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Ester Derivatives of Tournefolic Acid B Attenuate N-Methyl-D-aspartate-Mediated Excitotoxicity in Rat Cortical Neurons

National Research Institute of Chinese Medicine, Taipei, Taiwan, Republic of China (C.-N.W., Y.-L.L., Y.-J.S.); and Institute of Biopharmaceutical Science (H.-C.P., Y.-J.S.) and Institute of Pharmacology (C.-W.C.), National Yang-Ming University, Taipei, Taiwan, Republic of China

Received September 7, 2005; accepted December 19, 2005

	Abstract

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The effects of tournefolic acid B (TAB) and two ester derivatives, TAB methyl ester (TABM) and TAB ethyl ester (TABE), on N-methyl-D-aspartate (NMDA)-mediated excitotoxicity and the underlying mechanisms were investigated. Treatment with 50 µM NMDA elicited neuronal death by 48.7 ± 5.1%, coinciding with the appearance of injured morphology. TABM (50 µM) attenuated the NMDA-induced cell death by 60.9 ± 19.7%, and to a lesser extent by TABE. The NMDA-mediated activation of calpain was not affected by TABM and TABE, as determined by the cleavage of -spectrin. NMDA increased the activity of caspases 2, 3, 6, 8, and 9 and reached the maximum after 8-h treatment. TABM and TABE abrogated NMDA-induced activation of caspases 2, 3, 6, and 8 by approximately 80 to 90% and 50 to 60%, respectively, and to a higher extent for caspase 9. TABM and TABE also blocked the NMDA-mediated activation of caspase 12. Furthermore, TABM and TABE eliminated the NMDA-induced accumulation of superoxide anion (). NMDA evoked significant depolarization of mitochondria, whereas TABM elicited a mild decrease of mitochondrial membrane potential as determined by tetramethylrhodamine methyl ester perchlorate. NMDA treatment induced elevation of Ca²⁺ levels in cytosol, endoplasmic reticulum (ER), and mitochondria. TABM (50 µM) significantly diminished the NMDA-induced elevation of Ca²⁺ levels in mitochondria and ER but not cytosol. Therefore, TABM decreased mitochondrial membrane potential and attenuated the NMDA-mediated Ca²⁺-loading in ER and mitochondria. These events subsequently eliminated the accumulation of and blocked the activation of caspase cascade, thereby conferring their neuroprotective effects on NMDA-mediated excitotoxicity.

Binding of glutamate to NMDA receptors triggers a rapid influx of Ca²⁺ that culminates in neuron death. The extreme overload of Ca²⁺ in neurons and dysfunction of Ca²⁺ extrusion pathway consequently lead to failure of maintaining a low intracellular Ca²⁺ level (Bano et al., 2005). The excessive amount of Ca²⁺ is sequestered by mitochondria and endoplasmic reticulum (ER) (Nicholls and Akerman, 1982). Mitochondria uptake cytoplasmic Ca²⁺ via a uniporter using a proton electrochemical gradient as a driving force (Nicholls et al., 2003). The overload of calcium in mitochondria might have detrimental effects because of the enhanced production of reactive oxygen species (ROS), mitochondria depolarization, and bioenergetic failure (Patel et al., 1996; Schinder et al., 1996; White and Reynolds, 1996).

Alteration of Ca²⁺ homeostasis in ER has been reported to render neurons vulnerable to the excitotoxicity (Guo et al., 1999). Sustained elevations of intracellular Ca²⁺ induce apoptosis by activating caspase 12 in a calpain-dependent manner (Nakagawa and Yuan, 2000). Caspase 12 activity is induced by caspase 7 or the Ire1/tumor necrosis factor receptorassociated factor 2/c-Jun N-terminal kinase complex at the ER membrane. Caspase 12 plays a pivotal role in the progression of ER stress-induced cell death through cleaving procaspase 9 (Nakagawa and Yuan, 2000; Morishima et al., 2002).

Evidence has suggested that one of the most damaging consequences of excessive calcium is the generation of ROS (Coyle and Puttfarcken, 1993). Neurons are especially vul-nerable to ROS damage because of their high basal metabolic rate and relatively low levels of antioxidant defense (Halliwell, 1992). Activation of glutamate receptor increases the formation of superoxide anion () through a mitochondria-mediated pathway (Patel et al., 1996). Receptor-mediated elevation of Ca²⁺ also promotes the formation of nitric oxide (NO). through neuronal nitric-oxide synthase. Both NO and are involved. in the neurotoxic process. Furthermore, interaction of and NO generates a peroxynitrite ion that exhibits a much more damaging effect on neurons. The nuclear translocation of apoptosis-inducing factor, activation of poly(ADP)-ribose polymerase-1 (Yu et al., 2002), and increase in p53 and Bax (Djebaili et al., 2000) are also implicated in the NMDA-induced neuronal apoptosis.

