A highly sulfated chondroitin sulfate preparation, CS-E, prevents excitatory amino acid-induced neuronal cell death Yoshiaki Sato,* ,   , à Keiko Nakanishi,* Yoshihito Tokita,* Hiroko Kakizawa,* ,  Michiru Ida,* Hiroshi Maeda,§ Fumiko Matsui,* Sachiko Aono,* Akiko Saito,* ,   , à Yoshiyuki Kuroda,* Masahiro Hayakawa,¶ Seiji Kojima and Atsuhiko Oohira* *Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan  Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan àDepartment of Neonatology, Central Hospital, Aichi Human Service Center, Kasugai, Aichi, Japan §Central Research Laboratories, Seikagaku Corporation., Higashiyamato, Tokyo, Japan ¶Maternity and Perinatal Care Center, Nagoya University Hospital, Nagoya, Japan Chondroitin sulfate (CS) is the major constituent of the extracellular matrix of the CNS. Evidence has been accumulated to support the idea that CS is involved in various cellular events in the formation and maintenance of the neural network (Bandtlow and Zimmermann 2000; Sugahara et al. 2003; Ida et al. 2006). The CS chain consists of repeating disaccharide units of -4GlcUAb1- 3GalNAcb1-, which are commonly sulfated on GalNAc and rarely on GlcUA residues, and is highly heterogeneous in structure (Lamari and Karamanos 2006). There are six CS-disaccharide units with a different number and position of sulfation; O-, A-, B-, C-, D-, and E-unit (Nandini and Sugahara 2006). Of these disaccharide units, D- and E- units contain two sulfate residues, and CS polysaccharides rich in these highly sulfated disaccharides (CS-D and -E, respectively) have been shown to bind to several growth factors (Ueoka et al. 2000; Deepa et al. 2002) and to be involved in neurite outgrowth (Nadanaka et al. 1998; Clement et al. 1999); however, the neuroprotective activity of CS has not yet been examined. In addition, the structure–function relationship of CS has been understood very poorly. Received July 25, 2007; revised manuscript October 12, 2007; accepted October 22, 2007. Address correspondence and reprint requests to Atsuhiko Oohira, PhD, Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi 480-0392, Japan. E-mail: atsu48@aichi-med-u.ac.jp Yoshiaki Sato and Keiko Nakanishi contributed equally to this work. Abbreviations used: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxaz- olepropionic acid; CS, chondroitin sulfate; CSPG, chondroitin sulfate proteoglycan; DIV, days in vitro; LDH, lactate dehydrogenase; MAP2, microtubule-associated protein 2; PBS, phosphate-buffered saline; PC, positive control; TBS, tris-buffered saline. Abstract Chondroitin sulfate (CS) is a major microenvironmental mol- ecule in the CNS, and there have been few reports about its neuroprotective activity. As neuronal cell death by excitotox- icity is a crucial phase in many neuronal diseases, we examined the effect of various CS preparations on neuronal cell death induced by the excitotoxicity of glutamate analogs. CS preparations were added to cultured neurons before and after the administration of glutamate analogs. Then, the extents of both neuronal cell death and survival were esti- mated. Pre-administration of a highly sulfated CS preparation, CS-E, significantly reduced neuronal cell death induced by not only NMDA but also (S)-a-amino-3-hydroxy-5-methyl-4-isox- azolepropionic acid or kainate. Neither CS preparations other than CS-E nor other highly sulfated polysaccharides such as heparin and dextran sulfate exerted any neuroprotective effects. NMDA-induced current in neurons was not changed by pre-administration of CS-E, but the pattern of protein- tyrosine phosphorylation was changed. In addition, the ele- vation of caspase 3 activity was significantly suppressed in CS-E-treated neurons. These results indicate that CS-E pre- vents neuronal cell death mediated by various glutamate receptors, and suggest that phosphorylation-related intracel- lular signals and the suppression of caspase 3 activation are implicated in neuroprotection by CS-E. Keywords: caspase 3, chondroitin sulfate, excitotoxicity, glutamate receptor, neuronal death, neuroprotection. J. Neurochem. (2008) 104, 1565–1576. d JOURNAL OF NEUROCHEMISTRY | 2008 | 104 | 1565–1576 doi: 10.1111/j.1471-4159.2007.05107.x Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 1565–1576 1565