A highly sulfated chondroitin sulfate preparation ... - Wiley Online Library

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JOURNAL OF NEUROCHEMISTRY

| 2008 | 104 | 1565–1576

doi: 10.1111/j.1471-4159.2007.05107.x

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

Abstract Chondroitin sulfate (CS) is a major microenvironmental molecule in the CNS, and there have been few reports about its neuroprotective activity. As neuronal cell death by excitotoxicity 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 estimated. 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-isoxazolepropionic 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 proteintyrosine phosphorylation was changed. In addition, the elevation of caspase 3 activity was significantly suppressed in CS-E-treated neurons. These results indicate that CS-E prevents neuronal cell death mediated by various glutamate receptors, and suggest that phosphorylation-related intracellular 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.

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 -4GlcUAb13GalNAcb1-, 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 Eunits 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: [email protected] Yoshiaki Sato and Keiko Nakanishi contributed equally to this work. Abbreviations used: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic 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.

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 1565–1576

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The excitotoxicity of glutamate has been causally linked with the elevation of intracellular Ca2+ concentration in neurons (Choi 1987). Excessive glutamate leads to the overactivation of glutamate receptors and mediates neuronal degeneration. There are two classes of ionotropic glutamate receptor, namely NMDA and non-NMDA receptors. NonNMDA receptor is further subclassified into a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors (Nakanishi 1992; Meldrum 2000). The activation of non-NMDA type glutamate receptors mediates Na+ influx and membrane depolarization. Through voltagedependent Ca2+ channels, depolarization induces Ca2+ influx, while some non-NMDA receptors such as Ca2+-permeable non-NMDA receptors can mediate Ca2+ influx directly (Weiss and Sensi 2000). On the other hand, glutamate and previous membrane depolarization are required to open the NMDA channel, which mediates Ca2+ influx directly. Intracellular Ca2+ elevation initiates caspase activation and mediates neuronal cell death (Du et al. 1997). Such glutamate excitotoxicity has been reported to be pathologically linked to neurodegeneration disorders such as neonatal hypoxia–ischemia, stroke, spinal cord injury, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease (Choi 1988; Meldrum and Garthwaite 1990; Beal 1992; Lipton and Rosenberg 1994). Many patients suffer badly from these diseases; for example, perinatal hypoxia–ischemia leads to millions of neonatal deaths and neurological disadvantages (Levene et al. 1999; Lawn et al. 2005), although very few effective treatments have been established for these diseases and it is therefore urgent to develop a novel therapy for these diseases. Protection of neuronal cells from the excitotoxicity of glutamate, a major excitatory neurotransmitter, could be a useful method to prevent neurodegeneration in these diseases. In this work, we show that a highly sulfated CS preparation, CS-E, exerts a neuroprotective effect on neurons treated with various glutamate analogs.

Materials and methods Materials The following CS preparations were purchased from Seikagaku Corp. (Tokyo, Japan): CS-A from whale cartilage, CS-B (or dermatan sulfate) from pig skin, CS-C from shark cartilage, CS-D from shark cartilage, and CS-E from squid cartilage. Heparin from porcine intestinal mucosa was purchased from Sigma-Aldrich (St Louis, MO, USA). Dextran sulfate was also obtained from SigmaAldrich. Partial degradation of CS-E and fractionation of degradation products Chondroitin sulfate-E (75 kDa; 1 g) was dissolved in 50 mL of 50 mmol/L phosphate buffer, pH 5.3. This solution was incubated with 1 000 000 units (international turbidity reducing unit; Dorfman

1955) of hyaluronidase from sheep testes (type V; E.C. 3.2.1.35; Sigma-Aldrich) for 16 h at 37C. The reaction mixture was applied on a Dowex 1 · 2 column (bed volume = 100 mL; Dow Chemical Co., Midland, MI, USA), and the column was washed with 1 mol/L NaCl. Degradation products of CS-E were eluted from the column with a linear salt gradient from 1 to 3.5 mol/L NaCl. The amount of hexuronate in each fraction was measured by the carbazole method of Bitter and Muir (1962). The hexuronate-containing peak fraction was applied to HPLC with a sequential gel permeable column system composed of G4500PWXL, G3000PWXL, and G2500PWXL (Toso, Tokyo, Japan). Peaks of CS-E-degradation products were detected using both a UV monitor (at 210 nm) and a refractive index monitor. The molecular sizes of these degradation products were estimated from the elution positions of authentic CS preparation of 1, 7, and 10 kDa. Fractions with an average molecular size of 3 and 5 kDa were pooled individually and concentrated using a rotary evaporator. Each degradation product was applied to Sephadex G-25 (GE Healthcare, Uppsala, Sweden) and eluted by distilled water to remove salt. Then, the products were lyophilized. To prepare a degradation product of 25 kDa, CS-E (75 kDa; 1 g) dissolved in 50 mL of 50 mmol/L phosphate buffer, pH 5.3, was incubated with 5000 units of the hyaluronidase for 16 h at 37C. The molecular size of the product was confirmed by applying an aliquot of the reaction mixture to the gel permeable column-HPLC. The 25 kDa product was precipitated from the reaction mixture by adding 200 mL of ethanol at room temperature (20–25C). Primary culture of neocortical neurons This study was approved by the Animal Research Committee of the Institute for Developmental Research, Aichi Human Service Center, and was carried out according to the guidelines for animal research of the Neuroscience Society of Japan to minimize the number of animals used and their suffering. Neocortical neurons were cultured by the method described previously (Nakanishi et al. 1999) with a slight modification. Briefly, Sprague–Dawley rats at a gestational age of 16 days were deeply anesthetized with diethyl ether, and fetuses were transferred to Petri dishes containing ice-cold Hanks’ balanced salt solution with Mg2+ and Ca2+. Cerebral cortices of the fetuses were digested with 0.02% papain for 40 min at 37C. After digestion, the tissues were mechanically dissociated into single cell suspension by pipetting. The cells were plated on poly-L-lysinecoated 24-well plates at a density of 1.3 · 105 cells/cm2. The number of cells was counted immediately before plating. The cells were maintained in serum-free neurobasal medium (Invitrogen, Carlsbad, CA, USA) containing 2% B-27 supplement (Invitrogen), 50 U/mL penicillin and 25 lg/mL streptomycin (Brewer et al. 1993). For the initial plating of primary neurons, 25 lmol/L glutamate was added to the medium. After 48 h in culture, 5¢-fluoro2¢-deoxyuridine (Sigma-Aldrich) and uridine (Sigma-Aldrich) were added at a final concentration of 2 and 10 lg/mL, respectively, to arrest the further growth of non-neuronal cells. Cultures were fed twice a week. Assessment of neuroprotective activity To assess the neuroprotective activity of CS, we preparations to neuronal cell cultures 24 and immediately (5 min) before, and immediately administration of glutamate analogs, namely

