Molecular and Cellular Neuroscience 49 (2012) 300–310

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Neuroscience

j ourna l homepage: www.e lsev ie r .com/ locate /ymcne

The JNK inhibitor D-JNKI-1 blocks apoptotic JNK signaling in brain mitochondria

Yi Zhao a,b, Giada Spigolon c, Christophe Bonny d, Juraj Culman a, Alessandro Vercelli c, Thomas Herdegen a,⁎a Institute for Experimental and Clinical Pharmacology, University Hospital of Schleswig-Holstein, Campus Kiel, Hospital Strasse 4, 24105 Kiel, Germanyb Department of Nuclear Medicine, Molecular Image, Diagnostics and Therapy, University Hospital of Schleswig-Holstein, Campus Kiel, Arnold-Heller Strasse 3, House 23, 24105 Kiel, Germanyc Neuroscience Institute of the Cavalieri Ottolenghi Foundation, University of Torino, Regione Gonzole 10, 10043 Orbassano, Italyd Division of Medical Genetics, CHUV-University Hospital, 1011 Lausanne, Switzerland

⁎ Corresponding author. Fax: +49 431 597 3520.E-mail address: herdegen.office@pharmakologie.uni

1044-7431/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.mcn.2011.12.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 July 2011Revised 19 November 2011Accepted 14 December 2011Available online 21 December 2011

Keywords:JNKD-JNKI-1HippocampusMitochondriaRatSeizures

Kainic acid (KA) induced seizures provokes an extensive neuronal degeneration initiated by c-Jun N-terminalkinases (JNK) as central mediators of excitotoxicity. However, the actions of their individual isoforms in cel-lular organelles including mitochondria remain to be elucidated. Here, we have studied the activation ofJNK1, JNK2 and JNK3 and their activators, mitogen-activated protein kinase kinase (MKK) 4/7, in brain mito-chondria, cytosolic and nuclear fractions after KA seizures. In the mitochondrial fraction, KA significantly in-creased the presence of JNK1, JNK3 and MKK4 and stimulated their phosphorylation i.e. activation. The pro-apoptotic proteins, Bim and Bax were induced and, consequently, the ratio Bcl-2-Bax decreased. Thesechanges were paralleled by the release of cytochrome c and cleavage of poly(ADP-ribose)-polymerase(PARP).The JNK peptide inhibitor, D-JNKI-1 (XG-102) reversed these pathological events in the mitochondria and al-most completely abolished cytochrome c release and PARP cleavage. Importantly, JNK3, but not JNK1 or JNK2,was associated with Bim in mitochondria and D-JNKI-1 prevented the formation of this apoptotic complex.Apart from of the attenuation of c-Jun phosphorylation in the nucleus, D-JNKI-1 did not affect the level ofJNK3 isoform in the nuclear and cytosolic fractions. These findings provide novel insights into the mode ofaction of individual JNK isoforms in cell organelles and points to the JNK3 pool in mitochondria as a targetof the JNK inhibitor D-JNKI-1 to confer neuroprotection.

© 2011 Elsevier Inc. All rights reserved.

Introduction

The hippocampus belongs to vulnerable brain tissues with a par-ticularly high propensity to epileptic activity. Treatment of rats withthe powerful convulsant, kainic acid (KA), triggers temporal lobe ep-ilepsy with extensive degeneration of hippocampal pyramidal cells(Ben-Ari and Cossart, 2000; Castillo et al., 1997; Nadler et al., 1978;Olney et al., 1974). Activation of KA receptor stimulates different sig-nalling pathways resulting in the activation of various proteases, celland organelle swelling and necrotic cell death on one side, and the ac-tivation of intrinsic mitochondrial cell death pathway on the otherside (for review see Meldrum, 2002). KA induces changes in the ex-pression of pro-and anti-apoptotic molecules, including those of theBcl-2 family which are linked to mitochondrial dysfunction(Henshall et al., 2002; Murphy et al., 2007). Among the pro-apoptotic proteins, BH3-only proteins, like Bcl-2 interacting mediatorof cell death (Bim), play a critical role in the activation of intrinsic ap-optosis pathways in the CNS (Becker et al., 2004; Lei and Davis, 2003;Putcha et al., 2001).

-kiel.de (T. Herdegen).

rights reserved.

Within this pathological network, the c-Jun N-terminal kinases(JNK) hold a central position being effective mediators of neuronaldeath (Björkblom et al., 2008; Bogoyevitch et al., 2010; Haeusgen etal., 2009; Waetzig and Herdegen, 2005). Previously we have shownthat the KA-induced JNK activation and hippocampal neuronaldeath can be prevented by inhibition of JNK (Brecht et al., 1999;Mielke et al., 1999; Spigolon et al., 2010). In line with these findings,JNK3 knockout mice are resistant to KA-induced excitotoxicity in thehippocampus (Yang et al., 1997).

Although recent findings have convincingly demonstrated thatmitochondria are one of the primary targets of JNK mediated neuro-nal death (Chipuk and Green, 2008; Kuan et al., 2003; Pirianov etal., 2007), our understanding of the pathophysiological function of in-dividual JNK isoforms in mitochondria is rather limited (Zhao andHerdegen, 2009; Zhou et al., 2008).

In PC12 cells (expressing only JNK1 and JNK2 isoforms) exposed tooxidative damage, JNK2, but not JNK1, translocates into the mito-chondria. Transfection of an inactive JNK2 mutant prevented mito-chondrial translocation of JNK2, release of cytochrome c andexpression of Bim (Eminel et al., 2004). We have also demonstratedan increase in JNK3- and a decrease in JNK1 activity in mitochondriahomogenates from adult rat brain following cerebral ischemia (Zhaoand Herdegen, 2009). These findings clearly indicate the importance

301Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

of understanding the role of individual JNK isoforms in mitochondrialpathology.

The function of the individual JNK isoforms in the regulation of thepro- and/or anti-apoptotic mitochondrial pathways and proteins inthe adult brain remains elusive, as the pharmacological tools for

Fig. 1. Neuronal cell death in the hippocampus. Morphological changes in the hippocampal Ccontrols (control) or 24 hours after KA induced seizures (KA). (B) Fluoro-Jade C staining introcytes GFAP or the marker for neurons NeuN, DAPI, TUNEL and co-staining DAPI + TUNE

manipulating these pathways are limited. Neuroprotection by the cell-penetrating, protease-resistant peptide, D-JNKI-1, has been reportedfollowing cerebral ischemia, epileptic seizures, retinal excitotoxicity,hearing loss and neuropathic pain induced by spinal nerve ligation(Bessero et al., 2010; Borsello et al., 2003; Eshraghi et al., 2006; Liu et

A 3 and CA 4 subfields. (A) Cresyl-violet staining, c-Jun and phospho-c-Jun in untreatedcontrols (left) and after KA (right). Co-staining of Fluro-JADE C with the marker for as-L is shown following KA.

302 Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

al., 2010; Spigolon et al., 2010; Zhuang et al., 2006). Interestingly, D-JNKI-1 strongly interferes with JNK activationmainly upon pathologicalbut not in physiological conditions (Repici et al., 2009). The peptide in-terferes with the binding of JNKs to their substrates, but the mecha-nisms of its neuroprotective actions are only partially understood.

Using the KA-induced epilepsy model, we provide evidence thatindividual JNK isoforms are differentially activated in a defined organ-elle such as mitochondria and form specific up- and down-stream sig-nalosomes. The JNK inhibitor, D-JNKI-1, disrupts the formation ofharmful JNK-signalosomes such as the JNK3-Bim-complex in mito-chondria. Thus, the powerful neuroprotection by D-JNKI-1 mightnot be merely mediated by the inhibition of the common apoptoticend route (i.e. caspase 3 activation, PARP cleavage), but it rather im-plies specific actions such as inhibition of JNK intraneuronal traffick-ing (translocation) and formation of pro-apoptotic signalosomes.