Extensive evidence suggests that some antioxidants are capable of inhibiting glutamate-induced neurotoxicity (Lee et al., 2004; Kanada et al., 2005). A variety of polyphenolic compounds has been isolated from Tournefortia sarmentosa Lam. (Boraginaceae) (Chinese name: Teng Zi Dan), widely used in Taiwan as a detoxicant and anti-inflammatory agent (Lin et al., 2002). Most of these polyphenolic compounds possess antioxidative activity, including tournefolic acid B (TAB), TAB methyl ester (TABM), and TAB ethyl ester (TABE) (Fig. 1). The TAB structure-relative compounds also exhibit neuroprotective potency against glutamate-mediated neurotoxicity (Chi et al., 2005). In the present study, we therefore attempted to investigate the neuroprotective effects of TAB, TABM, and TABE on NMDA-mediated toxicity in primary cultures of rat cortical neurons. The underlying mechanisms by which TAB derivatives confer their effects were also elucidated. The results showed that TABM/TABE decreased mitochondrial membrane potential, attenuated the NMDA-mediated Ca²⁺ stress on ER and mitochondria, and subsequently blocked the activation. of caspase cascade and eliminated the accumulation of , thereby conferring their neuroprotective effects on NMDA-mediated neurotoxicity.

View larger version (21K): [in this window] [in a new window]   Fig. 1. The structure of TAB, TABM, and TABE.

	Materials and Methods

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Materials. All reagents for electrophoresis were purchased from Bio-Rad Laboratories (Hercules, CA). Medium and materials for cell cultures were obtained from Invitrogen (Carlsbad, CA). Enhanced chemiluminescence detection reagents, anti-rabbit IgG antibody conjugated with horseradish peroxidase, and anti-mouse IgG antibody conjugated with horseradish peroxidase were obtained from Amersham Bioscience (Buckinghamshire, UK). NMDA was purchased from Sigma (St. Louis, MO). Fluo-3 AM, rhod-2 AM, mag-fura-2 AM, TMRM, MitoTracker Green FM, ER-tracker Red, and dihydroethidium were purchased from Molecular Probes (Eugene, OR). Monoclonal anti-microtubule-associated protein 2 (MAP2) and anti--spectrin antibodies were obtained from Upstate (Charlottesville, VA) and Chemicon (Temecula, CA), respectively. Polyclonal anticaspase 12 antibody was purchased from BioVision (Mountain View, CA). Colorimetric substrates for caspases were obtained from BioSourse (Nivelles, Belgium). All other reagents were purchased from Sigma or Merck (Darmstadt, Germany).

Extraction and Isolation. Stems of T. samentosa Lam. were collected from Nei-Men, Kaohsiung County, Taiwan, in August, 1998. The plant was identified by comparison with voucher specimens deposited earlier at the Herbarium of Department of Botany, National Taiwan University, Taipei, Taiwan (no. TAI175693; collected April 1, 1979). The extraction of T. samentosa and the isolation and purification of bioactive polyphenolic compounds were described previously (Lin et al., 2002). TABM was the derivative of TAB during the hot-methanol extraction. The purity of all compounds was greater than 95% using high-performance liquid chromatography analysis.

Cell Culture. Primary cultures of neonatal cortical neurons were prepared as described previously (Wang et al., 2001). The Institutional Animal Care and Use Committee at the National Research Institute of Chinese Medicine had approved the animal protocol. In brief, the cortex isolated from Sprague-Dawley rat pup by decapitation was digested in 0.5 mg/ml papain at 37°C for 15 min. The tissue was dissociated in Hibernate A medium (containing B27 supplement) by aspirating trituration. Cells were plated and maintained in Neurobasal medium containing B27 supplement, 10 units/ml penicillin, 10 µg/ml streptomycin, and 0.5 µg/ml glutamine for 3 days. Cells were then exposed to cytosine--D-arabinofuranoside (5 µM) for 1 day to eliminate proliferation of non-neuronal cells. The cells were used for experiments on the 10th day.

Treatment of NMDA and Ester Derivatives of TAB. Cortical neurons were pretreated with vehicle [0.1% dimethyl sulfoxide (DMSO)], TABM, TABE, or TAB for 30 min. Afterward, NMDA was added directly into medium (containing vehicle, TAB, or the ester derivatives of TAB) to a final concentration of 50 µM and incubated for a specific period.

Measurement of Cell Viability. The reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and release of lactate dehydrogenase (LDH) were used to evaluate the cell viability. Cells were incubated with 0.5 mg/ml MTT for 1 h. The formazan particle was dissolved with DMSO. OD_600nm was measured by using an enzyme-linked immunosorbent assay reader. The activity of LDH was determined by the method described previously (Roth et al., 1999).

