Yoshiaki Sato and Keiko Nakanishi contributed equally to this work.
A highly sulfated chondroitin sulfate preparation, CS-E, prevents excitatory amino acid-induced neuronal cell death
Article first published online: 7 NOV 2007
© 2007 The Authors
Journal of Neurochemistry
Volume 104, Issue 6, pages 1565–1576, March 2008
How to Cite
Sato, Y., Nakanishi, K., Tokita, Y., Kakizawa, H., Ida, M., Maeda, H., Matsui, F., Aono, S., Saito, A., Kuroda, Y., Hayakawa, M., Kojima, S. and Oohira, A. (2008), A highly sulfated chondroitin sulfate preparation, CS-E, prevents excitatory amino acid-induced neuronal cell death. Journal of Neurochemistry, 104: 1565–1576. doi: 10.1111/j.1471-4159.2007.05107.x
- Issue published online: 7 NOV 2007
- Article first published online: 7 NOV 2007
- Received July 25, 2007; revised manuscript October 12, 2007; accepted October 22, 2007.
- caspase 3;
- chondroitin sulfate;
- glutamate receptor;
- neuronal death;
J. Neurochem. (2008) 104, 1565–1576.
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)-α-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 protein-tyrosine 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.
chondroitin sulfate proteoglycan
days in vitro
microtubule-associated protein 2
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 -4GlcUAβ1-3GalNAcβ1-, 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.
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. Non-NMDA receptor is further subclassified into α-amino-3-hydroxy-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 voltage-dependent 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
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 Sigma-Aldrich.
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. 184.108.40.206; Sigma-Aldrich) for 16 h at 37°C. 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 37°C. 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 (20–25°C).
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 37°C. After digestion, the tissues were mechanically dissociated into single cell suspension by pipetting. The cells were plated on poly-l-lysine-coated 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 μg/mL streptomycin (Brewer et al. 1993). For the initial plating of primary neurons, 25 μmol/L glutamate was added to the medium. After 48 h in culture, 5′-fluoro-2′-deoxyuridine (Sigma-Aldrich) and uridine (Sigma-Aldrich) were added at a final concentration of 2 and 10 μg/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 added various CS preparations to neuronal cell cultures 24 and 3 h, 30 min and immediately (5 min) before, and immediately (5 min) after the administration of glutamate analogs, namely NMDA (Tocris, 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 μg/mL and from 10 to 1000 μmol/L, respectively. In some experiments, CS-E was washed away from cultures pre-treated with CS-E (100 μg/mL) for 24 h twice with the fresh medium, then NMDA (20 μmol/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 μg/mL) and dextran sulfate (10 and 100 μg/mL) separately to neuronal cell cultures 24 h before administration of NMDA (20 μmol/L). Their neuroprotective activities were estimated by the method described above.
Cells were fixed for 30 min at room temperature (20–25°C) 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; Sigma-Aldrich) in TBS containing 0.1% bovine serum albumin and 0.1% Triton X-100 at 4°C. After washing with TBS, cultures were reacted with Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch Labs, West Grove, PA, USA) for 1 h.
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 μmol/L tetrodotoxin, 10 μmol/L LaCl3, and 10 μmol/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 (20–25°C).
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 MΩ 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 MΩ and was usually compensated by 60–85%. Recordings with a larger uncompensated series resistance than 15 MΩ were not included in this analysis.
To measure the NMDA-induced current, voltage steps from −80 to +40 mV were applied, and NMDA (50 μmol/L) was pressure applied at each membrane potential using a solenoid valve (FSS-03TBZC; 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 μg/mL) was compared. To observe the effect of chronic treatment of CS-E on neurons, CS-E (100 μg/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 μg/mL) on 13 DIV, the cells were washed with cold PBS and lysed in 200 μL 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 0°C 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-β-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 μg/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 μL/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.
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.
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 μg/mL) were added to neuronal cell cultures 24 h before NMDA administration (20 μmol/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 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.
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 μg/mL) 24 h after NMDA administration (20 μmol/L). LDH activities at concentrations between 10 and 100 μg/mL CS-E were significantly lower than that of the sample at 0.1 μg/mL (52.8 ± 6.8% of the sample treated with 1000 μmol/L NMDA for 24 h, in which no MAP2-positive cells survived) (p < 0.01, samples at 10, 50, and 100 μg/mL vs. sample at 0.1 μg/mL; Fig. 5a). This indicates that the effective concentration of CS-E for neuroprotection is 10–100 μg/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 dose-dependent manner as expected (Fig. 5b). CS-E (both 10 and 100 μg/mL) exerted neuroprotective effect against excitotoxicity of NMDA even at concentrations over 20 μmol/L.
Administration-time dependence of neuroprotective activity of CS-E
To determine the effective timing of CS-E administration for neuroprotection, CS-E (100 μg/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 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.
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 μg/mL) were measured at 24 h after AMPA addition (100 μmol/L; Fig. 7a). LDH activity of CS-E 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 μmol/L (Fig. 7b).
Chondroitin sulfate-E could also suppress the elevation of LDH activity in cultures induced by 100 μmol/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 μmol/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.
Effect of CS-E treatment on NMDA-induced currents
To obtain a clue about the neuroprotective mechanism of CS-E, we examined the effect of CS-E on neuronal response to NMDA. Neocortical neurons were voltage clamped at various potentials, and NMDA (50 μmol/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 μmol/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 CS-E (100 μg/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 and Di Carlo 2006). We examined whether CS-E treatment of neurons altered the profile of phosphotyrosine-containing protein bands. Western blot analysis for phosphotyrosine-containing 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 CS-E-treated cells increased to 136% of the sample treated with vehicle (PBS) or with 100 μg/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.
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 μmol/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 μg/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).
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 (Fernandes-Alnemri 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 heparin-binding 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 medium used in our culture system (Brewer et al. 1993). CS-E 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 CS-E 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 CS-E 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 μmol/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 μg/mL, which correspond to 0.13 and 1.3 μmol/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 up-regulated 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-2-mediated 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 γ-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-E-derived 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.
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.
- 2004) Chondroitin sulphate structure affects its immunological activities on murine splenocytes sensitized with ovalbumin. Biochem. J. 382, 269–278. , , , , and (
- 2000) Proteoglycans in the developing brain: new conceptual insights for old proteins. Physiol. Rev. 80, 1267–1290. and (
- 1998) Ischemic preconditioning and brain tolerance: temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression. Stroke 29, 1937–1950. , , , , , and (
- 1999) Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. J. Neurosci. 19, 1708–1716. , , , , , and (
- 1992) Mechanisms of excitotoxicity in neurologic diseases. FASEB J. 6, 3338–3344. (
- 1962) A modified uronic acid carbazole reaction. Anal. Biochem. 4, 330–334. and (
- 2002) Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416, 636–640. , , , , , , and (
- 1993) Optimized survival of hippocampal neurons in B27-supplemented neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576. , , and (
- 1987) Ionic dependence of glutamate neurotoxicity. J. Neurosci. 7, 369–379. (
- 1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron 1, 623–634. (
- 1987) Glutamate neurotoxicity in cortical cell culture. J. Neurosci. 7, 357–368. , and (
- 1999) Chondroitin sulfate E promotes neurite outgrowth of rat embryonic day 18 hippocampal neurons. Neurosci. Lett. 269, 125–128. , and (
- 1997) Caspases: the executioners of apoptosis. Biochem. J. 326, 1–16. (
- 2002) Specific molecular interactions of oversulfated chondroitin sulfate E with various heparin-binding growth factors. Implications as a physiological binding partner in the brain and other tissues. J. Biol. Chem. 277, 43707–43716. , , , and (
- 2004) Chondroitin sulfate chains on syndecan-1 and syndecan-4 from normal murine mammary gland epithelial cells are structurally and functionally distinct and cooperate with heparan sulfate chains to bind growth factors. A novel function to control binding of midkine, pleiotrophin, and basic fibroblast growth factor. J. Biol. Chem. 279, 37368–37376. , , , and (
- 1955) Mucopolysaccharidases. Meth. Enzymol. 1, 166–173. (
- 1997) Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons. Proc. Natl Acad. Sci. USA 94, 11657–11662. , , , , , , , and (
- 1994) CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J. Biol. Chem. 269, 30761–30764. , and (
- 1986) Synthesis and secretion of nerve growth factor by mouse astroglial cells in culture. Biochem. Biophys. Res. Commun. 136, 57–63. , , and (
- 2006) Sulfation patterns of glycosaminoglycans encode molecular recognition and activity. Nat. Chem. Biol. 2, 467–473. , , , , , , , and (
- 2006) Cerebral preconditioning and ischaemic tolerance. Nat. Rev. Neurosci. 7, 437–448. (
- 1999) Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding. Glycobiology 9, 143–155. , and (
- 2006) Identification and functions of chondroitin sulfate in the milieu of neural stem cells. J. Biol. Chem. 281, 5982–5991. , , et al. (