Results

Neuronal cell death in the hippocampus

KA- induced seizures initiate neuronal cell death in the vulnerableareas of the hippocampus as previously shown in detail (Spigolon etal., 2010). In the present study, we demonstrated the degenerative ef-fects of KA by semi-quantitative analysis. Neuronal cell death in theCA3 and DG areas was demonstrated by cresyl violet staining andFluoro-Jade C staining (Fig. 1). Apoptosis mainly occurred in neurons,but not astrocytes (Fig. 1B). The vulnerable CA3 and DG areas exhib-ited a pronounced expression and N-terminal phosphorylation of c-Jun (Fig. 1A) as an indicator of JNK activity.

Fig. 2. JNK isoforms and phosphorylated JNK inmitochondria.Mitochondria from the hip-pocampus were isolated from untreated controls (CO) or 3 h, 6 h and 12 h after KA injec-tion. Each lane gives a pooled sample from 2 rats. Light grey bar shows the 46 kDa band,dark grey bar the 54 kDa band. Values represent the mean±SEM. Statistical comparisonsto the control group at the respective time points: * pb0.05, ** pb0.01, *** pb0.001,calculated by one-way ANOVA followed by a post-hoc Dunnett test.

Distribution of JNK isoforms in mitochondria, cytoplasm and nucleus

Firstly, we studied the time course of the distribution of JNK iso-forms in various cellular compartments of the hippocampal tissue fol-lowing KA-induced seizures (Figs. 2–4). All biochemical procedureswere performed with hippocampal homogenates, i.e. we could nei-ther discriminate between neuronal and non-neuronal mitochondrianor between perikaryon and dendritic structures.

In mitochondria, JNK1 (46 kDa band) and to a less extent JNK3(54 kDa band) increased with peak levels between 6 and 12 h. JNK2did not change. The JNK phosphorylation was maximal 3 h after KAapplication (Fig. 2).

In the cytosolic fraction, JNK1 and JNK3 increased between 3 and6 h, and returned thereafter to basal levels. JNK2 decreased not before12 h (Fig. 3). In contrast to mitochondria, the activation of cytosolicJNK significantly increased at 3 h and reached its peak at 12 h afterKA injection.

In the nuclear fraction, the specific pattern of JNK changes consistedfrom a significant decrease in JNK1 at 6 h, a delayed rise of the 54 kDaband of JNK2 and a peak of JNK3 at 3 h (Fig. 4). In contrast to the cytosol,wheremainly JNK1 and JNK3 constituted the pool of total JNK, JNK2wasby far the predominate form in the nuclear JNK pool. Surprisingly, acti-vation of JNKs was not seen before 12 h, whereas phosphorylation oftheir high affinity substrate, c-Jun, reached its maximum already at 3 h.

Our data indicate pronounced compartment-specific changes fol-lowing KA-induced seizures which might indicate translocation ofisoforms among organelles: JNK1 translocates from the nucleus tothe cytosol and mitochondria, JNK2 translocates from the cytosol tothe nucleus and the JNK3 isoform increases in all cellularcompartments.

Fig. 3. JNK isoforms and phosphorylated JNK in the cytosol. Cytosolic preparations fromthe hippocampus were isolated from untreated controls (CO) or 3 h, 6 h and 12 h afterKA injection. Each group consists of 6 rats, each lane gives a pooled sample from 2 rats.Light grey bar shows the 46 kDa band, dark grey bar the 54 kDa band. Values representthe mean±SEM. Statistical comparisons to the control group at the respective timepoints: * pb0.05, ** pb0.01, *** pb0.001, calculated by one-way ANOVA followed bya post-hoc Dunnett test.

Fig. 4. JNK isoforms, phosphorylated JNK and phosphorylated c-Jun in the nuclear frac-tion. Nuclear fractions from the hippocampus were isolated from untreated controls(CO) or 3 h, 6 h and 12 h after KA injection. Each lane gives a pooled sample from 2rats. Light grey bar shows the 46 kDa band, dark grey bar the 54 kDa band. Values rep-resent the mean±SEM. Statistical comparisons to the control group at the respectivetime points: * pb0.05, ** pb0.01, *** pb0.001, calculated by one-way ANOVA followedby a post-hoc Dunnett test.

303Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

The up-stream activators of JNKs in mitochondria, MKK4 and MKK7

Activation of JNKs requires the interaction with their upstream ac-tivators MKK4 and/or MKK7. Both MAP2 kinases were present inbrain mitochondria with a transient, but significant increase after3 h (Fig. 5). The concentration of phosphorylated MKK4 gradually in-creased between 3 and 12 h and, consequently the relative level (the

Fig. 5. Upstream of JNK: MKK4 and MKK7. Mitochondria from the hippocampus wereisolated from untreated controls (CO) or 3 h, 6 h and 12 h after KA injection. Eachlane gives a pooled sample from 2 rats. Values represent the mean±SEM. Statisticalcomparisons to the control group at the respective time points: * pb0.05, ** pb0.01,***pb0.001, calculated by one-way ANOVA followed by a post-hoc Dunnett test.

ratio between the phosphorylated- and total level of MKK4) signifi-cantly increased. Due to technical reasons, we could not detect phos-phorylation of MKK7.

Bcl-2 proteins in mitochondria

The level of the pro-apoptotic Bim protein in mitochondria was sig-nificantly raised between 6 and 12 h. Its activation (phosphorylation)already peaked at 3 h (Fig. 6). Similarly, increased concentrations ofBax protein were observed between 3 h and 12 h. In contrast to Bimand Bax, the anti-apoptotic Bcl-2 did not change in the mitochondrialfraction. Consequently, the ratio Bcl-2/ Bax significantly dropped be-tween 3 and 12 h after KA injection (Fig. 6).

Cytochrome c release and PARP

The release of cytochrome c is an early event of the apoptotic cas-cade. We detected cytochrome c already 3 h after KA in the cytosoland cytochrome c remained elevated until 12 h (Fig. 7). In contrast,nuclear cleavage of PARP, a delayed apoptotic feature, did not in-crease before 12 h (Fig. 7).

The JNK inhibitor D-JNKI-1 reverses the KA induced alterations in JNKsignaling

Distribution and activation of JNK isoforms and MKKsIn the second part of this study, we investigated to what extent

the JNK inhibitor D-JNKI-1 is able to prevent the above reported traf-ficking and activation of individual JNK isoforms following KA-induced seizures. The effects of D-JNKI-1 were studied 6 h after KA,when the majority of significant changes had already developed.

In mitochondria, D-JNKI-1 significantly reduced the amount ofJNK1, the 46 kDa band of JNK2 and JNK3, while the 54 kDa-JNK2

Fig. 6. Bim, Bax and Bcl-2 inmitochondria. Mitochondria from the hippocampus were iso-lated from untreated controls (CO) or 3 h, 6 h and 12 h after KA injection. Each lane gives apooled sample from 2 rats. Values represent the mean±SEM. Statistical comparisons tothe control group at the respective timepoints: * pb0.05, ** pb0.01, *** pb0.001, calculatedby one-way ANOVA followed by a post-hoc Dunnett test.

Fig. 7. Cytochrome c release and cleaved PARP. Cytosolic fraction for the analysis of cy-tochrome c and nuclear fractions for the analysis of cleaved PARP were isolated fromthe hippocampi of untreated control rats (CO) or rats 3 h, 6 h and 12 h after KA injec-tion. Each lane gives a pooled sample from 2 rats. Values (arbitrary units) represent themean±SEM. Statistical comparisons to the control group at the respective time points:** pb0.01, *** pb0.001, calculated by one-way ANOVA followed by a post-hoc Dunnetttest.