Immunocytochemistry. Treated cells were fixed with 4% paraformaldehyde at room temperature for 15 min and permeabilized with 0.5% Triton X-100 for 10 min. Cells were blocked with 10% control donkey serum at room temperature for 2 h. Thereafter, cells were exposed to anti-MAP2 monoclonal antibody (1:100 dilution) at 4°C overnight. After wash, donkey anti-mouse IgG antibody conjugated with fluorescein (20 µg/ml) was applied to cells and incubated at 37°C for 1 h. Coverslips were mounted onto glass slides and detected by a Leica CS SP confocal fluorescence microscope (Wetzlar, Germany).

Measurement of Superoxide Anion (). The intracellular level of was measured by dihydroethidium assay (Zhang et al., 2003). Treated cells were loaded with 3.2 µM dihydroethidium at 37°C for 30 min and then observed by a Leica DMIRB fluorescence microscope. The intensity of fluorescence in nuclear position was measured by using MetaMorph software (Universal Imaging Co., West Chester, PA).

Measurement of Cellular Activity of Caspases. Treated cells were harvested in cell lysis buffer (50 mM HEPES, pH 7.4, 1 mM dithiothreitol, 0.1 mM EDTA, 0.1% Chaps, and 0.1% Triton X-100). The cellular lysates were prepared and subjected to assay of caspases activity. Intracellular activity of caspase 2, 3, 6, 8, and 9 was determined by the ability to cleavage Ac-VDVAD-pNA, Ac-DEVD-pNA, Ac-VEID-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA, respectively. The detailed experiments were performed according to the manufacturer's protocol. Immunoblotting was performed to verify the activation of caspase 12 by the disappearance of procaspase 12 (40 kDa).

Immunoblotting. Treated cells were washed twice with ice-cold phosphate-buffered saline and collected in harvest buffer (50 mM HEPES, pH 7.5, 2.5 mM EDTA, 1 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The cellular lysates were prepared and subjected to SDS-polyacrylamide gel electro-phoresis and immunoblotting. Rabbit anti-caspase 12 antibody (1: 3000 dilution) and mouse anti--spectrin antibody (1:3000 dilution) were used to detect the intracellular level of procaspase 12 and -spectrin, respectively. Fujifilm LAS-3000 (Tokyo, Japan) was used to detect and quantify the immunoreactive protein.

Determination of Mitochondrial Membrane Potential. Cells were loaded with 100 nM TMRM at 37°C for 20 min. After wash with Hanks' balanced salt solution (HBSS; 137 mM NaCl, 5.4 mM KCl, 0.4 mM KH₂PO₄, 4.2 mM NaHCO₃, 0.5 mM MgCl₂, 0.6 mM MgSO₄, and 5.6 mM D-glucose, pH 7.4), cells were incubated in HBSS containing 2 mM CaCl₂ and transferred to a microscope-equipped humidity chamber in a condition of 37°C and 5% CO₂. NMDA (50 µM) or 50 µM TABM were added into cell culture at 3-min time points and incubated for 27 min. The fluorescence of TMRM was detected by a Leica DMIRB fluorescence microscope with an excitation wavelength of 555 nm. The time-lapse images during 30 min were captured every 15 s using MetaFluor software (Universal Imaging Co.).

Measurement of Intracellular, Mitochondria, and ER Calcium. Intracellular free Ca²⁺ ([Ca²⁺]_i) was measured using fluo-3 AM. Cells were loaded with 1 µM fluo-3 AM at 37°C for 15 min. Cells were washed with HBSS and incubated in HBSS containing 2 mM CaCl₂. Cells were treated with vehicle or 50 µM TABM at 37°C for 30 min and then transferred to a microscope-equipped humidity chamber in a condition of 37°C and 5% CO₂. Cells were exposed to 50 µM NMDA and detected by a Leica DMIRB fluorescence microscope with an excitation wavelength of 484 nm. Free Ca²⁺ in mitochondria ([Ca²⁺]_mit) was quantified using rhod-2 AM. The cells were loaded with 1 µM rhod-2 AM at 37°C for 15 min. Cells were treated with vehicle or TABM as described above. Cells were transferred to the humidity chamber and were then exposed to NMDA and detected by a fluorescence microscope with an excitation wavelength of 555 nm. Free Ca²⁺ in ER ([Ca²⁺]_er) was determined by mag-fura-2 AM. Cells were loaded with 5 µM mag-fura-2 AM at 37°C for 60 min. Cells were incubated in Mg²⁺-free HBSS containing 2 mM CaCl₂ and treated with vehicle or TABM as described above. Cells were transferred to a humidity chamber and were then exposed to NMDA and detected by a fluorescence microscope with a dual excitation wavelength of 340 and 380 nm. The time-lapse images during 60 min were captured every 15 s using MetaFluor software. The validity of using mag-fura-2 and rhod-2 to measure [Ca²⁺]_er and [Ca²⁺]_mit was confirmed by the colocalization with ER and mitochondria tracker, respectively.

