Neuroprotection, Brain Damage and Behavior Inhibition of the JNK/AP-1 pathway reduces neuronal death and improves behavioral outcome after neonatal hypoxic–ischemic brain injury Cora H. Nijboer a , Michael A. van der Kooij a,b , Frank van Bel b , Frauke Ohl c,d , Cobi J. Heijnen a, * , Annemieke Kavelaars a a Laboratory of Psychoneuroimmunology, University Medical Center Utrecht, KC03.068.0, Lundlaan 6, 3584 EA Utrecht, The Netherlands b Department of Neonatology, University Medical Center Utrecht, Utrecht, The Netherlands c Department of Animals in Science and Society, University of Utrecht, Utrecht, The Netherlands d Rudolf Magnus Institute of Neurosciences, Utrecht, The Netherlands article info Article history: Received 25 August 2009 Received in revised form 11 September 2009 Accepted 12 September 2009 Available online 17 September 2009 Keywords: JNK MAP kinase Neonatal hypoxia–ischemia Neuroprotection Apoptosis Behavior Transduction peptide Smac/DIABLO Calpain AP-1 Inflammation abstract Perinatal hypoxic–ischemic (HI) brain damage continues to be a major clinical problem. We investigated the contribution of the MAP kinase c-Jun N-terminal kinase (JNK), to neonatal HI brain damage. JNK reg- ulates several transcriptional (via AP-1 activation) and non-transcriptional processes involved in brain damage such as inflammation and cell death/survival. P7 rats were subjected to HI by unilateral carotid artery occlusion and hypoxia. HI-induced activation of cerebral AP-1 peaked at 3–6 h post-HI. Intraperitoneal administration of the JNK-inhibitor TAT-JBD immediately after HI prevented AP-1 activation. TAT-JBD treatment within 3 h after HI reduced early neuronal damage by 30%. JNK/AP-1 inhibition did not reduce HI-induced cytokine/chemokine expres- sion. Analysis of indicators of apoptotic cell death revealed that TAT-JBD markedly reduced the HI- induced increase in active caspase 3. However, the upstream mediators of apoptosis: active caspase 8, cleaved Bid, mitochondrial cytochrome c release and caspase 9 cleavage were not reduced after TAT- JBD. TAT-JBD inhibited the HI-induced increase in Smac/DIABLO, an inhibitor of IAPs that prevent activa- tion of caspase 3. TAT-JBD treatment also reduced cleavage of a-fodrin, indicating that calpain-mediated brain damage was reduced. Neuroprotection by TAT-JBD treatment was long-lasting as gray- and white matter damage was diminished by 50% at 14 weeks post-HI concomitantly with marked improvement of sensorimotor behavior and cognitive functioning. In conclusion, JNK inhibition by TAT-JBD treatment reduced neonatal HI brain damage with a therapeu- tic window of 3 h and long-lasting anatomical and behavioral improvements. We propose that inhibition of mitochondrial Smac/DIABLO release and calpain activation contribute to neuroprotection by TAT-JBD. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Perinatal hypoxia–ischemia (HI) is a major risk factor for human neonatal mortality and development of major neurodevelopmental disabilities (Volpe, 2001; Ferriero, 2004). At present, no effective pharmacological treatment to combat perinatal HI brain injury is available for these infants (Perlman, 2006). Excitotoxicity, inflam- mation and apoptosis are the major pathophysiological mecha- nisms that contribute to cerebral injury after HI (Vexler and Ferriero, 2001). Mitogen-activated protein (MAP) kinases are essential kinases that respond to a plethora of extracellular stimuli and play a criti- cal role in the regulation of several cellular processes like growth, proliferation, differentiation and cell survival/apoptosis (Cobb, 1999). c-Jun N-terminal kinases (JNKs), also known as stress-acti- vated protein kinases (SAPKs) as their activation is associated with a wide variety of environmental stressors, represent an important subfamily of the MAP kinase group (Bogoyevitch et al., 2004; Karin and Gallagher, 2005). JNKs were originally identified by their abil- ity to phosphorylate c-Jun, the dominant component of the tran- scription factor AP-1, which is a DNA binding dimer composed of c-Jun combined with c-Fos, Maf or ATF subunits. JNK-mediated phosphorylation of c-Jun enhances the ability of c-Jun/AP-1 to in- duce transcription of numerous target genes involved in prolifera- tion, cell survival, cell death, DNA repair, metabolism and inflammation (Bogoyevitch and Kobe, 2006). Nowadays, JNKs are also acknowledged for their capacity to regulate cellular function through phosphorylation of non-nuclear proteins. For example, JNKs have been suggested to play a key role in regulation of apop- 0889-1591/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2009.09.008 * Corresponding author. Fax: +31 88 755 5311. E-mail address: C.Heijnen@umcutrecht.nl (C.J. Heijnen). Brain, Behavior, and Immunity 24 (2010) 812–821 Contents lists available at ScienceDirect Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi