evidence JNK3 is activatedfollowing excitotoxic stress and that JNK3 is involved incausing apoptotic cell death (Kuan et al. 2003; Centeno

et al. 2007). Identifying the isoform of JNK involved incausing necrotic cell death was beyond the scope of thisstudy, as it will require a more specific inhibitor than TAT-TIJIP or an alternate approach.

In summary, we have now shown that a peptide inhibitorof JNK is capable of preventing cell death by necrosis. Thisis significant because the protective effect of blocking theactivity of JNKs, either directly or via knockouts, haspreviously been ascribed to preventing the progression ofapoptosis (Borsello et al. 2003). Neuronal death by necrosisas well as apoptosis is observed following a wide range ofpathological insults to the brain. A neuroprotective agent ableto prevent both necrosis and apoptosis would be veryattractive clinically.

Acknowledgements

This study was funded by the Western Australian Neurotrauma

Research Program. We acknowledge the assistance of Ingrid Boehm

in the preliminary stages of this work, Bruno Meloni for providing

cortical neurons and Renae Barr for in vitro JNK inhibition assays.

G. Mattich was the recipient of a Woodside scholarship.

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