Statistical Analysis. Results are expressed as mean ± S.D. and were analyzed by analysis of variance with post hoc multiple comparisons with a Bonferroni test.

View larger version (53K): [in this window] [in a new window]   Fig. 2. TABM and TABE decreased NMDA-mediated cell death. Cortical neurons were treated with vehicle (0.1% DMSO, v/v) or 10-100 µM TAB (A), TABM (B), or TABE (C) for 30 min. Afterward, NMDA was added to a final concentration of 50 µM and incubated for 24 h. Cell viability was measured by MTT reduction. Cells were treated with vehicle, 50 µM TAB, TABM, or TABE for 30 min and then challenged without (open column) or with (closed column) 50 µM NMDA for 24 h. Cell viability was measured by LDH release (D). Results are means ± S.D. from five independent experiments for MTT reduction and four independent experiments for LDH release and are expressed relative to control cells for MTT reduction and relative to the intracellular LDH in control cells for LDH release. Significant differences between cells treated with NMDA and NMDA plus TAB derivatives are indicated by *, p < 0.05; **, p < 0.01; and *** p < 0.001. E, representative photographs of MAP2 immunofluorescent staining of neurons. Cells were treated with vehicle (a and d), 50 µM TABM (b and e), or 50 µM TABE (c and f) for 30 min and then exposed to 50 µM NMDA for 0 (a-c) and 4 h (d-f).

	Results

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TABM and TABE Abrogated the NMDA-Induced Activation of Caspases. Treatment with NMDA induced the activation of calpain and caspase 3 as determined by the degradation of -spectrin (Fig. 3) (Pang et al., 2003). The activation of calpain produced 145- and 150-kDa fragments of -spectrin, whereas activation of caspase 3 generated 120- and 150-kDa fragments of -spectrin. Both 150- and 145-kDa protein fragments appeared at 30 to 60 min after exposure to 50 µM NMDA (Fig. 3, C and D). The 120-kDa degraded fragment only significantly generated after 4 to 30 h (Fig. 3E). The generation of degraded -spectrin fragments was in parallel to the disappearance of 250-kDa -spectrin in a time-dependent manner (Fig. 3B). TABM and TABE did not show any effects on the activation of calpain (Fig. 4, C and D). TABM (50 µM) reduced the NMDA-mediated production of 120 kDa by 37.6 ± 7.1 and 25.3 ± 1.6% at 8 and 24 h, respectively (Fig. 4E). TABM attenuated the production of 120 kDa coincided with the accumulation of 250 kDa at 8 and 24 h (Fig. 4B). Fifty micromolar TABM and TABE diminished the degradation of -spectrin by 63.2 ± 12.6 and 35.8 ± 0.7% at 24 h, respectively.

View larger version (32K): [in this window] [in a new window]   Fig. 3. NMDA activated calpain and caspase 3. Cortical neurons were incubated with 50 µM NMDA for 0 to 30 h. Equal amounts of cell lysate were subjected to immunoblotting using anti--spectrin antibody. A, representative immunoblots of -spectrin and degraded fragments. The activation of calpain cleaved 250-kDa -spectrin (B) to produce 150- (C) and 145-kDa (D) fragments, whereas activation of caspase 3 generated 120-(E) and 150-kDa fragments of -spectrin. Results are means ± S.D. (where large enough to be shown) from three independent experiments and are expressed relative to control cells for 250 kDa or relative to cells treated with NMDA for 30 h for 150, 145, and 120 kDa. Significant differences between control cells and cells treated with NMDA are indicated by *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

View larger version (31K): [in this window] [in a new window]   Fig. 4. TABM and TABE attenuated NMDA-mediated activation of caspase 3. Cortical neurons were incubated with vehicle (open column), 50 µM TABM (closed column), or 50 µM TABE (dotted column) for 30 min and then challenged with 50 µM NMDA for 0 to 24 h. Immunoblotting was performed using anti--spectrin antibody to detect 250-kDa (B) -spectrin or 150- (C), 145- (D), and 120-kDa (E) degraded fragments of -spectrin. A, representative immunoblots of -spectrin and degraded fragments. Results are means ± S.D. (where large enough to be shown) from four independent experiments and are expressed relative to cells at the zero time point for 250 kDa or relative to cells treated with MNDA for 24 h for 150, 145, and 120 kDa. Significant differences between cells treated with NMDA alone and cells treated with NMDA plus TABM or TABE are indicated by *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

The in vitro assay was further conducted to evaluate the effects of TABM and TABE on the activity of caspases. NMDA treatment increased the caspase activity in a time-dependent manner and reached the maximum at 8 h (Fig. 5A). Treatment with 50 µM NMDA for 8 h increased the activity of caspases 2, 3, 6, and 8 to 9.7 ± 2.1-, 7.2 ± 0.4-, 7.3 ± 2.8-, and 3.0 ± 0.7-fold of control, respectively. The activity of caspase 9 was barely detected in control cells. NMDA treatment elevated the activity of caspase 9 to a maximum at 8 h. TABM (50 µM) attenuated the NMDA-mediated activity of caspases 2, 3, 6, 8, and 9 by 87.6 ± 13.5, 78.3 ± 10.6, 76.5 ± 14.1, 89.1 ± 18.8, and 101.8 ± 3.7% at 8 h, respectively (Fig. 5B). The equivalent values for TABE were 50.4 ± 14.5, 48.9 ± 9.9, 49.2 ± 10.1, 59.6 ± 13.6, and 78.3 ± 12.7%.

View larger version (23K): [in this window] [in a new window]   Fig. 5. TABM and TABE attenuated the NMDA-induced increase of caspases activities. Cells were treated with NMDA for 0 to 24 h (A). Cells were harvested to assay the activity of caspases 2 (open column), 3 (closed column), 6 (dotted column), 8 (hatched column), and 9 (crosshatched column). Results are means ± S.D. (where large enough to be shown) from three independent experiments. Significant differences between control cells and cells treated with NMDA are indicated by *, p < 0.05; **, p < 0.01; and ***, p < 0.001. Cells were treated with vehicle (open column), 50 µM TABM (closed column), or 50 µM TABE (dotted column) for 30 min and then exposed to 50 µM NMDA for 8 h (B). Cells were harvested to assay the activity of caspases 2, 3, 6, 8, and 9. Results are means ± S.D. (where large enough to be shown) from five independent experiments. Significant differences between cells treated with NMDA and cells treated with NMDA plus TAB ester derivative are indicated by **, p < 0.01; and ***, p < 0.001.

View larger version (22K): [in this window] [in a new window]   Fig. 6. TABM and TABE attenuated the activation of caspase 12 induced by NMDA. Cells were treated with 50 µM NMDA for 0 to 24 h (A). Equal amounts of cell lysate were subjected to immunoblotting using anticaspase 12 antibody to detect the level of 55-kDa (open column) and 40-kDa (closed column) procaspase 12. Cells were treated with vehicle, 50 µM TABM, or 50 µM TABE for 30 min and then exposed to 50 µM NMDA for 8 h (B). Equal amounts of cell lysate were subjected to immunoblotting using anticaspase 12 antibody. Activation of caspase 12 induced the cleavage of 40-kDa procaspase 12. Results are means ± S.D. from four independent experiments and are expressed relative to control cells. The top parts of each panel are the representative immunoblots of 55-kDa and 40-kDa procaspase 12. Significant differences between control cells and cells treated with NMDA are indicated by *, p < 0.05; and ***, p < 0.001. Significant differences between cells treated with NMDA and cells treated with NMDA plus TAB ester derivative are indicated by , p < 0.05; and , p < 0.01.

TABM and TABE Blocked the NMDA-Induced Accumulation of

View larger version (16K): [in this window] [in a new window]   Fig. 7. TABM and TABE diminished the NMDA-mediated accumulation. Cells were exposed to 50 µM NMDA for 0 to 8 h (A). Cells were treated with vehicle, 50 µM TABM, or 50 µM TABE for 30 min and then incubated without. or with 50 µM NMDA for 20 min (B). The level of intracellular was measured by dihydroethidium assay. Results are means ± S.D. from four independent experiments and are expressed relative to control cells. Significant differences between control cells and cells treated with NMDA in A or between cells treated with NMDA and cells treated with NMDA plus TABM or TABE in B, are indicated by ***, p < 0.001.

TABM Decreased the Mitochondrial Membrane Potential. TMRM was used to verify whether TABM directly modulated the mitochondrial membrane potential. The change profile of fluorescence after exposure to NMDA or TABM was similar during cell population in spite of the heterogeneous fluorescence intensity of each individual cell (Fig. 8). NMDA (50 µM) induced a slight decline of TMRM fluorescence at 0.5 min. A marked mitochondria depolarization occurred after 1 min and reached the plateau at approximately 10 min. In contrast to NMDA, TABM elicited significant mitochondria depolarization immediately. Treatment with 50 µM TABM for 0.5 min reduced the fluorescence intensity by 22.3 ± 4.6%. However, TABM-induced mitochondria depolarization was much slower than NMDA after 1 min. Treatment with NMDA and TABM for 7 min decreased the fluorescence intensity by 63.3 ± 4.2 and 41.8 ± 5.3%, respectively. Nevertheless, there was no significant difference in fluorescence intensity between cells treated with NMDA and TABM after 20 min.

View larger version (24K): [in this window] [in a new window]   Fig. 8. The effects of TABM and NMDA on the mitochondrial membrane potential. Cells were loaded with TMRM to monitor the mitochondrial membrane potential. TMRM-loaded cells were transferred to a microscope-equipped humidity chamber in a condition of 37°C and 5% CO2. Cells were exposed to 50 µM NMDA (open column) or 50 µM TABM (closed column) at a 3-min time point and detected by a fluorescence microscope with an excitation wavelength of 555 nm. The time-lapse images during 30 min were captured every 15 s using MetaFluor software. Results are means ± S.D. from four independent experiments and are expressed relative to cells at the first recording time point. A and B, representative time course of TMRM fluorescence recorded in cells treated with 50 µM NMDA and 50 µM TABM, respectively. C, relative average of fluorescence intensity in cells treated with NMDA or TABM. D, relative fluorescence intensity in cells treated with NMDA or TABM for 0, 0.5, 7, 17, and 27 min. Significant differences between cells treated with NMDA and TABM are indicated by **, p < 0.01.

View larger version (27K): [in this window] [in a new window]   Fig. 9. Effect of TABM on the NMDA-induced elevation of [Ca2+]i. Cells were loaded with fluo-3 AM to monitor the level of [Ca2+]i. Fluo-3 AM-loaded cells were treated with vehicle (open column) or 50 µM TABM (closed column) for 30 min and then transferred to a microscope-equipped humidity chamber in a condition of 37°C and 5% CO2. Cells were exposed to 50 µM NMDA at a 1-min time point and detected by a fluorescence microscope with excitation wavelength of 484 nm. The time-lapse images during 60 min were captured using MetaFluor software. Results are means ± S.D. from four independent experiments. A and B, representative time course of Ca2+-dependent fluorescence recorded in control cells and TABM-treated cells, respectively. C, average of time course effect of TABM on the NMDA-induced elevation of fluorescence. D, fluorescence intensity at 0, 5, 30, and 60 min. Significant differences between cells treated with NMDA and cells treated with NMDA plus TABM are indicated by **, p < 0.01; and ***, p < 0.001.

View larger version (23K): [in this window] [in a new window]   Fig. 10. TABM eliminated the NMDA-induced elevation of [Ca2+]er. Cells were loaded with mag-fura-2 AM to monitor the level of [Ca2+]er. Magfura-2 AM-loaded cells were treated with vehicle (open column) or 50 µM TABM (closed column) for 30 min and then transferred to a microscope-equipped humidity chamber in a condition of 37°C and 5% CO2. Cells were exposed to 50 µM NMDA at a 1-min time point and detected by a fluorescence microscope with a dual excitation wavelength of 340 and 380 nm. The time-lapse images during 60 min were captured using MetaFluor software. Results are means ± S.D. from four independent experiments. A and B, representative time course of ratiometric values (Ex 340 nm/Ex 380 nm) in control cells and TABM-treated cells, respectively. C, average of time course effect of TABM on the NMDA-induced elevation of ratiometric value. D, ratiometric value at 0, 5, 30, and 60 min. Significant differences between cells treated with NMDA and cells treated with NMDA plus TABM are indicated by **, p < 0.01.

The fluorescence of rhod-2 was elevated by NMDA to 2.17 ± 0.15-, 3.02 ± 0.19-, and 2.98 ± 0.09-fold of initial fluorescence intensity for 5, 30, and 60 min, respectively (Fig. 11). Treatment with 50 µM TABM diminished the basal fluorescence intensity by approximately 50%. TABM caused a delayed increase of rhod-2 fluorescence and reached the maximum at 30 min. The NMDA-mediated elevation of fluorescence intensity was also abrogated by TABM.

View larger version (26K): [in this window] [in a new window]   Fig. 11. TABM eliminated the NMDA-induced elevation of [Ca2+]mit. Cells were loaded with rhod-2 AM to monitor the level of [Ca2+]mit. Rhod-2 AM-loaded cells were treated with vehicle (open column) or 50 µM TABM (closed column) for 30 min and then transferred to a microscope-equipped humidity chamber in a condition of 37°C and 5% CO2. Cells were exposed to 50 µM NMDA at a 1-min time point and detected by a fluorescence microscope with an excitation wavelength of 555 nm. The time-lapse images during 60 min were captured using MetaFluor software. Results are means ± S.D. from four independent experiments (where large enough to be shown). A and B, representative time course of Ca2+-dependent fluorescence recorded in control cells and TABM-treated cells, respectively. C, average of time course effect of TABM on the NMDA-induced elevation of fluorescence. D, fluorescence intensity at 0, 5, 30, and 60 min. Significant differences between cells treated with NMDA and cells treated with NMDA plus TABM are indicated by **, p < 0.01; and ***, p < 0.001.

	Discussion

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In the present study, we demonstrated that TABM and TABE, but not TAB, abrogated NMDA-mediated excitotoxicity as determined by MTT reduction, LDH release, and morphological observation. TABM decreased mitochondrial membrane potential in parallel to attenuating the NMDA-mediated Ca²⁺-loading in ER and mitochondria. TABM and TABE diminished the accumulation of , blocked activation of the caspase cascade, and thereby protected neuron against NMDA-mediated neurotoxicity.

Both necrosis and apoptosis are implicated in excitotoxicity (Portera-Cailliau et al., 1997; Polster and Fiskum, 2004). Necrosis is characterized by the irreversible swelling of cytoplasm and its organelles. Loss of membrane integrity results in cell lysis and the release of noxious cellular constituents. Recent studies have suggested that calpain is involved in the necrotic and apoptotic processes during excitotoxicity (Wang, 2000; Pang et al., 2003). There are two isoforms of calpain named µ-calpain and m-calpain. µ-Calpain and m-calpain require micromolar or millimolar of Ca²⁺ level for the activation, respectively. Our results showed that NMDA triggered the leakiness of LDH, indicating that a necrotic event is involved in NMDA-mediated excitotoxicity. TABM and TABE significantly abrogated NMDA-mediated neuron necrosis as measured by LDH release. However, neither TABM nor TABE affected the NMDA-mediated activation of calpain. These results suggest that TABM and TABE might modulate other mechanisms to confer their antinecrotic effects. The study of Adamec et al. (1998) also showed that calpain I activation in rat hippocampal neurons is NMDA receptor-selective but not essential for excitotoxic cell death.

Apoptosis-mediated cell death in excitotoxicity has become the focus of intense interest. However, the underlying mechanisms remain unclear. Studies have shown that glutamate activates caspase 3 in cultured cortical neurons, striatal neurons, and mesencephalic neurons (Liu and Zhu, 1999; Madhavan et al., 2003). Our previous study also demonstrates that glutamate ubiquitously elicits the activation of caspases 2, 3, 6, 8, and 9 (Chi et al., 2005). The present study further provided evidence that NMDA extensively activated the initiative caspases (i.e., caspases 2, 8, 9, and 12) and the executive caspases (i.e., caspases 3 and 6). The attenuation of NMDA-induced activation of caspase activity may, at least in part, account for the neuroprotective effect of TABM and TABE.

Glutamate triggers a massive influx of Ca²⁺ via ionotropic receptor into the intracellular compartment. ER and mitochondria function as the intracellular stores to sequester the excessive amount of cytosolic Ca²⁺. It is well established that the disturbance of Ca²⁺ homeostasis in mitochondria ultimately causes the deregulation of ROS production and bioenergetic metabolism. The mitochondrion is the main target compartment for multiple antiapoptotic and proapoptotic molecules such as Bcl-2, Bax, Bad, Bid, etc. Furthermore, mitochondrion is involved in the activation of caspase 9 mediating by cytochrome c release. Therefore, the mitochondrion is a pivotal organelle implicating in apoptosis (Polster and Fiskum, 2004). Recently, ER has attracted attention as an intracellular compartment in which disturbance of calcium homeostasis may contribute to pathological processes culminating in neuronal injury (Mattson et al., 2000; Paschen, 2001; Scorrano et al., 2003). Calcium levels in ER are several orders of magnitude higher than that in cytoplasm. ER calcium homeostasis is controlled by the ryanodine receptor RyaR and IP3 receptor IP3R, which release calcium ions from ER stores upon activation, and the calcium pump SERCA, which pumps back calcium ions against a steep concentration gradient.