Fig. 8. Effect of D-JNKI-1 on JNK isoforms inmitochondria. (A)Mitochondria from the hip-pocampus were isolated 6 h after injection of KA with D-JNKI-1 (hatched bars) or vehicle(empty bars). Each lane gives one individual rat. Light grey bar shows the 46 kDa band,dark grey bar the 54 kDa band. Values represent the mean±SEM. Statistical comparisonsto the vehicle-treated group: * pb0.05, **pb0.01, ***pb0.001, calculatedwith Student´s t-test for unpaired samples. (B) Following the double-immunodepletion of two JNKisoforms, the remaining protein extract was blotted either against total-JNK- oderphospho-JNK antibodies. Each lane gives the pooled extracts from 6 hippocampi. Lane 1,control; lane 2, KA; lane 3, KA+D-JNK1-I. The columns show the respective densitometryvalues.

304 Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

isoform increased (Fig. 8A). In striking contrast to these effects, D-JNKI-1 enhanced the phosphorylation of JNKs. This paradoxical find-ing, however, is in line with the observation that the inhibition ofJNK activity induces their activation. To study the activation of eachindividual JNK isoform, we performed double immunodepletion andthe remaining single JNK isoform was blotted against the anti-phospho-JNK antibody (Fig. 8B). Importantly, D-JNKI-1 diminishedthe level of activated JNK3 in mitochondria whereas changes inJNK1 and JNK2 were less pronounced. The desitomitry analysis ofthe ratios between the individual phosphorylated JNK isoforms andthe corresponding JNK isoforms showed an increase in JNK1 activa-tion and a decrease in JNK3 activation after D-JNKI-1 treatment.Thus, the JNK inhibitor suppresses the activation of the harmfulJNK3 isoform, but leaves the activation of the physiological JNK1and 2 unaltered.

In the cytosol, D-JNKI-1 enhanced JNK1, but significantly loweredJNK2 compared to the vehicle-treated group (Fig. 9); JNK3 and thetotal JNK pool were not affected. Similarly to mitochondria,phospho-JNK was increased. Finally, D-JNKI-1 diminished JNK1 inthe nuclear fraction (Fig. 10). As with mitochondria and the cytosol,the inhibitor enhanced the JNK activation, but almost completelyinhibited activation of c-Jun (Fig. 10).

D-JNKI-1 did not alter the levels ofMKK4 andMKK7 inmitochondria,but similarly to the JNK inhibition, it enhanced the MKK4 activation(Fig. 11).

In summary, D-JNKI-1 prevented a variety of KA-induced changesin the JNK signaling cascade with a prominent inactivation of JNK3(Table 1).

Bcl-2/Bim signalingThe inhibition of JNK also affected the KA-induced alterations in

Bcl-2 proteins in mitochondrial preparations. It diminished theamount of Bim, phosphorylated Bim and Bax but tended to increasethe level of Bcl-2 (Fig. 12). As a consequence, D-JNKI-1 increasedthe ratio Bcl-2/Bax i.e. it shifted the ratio from the apoptotic to theanti-apoptotic signaling.

Using the immunoprecipitation technique, we also obtained novelinsights into the regulatory role of JNK isoforms in neurodegenerativeprocesses. JNK3, but not JNK1 or JNK2, formed a complex with thepro-apoptotic Bim in mitochondria (Fig. 13, lane 3). The formationof this complex was prevented by D-JNKI-1 (Fig. 13, lane 4). This find-ing suggests that D-JNKI-1 interferes with an organelle-specific actionof the apoptotic JNK isoform.

Cytochrome c and PARPThe pronounced release of cytochrome c and the cleavage of PARP

are the hallmarks of neuronal apoptosis. The inhibition of JNK activityby D-JNKI-1 almost completely and highly significantly abolishedthese changes (Fig. 14).

Fig. 9. D-JNKI-1 and JNK isoforms in the cytosol. Rats were treated with vehicle or D-JNKI-1 1 hour after KA injection. Six hours after KA application, the rats were killedfor the cytosol fraction isolation. Each lane gives an individual rat. Light grey barshows the 46 kDa, and dark grey bar the 54 KDa band. The columns represent thedensitometry values, which are expressed as the mean±SEM. * pb0.05, ** pb0.01,*** pb0.001, statistical comparison to vehicle-treated rats calculated with Student's t-test for unpaired samples.

Fig. 10. D-JNKI-1 and JNK isoforms in the nuclear fraction. Rats were treated with vehicleor D-JNKI-1 1 hour after KA injection. Six hours after KA application, the rats were killedfor the nuclear fraction isolation. Each lane gives an individual rat. Light grey bar showsthe 46 kDa, and dark grey bar the 54 kDa band. The columns represent the densitometryvalues, which are expressed as the mean±SEM. * pb0.05, ** pb0.01, *** pb0.001, statis-tical comparison to vehicle-treated rats calculated with the Student´s t-test for unpairedsamples.

Fig. 11. D-JNKI-1 and mitochondrial MKKs. Rats were treated with vehicle or D-JNKI-1 1hour after KA injection. Six hours after KA application, the rats were killed for the isolationof mitochondrial fraction. Each lane gives an individual rat. The columns represent thedensitometry values, which are expressed as the mean±SEM. ** pb0.01, statistical com-parison to the vehicle-treated group calculated with the Student's t-test for unpairedsamples.

305Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

Discussion

The present study addressed the effect of excitotoxic neurodegen-eration on the activation and distribution of individual c-Jun N-terminal kinase isoforms in hippocampal homogenates and the effectof the JNK inhibitor peptide, D-JNKI-1, on these pathophysiologicallyrelevant alterations.

We report for the first time on i) the activation and changes of JNKisoforms within the subcellular fractions with a particular focus onmitochondria, ii) the formation of complexes of individual JNK iso-forms with proapoptotic proteins in mitochondria and iii) the effectof the JNK-inhibitor, D-JNKI-1, on these events.

KA-induced excitotoxicity enhanced the presence and activationof JNK3 in mitochondria, whereas JNK1 was dramatically reduced inthe nuclear fraction with a parallel increase in JNK2 and JNK3. KA en-hanced the presence and/or activation of Bim and Bax in mitochon-dria and the specific formation of the JNK3-Bim complex. Moreover,the classical features of apoptosis, cytochrome c release and nuclearPARP were strongly increased. D-JNK1-I reversed most of these path-ological changes, in particular the changes of JNK3.

Neuronal death after epileptic seizures

In our preceding paper, we have shown that the JNK inhibitor D-JNKI-1 very efficiently protects against KA induced neuronal celldeath with a detailed quantification and time courses (Spigolon etal., 2010). Here we have extended these findings on the underlyingmechanisms. The JNK inhibitor almost completely suppressed the re-lease of cytochrome c into the cytoplasm and the presence of cleavedPARP in the nucleus. Both events are hallmarks of apoptosis inducedby excitotoxicity (Dawson and Dawson, 2004). About 3–5% of cells

identified as neurons in the hippocampal C3 region were TUNEL-positive cells 24 h after KA-injection. Western blotting demonstratedthe accumulation of pro-apoptotic proteins such as Bim, activated(phosphorylated) Bim and Bax in mitochondria.

Fig. 12. D-JNKI-1 and Bim, Bax and Bcl-2 in mitochondria. Rats were treated with vehi-cle or D-JNKI-1 1 hour after KA injection. Six hours after KA application, rats were killedfor the isolation of mitochondrial fraction. Each lane gives an individual rat. Thecolumns represent the densitometry values, which are expressed as the mean±SEM.* pb0.05, statistical comparison to the vehicle-treated group calculated with the Stu-dent´s t-test for unpaired samples.

Fig. 14. D-JNKI-1 on cytochrome c release and PARP. Rats were treated with vehicle or D-JNKI-1 1 hour after KA injection. Six hours following KA application, rats were killed andthe cytosol fraction (determination of cytochrome c) and nucler fraction (determinationof cleaved PARP) were isolated. Each lane gives the sample from an individual rat. Thecolumns represent the densitometry values, which are expressed as the mean±SEM.*** pb0.001, statistical comparison to the vehicle-treated group calculated with the Stu-dent's t-test for unpaired samples.

306 Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

Subcellular distribution of JNK isoforms

Our knowledge on the activation and function of individual JNKisoforms in the adult brain is rather limited. Some evidence indicatesthat different functions of individual JNK isoforms are linked to theirsubcellular localisation (Björkblom et al., 2008; Waetzig et al., 2009;Zhao and Herdegen, 2009).