- 1996) Midkine, a heparin-binding growth/differentiation factor, exhibits nerve cell adhesion and guidance activity for neurite outgrowth in vitro. J. Biochem. (Tokyo) 119, 1150–1156. , , , and (
- 2002) Ischemic tolerance. J. Cereb. Blood Flow Metab. 22, 1283–1296. (
- 1990) ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res. 528, 21–24. , , et al. (
- 2006) Structure of chondroitin sulfate, in Chondroitin Sulfate: Structure, Role and Pharmacological Activity, Advances in Pharmacology (VolpiN., ed.), volume 53, pp. 33–48. Academic Press, San Diego, CA. and (
- 2005) 4 million neonatal deaths: when? where? why? Lancet 365, 891–900. , and (
- 1999) An international network for evaluating neuroprotective therapy after severe birth asphyxia. Semin. Perinatol. 23, 226–233. , , and (
- 1994) Excitatory amino acids as a final common pathway for neurologic disorders. N. Engl. J. Med. 330, 613–622. and (
- 2003) Regulation of macrophage activation. Cell. Mol. Life Sci. 60, 2334–2346. , , , , , , and (
- 2004) Proteoglycans and injury of the central nervous system. Congenit. Anom. (Kyoto) 44, 181–188. and (
- 1994) A novel family of heparin-binding growth factors, pleiotrophin and midkine, is expressed in the developing rat cerebral cortex. Brain Res. Dev. Brain Res. 79, 229–241. , , , , and (
- 2000) Glutamate as a neurotransmitter in the brain: review of physiology and pathology. J. Nutr. 130, 1007S–1015S. (
- 1990) Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11, 379–387. and (
- 1991) Purification of recombinant midkine and examination of its biological activities: functional comparison of new heparin binding factors. Biochem. Biophys. Res. Commun. 177, 652–658. and (
- 1998) Characteristic hexasaccharide sequences in octasaccharides derived from shark cartilage chondroitin sulfate D with a neurite outgrowth promoting activity. J. Biol. Chem. 273, 3296–3307. , , , and (
- 1992) Molecular diversity of glutamate receptors and implications for brain function. Science 258, 597–603. (
- 1999) Dual intracellular recording of neocortical neurons in a neuron-glia co-culture system. Brain Res. Brain Res. Protoc. 4, 105–114. , and (
- 2006) Role of the sulfate pattern of chondroitin sulfate in its biological activities and in the binding of growth factors, in Chondroitin Sulfate: Structure, Role and Pharmacological Activity, Advances in Pharmacology (VolpiN., ed.), volume 53, pp. 253–279. Academic Press, San Diego, CA. and (
- 1993) The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27, 437–496. and (
- 2007) Functions of chondroitin sulfate/dermatan sulfate chains in brain development. Critical roles of E and iE disaccharide units recognized by a single chain antibody GD3G7. J. Biol. Chem. 282, 19442–19452. , , , , , , and (
- 1989) An 18-kDa heparin-binding protein of developing brain that is distinct from fibroblast growth factors. EMBO J. 8, 2933–2941. (
- 2004) Chondroitin sulphate proteoglycans: preventing plasticity or protecting the CNS? J. Anat. 204, 33–48. and (
- 2004) A disaccharide derived from chondroitin sulphate proteoglycan promotes central nervous system repair in rats and mice. Eur. J. Neurosci. 20, 1973–1983. , , , , , , , and (
- 2006) A sulfated disaccharide derived from chondroitin sulfate proteoglycan protects against inflammation-associated neurodegeneration. FASEB J. 20, 547–549. , , , , and (
- 2006) Cell signalling mechanisms and the control of cell life and death. Radiat. Prot. Dosimetry 122, 210–220. and (
- 2004) Developmental changes in the biochemical and immunological characters of the carbohydrate moiety of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan. Glycoconj. J. 20, 267–278. , , , , , and (
- 1994) Inhibitory molecules in development and regeneration. J. Neurol. 242, S22–S24. (
- 2007) Activation of phospholipase C pathways by a synthetic chondroitin sulfate-E tetrasaccharide promotes neurite outgrowth of dopaminergic neurons. J. Neurochem. 103, 749–760. , , , , , and (
- 2003) Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Curr. Opin. Struct. Biol. 13, 612–620. , , , , and (
- 1986) Synthesis and secretion of a nerve growth-stimulating factor by neonatal mouse astrocyte cells in vitro. Pediatr. Res. 20, 367–372. , and (
- 1996) Characterization of excitatory amino acid neurotoxicity in N-methyl-D-aspartate receptor-deficient mouse cortical neuronal cells. Eur. J. Neurosci. 8, 69–78. , , , , , and (
- 2001) Appican, the proteoglycan form of the amyloid precursor protein, contains chondroitin sulfate E in the repeating disaccharide region and 4-O-sulfated galactose in the linkage region. J. Biol. Chem. 276, 37155–37160. , , , , , , and (
- 2000) Neuronal cell adhesion, mediated by the heparin-binding neuroregulatory factor midkine, is specifically inhibited by chondroitin sulfate E. Structural and functional implications of the over-sulfated chondroitin sulfate. J. Biol. Chem. 275, 37407–37413. , , , , and (
- 2000) Ca2+–Zn2+ permeable AMPA or kainate receptors: possible key factors in selective neurodegeneration. Trends Neurosci. 23, 365–371. and (
- 1999) The neuroprotective potential of heat shock protein 70 (HSP70). Mol. Med. Today 5, 525–531. , , and (