Treatment with glutamate or NMDA evokes a rapid rise in [Ca²⁺]_i, which then shows a second phase of Ca²⁺ elevation (Duchen, 2000; Pivovarova et al., 2004; Bano et al., 2005). Glutamate-induced initial calcium spike is due mainly to the calcium influx through an NMDA receptor-gated channel. The influx of calcium through voltage-gated calcium channels and calcium intake due to the reverse mode of the Na⁺/Ca²⁺ exchanger may also partly contribute to the initial calcium increase (Tymianski et al., 1993). Our unpublished data show that a change of [Ca²⁺]_i was barely detected when the experiment was conducted using calcium-free buffer. Therefore, the release of calcium from intracellular stores may be not involved in the initial increase of [Ca²⁺]_i. induced by NMDA. The delayed increase of [Ca²⁺]_i is proposed to be the consequence of influx of Ca²⁺, outflow of Ca²⁺ from mitochondria, and calpain-dependent cleavage of Na⁺/Ca²⁺ exchanger (Randall and Thayer, 1992; Duchen, 2000; Bano et al., 2005). The present study showed that the profile of [Ca²⁺]_i displayed a mixed pattern rather than a typical two-phase elevation of Ca²⁺. It is conceivable that the outflow of [Ca²⁺]_i from mitochondria may be faster in our system. This speculation is supported by the result of mitochondrial membrane potential. NMDA elicited rapid depolarization of mitochondria, subsequently leading to the release of [Ca²⁺]_i from mitochondria. Treatment with TABM caused a higher level of [Ca²⁺]_i after 15 min compared with the control cells. The result suggests that TABM might retard the entrance of Ca²⁺ into mitochondria or ER. This assumption is further confirmed by the results of [Ca²⁺] and [Ca²⁺_er]_mit. NMDA elicited a significant elevation of [Ca²⁺] and [Ca²⁺_er]_mit. Cells treated with TABM exhibited a delayed increase of [Ca²⁺]_er. Individual TABM-treated cells exerted a heterogeneous time course of delayed increase of [Ca²⁺]_er. For [Ca²⁺]_mit, TABM delayed the time course of increase of [Ca²⁺]_mit and decreased the extent of maximum value. Therefore, the increased level of [Ca²⁺]_i in TABM-treated cells may reflect the impairment of Ca²⁺ sequestrating by ER and mitochondria. Furthermore, the relevance of delayed increase of [Ca²⁺]_er and the attenuated elevation of [Ca²⁺]_mit on the neuroprotection is supported by the subsequent antiapoptotic events. These events include the diminishment of ER-related activation of caspase 12, abolishment of mitochondria-mediated accumulation of , and blockade of mitochondria-involved activation of caspase 9 and caspase 3.

The Ca²⁺ homeostasis of ER is maintained by the balance between RyaR/IP3R-mediated release of Ca²⁺ and SERCA-related pump in of Ca²⁺. The TABM-mediated depolarization of mitochondria may be concomitant with the depletion of ATP (Starkov et al., 2004), thereby impairing ATP-dependent processes of ER Ca²⁺ accumulation (via SERCA). This may provide an explanation for the modulation of Ca²⁺ homeostasis in ER by TABM. Several studies have provided evidence that a mild decrease in mitochondrial membrane potential impairs the uptake of Ca²⁺, in turn, protecting neurons from cell death (Stout et al., 1998; Mattiasson et al., 2003; Pivovarova et al., 2004; Marks et al., 2005). Our results also demonstrated that TABM decreased mitochondrial membrane potential. The depolarization of mitochondria is associated with decreasing ROS production and blocking mitochondrial Ca²⁺ uptake (Starkov et al., 2004). This explains the result that the basal level of [Ca²⁺]_mit was prominently reduced by TABM. These results clearly demonstrated that TABM/TABE decreased mitochondrial membrane potential and attenuated the Ca²⁺ overload in both mitochondria and ER, consequently impaired NMDA-induced activation of caspases cascade, and eliminated the accumulation of , thereby conferring their neuroprotective effects on NMDA-mediated neurotoxicity.

	Footnotes

 
This study was supported by grant NSC-93-2323-B-077-001 from National Science Council, and grants 94DBCMR-2 and 94DBCMR-3 from National Research Institute of Chinese Medicine, Taiwan, Republic of China.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; ER, endoplasmic reticulum; ROS, reactive oxygen species; , superoxide anion; NO, nitric oxide; TAB, tournefolic acid B; TABM, tournefolic acid B methyl ester; TABE, tournefolic acid B ethyl ester; AM, acetoxymethyl ester; TMRM, tetramethylrhodamine methyl ester perchlorate; MAP2, microtubule-associated protein 2; DMSO, dimethyl sulfoxide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; LDH, lactate dehydrogenase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Ac, N-acetyl; pNA, p-nitroanilide; HBSS, Hanks' balanced salt solution; SERCA, sarco(endo)plasmic reticulum Ca²⁺ ATPase.

Address correspondence to: Dr. Young-Ji Shiao, National Research Institute of Chinese Medicine, No. 155-1. Sec. 2, LiNung St., Peitou, Taipei, Taiwan, Republic of China. E-mail: yshiao{at}nricm.edu.tw

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