Nuclear localization. The nuclear translocation of all JNK isoforms iscrucial for the induction of apoptosis of cerebellar granule neuronsafter trophic support withdrawal (Björkblom et al., 2008). FollowingKA, JNK2 and JNK3, but not JNK1, accumulated in hippocampal nucleiin which c-Jun was rapidly activated within 3 h after the application

Fig. 13. JNK3-Bim-complexes in mitochondria. Rats were treated with vehicle or D-JNKI-1 1 hour after KA injection. Six hours following KA application, rats were killedfor the isolation of mitochondrial fraction. Immunoprecipitation (IP) with specific an-tibodies against JNK1, JNK2 or JNK3 was followed by immunoblotting (IB) with anti-Bim antibody. Lanes 1, negative control (lysis buffer); lanes 2, control rats treatedwith vehicle; lanes 3, KA+vehicle; lanes 4, KA+D-JNKI-1. Lane C, total protein extrac-tion without IP; lane M, molecular weight marker.

of KA. The c-Jun/AP-1 transcriptional regulation of apoptotis confer-ing gene products is well established (Besirli et al., 2005; Dietrich etal., 2004; Ventura et al., 2006; reviewed by Waetzig and Herdegen,2005). The JNK inhibitor, D-JNKI-1, diminished the nuclear JNK1pool and almost completely blocked c-Jun phosphorylation. The re-duction of nuclear JNK1 from high basal level suggests a change ofthe JNK1-controlled transcription to a JNK2/3 mediated transcriptionof degenerative effector proteins (Björkblom et al., 2008). However,nuclear JNK1 may also mediate apoptosis after trophic withdrawalin cerebellar granule neurons when JNK2 and JNK3 are absent(Björkblom et al., 2008).

Cytosol location. Excessive neuronal activity provokes substantialchanges in the morphology and neurogenesis of granule cells in thedentate gyrus of the hippocampus (Jessberger et al., 2007). A numberof cytosolic proteins serve as substrates of JNK in the cytosol(Bogoyevitch et al., 2004; Waetzig et al., 2006). The significant in-crease in cytoplasmic JNK can be related to changes of axonal anddendritic architecture via microtubule regulator protein targets ofJNK, mainly of the cytosolic JNK1 (Barnat et al., 2010; Björkblom etal., 2005; Tararuk et al., 2006;Westerlund et al., 2008). We have dem-onstrated that JNK1 supports neurogenesis and neuronal differentia-tion in neuronal cell culture (Waetzig and Herdegen, 2003). Inaddition, the TAK1/JNK1 signal cascade activates IAP (inhibitor of ap-optosis family of proteins) which in turn suppresses ICE and TNF-αinduced apoptosis (Sanna et al., 2002). Importantly, D-JNKI-1 pre-vents the drop of JNK1 in the cytosol and significantly enhancesboth, the presence of JNK1 and the total JNK pool above basal levels.This specific reactivation of a JNK isoformmight contribute the neuro-protective effects of JNK inhibition.

Mitochondrial location. Mitochondria play a critical role in the con-trol of cell death promoting pathways, including the raise in intracellu-lar Ca2+, production of free radicals and regulation of Bcl-2 familyproteins (Liou et al., 2003). In the hippocampus, mitochondrial Ca2+

loading has long been known to occur after seizure activity (Griffithset al., 1984). Antiapoptotic members of the Bcl-2 family such as Bcl-2and Bcl-xl confer protection by acting directly on mitochondrial mem-brane integrity and Ca2+ mobilization (Cory and Adams, 2002; Polsterand Fiskum, 2004). Under physiological conditions, Bcl-2, an antiapop-totic protein, can oligomerize with Bax and thereby neutralizes its pro-apoptotic function. Inversely, the (stoichiometric) drop in Bcl-2 is acommon feature of apoptosis.

Here we show an increased ratio of Bax/Bcl-2 which is linked tothe accumulation of Bax in the mitochondria. This indicates an alteredbalance between Bax and Bcl-2, and a shift to Bax-Bak, thereby repla-cing the signaling of cell survival with that of cell death (Chipuk et al.,2010).

Functional Bcl-2 inhibition, which has been shown after KA-induced seizures in rats, is based on elevated phosphorylation levels(Korhonen et al., 2003). JNK1 is a stress-activated Bcl-2 kinase

307Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

which is co-localized with Bcl-2 in the mitochondria (Deng et al.,2001).

Bax as well as Bim particle clustering has also been visualized at theoutermembrane of themitochondriawithin 2 h of seizures (Henshall etal., 2002; Korhonen et al., 2003; Shinoda et al., 2004). Our study focusedon Bim regulation for two reasons: Although JNKs control expressionand activation of Bim (Murphy et al., 2010; Putcha et al., 2003), obser-vations in brain mitochondria ex vivo are rare. Secondly, we have previ-ously shown that dominant-negative JNK2 plasmids suppressed Bimexpression and JNK2 translocation to mitochondria in PC12 cellsstressed by 6-hydroxy-dopamine (Eminel et al., 2004).

The Bim-mediated cell death is regulated at several levels: i) tran-scriptional induction, ii) post-translational activation, and iii) neutral-ization of anti-apoptotic molecule, Bcl-w (Engel et al., 2011), with JNKas a central regulator. JNK induces the AP-1 mediated transcription ofBim, and this induction of Bim is specifically involved in neuronal celldeath but not in cell death in other cell types (Putcha et al., 2003).

We detected increases in Bim and phospho-Bim after seizures,whichmight be sufficient to kill the cells by interrupting themitochon-drial membrane integrity. The most powerful proapoptotic BH3-onlyproteins, Bim and PUMA, avidly bind all anti-apoptotic Bcl-2 family pro-teins and neutralize them to induce cytochrome c release from mito-chondria (Chen et al., 2005).

Therefore, we emphasized to ascertain whether blockade of the JNKbinding domain by D-JNKI-1 affects the interaction of JNK3-Bim com-plex formation.We report here for the first time on the complex forma-tion between an individual JNK isoform and Bim in brain mitochondriaex vivo. In our hands, JNK1 and JNK2 are not detected in the JNK-Bimcomplexes, whereas JNK3 is clearly seen in Bim containing protein ex-tracts frombrainmitochondria. Importantly, this complex does not lon-ger exist following application of D-JNKI-1. In parallel, JNK inhibitionprevents the increase in JNK levels in mitochondria. Consequently,JNKs were significantly reduced compared to saline treated rats.

Taken together, our results clearly demonstrate a simultaneoustranslocation of Bim, phospho-Bim and Bax and as well as activatedJNK3 into mitochondria. JNK3, but not JNK1 and JNK2, binds to Bim.This effect was reversed when the JNK binding domain was blockedwith D-JNKI-1. The obvious disruption of binding of the JNK3 isoformto a pro-apoptotic substrate such as Bim represents a selective modeof action of the peptide inhibitor, D-JNKI-1, which leaves many otherphysiological functions of JNK unchanged.

MKK — upstream of JNK

KA increased the activation of MKK4 in mitochondria and thismight be a prerequisite for the changes of JNK in mitochondria, inparticular activation of JNK3. Indeed, MKK4 is an activator of JNK3in COS-7 cells (Miller et al., 2001). Furthermore, the inhibitor, D-JNKI-1, increased the phosphorylation of MKK4. As MKK4 is upstreamof JNK, we assume that a negative feedback loop pathway compensa-tory reinforced phosphorylation of JNK and their upstream MKK ki-nases when the JNK binding domain is blocked by the inhibitor. Asrecently shown, D-JNKI-1 also triggers ERK activation via MEK1, sug-gesting a cross-talk between MKK-JNK signalling and MEK1-ERKpathway under both, normal conditions (Repici et al., 2009) and re-storative neurite formation (Waetzig and Herdegen, 2005). Again,this can also contribute to neuroprotective actions of D-JNKI-1.

