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Very low levels of methylmercury induce cell death of cultured rat cerebellar neurons via calpain activation
Toxicology 213 (2005) 97–106
Very low levels of methylmercury induce cell death of cultured rat cerebellar neurons via calpain activation Motoharu Sakaue ∗ , Maiko Okazaki, Shuntaro Hara Department of Public Health, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan Received 1 April 2005; received in revised form 18 May 2005; accepted 19 May 2005 Available online 27 June 2005
Abstract Methylmercury, an environmental neurotoxicant, induces the apoptotic death of cerebellar granule cells in vitro at a low concentration. To further understand the mechanism of cell death, we used a rat cerebellar granule cell culture system to investigate whether the calpain/cyclin-dependent kinase 5 (cdk5)/p35 cascade, an important cascade for neuronal apoptosis, is involved in the methylmercury-induced death. A noteworthy finding was that the cerebellar granular cell death was increased at a very low concentration of methylmercury, 30 nM, which is lower than that previously reported. The high sensitivity to methylmercury indicates that this culture system is useful for studying methylmercury toxicity at very low concentrations. Using this system, we here found that the methylmercury-induced death was inhibited by the calpain inhibitor II. Furthermore, it was shown that, in methylmercury-exposed cells, ␣-fodrin and tau, calpain substrates, were cleaved to the fragments that disappeared by treatment with the calpain inhibitor II. We next assayed and showed that the intracellular Ca2+ concentration in cerebellar granule cells increased after methylmercury exposure in a time- and dose-dependent manner, significantly even at 30 nM. These results indicated that a very low concentration of methylmercury causes the intracellular Ca2+ concentration to increase, activates calpain in the cells, and then induces cell death. We further found that the p35 protein was also processed to p25 that forms the cdk5–p25 complex, a hyperactive kinase for tau. However, an immunoblot using the anti-phosphorylated tau antibody showed that there was no increase of phosphorylated tau in methylmercury-exposed cells. These results suggested that methylmercury-induced cell death via calpain activation should not involve the stimulation of tau phosphorylation activity. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Methylmercury; Cerebellar granule neurons; p35; Cyclin-dependent kinase 5; Calpain; Very-low-concentration neurotoxicity
1. Introduction ∗
Corresponding author. Tel.: +81 3 5791 6266; fax: +81 3 3442 4146. E-mail address: [email protected] (M. Sakaue).
Methylmercury is widely recognized as a potent neurotoxicant. Intoxication by methylmercury causes pathological degeneration, specifically in the brain
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(Chang, 1977; Clarkson, 1987; Nagashima et al., 1996). Cerebellar granule cells, which are particularly vulnerable to methylmercury-induced damage in vivo, have provided a good model for analysis of methylmercury-induced neuronal cell death, apoptosis. Previous reports using the in vitro culture system of cerebellar granule cells have demonstrated methylmercury toxicity, and have shown that methylmercury can induce apoptotic death of the cells at lower concentrations, whereas it induces non-apoptotic cell death at higher concentrations (>1 M) (Chang, 1977; Dare et al., 2000; Kunimoto, 1994; Sakaue et al., 2003). Methylmercury causes neuronal cell death by disrupting the intracellular homeostasis, changing the intracellular Ca2+ concentration, inhibiting microtubule assembly and increasing reactive oxygen species production (Marty and Atchison, 1997; Miura and Imura, 1987; Sarafian and Verity, 1991; Sarafian, 1993) when exposed to the cells at sub M and higher. Even at lower concentrations (≤sub M), apoptotic death of cerebellar granule cells is induced via the caspaseindependent pathway (Chang, 1977; Dare et al., 2000; Kunimoto, 1994; Sakaue et al., 2003), but little is known about how a lower concentration of methylmercury induces neuronal cell death in a neuron-specific manner. Lee et al. reported that the calpain/cyclin-dependent kinase 5 (cdk5)/p35 cascade is important for the apoptosis of neuronal cells. A cdk5 activator p35 is specifically expressed in neuronal cells and a cleavage of p35 to p25, which is followed by the redistribution and overactivation of cdk5 after association with p25 (Lee et al., 2000; Lew et al., 1994; Tsai et al., 1994). Calpain, a calcium-dependent cysteine peptidase (Guroff, 1963; Wang, 1999; Yoshimura et al., 1983), is abundantly expressed in neurons (Wang, 1999; Hamakubo et al., 1986) and degrades specific substrates, for example, microtubule-associated protein 2, tau, ␣-fodrin and p35. After p35 is processed to p25 by calpain, p25 forms the complex of cdk5–p25, by which many proteins, including the tau protein and neurofilaments, are hyperphosphorylated (Nath et al., 2000). As tau acts as a stabilizer of microtubules, its degradation by calpain or hyperphosphorylation by cdk5–p25 causes its dysfunction, induces microtubule disruption, and then results in neuronal cell apoptosis (Lee et al., 2000; Nath et al., 2000; Kusakawa et al., 2000). Some models of neurodegenerative disorders, such as
Alzheimer’s disease and amyotrophic lateral sclerosis, include the activation of calpain and the processing of p35 to p25, which lead to neuronal apoptosis (Lee et al., 2000; Nguyen et al., 2001). We have reported that axonal protein 440 kDa ankyrinB , a calpain substrate of rat cerebellar granule cells, disappears with methylmercury treatment at a very low concentration, which suggests that calpain might contribute to the mechanism for specific neuronal cell death at a very low concentration of methylmercury (Sakaue et al., 2003). To gain a further understanding of the mechanism of methylmercury-induced cell death and to account for its neurotoxicity, we show, in the present study, the effects of a very low concentration of methylmercury on calpain activity in rat cerebellar granular cells, and detect the degradation of the calpain substrates, ␣-fodrin, tau and p35. Furthermore, we investigate and discuss whether processing p35 to p25 is involved in methylmercury-induced cell death at a very low concentration.
2. Materials and methods 2.1. Cell cultures Primary cultures of cerebellar granule neurons were prepared from Wistar rats (Jcl: Wistar; Clea Co., Tokyo, Japan) within 24 h after birth, as described previously (Kunimoto et al., 1992). Cerebella incubated with trypsin for 13 min at room temperature were minced by mild trituration with a Pasteur pipette. Cerebellar granule cells were seeded in Eagle’s minimal essential medium (Gibco BRL, Grand Island, NY) containing 1 mg/ml BSA, 10 g/ml bovin insulin, 0.1 nM thyroxine, 0.1 mg/mg human transferrin, 1 g/ml aprotinin, 30 nM Na2 SeO3 , 0.25% glucose, 100 units/mL penicillin and 135 g/mL streptomycin on poly-l-lysine-coated dishes and cultured for 2 days. All cell culture supplements were purchased from Sigma Chemical Company (St Louis, MO). Then, the cells were treated with methylmercuric chloride (Tokyo Kasei Kogyo. Co. Ltd., Tokyo, Japan) at 0–1 M for 24 or 48 h with or without calpain inhibitor II (Calbiochem San Diego, CA). All experiments were done in accordance with the Kitasato University Guidelines for Animal Care and Experimentation.