added various CS 3 h, 30 min and (5 min) after the NMDA (Tocris,


Chondroitin sulfate-E prevents neuronal cell death | 1567

Bristol, UK), (S)-AMPA (Tocris), and kainate (Sigma-Aldrich), on 14 days in vitro (DIV). The concentration ranges of CS preparations and glutamate analogs were from 0.1 to 100 lg/mL and from 10 to 1000 lmol/L, respectively. In some experiments, CS-E was washed away from cultures pre-treated with CS-E (100 lg/mL) for 24 h twice with the fresh medium, then NMDA (20 lmol/L) was added to the cultures. Cells were cultured for an additional 24 h, and the number of microtubule-associated protein 2 (MAP2)-positive cells, or surviving neurons, was counted. In addition, neuronal cell death was estimated by measuring the activity of lactate dehydrogenase (LDH) released from damaged or destroyed cells into culture media (Choi et al. 1987; Tokita et al. 1996). LDH activity was measured using an LDH-cytotoxic test kit according to the protocol recommended by the manufacturer (Wako, Osaka, Japan). To assess neuroprotective activities of highly sulfated polysaccharides other than CS-E, we added heparin (10 and 100 lg/mL) and dextran sulfate (10 and 100 lg/mL) separately to neuronal cell cultures 24 h before administration of NMDA (20 lmol/L). Their neuroprotective activities were estimated by the method described above. Immunocytochemistry Cells were fixed for 30 min at room temperature (20–25C) with 3% paraformaldehyde in phosphate-buffered saline (PBS) containing 1% sucrose, 1 mmol/L MgCl2, 0.1 mmol/L CaCl2, and 0.1% glutaraldehyde. The paraformaldehyde solution was washed away with tris-buffered saline (TBS). Cultures were exposed first to a blocking solution consisting of 2% bovine serum albumin, 2% horse serum, 2% goat serum, 0.1% Triton X-100, and 0.1% sodium azide in TBS for 1 h, followed by overnight incubation with a monoclonal antibody to MAP2, a neuronal marker protein, (1 : 200; SigmaAldrich) in TBS containing 0.1% bovine serum albumin and 0.1% Triton X-100 at 4C. After washing with TBS, cultures were reacted with Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch Labs, West Grove, PA, USA) for 1 h. Electrophysiology For whole-cell recording of NMDA-evoked currents, neocortical neurons were cultured on poly-L-lysine-coated glass coverslips for 14 days. The coverslips were transferred to a recording chamber on the stage of a microscope (IX70; Olympus, Tokyo, Japan) and continuously perfused with external solution at a flow rate of 2 mL/ min. The external solution contained the following: 135 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl2, 0.8 mmol/L MgCl2, 10 mmol/L glucose, 10 mmol/L HEPES, pH 7.3, 290 mOsm. During voltage-clamp recording, 0.3 lmol/L tetrodotoxin, 10 lmol/L LaCl3, and 10 lmol/L bicuculline methiodide were added to the external solution to block voltage-dependent Na+ channels, voltage-dependent Ca2+ channels, and GABAA receptors, respectively. Electrical recordings were conducted at room temperature (20–25C). Patch electrodes were fabricated from glass capillary tubes with an outer diameter of 1.5 mm (G-1.5; Narishige, Tokyo, Japan) using a puller (P-97; Sutter Inst., Novato, CA, USA). The pipette solution contained 120 mmol/L Cs-methasulfonate, 10 mmol/L CsCl, 1 mmol/L MgCl2, 10 mmol/L HEPES, 1 mmol/L CaCl2, 10 mmol/L EGTA, 3 mmol/L MgATP, and 0.3 mmol/L NaGTP (pH was adjusted to 7.2–7.3 with 1 N Tris-OH, 270–280 mOsm). The patch electrodes had a resistance of 3–5 MW when filled with the pipette solution.

Membrane currents were recorded using a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Inc., Union City, CA, USA) and monitored on an oscilloscope (5521; Kikusui, Yokohama, Japan). Data obtained were digitized using a digidata 1332A data-acquisition system (Axon Instruments) at a 5 kHz sampling rate through a 2 kHz low-pass filter. Data were acquired by means of pCLAMP 9 software (Axon Instruments) and stored on a hard disk for off-line analysis using Clampfit 9.0 (Axon Instruments). Series resistance, Rs, was between 6 and 40 MW and was usually compensated by 60–85%. Recordings with a larger uncompensated series resistance than 15 MW were not included in this analysis. To measure the NMDA-induced current, voltage steps from )80 to +40 mV were applied, and NMDA (50 lmol/L) was pressure applied at each membrane potential using a solenoid valve (FSS03TBZC; Flon Industry Co. Ltd, Tokyo, Japan) connected to a pulse generator (SEN-3301; Nihon Kohden, Tokyo, Japan) for 30 ms through a patch pipette closed to the soma of the recorded neurons. Peak current responses for each voltage were plotted, and the data were fitted using KyPlot software (Kyence Inc., Tokyo, Japan). Current amplitudes were normalized to cell capacitance in all cases to calculate current density. To observe the effect of the acute application of CS-E on neurons, NMDA-induced currents were continuously recorded every 5 min. The response between just before (pre) and 20 min after (post) perfusion of the external solution containing CS-E (20 lg/mL) was compared. To observe the effect of chronic treatment of CS-E on neurons, CS-E (100 lg/mL) was added to cultures on 13 DIV, and NMDA-induced currents were recorded on the next day (14 DIV). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting For the analysis of phosphotyrosine-containing proteins, neocortical neurons were seeded on 35 mm tissue culture dishes at a density of 1.3 · 105 cells/cm2 and cultured for 13 days. Fifteen minutes after treatment with CS-E (100 lg/mL) on 13 DIV, the cells were washed with cold PBS and lysed in 200 lL of 50 mmol/L HEPES, pH 7.5, containing 1% Triton X-100, 1 mmol/L sodium orthovanadate, 100 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 1 mmol/L EDTA, and 1% protease inhibitor cocktail (Sigma-Aldrich). After 60 min of incubation on ice, cell lysates were centrifuged at 14 000 g for 15 min, and the supernatants were used for western blot analyses. Proteins precipitated from each sample by adding three volumes of 95% ethanol containing 1.3% potassium acetate at 0C were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 5–20% gradient gel (Bio Craft, Tokyo, Japan). The proteins in the gel were transferred electrophoretically to a Hybond enhanced chemiluminescence membrane (GE Healthcare). The membrane was incubated sequentially with a monoclonal antibody to phosphotyrosine (PY-20; Sigma-Aldrich) and with peroxidase-conjugated anti-mouse IgG (Vector Lab, Burlingame, CA, USA), and immunoproducts were detected using the enhanced chemiluminescence system (Perkin-Elmer, Waltham, MA, USA). Immunoblotting using an anti-b-actin antibody (Sigma-Aldrich) was carried out to estimate the yield of proteins after stripping of the membrane. The quantification of each protein band was performed using the public domain NIH image program (National Institutes of Health, Bethesda, MD, USA).



Measurement of caspase 3 activity Neuronal cells were cultured in 35 mm tissue culture dishes at a density of 2.0 · 105 cells/cm2 as described above. We added CS-E to neuronal cell cultures at a final concentration of 10 or 100 lg/mL 24 h before administration of NMDA on 14 DIV. To measure caspase 3 activity, we used a caspase 3 cellular activity assay kit plus -AK-703 (Biomol, Plymouth Meeting, PA, USA). Cells were lysed in 300 lL/dish of a cell lysis buffer included in the assay kit at various time points ranging from 1 to 10 h after the administration of NMDA, and caspase 3 activities in lysates were measured. Statistics Numerical data are presented as the mean ± SD. Statistical analysis was performed by ANOVA followed by Scheffe’s post hoc test using SPSS statistics software (SPSS Inc., Chicago, IL, USA). A p-value of < 0.05 was considered significant.

Results Neuroprotective effect of various CSs and related polyanions against excitotoxicity of NMDA To examine the neuroprotective effect of CSs against excitotoxicity of NMDA, various CS preparations (10 lg/ mL) were added to neuronal cell cultures 24 h before NMDA administration (20 lmol/L). When cultured neurons were immunostained with the anti-MAP2 antibody 24 h after NMDA addition, only CS-E (75 kDa) appeared to reduce the loss of MAP2-positive cells in cultures (Fig. 1). The number of MAP2-positive cells in various CS-treated samples was counted 24 h after NMDA administration, and summarized in Fig. 2a. Cells in the CS-E-treated sample survived more than those in other samples (p < 0.01, CS-E vs. CS-A, B, C, D, and vehicle). To estimate neuronal cell death, we measured the activities of LDH in the culture media of CS-treated samples (Fig. 2b). The LDH activity of CS-E-treated samples was significantly lower than those of other samples (p < 0.01, CS-E vs. CS-A, B, C, D, and vehicle). These results indicate that only CS-E exerts a

Fig. 1 Microtubule-associated protein 2 (MAP2) immunocytochemistry of neocortical neurons in culture. Photomicrographs of cultured neurons immunostained for MAP2, 24 h after administration of NMDA (20 lmol/L) in the presence (a) or absence (b) of chondroitin sulfate (CS)-E (10 lg/mL). Note that the presence of CS-E appears to reduce the loss of MAP2-positive cells in cultures. Bar = 50 lm.