In summary, our data provide novel insights into the regulationand presence of individual JNK isoforms in the brain under neurode-generative conditions. Particularly, a great effort has been made toanalyze the presence and activation of JNK isoforms and their Bcl-2substrates in mitochondria. JNK3 was strongly increased and activat-ed in mitochondria and formed complex with the pro-apoptotic Bim.The JNK-inhibitor peptide, D-JNKI-1, reversed the majority of the KAinduced changes such as neuronal cell death in the hippocampus,the release of cytochrome c, the cleavage of PARP as well as the

increase of JNK3 in mitochondria and nucleus, the rise of Bim and re-stored the equilibrium between the anti-apoptotic Bcl-2 and the pro-apoptotic Bax proteins. Most importantly, D-JNKI-1 abolished thespecific complex formation of JNK3 and Bim in mitochondria. The bet-ter understanding of the compartment-specific alterations of individ-ual JNK isoforms are the prerequisite to develop specific molecularinterventions which blocks pathological events and leave physiologi-cal processes unaltered.

Experimental methods

Chemical regents

Kainic acid (KA), (Tocris Bioscience, Bristol, UK, 15 mg/kg), D-JNKI-1(XigenPharm. SA, Lausanne, Switzerland), Enhanced chemiluminescence(ECL) detection system, High performance chemiluminescence film(Amersham; Piscataway, NJ), Immobilon-P polyvinylidene difluoridemembrane (PVDF), Fluoro-Jade C (Millipore; Billerica, MA), PhosphataseInhibitor Cocktail II (Sigma-Aldrich; St. Louis, MO), protease inhibitor(complete), in Situ Cell Death Detection Kit (Roche; Indianapolis, IN),MedCode Reversible Protein Stain Kit, Restore Western Bolt StrippingBuffer, BCA™ protein assay kit, Pierce® mammalian Co-Immuno-precipitation Kit (Pierce; Rockford, IL), ExacraCruz ImmunopreciptationKit (Santa Cruz Biotech-nology; Santa Cruz, CA), Bovine Serum Albumin(BSA) (Boehringer; Mannheim, Germany), Biotinylated Protein LadderDetection Pack (Cell Signaling Techonolgy; Beverly, MA), prolong® goldantifade reagent (Invitrogen, Karlsruhe, Germany). All other chemicalswhich are not explicitly mentioned, were purchased from Sigma orMerck companies.

Antibodies

Rabbit anti-phosphorylated JNK polyclonal antibody, rabbit anti-PARP p85 fragment polyclonal antibody (Promega; Madison, WI);mouse anti-JNK1monoclonal antibody, rabbit anti-BIM polyclonal anti-body, mouse anti-Cytochrome C (BD Pharmingen; San Diego, CA), rab-bit anti-JNK2monoclonal antibody (Epi-tomics; Burlingame, CA), rabbitanti-JNK3/SAPK1b monoclonal antibody, rabbit anti Bim/BOK antibody(Upstate; Lake Placid, NY), rabbit anti-phospho-Bim (ser55) polyclonalantibody, rabbit anti-MKK4 polyclonal antibody, rabbit anti-SAPK/JNKpolyclonal antibody, rabbit anti-MKK7 polyclonal antibody, rabbitanti-phospho-MKK4 polyclonal antibody and rabbit anti-phospho-MKK7 polyclonal antibody, rabbit anti phospho-c-Jun (Cell SignalingTechnology; Beverly, MA), rabbit anti Bcl-2 antibody, mouse anti Baxantibody, rabbit anti c-Jun, rabbit anti-Glial Fibrillary Acidic Protein(GFAP) polyclonal antibody (Santa Cruz Biotechnology; Santa Cruz,CA), mouse anti-β-actin monoclonal antibody (Sigma-Aldrich; St.Louis, MO), mouse anti-neuronal nuclei (NeuN) monoclonal antibody(Millipore, Billerica, MA), horseradish peroxidase-linked donkey anti-rabbit antibody, horseradish peroxidase-linked sheep anti-mouse anti-body (Amersham; Piscataway, NJ), Alexa Fluor®546 -conjugated goatanti-rabbit antibody; Alexa Fluor®546 conjugated goat anti-mouse an-tibody (Molecular Probes, Leiden, TheNetherlands). The concentrationsare given in the respective legends.

Animal experiments

The experiments were carried out at the Institute of Experimentaland Clinical Pharmacology, University Hospital of Schleswig-HolsteinCampus Kiel (Germany) or at the Department of Anatomy, Pharmacol-ogy and ForensicMedicine of theUniversity of Torino (Italy). AdultmaleSprague–Dawley rats (weight 270–320 g)were used in all experiments.Animals had free access to food and water. The experiments were car-ried out according to the European Communities Council Directive of24 November 1986 (86/609/EEC). All efforts were made to minimizethe number of animals and their suffering.

308 Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

Experiment 1 Control animals were injected intraperitoneally (i.p.)with 0.9% saline (n=3). KA (15 mg/kg, n=3) was injected to triggerseizures. Twenty-four hours later, rats were deeply anaesthetizedwith an over-dose of chloral hydrate and intracardially perfusedwith phosphate-buffered saline (PBS) (pH 7.4) followed by 4% para-formaldehyde. The brains were removed, post-fixed overnight, andcryoprotected in 30% sucrose for 72 h at 4 °C. Brain sections (10 μmor 25 μm) were cut on a cryostat and used for the analysis of celldeath (staining with cresyl violet), apoptosis (staining for deoxynu-cleotidyl transferase-mediated biotinylated UTP nick end labeling,TUNEL), neurodegeneration (staining with Fluoro-Jade C) as well asfor c-Jun and phosphorylated (activated) c-Jun.

Experiment 2 Rats were randomly divided in 4 groups. Controls(n=6) were injected i.p.with 0.9% saline. Three groups of rats (eachn=6) were injected with KA (15 mg/kg, i.p.). Three, 6 and 12 hafter KA-injection, rats were deeply anaesthetised and decapitated.The heads were immediately put into ice-cold water for 3 min, thebrains removed and the hippocampus (both sides) was quickly dis-sected, frozen and stored at −80 °C until being used for Westernblot analysis.

Experiment 3 Rats were randomly divided in 3 groups (each groupn=6). Controls were injected with 0.9% saline (i.p). The secondgroup of rats received KA injection (15 mg/kg, i.p.) and the thirdgroup was treated with D-JNKI-1 (0.3 mg/kg, i.p.) 1 h after KA injec-tion (15 mg/kg, i.p). Twelve hours thereafter all rats were decapitatedin deep anaesthesia. The hippocampus was quickly removed and pro-cessed as described above.

In each experiments, epileptic seizures induced by i.p injection ofKA were quantified using the Racine scale (Ben-Ari, 1985; Racine,1972): stage 0, no seizures; stage 2, head nodding; stage 3, forelimbclonus; stage 4, rearing in addition to severe forelimb clonus; stage5, rearing and falling in addition to severe forelimb clonus. Onehour after the KA injection rats showed the first symptoms of epilepsy(immobility, facial myoclonus, head nodding). Only animals whichreached the stages 4 and 5 of were included into the study(Spigolon et al., 2010).

Immunohistochemistry

c-Jun and phospho-c-Jun. The brains were cut into coronal 25 μm-thick, free-floating sections, and stored in a cryoprotective solutionat −20 °C until staining for c-Jun and phospho-c-Jun as previouslydescribed (Zhao et al., 2006).

TUNEL staining. The In Situ Cell Death Detection Kit was used accord-ing to the manufacturer's protocol (Roche, Mannheim, Germany).Briefly, the brain sections (10 μM) were washed with PBS and permea-bilised in 0.1% Triton-100 of 0.1% sodium citrate solution for 5 min atroom temperature (RT). Afterwards, the sections were incubated in aTUNEL reaction mixture (1:9) for 1 h at RT, washed with PBS andmounted in prolong® gold antifade reagent.