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Rat neuroblastoma B35 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. The medium was changed every second day. The cells were treated with methylmercury chloride after 24 h preincubation. The number of viable cerebellar granule cells in the culture or B35 cells was estimated by crystal violet staining, as described previously (Sakaue et al., 2003). 2.2. SDS-PAGE and immunoblotting After being washed three times with PBS (pH 7.4), the cultured cells were harvested in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 0.1% Nonident P-40, 5 mM dithiothreitol, 10 mM NaF, 1 mM PMSF, 1 g/mL aprotinin, and 1 g/ml leupeptin. Twenty-five microgram or 50 g of each lysate were electrophoresized on SDS-PAGE gels and immunoblotted using rabbit polyclonal antibodies to p35 (Sigma) or cdk5 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal antibodies against glyceraldehydes-3-phosphate dehydrogenase (GAPDH; Sigma), ␣-fodrin (MP Biochemicals, Inc., Irvine, CA) or tau (Tau-1, BM; AT8, Endogen, Woburn, MA; 5E2, Upstate Biotechnology, Lake Placid, NY) after electrophoretic transfer to the Immobilon P membrane (Millipore Corp., Bedford, MA). The anti-p35 antibody detects p35 and p25, the calboxy-terminal proteolytic product of p35. Densitometric quantification of immunoblots was done using ImageJ (version 1.31) software (NIH, Bethesda, MD). 2.3. Intracellular calcium concentration assay using Fluo-3 AM The intracellular calcium concentration of cerebellar granule cells seeded in 24 well plates was assayed using a fluorescent Ca2+ indicator, Fluo-3 acetoxymethyl ester (Fluo-3 AM; Dojindo, Tokyo, Japan), which specifically binds a calcium ion (Kao et al., 1989; Minta et al., 1989). In the first experiment, after 2 or 3 days in the subculture, the cerebellar granule cells were washed with Tyrode’s salt solution (Sigma) and incubated in Fluo-3 AM Tyrode’s salt solution (4 M Fluo-3 AM, 0.018% Pluronic F-127) for 1 hr in 37 ◦ C in a 5% CO2 , 95% humidity atmosphere. Fluo-3 AM was taken up by the cells, and entrapped intracellularly after hydrolysis to Fluo-3 by neuronal
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esterases. The intracellular Fluo-3 was excited at 488 nm and emits in wavelength of 530 nm range. Fluorescence readings were taken once in every 3 min for 9 min prior to the methylmercury additions as their basal fluorescent intensity, and then every 3 min for 120 min following methylmercury exposure at room temperature in atmosphere using Fluoroskan Ascent type 347 (Labsystems, Helsinki, Finland). In the second experiment, cerebellar granule cells cultured in a lower methylmercury concentration medium for 3, 6 or 24 h were washed with Tyrode’s salt solution and assayed after loading Fluo-3 AM, and then were treated in the same manner as described above. 2.4. Statistical analysis Statistical significances between groups were assessed by one-way analysis of variance (ANOVA). Bonferroni/Dunn test was employed to compare individual means as a post-hoc test.
3. Results 3.1. Inhibition of methylmercury-induced cerebellar granule cell death by calpain inhibitor The viability of cerebellar granule cells was assayed using crystal violet staining 48 h after the methylmercury treatment (Fig. 1). The methylmercury-induced death of the cerebellar granule cells increased dosedependently (Fig. 1A). It was noteworthy that the cell death significantly increased at a very low concentration of methylmercury, 30 nM, which was much lower than that reported previously (Kunimoto, 1994). We further found that the viability of the cells treated with 30 or 100 nM methylmercury was statistically restored from 76 and 43% to 89 and 60% by treatment of calpain inhibitor II 1 hr before methylmercury exposure, respectively (Fig. 1B). The inhibitory effect of calpain inhibitor II on the methylmercury neurotoxicity was significant but incomplete. The effect of methylmercury treatment for 48 h on rat neuroblastoma B35 cells was also examined. The sensitivity of B35 cells towards methylmercury was lower than that of cerebellar granule cells. The viability of B35 cells was also significantly decreased only above 300 nM. The value of the viability was 94, 92, 65 and 1% at
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Fig. 1. The effect of the calpain inhibitor II on cerebellar granule cell death induced through a very low concentration of methylmercury. Cerebellar granule cell cultures and rat neuroblastoma cell line, B35 cells were exposed to methylmercury for 48 h and assayed for viability, as described in Section 2. (A) The concentration-response relationship for the cell death elicited by methylmercury in cerebellar granule cell culture; and (B) its prevention by the 2 M calpain inhibitor II (C.I.II). (C) The cell viability of B35 cells treated with methylmercury. Data are expressed as the percentage of cell viability with respect to the control value, and are mean ± S.D. from n = 4 replicates per treatment group. Asterisks indicate the data that was statistically significantly different from control group (A, C) or between treatment groups (B) (p < 0.05).
0.3, 1, 3 and 10 M of methylmercury, respectively (Fig. 1C). 3.2. Degradation of calpain substrates through methylmercury exposure To determine whether calpain was activated in the cerebellar granule cells 48 h after methylmercury exposure, the cleavage of non-erythroid ␣-spectrin
known as ␣-fodrin, one of the well-characterized calpain substrates (Dare et al., 2000; Lee et al., 2000; Wang, 1999; Nath et al., 2000; Siman et al., 1989), was examined. Methylmercury caused cleavage of ␣-fodrin into a 150 kDa fragment in a dose-dependent manner. The calpain inhibitor II inhibited this cleavage of tau (Fig. 2A), another calpain substrate, and one of the major microtubule-associated proteins (Wang, 1999; Canu et al., 1998; Johnson et al., 1989). As
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calpain inhibitor II (Fig. 2A). The inhibitor treatment also caused an increase in the full length tau and bands 5–10 kDa smaller than the full length tau. 3.3. Methylmercury-induced alteration of Intracellular Ca2+ concentration
Fig. 2. Proteolytic cleavage of calpain substrates in cerebellar granule cells is induced by treatment with very low concentrations of methylmercury, and is inhibited by calpain inhibitor II (C.I.II). Primary cerebellar granule cell cultures were exposed to methylmercury for 48 h. (A) Cell lysates were subjected to immunoblotting and probed with anti-␣-fodrin (upper) or the anti-tau (clone, Tau-1) (lower) antibody. (B) Relative intensities of Tau-1 antibody reacted signals, 55 (intact tau protein) and 17 kDa (processed tau protein) bands. Data are given as the mean ± S.D. (n = 3). An asterisk indicated statistically significant differences in means from control group (p < 0.05).
shown in Fig. 2A, Western blot analysis on the state of tau revealed the appearance of a 17 kDa fragment, which was inhibited by the calpain inhibitor II, and was also obtained after 48 h of 30 nM methylmercury exposure. Densitometric analysis on the Western blot showed that the level of the 17 kDa fragment about 5-fold increased by methylmercury treatment (Fig. 2B). The inhibition of the disappearance of full length tau and the formation of the 17 kDa fragment was seen when simultaneously treating with the
Calpain is a Ca2+ -dependent protease (Guroff, 1963; Wang, 1999; Yoshimura et al., 1983). We next measured changes in the fluorescence of the Ca2+ indicator in order to confirm whether methylmercury exposure increases the intracellular Ca2+ concentration of the cerebellar granule cells (Fig. 3). In the initial experiment to examine the rapid response of the cerebellar granule cells to a higher concentration of methylmercury, the fluorescence intensities were measured in the course of time immediately after the methylmercury exposure (Fig. 3A). Methylmercury increased the fluorescence of Fluo-3 in the cerebellar granule cells dose- and time-dependently after exposure to the cells at 0.5 M and higher, but not at 0.1 and 0 M methylmercury. Furthermore, in the secondary experiment conducted to detect alterations of the intracellular Ca2+ concentration at lower concentrations of methylmercury, the cerebellar granule cells were incubated for 3–24 h in culture medium containing 3–300 nM methylmercury. Because cell death was induced by 48 h incubation in the neurotoxicant, the incubation times were selected for assay for the fluorescence signal (Fig. 3B). At a 10 nM and higher concentration of methylmercury, the fluorescence intensity statistically increased in a dose-dependent manner. 3.4. Processing p35 to p25 through methylmercury exposure The protein levels of p35, cdk5 and GAPDH were detected using Western blotting in cerebellar granule cells 48 h after the methylmercury treatment (Fig. 4). The immunoreactivity of p25 increased and that of p35 decreased in a methylmercury-dose-dependent manner, which was partially inhibited by the calpain inhibitor II in the cells. In addition, there was no significant difference in the levels of GAPDH as an internal standard and cdk5 between the control and the cells treated with methylmercury or methylmercury and the calpain inhibitor II.
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Fig. 3. Effect of methylmercury on intracellular Ca2+ concentration of rat cerebellar granule cells following in vitro exposure to methylmercury. (A) Time-dependent alterations in Fluo-3 fluorescence intensity. The arrowhead indicates where methylmercury was added. (B) Changes of fluorescence intensity after incubation for 3–24 h at lower concentrations of methylmercury. Data are mean ± S.D. from n = 4 replicates per treatment group. Asterisks indicate the data that was statistically significantly different from 0 nM treatment group at the same incubation time (p < 0.05).
Fig. 4. Proteolytic cleavage of p35 to p25 in cerebellar granule cells was induced by treatment with very low concentrations of methylmercury and was inhibited by calpain inhibitor II (C.I.II). Primary cerebellar granule cell cultures were exposed to methylmercury for 48 h. (A) Cell lysates were subjected to immunoblotting and probed with anti-p35 C-terminus, anti-cdk5 or anti-GAPDH antibodies. (B) Relative intensities of p25 signals that were detected by anti-p35 antibody. Data are given as the mean ± S.D. (n = 3). An asterisk indicate statistically significant differences in means from control group (p < 0.05).