Fig. 2 Neuroprotective effects of various chondroitin sulfate (CS) against excitotoxicity of NMDA. CS preparations (10 lg/mL) (n = 3) were added to neuronal cell cultures 24 h before administration of NMDA (20 lmol/L). Data represent the mean ± SD. (a) The number of microtubule-associated protein 2 (MAP2)-positive cells in cultures 24 h after administration of NMDA. Data represent the number of MAP2-positive cells counted using a microscope in five fields. The number of MAP2-positive cells in the CS-E treated sample is significantly larger than those in other samples (*p < 0.01, vs. CS-A, -B, -C, -D, and vehicle). (b) The activity of lactate dehydrogenase (LDH) in culture media 24 h after NMDA administration. Date represent the percentage of the sample treated with 1000 lmol/L NMDA for 24 h, in which no MAP2-positive cells survived. The LDH level of the CS-Etreated sample is significantly lower than those of other samples (*p < 0.01, vs. CS-A, -B, -C, -D, and vehicle).

neuroprotective effect at this concentration against NMDA excitotoxicity. We then examined whether other highly sulfated polysaccharides such as heparin and dextran sulfate exerted neuroprotective effect against excitotoxicity of NMDA. Figure 3(a) (results of the assay for neuronal cell survival) and Fig. 3b (results of the assay for neuronal cell death) show that neither heparin nor dextran sulfate exerted neuroprotective effect at this concentration against NMDA excitotoxicity. As there was a good inverse correlation between the number of MAP2-positive cells and LDH activity in these experiments (Figs. 2a,b and 3a,b), we evaluated the neuroprotective effect of CS only by measuring LDH activity in the following experiments.



Fig. 4 Molecular-size dependence of neuroprotective effect of chondroitin sulfate (CS)-E against excitotoxicity of NMDA. Lactate dehydrogenase (LDH) activities in neuronal cell cultures pre-treated for 24 h with CS-E (100 lg/mL) with molecular sizes of 3 kDa (3K), 5 kDa (5K), 25 kDa (25K), and 75 kDa (75K) were measured 24 h after NMDA administration (20 lmol/L). CS-E-degradation products whose molecular sizes are over 5 kDa have a neuroprotective effect against NMDA excitotoxicity (20 lmol/L) (*p < 0.01, vs. vehicle) (n = 3). Data are shown as a percentage of the sample treated with 1000 lmol/L NMDA for 24 h. Data represent the mean ± SD.

Fig. 3 Neuroprotective effect of heparin and dextran sulfate against excitotoxicity of NMDA. Heparin (H10 and H100; 10 and 100 lg/mL, respectively), dextran sulfate (D10 and D100; 10 and 100 lg/mL, respectively), and chondroitin sulfate (CS)-E (E, 100 lg/mL) (n = 4, each) were added to neuronal cell cultures 24 h before administration of NMDA (20 lmol/L). Data represent the mean ± SD. (a) The number of microtubule-associated protein 2 (MAP2)-positive cells in cultures 24 h after administration of NMDA. Data represent the number of MAP2-positive cells counted using a microscope in five fields. The number of MAP2-positive cells in the CS-E-treated sample is significantly larger than that in other samples (**p < 0.01, vs. H10, 100 and vehicle. p < 0.05, vs. D100). (b) The activity of lactate dehydrogenase (LDH) in culture media 24 h after NMDA administration. Data represent the percentages of the sample treated with 1000 lmol/L NMDA for 24 h, in which no MAP2-positive cells survived. The LDH level of the CS-E-treated sample is significantly lower than those of other samples (*p < 0.01, vs. H10, H100, and vehicle). The LDH assay of dextran sulfate-treated samples could not be performed because dextran sulfate interferes the reaction of coloring agents.

Molecular-size dependence of neuroprotective activity of CS-E Chondroitin sulfate-E is a linear polysaccharide with a large molecular size (75 kDa). To determine the minimum size of CS-E required for neuroprotective activity, we prepared several CS-E-derived polysaccharides with smaller molecular sizes (3–25 kDa) by hyaluronidase digestion and determined their neuroprotective activities by the same method described above. The polysaccharide of 3 kDa did not reduce the

neuronal cell death induced by NMDA, whereas polysaccharides with a molecular size over 5 kDa significantly reduced the excitatory cell death of neurons (Fig. 4; p < 0.01, vs. vehicle). This indicates that around 5 kDa is the minimum size of CS-E required for biological activity. Dose dependence of neuroprotective activity of CS-E Lactate dehydrogenase activities were measured in cultures treated with CS-E (75 kDa) at various concentrations (0.1– 100 lg/mL) 24 h after NMDA administration (20 lmol/L). LDH activities at concentrations between 10 and 100 lg/mL CS-E were significantly lower than that of the sample at 0.1 lg/mL (52.8 ± 6.8% of the sample treated with 1000 lmol/L NMDA for 24 h, in which no MAP2-positive cells survived) (p < 0.01, samples at 10, 50, and 100 lg/mL vs. sample at 0.1 lg/mL; Fig. 5a). This indicates that the effective concentration of CS-E for neuroprotection is 10– 100 lg/mL. Then, we examined the neuroprotective effect of CS-E at various concentrations of NMDA. Neuronal cell death was induced by treatment with NMDA in a dosedependent manner as expected (Fig. 5b). CS-E (both 10 and 100 lg/mL) exerted neuroprotective effect against excitotoxicity of NMDA even at concentrations over 20 lmol/L. Administration-time dependence of neuroprotective activity of CS-E To determine the effective timing of CS-E administration for neuroprotection, CS-E (100 lg/mL) was administered to cultures at various times from 24 h before to 5 min after NMDA addition (Fig. 6a). LDH activities in cultures were measured 24 h after NMDA addition. CS-E administration prior to NMDA addition significantly reduced neuronal cell death (p < 0.01, vs. vehicle). This result indicates that the