Fluoro-Jade C. The Fluoro-Jade C staining was carried out as de-scribed elsewhere (Schmued et al., 2005) with slight modifications.Dried brain sections (10 μm) mounted onto gelatine coated slideswere immersed in 100% xylen for 10 minutes twice and subsequentlyexposed to decreasing concentrations of ethanol (100%, 50% and 10%)for 2 min each. The slices were exposed to 0.06% potassium perman-ganate (10 min), rinsed in distilled water (2 min) and immersed in0.0001% Fluoro-Jade C in 0.1% acetic acid solution for 10 min. Afterwashing three times in distilled water, the sections were dried andimmersed in xylene for 1 min, and then covered with D.P.X mountingmedia. In the co-localisation experiments, sections were first stainedfor NeuN (neuronal marker) or GFAP (marker for astrocytes) as de-scribed before (Zhao et al., 2006). After incubation with the secondaryantibody, the sections were shortly washed in distilled water, trans-ferred into 0.06% potassium permanganate and stained with Fluoro-Jade C as described above.

Isolation of organelles

Mitochondria and cytoplasmic fraction. Proteins from both, the cy-tosolic and the mitochondrial fractions were extracted according tothe slightly modified multiple centrifugation method described else-where (Schild et al., 2003; Zhao and Herdegen, 2009). Briefly, hippo-campal tissue washed twice with ice-cold PBS was gently dounced 10times in a glass Dounce Homogeniser (Wheaton, Millville, NJ) with3 ml of ice-cold isolation medium (250 mM Mannitol, 20 mM Tris,1 mM EGTA, 1 mM EDTA and 0.3% w/v BSA containing 1% phospha-tase and protease inhibitors). The homogenates were stored on icefor 20 min, centrifuged at 450 g for 10 min and the pellets wereused for nuclear protein isolation as described below. The centrifuga-tion of supernatants (22,000 g for 10 min) yielded the cytosolic andmitochondrial fractions. The pellets were washed twice with isolationmedium and then re-suspended in a denaturing lysis buffer (250 mMNaCl, Tris 20 mM, EDTA 3 mM, EGTA 3 mM, Nonidet 0.5%, 1% phos-phatase and protease inhibitor) and incubated on ice for 30 min.The mitochondria suspension was briefly sonicated and centrifugedto remove insoluble material (16,000 g for 15 min). Supernatantswere normalized for total mitochondrial proteins content (BCA™ pro-tein assay kit; Pierce, Rockford, IL) and stored at −80 °C.

Nuclear protein isolation. The crude nuclear fraction was washed inPBS and incubated in buffer containing 10 mM HEPES (pH 7.9),10 mM KCl, 1.5 mM MgCl2, 1% phosphatase and protease inhibitorson ice for 10 min. After centrifugation (12,000 g for 5 min), nuclearpellets resuspended in a lysis buffer (see above) were shortly incu-bated at 95 °C for 5 min. The lysates were briefly sonicated and centri-fuged (15,000 g at 4 °C for 15 min) to remove insoluble materials.

Immunoprecipitation

All steps were carried out at 4 °C if not otherwise mentioned.500 μg proteins from mitochondria preparations (pooled specimenfrom 6 rats of each group) were used for immunoprecipitation (IP)with the IP Kit according the manufacturer's instruction (Pierce,Rockford, IL). 200 μl of immobilized Protein G gel (50%) were addedinto the HandeeTM Spin Cup Columns and washed with binding/washing buffer (b/w buffer). JNK1, JNK2 or JNK3 antibody (1:100)was added to the pre-cleared gel, incubated 1 h at 4 °C on a rockerto form an antibody-gel complex. The antibody-gel complex waswashed with b/w buffer, then 12.5 μl of DSS solution was added andincubated for 30 min at RT to fix the antibody to the gel. After wash-ing with the elution buffer, the complex was incubated with dilutedsamples overnight at 4 °C. The negative control was prepared by incu-bation of the antibody-gel complex with the lysis buffer. On the sec-ond day, the sample-antibody-gel complexes were washed 5 timeswith b/w buffer and the samples were eluted by elution buffer. Theproteins were dissolved in the electrophoresis buffer and loadedonto 12% SDS–polyacrylamide for Western blotting.

Immunodepletion

For the visualization of the individual phosphorylated JNK iso-forms, we carried out double immunodepletion (ID) experiments, asdescribed in the method section of the recent publication (Zhao andHerdegen, 2009). This preparation removes two JNK isoforms andthe remaining one is blotted with anti-phospho-JNK antibody.150 μg of proteins from pooled mitochondria samples (n=6) wereused for ID. All steps were performed at 4 °C.

The samples were added to appropriate Preclearing Matrix for30 min and centrifuged The antibody-IP matrix complex was formedby incubating 1 μg primary antibody with 40 μl of IP matrix in 500 μlof PBS for 2 h. The antibody-IP matrix complex was then washedtwice with cold PBS and incubated with the pre-cleared samplesovernight. After centrifugation, the supernatants were incubated

309Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

with the secondary antibody-IP matrix complex overnight. The pel-lets were removed by centrifugation, the supernatants of double-IDsamples were collected and 15 μg of total double-ID proteins perlane were equally loaded and separated on 12% SDS-polyacrylamidegels for Western blotting. The negative control was prepared by incu-bation of the antibody-IP matrix complex with the lysis buffer.

Western blotting

Fifteen μg of total proteins per lane were loaded and separated on12% SDS-polyacrylamide gels and transferred to PVDF (Millipore) asdescribed in detail elsewhere (Zhao and Herdegen, 2009). The pro-tein levels were expressed as count units per mm2.

Statistics

All experiments and measurements were replicated at least threetimes. Data are expressed as means±SEM. Statistical analysis wascarried out by one-way ANOVA followed by a post-hoc Dunnett testfor comparisons of individual groups against the control group. Stu-dent's t-test for unpaired samples was used for statistical compari-sons between two groups. Statistical significance was accepted atpb0.05.

Acknowledgements

The authors thank Angela Schultz for her excellent technical assis-tance. The work was supported various national and internationalgrants (COST B30, SFB 415-A12).

References

Barnat, M., Enslen, H., Propst, F., Davis, R.J., Soares, S., Nothias, F., 2010. Distinct roles ofc-Jun N-terminal Kinase isoforms in neurite initiation and elongation during axo-nal regeneration. J. Neurosci. 30 (23), 7804–7816.

Becker, E.B., Howell, J., Kodama, Y., Barker, P.A., Bonni, A., 2004. Characterization of thec-Jun N-terminal kinase-BimEL signaling pathway in neuronal apoptosis. J. Neu-rosci. 24 (40), 8762–8770.

Ben-Ari, Y., 1985. Limbic seizure and brain damage produced by kainic acid: mecha-nisms and relevance to human temporal lobe epilepsy. Neuroscience 14 (2),375–403.

Ben-Ari, Y., Cossart, R., 2000. Kainate, a double agent that generates seizures: two de-cades of progress. Trends Neurosci. 23 (11), 580–587.

Besirli, C.G., Wagner, E.F., Johnson Jr., E.M., 2005. The limited role of NH2-terminal c-Junphosphorylation in neuronal apoptosis: identification of the nuclear pore complexas a potential target of the JNK pathway. J. Cell Biol. 170 (3), 401–411.

Bessero, A.C., Chiodini, F., Rungger-Brändle, E., Bonny, C., Clarke, P.G., 2010. Role of thec-Jun N-terminal kinase pathway in retinal excitotoxicity, and neuroprotection byits inhibition. J. Neurochem. 113 (5), 1307–1318.

Björkblom, B., Ostman, N., Hongisto, V., Komarovski, V., Filén, J.J., Nyman, T.A., Kallunki,T., Courtney, M.J., Coffey, E.T., 2005. Constitutively active cytoplasmic c-Jun N-terminal kinase 1 is a dominant regulator of dendritic architecture: role ofmicrotubule-associated protein 2 as an effector. J. Neurosci. 25 (27), 6350–6361.