3.5. The phosphorylation state of tau during methylmercury-induced apoptosis
examined the effect of methylmercury on the cdk5 activity in cerebellar granule cells by immunoblot analysis using the anti-tau monoclonal antibodies, 5E2, AT8 and Tau-1. Tau is phosphorylated by several kinases, including cdk5, GSK3␣, , PKA and PKC (Billingsley and Kincaid, 1997). Ser 193 and Thr 196
Processing p35 to p25 results in the over-activation of cdk5 that phosphorylates tau (Lee et al., 2000; Nath et al., 2000; Kusakawa et al., 2000). We next
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are phosphorylated by cdk5 (Billingsley and Kincaid, 1997). Antibody 5E2 recognizes both phosphorylated and non-phosphorylated tau, and Tau-1 recognizes non-phosphorylated tau. However, AT8 recognizes tau phosphorylated at Ser 193 and Thr 196 by cdk5 and others (Billingsley and Kincaid, 1997). As shown in Figs. 2 and 5, tau protein immunoreacted with 5E2 and Tau-1 was decreased after 48 h exposure to methylmercury in a dose-dependent manner, and the decrement of immunoreactivity was inhibited by the calpain inhibitor II. The 17 kDa fragments of tau were also recognized by Tau-1, and increased by the methylmercury treatment. The effect of methylmercury was not observed within 24 h (Fig. 5A). We found that the effect of methylmercury on the levels of protein immunoreacted with AT8
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was similar to that with 5E2 and Tau-1 (Fig. 5A) and that no significant difference was statistically detected in the levels of AT8 or Tau-1 immunoreactivity between control and methylmercury-treated cells (Fig. 5B). AT8 immunoreactive tau was also decreased after 48 h exposure to methylmercury in a dose-dependent manner (Fig. 5A). These results indicated that the methylmercury-induced cleavage of p35 did not result in the increment of the cdk5-phosphorylation of tau.
4. Discussion This paper showed that the methylmercury-induced death of neuronal cells occurs even at a very low concentration, 30 nM. The cell death inhibition by the calpain inhibitor indicates that calpain activation is included in cerebellar granule cell death induced at a very low concentration of methylmercury. Moreover, we detected that specifically expressed p35 in neuronal cells was cleaved to p25 through methylmercury treatment. 4.1. Methylmercury induced cell death and calpain activation
Fig. 5. Detection of phosphorylated tau in rat cerebellar granule cells following exposure to a very low concentration of methylmercury. CI II indicates the calpain inhibitor II. (A) Transferred membranes probed with anti-tau monoclonal antibodies, anti-phosphorylated tau antibody (AT8), anti-dephosphorylated tau antibody (Tau-1) or antiphosphorylated and dephosphorylated tau antibody (5E2). (B) Relative intensities of signals that were detected by antibodies, AT8 and Tau-1, in lysate of cerebellar granule cells non- or methylmercurytreated for 48 h. The signal intensities were quantified by standardizing with the intensity detected by 5E2 antibody. Data are given as the mean ± S.D. (n = 3).
It is preferable to use ␣-fodrin and tau as calpain substrates endogenously for the detection of calpain activation. The production of the 150 and 17 kDa fragmented products of ␣-fodrin and tau, respectively, have been used as a specific indicator of calpain activation (Dare et al., 2000; Lee et al., 2000; Wang, 1999; Nath et al., 2000; Siman et al., 1989; Canu et al., 1998; Johnson et al., 1989). In the present study, methylmercury decreased the amounts of the complete length ␣-fodrin and tau, and then increased the calpain-specific fragments, which were inhibited by the calpain inhibitor (Fig. 2). AnkyrinB and microtubule-associated protein 2 (MAP-2), other calpain substrates (Sakaue et al., 2003; Harada et al., 1997), are also cleaved by very low concentrations of methylmercury (Sakaue et al., 2003). Several investigators have reported that methylmercury exposure during neuronal development induced the degradation of ␣-fodrin to the 150 kDa fragments in the brain (Zhang et al., 2003), and the degradation of ␣-fodrin in cerebellar granule cells treated with the neurotoxicant (Dare et al., 2000). These reports supported
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our findings that revealed the induction of calpain activation in methylmercury-treated neuronal cells. Calpain is activated by increments of intracellular calcium concentrations (Guroff, 1963; Wang, 1999; Yoshimura et al., 1983). The intracellular calcium concentration assay showed that calcium ions in the cerebellar granule cells increased immediately after treatment with a high concentration of methylmercury (Fig. 3). These results are consistent with those in previous reports, in which high concentrations of methylmercury, sub M and higher were used (Marty and Atchison, 1997; Sarafian, 1993; Marty and Atchison, 1998). However, we have found that cell death was induced even at a concentration of 30 nM of methylmercury (Fig. 1). Therefore, we further investigated the effect of a lower concentration of methylmercury on the intracellular calcium concentration of cerebellar granule cells. The assays detected significant alterations in the Fluo-3 fluorescence intensity for cerebellar granule cells incubated in 30 nM methylmercury and higher for 3–6 h after the addition of the chemical. This result indicates that a very low concentration of methylmercury, 30 nM, causes an increase in the intracellular calcium concentration, which should induce the calpain activation in the cells. Although it was indicated that a very low dose of methylmercury activates calpain and leads to the death of neurons, the calpain inhibitor could not completely inhibit methylmercury from inducing neuronal cell death in the present study. Several mechanisms of methylmercuric toxicity via increments of intracellular Ca2+ concentration have been hypothesized. For example, it has been reported that the methylmercuryinduced increment of cytosolic Ca2+ concentration causes mitochondrial matrix Ca2+ to elevate, which could interfere with the respiratory chain and the tricarboxylic acid cycle and open the mitochondrial permeability transition pore, a contributor for releasing the proapoptotic factor into the cytosol (Limke et al., 2003). Methylmercury-induced cell death at very low concentrations may be caused through the alterations of mitochondrial function. 4.2. P35 processing and tau phosphorylation by cdk5 Neuron specific protein p35 is a regulator of cdk5 kinase activity binding to cdk5 and is a calpain
substrate (Lee et al., 2000; Lew et al., 1994; Nath et al., 2000; Kusakawa et al., 2000). The p25–cdk5 complex hyper-phosphorylates tau and neurofilaments more than p35–cdk5 complex, which contributes to the formation of neurofibrillary tangles and to the disruption of the physiological functions of the cytoskeletal proteins, thereby inducing cell death (Lee et al., 2000; Lew et al., 1994; Nath et al., 2000). In the present study, methylmercury treatment at very low concentrations caused calpain to cleave p35 to p25 in cerebellar granule cells (Fig. 4), but tau phosphorylation was not altered in the cells treated with the neurotoxicant (Fig. 5). An immunohistochemical study using anti-tau and anti--peptide antibodies indicated that there was no significant difference in the number of neurofibrillary tangles in the brains of patients suffering from methylmercury intoxication compared to the control subjects’ brains (Oyanagi and Ikuta, 1993). Tau dephosphorylation is stimulated in the death of the primary culture of hippocampal neurons (Kerokoski et al., 2001, 2002) and in the apoptotic death of cerebellar granule cells (Canu et al., 1998). Tau could not only be phosphorylated by cdk5 but also be dephosphorylated simultaneously, which could result in no alteration of the amount of phosphorylated tau. In any case, it is improbable that the phosphorylation of tau contributed to methylmercury-induced cell death at a very low concentration. In addition to p35, many intracellular proteins are calpain substrates. Thus, the proteins containing cytoskeletons seem to degrade and cause dysfunction via calpain-digestion in the neuronal cells treated with a very low dose of methylmercury (Sakaue et al., 2003), which probably causes the cytotoxicity. Furthermore, although previous reports indicated that methylmercury-induced cell death occurred through the disruption of microtubule polymerization (Miura and Imura, 1987), the cleavage of calpain substrates, ␣-/-tubulins and microtubule stabilizers, MAP-1, -2 and tau, may also contribute to the instability of the cytoskeleton protein by a very low concentration of the neurotoxicant. It should be noted that neurotoxicity at a very low concentration of methylmercury, 30 nM, was observed. This concentration is lower than the geometric average cord blood mercury concentration of children who showed neurobehavioral/neuropsychological deficits at about 7 years of age in a previous report (Grandjean et al., 1997). Cerebellar granule neuronal cells require
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at least 7 days preculture for morphological and physiological differentiation in vitro (Beale et al., 1980; Gallo et al., 1982; Viani et al., 1997), but the preculture term was only 2 days in the present study, so as to mimic the neuronal differentiation in vivo. Accordingly, the culture system in this study reflects the actual prenatal methylmercury exposure state of humans. Furthermore, this system obviously had a higher sensitivity to methylmercury than that using cultured neuroblastoma cell line, B35 cells (Fig. 1C), and the cerebellar granule cells precultured for 7 days (Kunimoto, 1994). The cerebellar granule cell culture system used in this study was very useful for studying the toxicity and mechanisms of the direct effects of methylmercury at a very low concentration on neurons. In conclusion, the data from the present study indicates that the calpain activation via methylmercuryinduced calcium release into the cytosol is included in the mechanisms of neurotoxicity by a very low concentration of methylmercury using the detection of the cleavage of its endogenous substrates and the calpain inhibitor. Calpain and its substrates are abundantly distributed in the brain, particularly in neurons (Wang, 1999; Hamakubo et al., 1986). Protein degradation via the activation of calpain may contribute to the high sensitivity of neurons to methylmercury.
References Beale, R., Dutton, G.R., Currie, D.N., 1980. An ion flux assay of action potential sodium channels in neuron- and glia-enriched cultures of cells dissociated from rat cerebellum. Brain Res. 183, 241–246. Billingsley, M.L., Kincaid, R.L., 1997. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration. Biochem. J. 323, 577–591. Canu, N., Dus, L., Barbato, C., Ciotti, M.T., Brancolini, C., Rinaldi, A.M., Novak, M., Cattaneo, A., Bradbury, A., Calissano, P., 1998. Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis. J. Neurosci. 18, 7061–7074. Chang, L.W., 1977. Neurotoxic effects of mercury: a review. Environ. Res. 14, 329–373. Clarkson, T.W., 1987. Metal toxicity in the central nervous system. Environ. Health Perspect. 75, 59–64. Dare, E., Gotz, M.E., Zhivotovsky, B., Manzo, L., Ceccatelli, S., 2000. Antioxidants J811 and 17-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death. J. Neurosci. Res. 62, 557–565.
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Gallo, V., Ciotti, M.T., Coletti, F., Aloisi, F., Levi, G., 1982. Selective release of glutamate from cerebellar granule cells differentiating in culture. Proc. Natl. Acad. Sci. USA 79, 7919–7923. Grandjean, P., Weihe, P., White, R.F., Debes, F., Araki, S., Yokoyama, K., Murata, K., Sorensen, N., Dahl, R., Jorgensen, P.J., 1997. Cognitive deficit in 7-year-old children with prenatal exposure to methylmercury. Neurotoxicol. Teratol. 19, 417–428. Guroff, G., 1963. A neutral, calcium-activated proteinase from the soluble fraction of rat brain. J. Biol. Chem. 239, 149–155. Hamakubo, T., Kannagi, R., Murachi, T., Matus, A., 1986. Distribution of calpains I and II in rat brain. J. Neurosci. 6, 3103– 3111. Harada, K., Fukuda, S., Kunimoto, M., Yoshida, K., 1997. Distribution of ankyrin isoforms and their proteolysis after ischemia and reperfusion in rat brain. J. Neurochem. 69, 371–376. Johnson, G.V., Jope, R.S., Binder, L.I., 1989. Proteolysis of tau by calpain. Biochem. Biophys. Res. Commun. 163, 1505– 1511. Kao, J.P.Y., Harootunian, A.T., Tsien, Y.R., 1989. Photochemically generated cytosolic calcium pulses and their detection by Fluo-3. J. Biol. Chem. 264, 8179–8186. Kerokoski, P., Suuronen, T., Salminen, A., Soininen, H., Pirttila, T., 2002. Cleavage of the cyclin-dependent kinase 5 activator p35 to p25 does not induce tau hyperphosphorylation. Biochem. Biophys. Res. Commun. 298, 693–698. Kerokoski, P., Suuronen, T., Salminen, A., Soininen, H., Pirttila, T., 2001. The levels of cdk5 and p35 proteins and tau phosphorylation are reduced during neuronal apoptosis. Biochem. Biophys. Res. Commun. 280, 998–1002. Kunimoto, M., 1994. Methylmercury induces apoptosis of rat cerebellar neurons in primary culture. Biochem. Biophys. Res. Commun. 204, 310–317. Kunimoto, M., Aoki, Y., Shibata, K., Miura, T., 1992. Differential cytotoxic effects of methylmercury and organotin compounds on mature and immature neuronal cells and non-neuronal cells in vitro. Toxicol. In Vitro 4, 349–355. Kusakawa, G., Saito, T., Onuki, R., Ishiguro, K., Kishimoto, T., Hisanaga, S., 2000. Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25. J. Biol. Chem. 275, 17166–17172. Lee, M.S., Kwon, Y.T., Li, M., Peng, J., Friedlander, R.M., Tsai, L.H., 2000. Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature 405, 360–364. Lew, J., Huang, Q., Qi, Z., Winkfein, R.J., Aebersold, R., Hunt, T., Wang, J.H., 1994. A brain-specific activator of cyclin-dependent kinase 5. Nature 371, 423–426. Limke, T.L., Otero-Montanez, J., Atchison, W.D., 2003. Evidence for interactions between intracellular calcium stores during methylmercury-induced intracellular calcium dysregulation in rat cerebellar granule neurons. J. Pharm. Exp. Ther. 304, 949–958. Marty, M.S., Atchison, W.D., 1997. Pathways mediating Ca2+ entry in rat cerebellar granule cells following in vitro exposure to methylmercury. Toxicol. Appl. Pharmacol. 147, 319– 330. Marty, M.S., Atchison, W.D., 1998. Elevations of intracellular Ca2+ as a probable contributor to decreased viability in cerebellar
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granule cells following acute exposure to methylmercury. Toxicol. Appl. Pharmacol. 150, 98–105. Minta, A., Kao, J.P.Y., Tsien, R.Y., 1989. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178. Miura, K., Imura, N., 1987. Mechanism of methylmercury cytotoxicity. Crit. Rev. Toxicol. 18, 161–188. Nagashima, K., Fujii, Y., Tsukamoto, T., Nukuzuma, S., Satoh, M., Fujita, M., Fujioka, Y., Akagi, H., 1996. Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats. Acta Neuropathol. 91, 72–77. Nath, R., Davis, M., Probert, A.W., Kupina, N.C., Ren, X., Schielke, G.P., Wang, K.K.W., 2000. Processing of cdk5 activator p35 to its truncated form (p25) by calpain in acutely injured neuronal cells. Biochem. Biophys. Res. Commun. 274, 16–21. Nguyen, M.D., Lariviere, R.C., Julien, J.P., 2001. Deregulation of cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron 30, 135–147. Oyanagi, K., Ikuta, F., 1993. The aging of central nervous system in methylmercury intoxication in Niigata, Japan—quantitative investigation of neurofibrillary tangles and senile plaques. No to Shinkei 45, 241–244. Sakaue, M., Takanaga, H., Adachi, T., Hara, S., Kunimoto, M., 2003. Selective disappearance of an axonal protein, 440-kDa ankyrinB , associated with neuronal degeneration induced by methylmercury. J. Neurosci. Res. 73, 831–839.
Sarafian, T.A., 1993. Methyl mercury increases intracellular Ca2+ and inositol phosphate levels in cultured cerebellar granule neurons. J. Neurochem. 61, 648–657. Sarafian, T.A., Verity, M.A., 1991. Oxidative mechanisms underlying methyl mercury neurotoxicity. Int. J. Dev. Neurosci. 9, 147–153. Siman, R., Baudry, M., Lynch, G., 1989. Brain fodrin: Substrate for calpain I, an endogenous calcium-activated protease. Proc. Natl. Acad. Sci. USA 81, 3572–3576. Tsai, L.H., Delalle, I., Caviness, V.S., Chae, T., Harlow, E., 1994. p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature 371, 419–423. Viani, P., Giussani, P., Riboni, L., Bassi, R., Tettamanti, G., 1997. Behaviour of nitric oxide synthase in rat cerebellar granule cells differentiating in culture. FEBS Lett. 408, 131–134. Wang, K.K.W., 1999. Calpain substrates, assay methods, regulation and its inhibitory agents. In: Wang, K.K.W., Yuen, P. (Eds.), CALPAIN: Pharmacology and Toxicology of CalciumDependent Protease. Taylor & Francis, Philadelphia, pp. 77–101. Yoshimura, N., Kikuchi, T., Sasaki, T., Kitahara, A., Hatanaka, M., Murachi, T., 1983. Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney. J. Biol. Chem. 258, 8883–8889. Zhang, J., Miyamoto, K., Hashioka, S., Hao, H.P., Murao, K., Saido, T.C., Nakanishi, H., 2003. Activation of mu-calpain in developing cortical neurons following methylmercury treatment. Dev. Brain Res. 142, 105–110.