Fig. 5 Dose dependence of neuroprotective activity of chondroitin sulfate (CS)-E. (a) Cultured neurons were pre-treated with CS-E (n = 4) at various concentrations (0.1–100 lg/mL) for 24 h and lactate dehydrogenase (LDH) activities were measured 24 h after NMDA administration (20 lmol/L). The effective concentration of CS-E is 10– 100 lg/mL (*p < 0.01, vs. sample at 0.1 lg/mL). (b) LDH activities in neuronal cell cultures pre-treated for 24 h with CS-E at various concentrations (0–100 lg/mL) were measured at 24 h after NMDA administration at various concentrations (10–100 lmol/L) (n = 4); *p < 0.01, vs. the sample without CS-E at the same concentration of NMDA. Data represent the percentages of the sample treated with 1000 lmol/L NMDA for 24 h, and the values are shown as the mean ± SD in both figures.

earlier the addition of CS-E, the more effective its neuroprotection. The administration of CS-E immediately after NMDA addition tended to reduce neuronal cell death, but not significantly (Fig. 6a). The results shown in Fig. 6a suggest that neuronal cells become tolerant to NMDA excitotoxicity during pre-incubation with CS-E. To examine whether coexistence of CS-E with NMDA is essential to exert the neuroprotective effect, we added NMDA to cultures pre-treated with CS-E for 24 h after removal of CS-E by washing and evaluated neuronal cell death 24 h after NMDA administration (Fig. 6b). The LDH activity of the culture from which CS-E had been washed away (E/wash) was almost identical to that of the sample without washing (E), and was significantly lower than those of control cultures without CS-E pre-treatment (p < 0.01, vs. vehicle and V/wash). This indicates that neurons are tolerant to NMDA excitotoxicity even after removal of CS-E.

Fig. 6 Changes in neuroprotective effect of chondroitin sulfate (CS)-E depending on its administration time and neuroprotective effect after removal of CS-E from cultures prior to NMDA administration. (a) Lactate dehydrogenase (LDH) activities were measured 24 h after addition of NMDA (20 lmol/L) to cultures treated with CS-E (100 lg/ mL) at various time points: 24 h ()24 h), 3 h ()3 h), 30 min ()30 m), and immediately ()5 m) before, and immediately after (+5 m) NMDA addition. (b) Cultures were pre-treated with CS-E (100 lg/mL) or vehicle (phosphate-buffered saline) for 24 h, then NMDA (20 lmol/L) was added to the cultures (E and vehicle). To some cultures pretreated with CS-E or vehicle for 24 h, NMDA was added after washing with the fresh medium twice, and culture was continued in the medium without CS-E (E/wash and V/wash). LDH activities in culture media were measured 24 h after NMDA administration. Data represent percentages of the sample treated with 1000 lmol/L NMDA for 24 h, and the values are shown as the mean ± SD. (n = 4); *p < 0.01, vs. vehicle.

Neuroprotective effect of CS-E against excitotoxicity of other glutamate analogs There are three ionotropic receptors of glutamate, namely, NMDA, AMPA, and kainate receptors (Nakanishi 1992; Meldrum 2000) as described above. NMDA, a glutamate analog, binds to and activates the NMDA subtype of glutamate receptor. To examine the effect of CS-E on neuronal cell death mediated by other subtypes of glutamate receptor, we administered AMPA and kainate to neuronal cell cultures as glutamate analogs which specifically activate the AMPA and kainate subtypes of glutamate receptor, respectively. The activities of LDH in cultures treated with various CS preparations (100 lg/mL) were measured at 24 h after



Fig. 7 Neuroprotective effect of chondroitin sulfate (CS)-E against excitotoxicity of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). (a) Activities of lactate dehydrogenase (LDH) in cultures treated with various CS preparations (100 lg/mL) 24 h before AMPA addition (100 lmol/L) were measured at 24 h after AMPA administration (n = 3); *p < 0.01, versus vehicle (b) LDH activities in neuronal cell cultures pre-treated for 24 h with CS-E at various concentrations (0–100 lg/mL) were measured at 24 h after AMPA administration at various concentrations (10–1000 lmol/L) (n = 2); *p < 0.01 vs. sample without CS-E at the same concentration of AMPA. Data represent the percentage of the sample treated with 1000 lmol/L NMDA for 24 h, and the values are shown as the mean ± SD in both figures.