Björkblom, B., Vainio, J.C., Hongisto, V., Herdegen, T., Courtney, M.J., Coffey, E.T., 2008.All JNKs can kill, but nuclear localization is critical for neuronal death. J. Biol.Chem. 283 (28), 19704–19713.

Bogoyevitch, M.A., Boehm, I., Oakley, A., Ketterman, A.J., Barr, R.K., 2004. Targeting theJNK MAPK cascade for inhibition: basic science and therapeutic potential. Biochim.Biophys. Acta 1697 (1–2), 89–101.

Bogoyevitch, M.A., Ngoei, K.R., Zhao, T.T., Yeap, Y.Y., Ng, D.C., 2010. c-Jun N-terminal ki-nase (JNK) signaling: recent advances and challenges. Biochim. Biophys. Acta 1804(3), 463–475 Mar.

Borsello, T., Clarke, P.G., Hirt, L., Vercelli, A., Repici, M., Schorderet, D.F., Bogousslavsky,J., Bonny, C., 2003. A peptide inhibitor of c-Jun N-terminal kinase protects againstexcitotoxicity and cerebral ischemia. Nat. Med. 9 (9), 1180–1186.

Brecht, S., Simler, S., Vergnes, M., Mielke, K., Marescaux, C., Herdegen, T., 1999. Repet-itive electroconvulsive seizures induce activity of c-Jun N-terminal kinase andcompartment-specific desensitization of c-Jun phosphorylation in the rat brain.Brain Res. Mol. Brain Res. 68 (1–2), 101–108.

Castillo, P.E., Malenka, R.C., Nicoll, R.A., 1997. Kainate receptors mediate a slow post-synaptic current in hippocampal CA3 neurons. Nature 388 (6638), 182–186.

Chen, L., Willis, S.N., Wei, A., Smith, B.J., Fletcher, J.I., Hinds, M.G., Colman, P.M., Day,C.L., Adams, J.M., Huang, D.C., 2005. Differential targeting of prosurvival Bcl-2 pro-teins by their BH3-only ligands allows complementary apoptotic function. Mol.Cell 17 (3), 393–403.

Chipuk, J.E., Green, D.R., 2008. How do BCL-2 proteins induce mitochondrial outermembrane permeabilization? Trends Cell Biol. 18 (4), 157–164.

Chipuk, J.E., Moldoveanu, T., Llambi, F., Parsons, M.J., Green, D.R., 2010. The BCL-2 fam-ily reunion. Mol. Cell 37 (3), 299–310.

Cory, S., Adams, J.M., 2002. The Bcl2 family: regulators of the cellular life-or-deathswitch. Nat. Rev. Cancer 2 (9), 647–656.

Dawson, V.L., Dawson, T.M., 2004. Deadly conversations: nuclear-mitochondrial cross-talk. J. Bioenerg. Biomembr. 36 (4), 287–294.

Deng, X., Xiao, L., Lang, W., Gao, F., Ruvolo, P., May Jr., W.S., 2001. Novel role for JNK as astress-activated Bcl2 kinase. J. Biol. Chem. 276 (26), 23681–23688.

Dietrich, N., Thastrup, J., Holmberg, C., Gyrd-Hansen, M., Fehrenbacher, N., Lademann,U., Lerdrup, M., Herdegen, T., Jäättelä, M., Kallunki, T., 2004. JNK2 mediates TNF-induced cell death in mouse embryonic fibroblasts via regulation of both caspaseand cathepsin protease pathways. Cell Death Differ. 11 (3), 301–313.

Eminel, S., Klettner, A., Roemer, L., Herdegen, T., Waetzig, V., 2004. JNK2 translocates tothe mitochondria and mediates cytochrome c release in PC12 cells in response to6-hydroxydopamine. J. Biol. Chem. 279 (53), 55385–55392.

Engel, T., Plesnila, N., Prehn, J.H., Henshall, D.C., 2011. In vivo contributions of BH3-onlyproteins to neuronal death following seizures, ischemia, and traumatic brain inju-ry. J. Cereb. Blood Flow Metab. 31 (5), 1196–1210.

Eshraghi, A.A., He, J., Mou, C.H., Polak, M., Zine, A., Bonny, C., Balkany, T.J., Van DeWater,T.R., 2006. D-JNKI-1 treatment prevents the progression of hearing loss in a modelof cochlear implantation trauma. Otol. Neurotol. 27 (4), 504–511.

Griffiths, T., Evans, M.C., Meldrum, B.S., 1984. Status epilepticus: the reversibility of cal-cium loading and acute neuronal pathological changes in the rat hippocampus.Neuroscience 12 (2), 557–567.

Haeusgen, W., Boehm, R., Zhao, Y., Herdegen, T., Waetzig, V., 2009. Specific activities ofindividual c-Jun N-terminal kinases in the brain. Neuroscience 161 (4), 951–959.

Henshall, D.C., Araki, T., Schindler, C.K., Lan, J.Q., Tiekoter, K.L., Taki, W., Simon, R.P.,2002. Activation of Bcl-2-associated death protein and counter-response of Aktwithin cell populations during seizure-induced neuronal death. J. Neurosci. 22(19), 8458–8465.

Jessberger, S., Zhao, C., Toni, N., Clemenson Jr., G.D., Li, Y., Gage, F.H., 2007. Seizure-associated, aberrant neurogenesis in adult rats characterized with retrovirus-mediated cell labeling. J. Neurosci. 27 (35), 9400–9407.

Korhonen, L., Belluardo, N., Mudo, G., Lindholm, D., 2003. Increase in Bcl-2 phosphory-lation and reduced levels of BH3-only Bcl-2 family proteins in kainic acid-mediatedneuronal death in the rat brain. Eur. J. Neurosci. 18 (5), 1121–1134.

Kuan, C.Y., Whitmarsh, A.J., Yang, D.D., Liao, G., Schloemer, A.J., Dong, C., Bao, J., Bana-siak, K.J., Haddad, G.G., Flavell, R.A., Davis, R.J., Rakic, P., 2003. A critical role ofneural-specific JNK3 for ischemic apoptosis. Proc. Natl. Acad. Sci. 100 (25),15184–15189.

Lei, K., Davis, R.J., 2003. JNK phosphorylation of Bim-related members of the Bcl2 familyinduces Bax-dependent apoptosis. Proc. Natl. Acad. Sci. U. S. A. 100 (5), 2432–2437.

Liou, A.K., Clark, R.S., Henshall, D.C., Yin, X.M., Chen, J., 2003. To die or not to die forneurons in ischemia, traumatic brain injury and epilepsy: a review on the stress-activated signaling pathways and apoptotic pathways. Prog. Neurobiol. 69 (2),103–142.

Liu, J.R., Zhao, Y., Patzer, A., Staak, N., Boehm, R., Deuschl, G., Culman, J., Bonny, C., Herdegen,T., Eschenfelder, C., 2010. The c-Jun N-terminal kinase (JNK) inhibitor XG-102 en-hances the neuroprotection of hyperbaric oxygen after cerebral ischaemia in adultrats. Neuropathol. Appl. Neurobiol. 36 (3), 211–224.

Meldrum, B.S., 2002. Implications for neuroprotective treatments. Prog. Brain Res. 135,487–495.

Mielke, K., Brecht, S., Dorst, A., Herdegen, T., 1999. Activity and expression of JNK1, p38and ERK kinases, c-Jun N-terminal phosphorylation, and c-jun promoter binding inthe adult rat brain following kainate-induced seizures. Neuroscience 91 (2),471–483.

Miller, W.E., McDonald, P.H., Cai, S.F., Field, M.E., Davis, R.J., Lefkowitz, R.J., 2001. Iden-tification of a motif in the carboxyl terminus of beta-arrestin2 responsible for acti-vation of JNK3. J. Biol. Chem. 276 (30), 27770–27777.

Murphy, B., Dunleavy,M., Shinoda, S., Schindler, C., Meller, R., Bellver-Estelles, C., Hatazaki,S., Dicker, P., Yamamoto, A., Koegel, I., Chu, X., Wang,W., Xiong, Z., Prehn, J., Simon, R.,Henshall, D., 2007. Bcl-w protects hippocampus during experimental status epilepti-cus. Am. J. Pathol. 171 (4), 1258–1268.

Murphy, B.M., Engel, T., Paucard, A., Hatazaki, S., Mouri, G., Tanaka, K., Tuffy, L.P., Jime-nez-Mateos, E.M., Woods, I., Dunleavy, M., Bonner, H.P., Meller, R., Simon, R.P.,Strasser, A., Prehn, J.H., Henshall, D.C., 2010. Contrasting patterns of Bim inductionand neuroprotection in Bim-deficient mice between hippocampus and neocortexafter status epilepticus. Cell Death Differ. 17 (3), 459–468.

Nadler, J.V., Perry, B.W., Cotman, C.W., 1978. ntraventricular kainic acid preferentiallydestroys hippocampal pyramidal cells. Nature 271 (5646), 676–677.

Olney, J.W., Rhee, V., Ho, O.L., 1974. Kainic acid: a powerful neurotoxic analogue of glu-tamate. Brain Res. 77 (3), 507–512.

Pirianov, G., Brywe, K.G., Mallard, C., Edwards, A.D., Flavell, R.A., Hagberg, H., Mehmet,H., 2007. Deletion of the c-Jun N-terminal kinase 3 gene protects neonatal miceagainst cerebral hypoxic-ischaemic injury. J. Cereb. Blood Flow Metab. 27 (5),1022–1032.

Polster, B.M., Fiskum, G., 2004. Mitochondrial mechanisms of neural cell apoptosis. J.Neurochem. 90 (6), 1281–1289.

Putcha, G.V., Moulder, K.L., Golden, J.P., Bouillet, P., Adams, J.A., Strasser, A., Johnson,E.M., 2001. Induction of BIM, a proapoptotic BH3-only BCL-2 family member, iscritical for neuronal apoptosis. Neuron 29 (3), 615–628.

Putcha, G.V., Le, S., Frank, S., Besirli, C.G., Clark, K., Chu, B., Alix, S., Youle, R.J., LaMarche,A., Maroney, A.C., Johnson Jr., E.M., 2003. JNK-mediated BIM phosphorylation po-tentiates BAX-dependent apoptosis. Neuron 38 (6), 899–914.

310 Y. Zhao et al. / Molecular and Cellular Neuroscience 49 (2012) 300–310

Racine, R.J., 1972. Modification of seizure activity by electrical stimulation. II. Motor aseizure. Electroencephalogr. Clin. Neurophysiol. 32 (3), 281–294.

Repici, M., Mare, L., Colombo, A., Ploia, C., Sclip, A., Bonny, C., Nicod, P., Salmona, M.,Borsello, T., 2009. c-Jun N-terminal kinase binding domain-dependent phosphory-lation of mitogen-activated protein kinase kinase 4 and mitogen-activated proteinkinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and ex-tracellular signal-regulated kinase pathways in cortical neurons. Neuroscience 159(1), 94–103.

Sanna, M.G., da Silva Correia, J., Ducrey, O., Lee, J., Nomoto, K., Schrantz, N., Deveraux,Q.L., Ulevitch, R.J., 2002. IAP suppression of apoptosis involves distinct mecha-nisms: the TAK1/JNK1 signaling cascade and caspase inhibition. Mol. Cell. Biol. 22(6), 1754–1766.

Schild, L., Reinheckel, T., Reiser, M., Horn, T.F., Wolf, G., Augustin, W., 2003. Nitric osideproduced in rat liver mitochondria causes oxidative stress and impairment of res-piration after transient hypoxia. FASEB J. 17 (15), 2194–2201.

Schmued, L.C., Stowers, C.C., Scallet, A.C., Xu, L., 2005. Fluoro-Jade C results in ultrahigh resolution and contrast labeling of degerating neurons. Brain Res. 1035(1), 24–31.

Shinoda, S., Schindler, C.K., Meller, R., So, N.K., Araki, T., Yamamoto, A., Lan, J.Q., Taki,W., Simon, R.P., Henshall, D.C., 2004. Bim regulation may determine hippocampalvulnerability after injurious seizures and in temporal lobe epilepsy. J. Clin. Invest.113 (7), 1059–1068.

Spigolon, G., Veronesi, C., Bonny, C., Vercelli, A., 2010. c-Jun N-terminal kinase signalingpathway in excitotoxic cell death following kainic acid-induced status epilepticus.Eur. J. Neurosci. 31 (7), 1261–1272.

Tararuk, T., Ostman, N., Li, W., Björkblom, B., Padzik, A., Zdrojewska, J., Hongisto, V.,Herdegen, T., Konopka, W., Courtney, M.J., Coffey, E.T., 2006. JNK1 phosphorylationof SCG10 determines microtubule dynamics and axodendritic length. J. Cell Biol.173 (2), 265–277.

Ventura, J.J., Hübner, A., Zhang, C., Flavell, R.A., Shokat, K.M., Davis, R.J., 2006. Chemicalgenetic analysis of the time course of signal transduction by JNK. Mol. Cell. 21 (5),701–710.

Waetzig, V., Herdegen, T., 2003. The concerted signaling of ERK1/2 and JNKs is essentialfor PC12 cell neuritogenesis and converges at the level of target proteins. Mol. Cell.Neurosci. 24 (1), 238–249.

Waetzig, V., Herdegen, T., 2005. Context-specific inhibition of JNKs: overcoming the di-lemma of protection and damage. Trends Pharmacol. Sci. 26 (9), 455–461.

Waetzig, V., Zhao, Y., Herdegen, T., 2006. The bright side of JNKs-Multitalented media-tors in neuronal sprouting, brain development and nerve fiber regeneration. Prog.Neurobiol. 80 (2), 84–97.

Waetzig, V.,Wacker, U., Haeusgen,W., Björkblom, B., Courtney,M.J., Coffey, E.T., Herdegen,T., 2009. Concurrent protective and destructive signaling of JNK2 in neuroblastomacells. Cell. Signal. 21 (6), 873–880.

Westerlund, N., Zdrojewska, J., Courtney, M.J., Coffey, E.T., 2008. Superior cervicalganglion-10 protein as a molecular effector of c-Jun N-terminal kinase 1: implica-tions for the therapeutic targeting of Jun N-terminal kinase in nerve regeneration.Expert Opin. Ther. Targets 12 (1), 31–43.

Yang, D.D., Kuan, C.Y., Whitmarsh, A.J., Rincón, M., Zheng, T.S., Davis, R.J., Rakic, P., Flavell,R.A., 1997. Absence of excitotoxicity-induced apoptosis in the hippocampus of micelacking the Jnk3 gene. Nature 389 (6653), 865–870.

Zhao, Y., Herdegen, T., 2009. Cerebral ischemia provokes a profound exchange of acti-vated JNK isoforms in brain mitochondria. Mol. Cell. Neurosci. 41 (2), 186–195.

Zhao, Y., Patzer, A., Herdegen, T., Gohlke, P., Culman, J., 2006. Activation of cerebral per-oxisome proliferator-activated receptors gamma promotes neuroprotection by at-tenuation of neuronal cyclooxygenase-2 overexpression after focal cerebralischemia in rats. FASEB J. 20 (8), 1162–1175.

Zhou, Q., Lam, P.Y., Han, D., Cadenas, E., 2008. c-Jun N-terminal kinase regulates mito-chondrial bioenergetics by modulating pyruvate dehydrogenase activity in prima-ry cortical neurons. J. Neurochem. 104 (2), 325–335.

Zhuang, Z.Y., Wen, Y.R., Zhang, D.R., Borsello, T., Bonny, C., Strichartz, G.R., Decosterd, I.,Ji, R.R., 2006. A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanicalallodynia after spinal nerve ligation: respective roles of JNK activation in primarysensory neurons and spinal astrocytes for neuropathic pain development andmaintenance. J. Neurosci. 26 (13), 3551–3560.