Very low levels of methylmercury induce cell death of cultured rat cerebellar neurons via calpain activation Motoharu Sakaue ∗ , Maiko Okazaki, Shuntaro Hara Department of Public Health, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan Received 1 April 2005; received in revised form 18 May 2005; accepted 19 May 2005 Available online 27 June 2005
Abstract Methylmercury, an environmental neurotoxicant, induces the apoptotic death of cerebellar granule cells in vitro at a low concentration. To further understand the mechanism of cell death, we used a rat cerebellar granule cell culture system to investigate whether the calpain/cyclin-dependent kinase 5 (cdk5)/p35 cascade, an important cascade for neuronal apoptosis, is involved in the methylmercury-induced death. A noteworthy finding was that the cerebellar granular cell death was increased at a very low concentration of methylmercury, 30 nM, which is lower than that previously reported. The high sensitivity to methylmercury indicates that this culture system is useful for studying methylmercury toxicity at very low concentrations. Using this system, we here found that the methylmercury-induced death was inhibited by the calpain inhibitor II. Furthermore, it was shown that, in methylmercury-exposed cells, ␣-fodrin and tau, calpain substrates, were cleaved to the fragments that disappeared by treatment with the calpain inhibitor II. We next assayed and showed that the intracellular Ca2+ concentration in cerebellar granule cells increased after methylmercury exposure in a time- and dose-dependent manner, significantly even at 30 nM. These results indicated that a very low concentration of methylmercury causes the intracellular Ca2+ concentration to increase, activates calpain in the cells, and then induces cell death. We further found that the p35 protein was also processed to p25 that forms the cdk5–p25 complex, a hyperactive kinase for tau. However, an immunoblot using the anti-phosphorylated tau antibody showed that there was no increase of phosphorylated tau in methylmercury-exposed cells. These results suggested that methylmercury-induced cell death via calpain activation should not involve the stimulation of tau phosphorylation activity. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Methylmercury; Cerebellar granule neurons; p35; Cyclin-dependent kinase 5; Calpain; Very-low-concentration neurotoxicity
1. Introduction ∗
Corresponding author. Tel.: +81 3 5791 6266; fax: +81 3 3442 4146. E-mail address: [email protected] (M. Sakaue).
Methylmercury is widely recognized as a potent neurotoxicant. Intoxication by methylmercury causes pathological degeneration, specifically in the brain
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(Chang, 1977; Clarkson, 1987; Nagashima et al., 1996). Cerebellar granule cells, which are particularly vulnerable to methylmercury-induced damage in vivo, have provided a good model for analysis of methylmercury-induced neuronal cell death, apoptosis. Previous reports using the in vitro culture system of cerebellar granule cells have demonstrated methylmercury toxicity, and have shown that methylmercury can induce apoptotic death of the cells at lower concentrations, whereas it induces non-apoptotic cell death at higher concentrations (>1 M) (Chang, 1977; Dare et al., 2000; Kunimoto, 1994; Sakaue et al., 2003). Methylmercury causes neuronal cell death by disrupting the intracellular homeostasis, changing the intracellular Ca2+ concentration, inhibiting microtubule assembly and increasing reactive oxygen species production (Marty and Atchison, 1997; Miura and Imura, 1987; Sarafian and Verity, 1991; Sarafian, 1993) when exposed to the cells at sub M and higher. Even at lower concentrations (≤sub M), apoptotic death of cerebellar granule cells is induced via the caspaseindependent pathway (Chang, 1977; Dare et al., 2000; Kunimoto, 1994; Sakaue et al., 2003), but little is known about how a lower concentration of methylmercury induces neuronal cell death in a neuron-specific manner. Lee et al. reported that the calpain/cyclin-dependent kinase 5 (cdk5)/p35 cascade is important for the apoptosis of neuronal cells. A cdk5 activator p35 is specifically expressed in neuronal cells and a cleavage of p35 to p25, which is followed by the redistribution and overactivation of cdk5 after association with p25 (Lee et al., 2000; Lew et al., 1994; Tsai et al., 1994). Calpain, a calcium-dependent cysteine peptidase (Guroff, 1963; Wang, 1999; Yoshimura et al., 1983), is abundantly expressed in neurons (Wang, 1999; Hamakubo et al., 1986) and degrades specific substrates, for example, microtubule-associated protein 2, tau, ␣-fodrin and p35. After p35 is processed to p25 by calpain, p25 forms the complex of cdk5–p25, by which many proteins, including the tau protein and neurofilaments, are hyperphosphorylated (Nath et al., 2000). As tau acts as a stabilizer of microtubules, its degradation by calpain or hyperphosphorylation by cdk5–p25 causes its dysfunction, induces microtubule disruption, and then results in neuronal cell apoptosis (Lee et al., 2000; Nath et al., 2000; Kusakawa et al., 2000). Some models of neurodegenerative disorders, such as
Alzheimer’s disease and amyotrophic lateral sclerosis, include the activation of calpain and the processing of p35 to p25, which lead to neuronal apoptosis (Lee et al., 2000; Nguyen et al., 2001). We have reported that axonal protein 440 kDa ankyrinB , a calpain substrate of rat cerebellar granule cells, disappears with methylmercury treatment at a very low concentration, which suggests that calpain might contribute to the mechanism for specific neuronal cell death at a very low concentration of methylmercury (Sakaue et al., 2003). To gain a further understanding of the mechanism of methylmercury-induced cell death and to account for its neurotoxicity, we show, in the present study, the effects of a very low concentration of methylmercury on calpain activity in rat cerebellar granular cells, and detect the degradation of the calpain substrates, ␣-fodrin, tau and p35. Furthermore, we investigate and discuss whether processing p35 to p25 is involved in methylmercury-induced cell death at a very low concentration.
2. Materials and methods 2.1. Cell cultures Primary cultures of cerebellar granule neurons were prepared from Wistar rats (Jcl: Wistar; Clea Co., Tokyo, Japan) within 24 h after birth, as described previously (Kunimoto et al., 1992). Cerebella incubated with trypsin for 13 min at room temperature were minced by mild trituration with a Pasteur pipette. Cerebellar granule cells were seeded in Eagle’s minimal essential medium (Gibco BRL, Grand Island, NY) containing 1 mg/ml BSA, 10 g/ml bovin insulin, 0.1 nM thyroxine, 0.1 mg/mg human transferrin, 1 g/ml aprotinin, 30 nM Na2 SeO3 , 0.25% glucose, 100 units/mL penicillin and 135 g/mL streptomycin on poly-l-lysine-coated dishes and cultured for 2 days. All cell culture supplements were purchased from Sigma Chemical Company (St Louis, MO). Then, the cells were treated with methylmercuric chloride (Tokyo Kasei Kogyo. Co. Ltd., Tokyo, Japan) at 0–1 M for 24 or 48 h with or without calpain inhibitor II (Calbiochem San Diego, CA). All experiments were done in accordance with the Kitasato University Guidelines for Animal Care and Experimentation.
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Rat neuroblastoma B35 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. The medium was changed every second day. The cells were treated with methylmercury chloride after 24 h preincubation. The number of viable cerebellar granule cells in the culture or B35 cells was estimated by crystal violet staining, as described previously (Sakaue et al., 2003). 2.2. SDS-PAGE and immunoblotting After being washed three times with PBS (pH 7.4), the cultured cells were harvested in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 0.1% Nonident P-40, 5 mM dithiothreitol, 10 mM NaF, 1 mM PMSF, 1 g/mL aprotinin, and 1 g/ml leupeptin. Twenty-five microgram or 50 g of each lysate were electrophoresized on SDS-PAGE gels and immunoblotted using rabbit polyclonal antibodies to p35 (Sigma) or cdk5 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal antibodies against glyceraldehydes-3-phosphate dehydrogenase (GAPDH; Sigma), ␣-fodrin (MP Biochemicals, Inc., Irvine, CA) or tau (Tau-1, BM; AT8, Endogen, Woburn, MA; 5E2, Upstate Biotechnology, Lake Placid, NY) after electrophoretic transfer to the Immobilon P membrane (Millipore Corp., Bedford, MA). The anti-p35 antibody detects p35 and p25, the calboxy-terminal proteolytic product of p35. Densitometric quantification of immunoblots was done using ImageJ (version 1.31) software (NIH, Bethesda, MD). 2.3. Intracellular calcium concentration assay using Fluo-3 AM The intracellular calcium concentration of cerebellar granule cells seeded in 24 well plates was assayed using a fluorescent Ca2+ indicator, Fluo-3 acetoxymethyl ester (Fluo-3 AM; Dojindo, Tokyo, Japan), which specifically binds a calcium ion (Kao et al., 1989; Minta et al., 1989). In the first experiment, after 2 or 3 days in the subculture, the cerebellar granule cells were washed with Tyrode’s salt solution (Sigma) and incubated in Fluo-3 AM Tyrode’s salt solution (4 M Fluo-3 AM, 0.018% Pluronic F-127) for 1 hr in 37 ◦ C in a 5% CO2 , 95% humidity atmosphere. Fluo-3 AM was taken up by the cells, and entrapped intracellularly after hydrolysis to Fluo-3 by neuronal
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esterases. The intracellular Fluo-3 was excited at 488 nm and emits in wavelength of 530 nm range. Fluorescence readings were taken once in every 3 min for 9 min prior to the methylmercury additions as their basal fluorescent intensity, and then every 3 min for 120 min following methylmercury exposure at room temperature in atmosphere using Fluoroskan Ascent type 347 (Labsystems, Helsinki, Finland). In the second experiment, cerebellar granule cells cultured in a lower methylmercury concentration medium for 3, 6 or 24 h were washed with Tyrode’s salt solution and assayed after loading Fluo-3 AM, and then were treated in the same manner as described above. 2.4. Statistical analysis Statistical significances between groups were assessed by one-way analysis of variance (ANOVA). Bonferroni/Dunn test was employed to compare individual means as a post-hoc test.
3. Results 3.1. Inhibition of methylmercury-induced cerebellar granule cell death by calpain inhibitor The viability of cerebellar granule cells was assayed using crystal violet staining 48 h after the methylmercury treatment (Fig. 1). The methylmercury-induced death of the cerebellar granule cells increased dosedependently (Fig. 1A). It was noteworthy that the cell death significantly increased at a very low concentration of methylmercury, 30 nM, which was much lower than that reported previously (Kunimoto, 1994). We further found that the viability of the cells treated with 30 or 100 nM methylmercury was statistically restored from 76 and 43% to 89 and 60% by treatment of calpain inhibitor II 1 hr before methylmercury exposure, respectively (Fig. 1B). The inhibitory effect of calpain inhibitor II on the methylmercury neurotoxicity was significant but incomplete. The effect of methylmercury treatment for 48 h on rat neuroblastoma B35 cells was also examined. The sensitivity of B35 cells towards methylmercury was lower than that of cerebellar granule cells. The viability of B35 cells was also significantly decreased only above 300 nM. The value of the viability was 94, 92, 65 and 1% at
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Fig. 1. The effect of the calpain inhibitor II on cerebellar granule cell death induced through a very low concentration of methylmercury. Cerebellar granule cell cultures and rat neuroblastoma cell line, B35 cells were exposed to methylmercury for 48 h and assayed for viability, as described in Section 2. (A) The concentration-response relationship for the cell death elicited by methylmercury in cerebellar granule cell culture; and (B) its prevention by the 2 M calpain inhibitor II (C.I.II). (C) The cell viability of B35 cells treated with methylmercury. Data are expressed as the percentage of cell viability with respect to the control value, and are mean ± S.D. from n = 4 replicates per treatment group. Asterisks indicate the data that was statistically significantly different from control group (A, C) or between treatment groups (B) (p < 0.05).
0.3, 1, 3 and 10 M of methylmercury, respectively (Fig. 1C). 3.2. Degradation of calpain substrates through methylmercury exposure To determine whether calpain was activated in the cerebellar granule cells 48 h after methylmercury exposure, the cleavage of non-erythroid ␣-spectrin
known as ␣-fodrin, one of the well-characterized calpain substrates (Dare et al., 2000; Lee et al., 2000; Wang, 1999; Nath et al., 2000; Siman et al., 1989), was examined. Methylmercury caused cleavage of ␣-fodrin into a 150 kDa fragment in a dose-dependent manner. The calpain inhibitor II inhibited this cleavage of tau (Fig. 2A), another calpain substrate, and one of the major microtubule-associated proteins (Wang, 1999; Canu et al., 1998; Johnson et al., 1989). As
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calpain inhibitor II (Fig. 2A). The inhibitor treatment also caused an increase in the full length tau and bands 5–10 kDa smaller than the full length tau. 3.3. Methylmercury-induced alteration of Intracellular Ca2+ concentration
Fig. 2. Proteolytic cleavage of calpain substrates in cerebellar granule cells is induced by treatment with very low concentrations of methylmercury, and is inhibited by calpain inhibitor II (C.I.II). Primary cerebellar granule cell cultures were exposed to methylmercury for 48 h. (A) Cell lysates were subjected to immunoblotting and probed with anti-␣-fodrin (upper) or the anti-tau (clone, Tau-1) (lower) antibody. (B) Relative intensities of Tau-1 antibody reacted signals, 55 (intact tau protein) and 17 kDa (processed tau protein) bands. Data are given as the mean ± S.D. (n = 3). An asterisk indicated statistically significant differences in means from control group (p < 0.05).
shown in Fig. 2A, Western blot analysis on the state of tau revealed the appearance of a 17 kDa fragment, which was inhibited by the calpain inhibitor II, and was also obtained after 48 h of 30 nM methylmercury exposure. Densitometric analysis on the Western blot showed that the level of the 17 kDa fragment about 5-fold increased by methylmercury treatment (Fig. 2B). The inhibition of the disappearance of full length tau and the formation of the 17 kDa fragment was seen when simultaneously treating with the
Calpain is a Ca2+ -dependent protease (Guroff, 1963; Wang, 1999; Yoshimura et al., 1983). We next measured changes in the fluorescence of the Ca2+ indicator in order to confirm whether methylmercury exposure increases the intracellular Ca2+ concentration of the cerebellar granule cells (Fig. 3). In the initial experiment to examine the rapid response of the cerebellar granule cells to a higher concentration of methylmercury, the fluorescence intensities were measured in the course of time immediately after the methylmercury exposure (Fig. 3A). Methylmercury increased the fluorescence of Fluo-3 in the cerebellar granule cells dose- and time-dependently after exposure to the cells at 0.5 M and higher, but not at 0.1 and 0 M methylmercury. Furthermore, in the secondary experiment conducted to detect alterations of the intracellular Ca2+ concentration at lower concentrations of methylmercury, the cerebellar granule cells were incubated for 3–24 h in culture medium containing 3–300 nM methylmercury. Because cell death was induced by 48 h incubation in the neurotoxicant, the incubation times were selected for assay for the fluorescence signal (Fig. 3B). At a 10 nM and higher concentration of methylmercury, the fluorescence intensity statistically increased in a dose-dependent manner. 3.4. Processing p35 to p25 through methylmercury exposure The protein levels of p35, cdk5 and GAPDH were detected using Western blotting in cerebellar granule cells 48 h after the methylmercury treatment (Fig. 4). The immunoreactivity of p25 increased and that of p35 decreased in a methylmercury-dose-dependent manner, which was partially inhibited by the calpain inhibitor II in the cells. In addition, there was no significant difference in the levels of GAPDH as an internal standard and cdk5 between the control and the cells treated with methylmercury or methylmercury and the calpain inhibitor II.
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Fig. 3. Effect of methylmercury on intracellular Ca2+ concentration of rat cerebellar granule cells following in vitro exposure to methylmercury. (A) Time-dependent alterations in Fluo-3 fluorescence intensity. The arrowhead indicates where methylmercury was added. (B) Changes of fluorescence intensity after incubation for 3–24 h at lower concentrations of methylmercury. Data are mean ± S.D. from n = 4 replicates per treatment group. Asterisks indicate the data that was statistically significantly different from 0 nM treatment group at the same incubation time (p < 0.05).
Fig. 4. Proteolytic cleavage of p35 to p25 in cerebellar granule cells was induced by treatment with very low concentrations of methylmercury and was inhibited by calpain inhibitor II (C.I.II). Primary cerebellar granule cell cultures were exposed to methylmercury for 48 h. (A) Cell lysates were subjected to immunoblotting and probed with anti-p35 C-terminus, anti-cdk5 or anti-GAPDH antibodies. (B) Relative intensities of p25 signals that were detected by anti-p35 antibody. Data are given as the mean ± S.D. (n = 3). An asterisk indicate statistically significant differences in means from control group (p < 0.05).
3.5. The phosphorylation state of tau during methylmercury-induced apoptosis
examined the effect of methylmercury on the cdk5 activity in cerebellar granule cells by immunoblot analysis using the anti-tau monoclonal antibodies, 5E2, AT8 and Tau-1. Tau is phosphorylated by several kinases, including cdk5, GSK3␣, , PKA and PKC (Billingsley and Kincaid, 1997). Ser 193 and Thr 196
Processing p35 to p25 results in the over-activation of cdk5 that phosphorylates tau (Lee et al., 2000; Nath et al., 2000; Kusakawa et al., 2000). We next
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are phosphorylated by cdk5 (Billingsley and Kincaid, 1997). Antibody 5E2 recognizes both phosphorylated and non-phosphorylated tau, and Tau-1 recognizes non-phosphorylated tau. However, AT8 recognizes tau phosphorylated at Ser 193 and Thr 196 by cdk5 and others (Billingsley and Kincaid, 1997). As shown in Figs. 2 and 5, tau protein immunoreacted with 5E2 and Tau-1 was decreased after 48 h exposure to methylmercury in a dose-dependent manner, and the decrement of immunoreactivity was inhibited by the calpain inhibitor II. The 17 kDa fragments of tau were also recognized by Tau-1, and increased by the methylmercury treatment. The effect of methylmercury was not observed within 24 h (Fig. 5A). We found that the effect of methylmercury on the levels of protein immunoreacted with AT8
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was similar to that with 5E2 and Tau-1 (Fig. 5A) and that no significant difference was statistically detected in the levels of AT8 or Tau-1 immunoreactivity between control and methylmercury-treated cells (Fig. 5B). AT8 immunoreactive tau was also decreased after 48 h exposure to methylmercury in a dose-dependent manner (Fig. 5A). These results indicated that the methylmercury-induced cleavage of p35 did not result in the increment of the cdk5-phosphorylation of tau.
4. Discussion This paper showed that the methylmercury-induced death of neuronal cells occurs even at a very low concentration, 30 nM. The cell death inhibition by the calpain inhibitor indicates that calpain activation is included in cerebellar granule cell death induced at a very low concentration of methylmercury. Moreover, we detected that specifically expressed p35 in neuronal cells was cleaved to p25 through methylmercury treatment. 4.1. Methylmercury induced cell death and calpain activation
Fig. 5. Detection of phosphorylated tau in rat cerebellar granule cells following exposure to a very low concentration of methylmercury. CI II indicates the calpain inhibitor II. (A) Transferred membranes probed with anti-tau monoclonal antibodies, anti-phosphorylated tau antibody (AT8), anti-dephosphorylated tau antibody (Tau-1) or antiphosphorylated and dephosphorylated tau antibody (5E2). (B) Relative intensities of signals that were detected by antibodies, AT8 and Tau-1, in lysate of cerebellar granule cells non- or methylmercurytreated for 48 h. The signal intensities were quantified by standardizing with the intensity detected by 5E2 antibody. Data are given as the mean ± S.D. (n = 3).
It is preferable to use ␣-fodrin and tau as calpain substrates endogenously for the detection of calpain activation. The production of the 150 and 17 kDa fragmented products of ␣-fodrin and tau, respectively, have been used as a specific indicator of calpain activation (Dare et al., 2000; Lee et al., 2000; Wang, 1999; Nath et al., 2000; Siman et al., 1989; Canu et al., 1998; Johnson et al., 1989). In the present study, methylmercury decreased the amounts of the complete length ␣-fodrin and tau, and then increased the calpain-specific fragments, which were inhibited by the calpain inhibitor (Fig. 2). AnkyrinB and microtubule-associated protein 2 (MAP-2), other calpain substrates (Sakaue et al., 2003; Harada et al., 1997), are also cleaved by very low concentrations of methylmercury (Sakaue et al., 2003). Several investigators have reported that methylmercury exposure during neuronal development induced the degradation of ␣-fodrin to the 150 kDa fragments in the brain (Zhang et al., 2003), and the degradation of ␣-fodrin in cerebellar granule cells treated with the neurotoxicant (Dare et al., 2000). These reports supported
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our findings that revealed the induction of calpain activation in methylmercury-treated neuronal cells. Calpain is activated by increments of intracellular calcium concentrations (Guroff, 1963; Wang, 1999; Yoshimura et al., 1983). The intracellular calcium concentration assay showed that calcium ions in the cerebellar granule cells increased immediately after treatment with a high concentration of methylmercury (Fig. 3). These results are consistent with those in previous reports, in which high concentrations of methylmercury, sub M and higher were used (Marty and Atchison, 1997; Sarafian, 1993; Marty and Atchison, 1998). However, we have found that cell death was induced even at a concentration of 30 nM of methylmercury (Fig. 1). Therefore, we further investigated the effect of a lower concentration of methylmercury on the intracellular calcium concentration of cerebellar granule cells. The assays detected significant alterations in the Fluo-3 fluorescence intensity for cerebellar granule cells incubated in 30 nM methylmercury and higher for 3–6 h after the addition of the chemical. This result indicates that a very low concentration of methylmercury, 30 nM, causes an increase in the intracellular calcium concentration, which should induce the calpain activation in the cells. Although it was indicated that a very low dose of methylmercury activates calpain and leads to the death of neurons, the calpain inhibitor could not completely inhibit methylmercury from inducing neuronal cell death in the present study. Several mechanisms of methylmercuric toxicity via increments of intracellular Ca2+ concentration have been hypothesized. For example, it has been reported that the methylmercuryinduced increment of cytosolic Ca2+ concentration causes mitochondrial matrix Ca2+ to elevate, which could interfere with the respiratory chain and the tricarboxylic acid cycle and open the mitochondrial permeability transition pore, a contributor for releasing the proapoptotic factor into the cytosol (Limke et al., 2003). Methylmercury-induced cell death at very low concentrations may be caused through the alterations of mitochondrial function. 4.2. P35 processing and tau phosphorylation by cdk5 Neuron specific protein p35 is a regulator of cdk5 kinase activity binding to cdk5 and is a calpain
substrate (Lee et al., 2000; Lew et al., 1994; Nath et al., 2000; Kusakawa et al., 2000). The p25–cdk5 complex hyper-phosphorylates tau and neurofilaments more than p35–cdk5 complex, which contributes to the formation of neurofibrillary tangles and to the disruption of the physiological functions of the cytoskeletal proteins, thereby inducing cell death (Lee et al., 2000; Lew et al., 1994; Nath et al., 2000). In the present study, methylmercury treatment at very low concentrations caused calpain to cleave p35 to p25 in cerebellar granule cells (Fig. 4), but tau phosphorylation was not altered in the cells treated with the neurotoxicant (Fig. 5). An immunohistochemical study using anti-tau and anti--peptide antibodies indicated that there was no significant difference in the number of neurofibrillary tangles in the brains of patients suffering from methylmercury intoxication compared to the control subjects’ brains (Oyanagi and Ikuta, 1993). Tau dephosphorylation is stimulated in the death of the primary culture of hippocampal neurons (Kerokoski et al., 2001, 2002) and in the apoptotic death of cerebellar granule cells (Canu et al., 1998). Tau could not only be phosphorylated by cdk5 but also be dephosphorylated simultaneously, which could result in no alteration of the amount of phosphorylated tau. In any case, it is improbable that the phosphorylation of tau contributed to methylmercury-induced cell death at a very low concentration. In addition to p35, many intracellular proteins are calpain substrates. Thus, the proteins containing cytoskeletons seem to degrade and cause dysfunction via calpain-digestion in the neuronal cells treated with a very low dose of methylmercury (Sakaue et al., 2003), which probably causes the cytotoxicity. Furthermore, although previous reports indicated that methylmercury-induced cell death occurred through the disruption of microtubule polymerization (Miura and Imura, 1987), the cleavage of calpain substrates, ␣-/-tubulins and microtubule stabilizers, MAP-1, -2 and tau, may also contribute to the instability of the cytoskeleton protein by a very low concentration of the neurotoxicant. It should be noted that neurotoxicity at a very low concentration of methylmercury, 30 nM, was observed. This concentration is lower than the geometric average cord blood mercury concentration of children who showed neurobehavioral/neuropsychological deficits at about 7 years of age in a previous report (Grandjean et al., 1997). Cerebellar granule neuronal cells require
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at least 7 days preculture for morphological and physiological differentiation in vitro (Beale et al., 1980; Gallo et al., 1982; Viani et al., 1997), but the preculture term was only 2 days in the present study, so as to mimic the neuronal differentiation in vivo. Accordingly, the culture system in this study reflects the actual prenatal methylmercury exposure state of humans. Furthermore, this system obviously had a higher sensitivity to methylmercury than that using cultured neuroblastoma cell line, B35 cells (Fig. 1C), and the cerebellar granule cells precultured for 7 days (Kunimoto, 1994). The cerebellar granule cell culture system used in this study was very useful for studying the toxicity and mechanisms of the direct effects of methylmercury at a very low concentration on neurons. In conclusion, the data from the present study indicates that the calpain activation via methylmercuryinduced calcium release into the cytosol is included in the mechanisms of neurotoxicity by a very low concentration of methylmercury using the detection of the cleavage of its endogenous substrates and the calpain inhibitor. Calpain and its substrates are abundantly distributed in the brain, particularly in neurons (Wang, 1999; Hamakubo et al., 1986). Protein degradation via the activation of calpain may contribute to the high sensitivity of neurons to methylmercury.