AMPA addition (100 lmol/L; Fig. 7a). LDH activity of CSE treated samples was significantly lower than those of the vehicle, CS-A and -C (p < 0.01, CS-E vs. vehicle; p < 0.05, CS-E vs. CS-A, -C). CS preparations other than CS-E tended to reduce AMPA-induced neuronal cell death, but the extents were not statistically significant (p > 0.05, vs. vehicle). In addition, LDH activity decreased depending on the concentration of CS-E added to cultures treated with AMPA at various concentrations up to 1000 lmol/L (Fig. 7b). Chondroitin sulfate-E could also suppress the elevation of LDH activity in cultures induced by 100 lmol/L kainate (Fig. 8a). The LDH activity of the CS-E-treated sample was significantly lower than those of the vehicle, CS-A, -B, and -C (p < 0.01, CS-E vs. vehicle and CS-A; p < 0.05, CS-E vs. CS-B, -C). CS preparations other than CS-E tended to reduce kainate-induced neuronal cell death, but the extents were not statistically significant (p > 0.05, vs. vehicle). LDH activity decreased depending on the concentration of CS-E added to cultures treated with kainate at various concentrations up to 1000 lmol/L (Fig. 8b). These results suggest that only CS-E exerts a neuroprotective effect against excitotoxicity of not only NMDA, but also AMPA or kainate; in other words, on neuronal cell death mediated by various glutamate receptors.

Fig. 8 Neuroprotective effect of chondroitin sulfate (CS)-E against kainate excitotoxicity. (a) Lactate dehydrogenase (LDH) activities in cultures treated with various CS preparations (100 lg/mL) 24 h before kainate addition (100 lmol/L) were measured at 24 h after kainate administration (n = 3); *p < 0.01, versus vehicle (b) LDH activities in neuronal cell cultures pre-treated for 24 h with CS-E at various concentrations (0–100 lg/mL) were measured at 24 h after kainate administration at various concentrations (10–1000 lmol/L) (n = 2); *p < 0.01, p < 0.05 vs. sample without CS-E at the same concentration of kainate. Data represent the percentage of the sample treated with 1000 lmol/L NMDA for 24 h, and the values are expressed as the mean ± SD in both figures.

Effect of CS-E treatment on NMDA-induced currents To obtain a clue about the neuroprotective mechanism of CSE, we examined the effect of CS-E on neuronal response to NMDA. Neocortical neurons were voltage clamped at various potentials, and NMDA (50 lmol/L) was pressure applied to the neurons during the voltage steps. First, we examined NMDA-induced current density just before and 20 min after treatment with CS-E (20 lmol/L) (Fig. 9a, left panel) or the application of an NMDA antagonist, AP5 (Fig. 9a, right panel). NMDA-induced currents were not changed by treatment with CS-E, while the currents almost disappeared by AP5 application. We next measured the NMDA-induced current density of neurons treated with CSE (100 lg/mL) for 24 h (Fig. 9b). NMDA-induced currents in CS-E-treated neurons were almost comparable with those of control neurons. These results indicate that CS-E does not function as an NMDA blocker. Effect of CS-E treatment on protein-tyrosine phosphorylation and caspase 3 activity Protein phosphorylation and dephosphorylation are pivotal steps in intracellular signal transduction that mediate many cellular processes including cell death and survival (Sapora



It is known that caspase 3 is activated during the degeneration of neurons treated with glutamate (Du et al. 1997). We then examined whether the activation of caspase 3 induced by NMDA was suppressed by treatment of neurons with CS-E. Figure 10(b) shows the time course of changes in caspase 3 activity following NMDA administration (20 lmol/L). Caspase 3 activities almost linearly elevated up to at least 10 h after NMDA administration (positive control; PC, Fig. 10b). Caspase 3 activities of CS-E-treated samples (10 and 100 lg/mL) were less elevated than that of the non-treatment sample (PC). Caspase 3 activity of neurons 5 h after NMDA administration (PC, Fig. 10c) was then compared with those of neurons cultured in the absence of NMDA and CS-E (negative control, Fig. 10c) and cultured with NMDA in the presence of CS-E (CS-E, Fig. 10c). Enzymatic activity in the CS-E-treated sample was significantly suppressed (p < 0.01, vs. PC).

Discussion

Fig. 9 Effects of chondroitin sulfate (CS)-E on NMDA-induced currents. Neocortical neurons were voltage-clamped at various potentials (VH = )60 mV with voltage steps from )80 to 40 mV), and NMDA (50 lmol/L) was pressure applied (30 ms) to neurons. (a) Left panel. Averaged NMDA-induced current densities were plotted as a function of membrane potential (current–voltage relationship, I–V relationship) just before (pre: open circles) and 20 min after (post: closed circles) treatment with CS-E (20 lg/mL) (n = 6). CS-E did not reduce NMDAinduced currents. Inset shows representative traces obtained before (pre) and after (post) application of CS-E. Error bars represent SD. Right panel: I–V relationship before (pre) and after (post) application of an NMDA antagonist, AP5. Insets show representative traces obtained before (pre: open square) and after (post: closed square) the application of AP5. (b) Averaged NMDA-induced current densities were plotted as a function of membrane potential (I–V relationship) for control (open circles; n = 6) and neurons treated with CS-E for 24 h (closed circles, n = 5).

and Di Carlo 2006). We examined whether CS-E treatment of neurons altered the profile of phosphotyrosine-containing protein bands. Western blot analysis for phosphotyrosinecontaining proteins in neurons revealed that the intensities of several bands were changed by the CS-E treatment (Fig. 10a). For example, the intensity of 30 kDa band in CSE-treated cells increased to 136% of the sample treated with vehicle (PBS) or with 100 lg/mL CS-A. The intensity of 22 kDa band, conversely, decreased to 67% of the control samples. This result indicates that CS-E treatment of neurons modifies intracellular signal transduction.

