Mitogen and stress response kinase-1 (MSK1) mediates excitotoxic induced death of hippocampal neurones Jane P. Hughes,* ,1 Penny C. Staton,* ,1 Marc G. Wilkinson,* ,2 Paul J. L. M. Strijbos,* Stephen D. Skaper,* J. Simon C. Arthur and Alastair D. Reithà *Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, UK  MRC Protein Phosphorylation Unit, MSI/WTB Complex, School of Life Sciences, University of Dundee, Dundee, UK àHigh Throughput Biology, Discovery Research, GlaxoSmithKline Medicines Resarch Centre, Stevenage, Hertfordshire, UK Abstract Activation of the mitogen-activated protein kinase (MAPK/ ERK) signal transduction pathway may mediate excitotoxic neuronal cell death in vitro and during ischemic brain injury in vivo. However, little is known, of the upstream regulation or downstream consequences of ERK activation under these conditions. Magnesium removal has been described to induce hyperexcitability and degeneration in cultured hippocampal neurones. Here, we show that neurotoxicity evoked by Mg 2+ removal in primary hippocampal neurones stimulates ERK, but not p38, phosphorylation. Removal of Mg 2+ also resulted in induction of the MAPK/ERK substrate mitogen- and stress- response kinase 1 (MSK1) and induced phosphorylation of the MSK1 substrate, the transcription factor cAMP response ele- ment binding protein (CREB). Neuronal death and phos- phorylation of components in this cascade were inhibited by the Raf inhibitor SB-386023, by the MEK inhibitor U0126, or by the MSK1 inhibitors H89 and Ro318220. Importantly, this form of cell death was inhibited in hippocampal neurones cultured from MSK1–/– mice and inhibitors of Raf or MEK had no additive neuroprotective effect. Together, these data indi- cate that MSK1 is a physiological kinase for CREB and that this activity is an essential component of activity-dependent neuronal cell death. Keywords: cAMP-responsive element binding protein, excitotoxicity, hippocampal neurones, mitogen and stress- response kinase-1, mitogen-activated protein kinase, neuro- protection. J. Neurochem. (2003) 86, 25–32. Excessive glutamatergic neurotransmission, particularly when mediated by the NMDA subtype of glutamate recep- tors, is thought to underlie neuronal death in several types of acute brain injury, including cerebral ischaemia, hypogly- cemia, seizures and mechanical trauma (Rothman and Olney 1987; Choi 1988). While the immediate events in excitotoxic injury, such as NMDA receptor activation and consequent Ca 2+ influx, are well-established (Tymianski et al. 1993; Choi 1994) the subsequent downstream events that result in neuronal death remain less clear. Signal transduction path- ways that relay extracellular signals to the nucleus via a series of phosphorylation events are strong candidates for mediating the downstream effects of excitotoxic injury. Amongst these, evidence specifically suggests a role for MAPK/ERK signalling pathways in this process. MAPK/ ERK phosphorylation has been shown to occur concomit- antly with excitotoxic injury in neurones both in vivo (Kurino et al. 1995; Alessandrini et al. 1999; Namura et al. 2001) and in vitro (Murray et al. 1998; Skaper et al. 2001). Whilst MAPK activation has been reported to protect certain populations of neurones against specific insults (Meyer- Franke et al. 1995; Anderson and Tolkovsky 1999; Received May 29, 2002; revised manuscript received February 17, 2003; accepted February 20, 2003. Address correspondence and reprint requests to Alastair D. Reith, Discovery Research, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. E-mail: Alastair_D_Reith@gsk.com 1 These authors contributed equally to this work. 2 The present address of Marc G. Wilkinson is Millenium Pharmaceu- ticals, Granta Park, Cambridge, UK. Abbreviations used: CREB, cAMP-responsive element binding pro- tein; DIC, days in culture; GSK-3, glycogen synthase kinase 3; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MAPKAP-K1/ 2/3, MAPK-activated protein kinase 1/2/3; MSK1, mitogen and stress- response kinase-1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetr- azolium bromide; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; ROCK, Rho-dependent protein kinase; S6K, p70 S6 kinase; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline. Journal of Neurochemistry, 2003, 86, 25–32 doi:10.1046/j.1471-4159.2003.01830.x Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 25–32 25