S100B POTENTLY ACTIVATES p65/c-Rel TRANSCRIPTIONAL COMPLEXES IN HIPPOCAMPAL NEURONS: CLINICAL IMPLICATIONS FOR THE ROLE OF S100B IN EXCITOTOXIC BRAIN INJURY D. KÖGEL, a * M. PETERS, b H.-G. KÖNIG, a S. M. A. HASHEMI, a N. T. BUI, c V. AROLT, b M. ROTHERMUNDT b AND J. H. M. PREHN a,d a Center for Neurology and Neurosurgery, Experimental Neurosurgery, Johann Wolfgang Goethe University Clinics, D-60590 Frankfurt, Germany b Department of Psychiatry and Psychotherapy, University Muenster Clinics, D-48149 Muenster, Germany c Interdisciplinary Center of Clinical Research, University Muenster Clinics, D-48149 Muenster, Germany d Department of Physiology, Royal College of Surgeons in Ireland, Dublin 2, Ireland Abstract —Increased serum levels of S100B are positively correlated with multiple forms of CNS damage, such as stroke, CNS trauma and neurodegenerative diseases, but also in psychiatric disorders. However, it is currently not known whether increased serum levels of S100B reflect a neuroregenerative or neurodegenerative response. Since glutamate receptor overactivation (excitotoxicity) may con- tribute to neuronal pathology in psychiatric disorders, we investigated the effect of S100B on N-methyl-D-aspartate (NMDA)-induced neuronal cell death. Here we demonstrate that very low concentrations of S100B significantly protect primary rat hippocampal neurons against NMDA toxicity by activation of transcription factors of the Rel/nuclear factor B (NF-B) family. Further experiments suggest that i) S100B activated expression of the receptor of advanced glycation products (RAGE) gene in neurons and ii) S100B induced a unique composition of the active NF-B complex consisting of the p65 and c-Rel subunits suggesting a novel mechanism for NF-B activation involved in S100B-mediated neuropro- tection. Our data suggest that S100B secreted during the glial response to brain injury potently activates p65/c-Rel in a RAGE-dependent manner and may exert neuroprotective and neuroregenerative effects in psychiatric disorders. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hippocampus, nerve tissue protein S100, nuclear factor-B, glutamate. S100B, a member of the S100 family of EF-hand Ca 2+ - binding proteins, is produced in the brain mainly by astro- cytes and exerts paracrine and autocrine effects on neu- rons and glia (Zimmer et al., 1995; Donato, 2001, 2003; Rothermundt et al., 2003). Increased serum levels of S100B are associated with a wide variety of acute and chronic forms of CNS damage, including ischemic stroke, CNS trauma and neurodegenerative diseases such as Alzheimer’s disease, Creutzfeld-Jacob disease and amyo- trophic lateral sclerosis (Rothermundt et al., 2003). Inter- estingly, significantly elevated S100B serum levels have also been detected in psychiatric disorders, such as schizophrenia and major depression (Rothermundt et al., 2001, 2003). Depending on its concentration, secreted glial S100B has been suggested to exert both neurotrophic and neurotoxic effects (Huttunen et al., 2000). The N- methyl-D-aspartate (NMDA) receptor is a receptor for glu- tamate, the major excitatory neurotransmitter in the CNS, and is involved in a wide range of processes in the brain, such as neuroplasticity, synaptic remodelling, memory, but also in excitotoxic cell death caused by NMDA receptor overactivation and subsequent Ca 2+ overloading of neu- rons (Goff and Wine, 1997). Dysfunction of the NMDA receptor may be implicated in the pathophysiology of psychiatric diseases, such as schizophrenia, major depression, posttraumatic stress disorder, and alcoholism (Heresco-Levy and Javitt, 1998; Mohn et al., 1999; Olney et al., 1999). In this study, we show that 1) very low concentrations of S100B are sufficient to exert potent, nuclear factor B (NFB)-dependent neuroprotective ef- fects against NMDA toxicity and 2) S100B induces neuro- nal NFB transcriptional complexes with a unique p65/c- Rel subunit composition. EXPERIMENTAL PROCEDURES Materials S100B (Sigma-Aldrich, Deisenhofen, Germany) was dissolved in phosphate-buffered saline (PBS) at a final concentration of 10 mg/ml and stored in aliquots at -80 °C. NMDA, LY294002, TNF- and Hoechst 33258 were also purchased from Sigma- Aldrich. Trypan blue was from Bio-Whittaker (Wokingham, UK), lactacystin from Biomol (Hamburg, Germany) and PD98059 and A23187 were from Alexis (Grünberg, Germany). [- 32 P] ATP (10 MBq) was purchased from Hartmann Analytics (Braunschweig, Germany). T4 polynucleotide kinase and NF-B oligo- nucleotide (1.75 pmol/l) were from Promega (Mannheim, Germany). Poly(dI-dC) was purchased from Roche Diagnostics GmbH (Mannheim, Germany). The RelA/p65 (sc-109x) and c-Rel (sc-70x) antibodies were from Santa Cruz Biotechnology (Heidel- berg, Germany). All other chemicals came in analytical grade purity from Merck (Darmstadt, Germany), Promega or Roth (Karlsruhe, Germany). *Correspondence to: D. Kögel, Experimental Neurosurgery, Center for Biological Chemistry, Johann Wolfgang Goethe University Clinics, Theodor-Stern-Kai 7, HS 25 B, D-60590 Frankfurt am Main, Germany. Tel: +49-69-6301-6923; fax: +49-69-6301-5575. E-mail address: koegel@em.uni-frankfurt.de (D. Kögel). Abbreviations: EMSA, electrophoretic mobility shift assay; ERP, event- related potential; HBS, HEPES-buffered saline; NF-B, nuclear factor B; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; RAGE, receptor of advanced glycation products; RT-PCR, reverse transcription–polymerase chain reaction. Neuroscience 127 (2004) 913–920 0306-4522/04$30.00+0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.06.013 913