In the present study, we demonstrated that CS-E, a highly sulfated CS preparation, prevented neuronal cell death induced by not only NMDA but also AMPA and kainate (Figs. 4, 7, and 8), which means that CS-E can prevent excitatory cell death of neurons mediated by various glutamate receptors. To our knowledge, this is the first report to show that CS-E prevents neuronal cell death in neocortical neurons. Possible mechanism of neuroprotection by CS-E Chondroitin sulfate-E suppressed the activation of caspase 3 in neurons induced by NMDA (Fig. 10b and c). Caspase 3 is well known as an apoptosis-related protein (FernandesAlnemri et al. 1994; Cohen 1997), so anti-apoptosis could be implicated in neuroprotection by CS-E. It is generally considered that intracellular protein phosphorylation and dephosphorylation mediate various cellular processes including apoptosis (Sapora and Di Carlo 2006). We demonstrated that CS-E treatment of neurons altered the profile of phosphotyrosine-containing protein bands (Fig. 10a). This finding suggests that CS-E gives rise to a certain intracellular signal related to apoptosis in cultured neurons. Chondroitin sulfate-E can interact with some heparinbinding trophic factors including midkine and pleiotrophin (Ueoka et al. 2000; Deepa et al. 2002; Gama et al. 2006; Sotogaku et al. 2007), which have biological activities for promoting neurite outgrowth and nerve cell migration, and neuroprotection (Rauvala 1989; Muramatsu and Muramatsu 1991; Kaneda et al. 1996). These trophic factors are expressed by embryonic rat cortical neurons (Matsumoto et al. 1994). CS-E may exert a neuroprotective effect via interaction with these trophic factors. In addition, neuroprotective materials such as hormones, anti-oxidants and growth factors are included in B27 supplement added to neurobasal



Fig. 10 Effect of chondroitin sulfate (CS)-E treatment on proteintyrosine phosphorylation and caspase 3 activity. (a) Neocortical neurons were cultured for 13 days and then treated for 15 min with vehicle (phosphate-buffered saline), CS-E (100 lg/mL), or CS-A (100 lg/mL). Each cell lysate was immunoblotted using an antibody against phosphotyrosine or b-actin. The positions of molecular mass markers are indicated at the left of the panel. Note that the intensity of 30 kDa band (arrow) increases upon CS-E treatment, and that the intensity of 22 kDa band (*) conversely decreases. (b) Changes in caspase 3 activity following NMDA administration. Open diamonds indicate positive controls (PC), which are samples treated with only

NMDA (20 lmol/L). Closed squares and closed triangles indicate samples pre-treated with 10 and 100 lg/mL of CS-E, respectively, for 24 h before NMDA administration (20 lmol/L). Open triangles indicate negative controls (NC), which are samples cultured in the absence of NMDA and CS-E. Note that the activation of caspase 3 is suppressed in the presence of CS-E. (c) Caspase 3 activities 5 h after NMDA administration. We added 100 lg/mL of CS-E to cultures 24 h before NMDA administration (20 lmol/L). Note that CS-E significantly suppresses the increase of caspase 3 activity following NMDA administration (*p < 0.01, vs. PC) (n = 4). Data represent the mean ± SD.

medium used in our culture system (Brewer et al. 1993). CSE may stabilize and present some of these trophic factors through binding to them. Glial cells are known to synthesize and secrete both CS proteoglycans (CSPGs) and some trophic factors (Furukawa et al. 1986; Tarris et al. 1986). Therefore, contaminating glial cells in neuronal cultures may also contribute to neuronal cell survival to some extents. To examine whether CS-E can exert neurotrophic activity even in the presence of glial cells, we evaluated the extent of neuroprotection by CSE from NMDA excitotoxicity using neuronal cell cultures on astrocyte monolayers (Nakanishi et al. 1999). Our preliminary experiments showed that CS-E prevented neuronal cell death in the neuron-glia co-culture system to an extent almost identical to that in the neuronal cell culture system used in the present work, indicating that the presence of astrocytes does not affect the neuroprotective effect of CS-E against NMDA excitotoxicity. Incidentally, B27-supplemented neurobasal medium used in the present work allows very few glial cells to grow (< 0.5% of total cells) (Brewer et al. 1993). Another possible explanation for the effect of neuroprotection is related to ‘brain tolerance’ (Kitagawa et al. 1990;

Barone et al. 1998; Kirino 2002; Gidday 2006). In the present study, the earlier the addition of CS-E, the more effective the protection from neuronal cell death induced by NMDA (Fig. 6a). In addition, neuronal cell death induced by NMDA was significantly reduced in cultures pre-treated with CS-E for 24 h even after the removal of CS-E prior to NMDA administration (Fig. 6b). Pre-treatment with CS-E may be a mild insult to neuronal cells and, from that conditioning, cells may prepare a self-defense mechanism such as the expression of some stress proteins to severer insults. To examine this possibility, we evaluated the expression levels of heat shock protein 70 and 27 by western blot analysis, as elevated expression of these proteins is considered to render cells tolerant to some stresses (Parsell and Lindquist 1993; Yenari et al. 1999); however, CS-E could not induce the up-regulation of these proteins (data not shown). There is a possibility that CS-E directly binds to and captures NMDA to prevent neurons from excitatory cell death induced by the reagent. Then, we examined by the Quartz Crystal Microbalance technique using Affinix Q (Initium Co., Tokyo, Japan) whether CS-E could bind to NMDA, but did not obtain the results showing binding of



