S100B IS EXPRESSED IN, AND RELEASED FROM, OLN-93 OLIGODENDROCYTES: INFLUENCE OF SERUM AND GLUCOSE DEPRIVATION J. STEINER, a * H.-G. BERNSTEIN, a B. BOGERTS, a T. GOS, a,b C. RICHTER-LANDSBERG, c M. T. WUNDERLICH a,d AND G. KEILHOFF e a Department of Psychiatry, University of Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany b Institute of Forensic Medicine, Medical University of Gdansk, ul. Debowa 23, 80-204 Gdansk, Poland c Department of Biology and Molecular Neurobiology, University of Oldenburg, 26111 Oldenburg, Germany d Department of Neurology, University of Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany e Institute of Medical Neurobiology, University of Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany Abstract—S100B (member of a family of proteins that are 100% soluble in ammonium sulfate at neutral pH) has been widely used as astrocyte marker in animal models and in human brain diseases. Recent studies revealed S100B-immunopositivity in oligodendrocytes and O2A oligodendroglial progenitor cells. It is unknown, however, if oligodendrocytes produce S100B themselves, or if the S100B-immunolabeling is caused by bind- ing or absorption of the protein. To address this question, S100B expression and protein release were analyzed in a highly pure oligodendrocytic OLN-93 cell line (from rat), in the astrocytic C6 cell line (from rat) and primary astrocytes. S100B was gene expressed in all cultures, as revealed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. OLN-93 cells and glial fibrillary acidic pro- tein (GFAP)-negative astrocytes expressed the multiligand re- ceptor for advanced glycation end products (RAGE). S100B protein levels were determined in supernatants and cell homog- enates by immunoluminometry under normal conditions and after serum and glucose deprivation (SGD). SGD led to a sev- eral-fold increased release of S100B (after 6 and 24 h), which was particularly pronounced in primary astrocytes. Increased S100B in cell homogenates was most notable in OLN-93 cells under SGD, indicating activated S100B synthesis. These cells also showed the highest percentage of dead cells, as deter- mined by propidium iodide-positivity, after SGD. Incubation with 0.5, 2 and 5 g/l exogenous S100B was not toxic to OLN-93 cells. In conclusion, OLN-93 cells produce more S100B under SGD than astrocytes and are more susceptible to cell death upon SGD, which provokes leakage of S100B. Our data indicate ac- tive S100B secretion from astrocytes under SGD since highly elevated levels of S100B were detected in the supernatant de- spite a low percentage of dead cells. The experimental results provide further evidence for a production/release of S100B in/ from oligodendrocytes, e.g. in metabolic stress conditions like cerebral ischemia. Studies on S100B in bodily fluids should be carefully interpreted in order to avoid misleading hypotheses concerning the specific involvement of astrocytes, due to the various cellular sources of S100B. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: astrocytes, oligodendrocytes, OLN-93, RAGE, S-100B, S100 beta. S100B (10.5 kDa; gene locus on human chromosome 21q22.3; 100% soluble in ammonium sulfate at neutral pH) is a calcium, copper and zinc ion binding protein that is a member of the S100-calmodulin-troponin superfamily, and was primarily found in high abundance within the nervous system (Moore, 1965; Moore and Perez, 1967). A number of intracellular growth-associated target proteins have been identified for S100B, such as growth-associated pro- tein 43, the regulatory domain of protein kinase C, the anti-apoptotic factor Bcl-2 (product of the B-cell lymphoma/ leukemia 2 gene) and the tumor-suppressor protein P53 (with a molecular mass of 53,000 Da) (Donato, 2001). S100B also regulates protein ubiquitination and the as- sembly of cytoskeleton components such as microtubules, glial fibrillary acidic protein (GFAP) and vimentin (Bianchi et al., 1994; Donato, 2001; Nowotny et al., 2003). Addi- tionally, S100B is a secretory protein, and it exhibits cyto- kine-like activities that mediate interactions among glial cells and between glial cells and neurons. Nanomolar lev- els of S100B stimulate neurite growth and promote neuro- nal survival, while micromolar levels result in opposite effects (van Eldik and Wainwright, 2003). This cytokine- like activity is transduced, in part, by the receptor for ad- vanced glycation end products (RAGE) and nuclear factor kappa B (Donato, 2001). Elevated levels of S100B in cerebrospinal fluid and peripheral blood have been observed in Alzheimer’s dis- ease, stroke, traumatic brain injury, meningoencephalitis, mood disorders and schizophrenia (Lins et al., 2005; Pelinka et al., 2004; Peskind et al., 2001; Schroeter et al., 2002; Steiner et al., 2006; Wunderlich et al., 2004). In these contexts, high S100B levels in body fluids have been thought to be the result of elevated secretion from astro- *Corresponding author. Tel: +49-391-6715019; fax: +49-391-6715223. E-mail address: johann.steiner@med.ovgu.de (J. Steiner). Abbreviations: C6, an astrocytic cell line (from rat); DAPI stain, a nuclear staining with 4=,6-diamidin-2=-phenylindol-dihydrochloride; DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediami- netetraacetic acid; GAPDH, glyceraldehyde-3-phosphate dehydroge- nase; GFAP, glial fibrillary acidic protein; LDH, lactate dehydrogenase; OLN-93, an oligodendrocytic cell line (from rat); O2A progenitor cells, immature oligodendrocytes; p75 NTR , p75 neurotrophin receptor; PI, propidium iodide; RAGE, receptor for advanced glycation end prod- ucts; RT-PCR, reverse transcriptase polymerase chain reaction; SGD, serum and glucose deprivation; S100B, member of a family of proteins that are 100% soluble in ammonium sulfate at neutral pH. Neuroscience 154 (2008) 496 –503 0306-4522/08$32.00+0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.03.060 496