CS-E to NMDA (data not shown). CS-E reduced neuronal cell death induced by glutamate receptor agonists by a similar percentage regardless of the concentration of an agonist (Fig. 5b for NMDA, Fig. 7b for AMPA, and Fig. 8b for kainite). This also supports the idea that CS-E exerts its neuroprotective effect through a mechanism rather than the direct binding to these analogs. The direct effect of CS-E on neurons may be a key of the neuroprotection by this sulfated polysaccharide. Protein neosynthesis is often required for cells to acquire a new property. To examine the requirement of protein synthesis for neurons to become tolerant to excitatory cell death by CS-E treatment, cycloheximide, an inhibitor of protein biosynthesis, was added, 3 h prior to CS-E, to neuronal cell cultures 24 h before NMDA administration, and the extent of cell death was estimated 24 h after NMDA administration. NMDA-induced cell death was effectively reduced by CS-E treatment even in the presence of cycloheximide as well as in the absence of the inhibitor, indicating that protein neosynthesis is not required in this case (data not shown). Minimum size of CS-E for neuroprotection Chondroitin sulfate-E is an unbranched long polysaccharide (75 kDa) with structural heterogeneity. This large molecule is a disadvantage to using CS-E as a treatment for neuronal degeneration. We tried to determine the minimum size of CSE required for neuroprotective activity against the excitotoxicity of NMDA. Products over 5 kDa, but not 3 kDa, have neuroprotective activity similar to that of the CS-E polysaccharide (Fig. 4). To develop a neuroprotective medicine derived from CS-E, the structure of the 5 kDa product should be determined. Rolls et al. (2004) have shown that disaccharide degradation products of CSPG by chondroitinase ABC have a neuroprotective effect against the excitotoxicity of glutamate on rat PC12 pheochromocytoma cells. We examined whether various CS disaccharides including disaccharide-E derived from CS-E have a neuroprotective effect against NMDA toxicity on primary cultured neurons; however, we could not demonstrate the neuroprotective effect of any CS disaccharides (data not shown). The discrepancy may be attributed to differences in the cell types used. CS-E in the CNS and possibility of CS-E derivatives as a curative The commercial preparation of CS-E used in the present work is mostly composed of an oversulfated CS-disaccharide unit named E-unit (70% of total). The content of E-unit in CS disaccharides derived from total CS in the developing brain is 1.2–2.0% of total (Ueoka et al. 2000; Ida et al. 2006). Several brain proteoglycans such as neuroglycan C (Shuo et al. 2004), appican (Tsuchida et al. 2001), and syndecans (Deepa et al. 2004) bear E-unit-containing CS side chains. Herndon et al. (1999) reported that the CS

concentration in the brain is 60 lmol/L, and if one assumes that most of these CSs were restricted to the extracellular space or the cell surface, then the local concentration could be five to 10-fold higher in those locations. We added CS-E (75 kDa) to the culture medium at the concentrations of 10 and 100 lg/mL, which correspond to 0.13 and 1.3 lmol/L, respectively. Therefore, the concentrations of CS-E we used in our research are biologically relevant. Many recent studies have shown that some CSPGs are upregulated after CNS injury, that the CSPGs inhibit axonal growth, and that depletion of CSPGs at injured sites promotes axonal regeneration in the CNS (Silver 1994; Bradbury et al. 2002; Matsui and Oohira 2004; Rhodes and Fawcett 2004). The CS-disaccharide composition of these injury-induced CSPGs remains to be determined. In contrast to their inhibitory effects on axonal growth and regeneration, CS chains with particular oversulfated structures are involved in neuronal adhesion, migration, and neuritogenesis (Sugahara et al. 2003). CS-E promotes the fibroblast growth factor-2mediated proliferation of neural stem/progenitor cells (Ida et al. 2006) and neurite outgrowth from hippocampal neurons, mesencephalic dopaminergic neurons, and dorsal root ganglion neurons (Clement et al. 1999; Gama et al. 2006; Sotogaku et al. 2007). In addition, CS-E promotes Th1 cell immune response, namely, elevates the production of some anti-inflammatory cytokines such as c-interferon and interleukin-2 (Akiyama et al. 2004). Th1 cells activate macrophages (Ma et al. 2003), which begin to actively express glial cell line-derived neurotrophic factor (Batchelor et al. 1999). CS relatively rich in E-unit is expressed in the cerebellum and also in the hippocampus, olfactory bulb, and striatum, suggesting that E-unit-rich CS is involved in neurogenesis, axon guidance, and/or neuronal survival (Purushothaman et al. 2007). Considering that CS-E has these desirable activities for neuroprotection and neuronal generation, CS-Ederived medicines and -related materials could be developed for the therapeutic treatment of various neuronal diseases and injuries in the future. Actually, Rolls et al. (2004) have shown that intravenous injection with CS disaccharides exerts a neuroprotective effect against glutamate toxicity in retinal ganglion cells in rodents, and promotes recovery from immune-induced neuropathologies of the CNS such as experimental autoimmune encephalomyelitis and experimental autoimmune uveitis (Rolls et al. 2006). In conclusion, we demonstrated that CS-E had neuroprotective activity against the excitotoxicity of excitatory molecules. The detailed mechanism underlying neuroprotection by CS-E and the structure of CS-E required for neuroprotective activity remain to be investigated.

Acknowledgements We thank Dr H. Ito for helpful discussion, and Dr H. Yatsuya for advice about the statistical analysis. This work was supported in part



by grants-in-aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan, and from the Japan Society for the Promotion of Science.

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