References Beale, R., Dutton, G.R., Currie, D.N., 1980. An ion flux assay of action potential sodium channels in neuron- and glia-enriched cultures of cells dissociated from rat cerebellum. Brain Res. 183, 241–246. Billingsley, M.L., Kincaid, R.L., 1997. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration. Biochem. J. 323, 577–591. Canu, N., Dus, L., Barbato, C., Ciotti, M.T., Brancolini, C., Rinaldi, A.M., Novak, M., Cattaneo, A., Bradbury, A., Calissano, P., 1998. Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis. J. Neurosci. 18, 7061–7074. Chang, L.W., 1977. Neurotoxic effects of mercury: a review. Environ. Res. 14, 329–373. Clarkson, T.W., 1987. Metal toxicity in the central nervous system. Environ. Health Perspect. 75, 59–64. Dare, E., Gotz, M.E., Zhivotovsky, B., Manzo, L., Ceccatelli, S., 2000. Antioxidants J811 and 17-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death. J. Neurosci. Res. 62, 557–565.
105
Gallo, V., Ciotti, M.T., Coletti, F., Aloisi, F., Levi, G., 1982. Selective release of glutamate from cerebellar granule cells differentiating in culture. Proc. Natl. Acad. Sci. USA 79, 7919–7923. Grandjean, P., Weihe, P., White, R.F., Debes, F., Araki, S., Yokoyama, K., Murata, K., Sorensen, N., Dahl, R., Jorgensen, P.J., 1997. Cognitive deficit in 7-year-old children with prenatal exposure to methylmercury. Neurotoxicol. Teratol. 19, 417–428. Guroff, G., 1963. A neutral, calcium-activated proteinase from the soluble fraction of rat brain. J. Biol. Chem. 239, 149–155. Hamakubo, T., Kannagi, R., Murachi, T., Matus, A., 1986. Distribution of calpains I and II in rat brain. J. Neurosci. 6, 3103– 3111. Harada, K., Fukuda, S., Kunimoto, M., Yoshida, K., 1997. Distribution of ankyrin isoforms and their proteolysis after ischemia and reperfusion in rat brain. J. Neurochem. 69, 371–376. Johnson, G.V., Jope, R.S., Binder, L.I., 1989. Proteolysis of tau by calpain. Biochem. Biophys. Res. Commun. 163, 1505– 1511. Kao, J.P.Y., Harootunian, A.T., Tsien, Y.R., 1989. Photochemically generated cytosolic calcium pulses and their detection by Fluo-3. J. Biol. Chem. 264, 8179–8186. Kerokoski, P., Suuronen, T., Salminen, A., Soininen, H., Pirttila, T., 2002. Cleavage of the cyclin-dependent kinase 5 activator p35 to p25 does not induce tau hyperphosphorylation. Biochem. Biophys. Res. Commun. 298, 693–698. Kerokoski, P., Suuronen, T., Salminen, A., Soininen, H., Pirttila, T., 2001. The levels of cdk5 and p35 proteins and tau phosphorylation are reduced during neuronal apoptosis. Biochem. Biophys. Res. Commun. 280, 998–1002. Kunimoto, M., 1994. Methylmercury induces apoptosis of rat cerebellar neurons in primary culture. Biochem. Biophys. Res. Commun. 204, 310–317. Kunimoto, M., Aoki, Y., Shibata, K., Miura, T., 1992. Differential cytotoxic effects of methylmercury and organotin compounds on mature and immature neuronal cells and non-neuronal cells in vitro. Toxicol. In Vitro 4, 349–355. Kusakawa, G., Saito, T., Onuki, R., Ishiguro, K., Kishimoto, T., Hisanaga, S., 2000. Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25. J. Biol. Chem. 275, 17166–17172. Lee, M.S., Kwon, Y.T., Li, M., Peng, J., Friedlander, R.M., Tsai, L.H., 2000. Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature 405, 360–364. Lew, J., Huang, Q., Qi, Z., Winkfein, R.J., Aebersold, R., Hunt, T., Wang, J.H., 1994. A brain-specific activator of cyclin-dependent kinase 5. Nature 371, 423–426. Limke, T.L., Otero-Montanez, J., Atchison, W.D., 2003. Evidence for interactions between intracellular calcium stores during methylmercury-induced intracellular calcium dysregulation in rat cerebellar granule neurons. J. Pharm. Exp. Ther. 304, 949–958. Marty, M.S., Atchison, W.D., 1997. Pathways mediating Ca2+ entry in rat cerebellar granule cells following in vitro exposure to methylmercury. Toxicol. Appl. Pharmacol. 147, 319– 330. Marty, M.S., Atchison, W.D., 1998. Elevations of intracellular Ca2+ as a probable contributor to decreased viability in cerebellar
106
M. Sakaue et al. / Toxicology 213 (2005) 97–106
granule cells following acute exposure to methylmercury. Toxicol. Appl. Pharmacol. 150, 98–105. Minta, A., Kao, J.P.Y., Tsien, R.Y., 1989. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178. Miura, K., Imura, N., 1987. Mechanism of methylmercury cytotoxicity. Crit. Rev. Toxicol. 18, 161–188. Nagashima, K., Fujii, Y., Tsukamoto, T., Nukuzuma, S., Satoh, M., Fujita, M., Fujioka, Y., Akagi, H., 1996. Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats. Acta Neuropathol. 91, 72–77. Nath, R., Davis, M., Probert, A.W., Kupina, N.C., Ren, X., Schielke, G.P., Wang, K.K.W., 2000. Processing of cdk5 activator p35 to its truncated form (p25) by calpain in acutely injured neuronal cells. Biochem. Biophys. Res. Commun. 274, 16–21. Nguyen, M.D., Lariviere, R.C., Julien, J.P., 2001. Deregulation of cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron 30, 135–147. Oyanagi, K., Ikuta, F., 1993. The aging of central nervous system in methylmercury intoxication in Niigata, Japan—quantitative investigation of neurofibrillary tangles and senile plaques. No to Shinkei 45, 241–244. Sakaue, M., Takanaga, H., Adachi, T., Hara, S., Kunimoto, M., 2003. Selective disappearance of an axonal protein, 440-kDa ankyrinB , associated with neuronal degeneration induced by methylmercury. J. Neurosci. Res. 73, 831–839.
Sarafian, T.A., 1993. Methyl mercury increases intracellular Ca2+ and inositol phosphate levels in cultured cerebellar granule neurons. J. Neurochem. 61, 648–657. Sarafian, T.A., Verity, M.A., 1991. Oxidative mechanisms underlying methyl mercury neurotoxicity. Int. J. Dev. Neurosci. 9, 147–153. Siman, R., Baudry, M., Lynch, G., 1989. Brain fodrin: Substrate for calpain I, an endogenous calcium-activated protease. Proc. Natl. Acad. Sci. USA 81, 3572–3576. Tsai, L.H., Delalle, I., Caviness, V.S., Chae, T., Harlow, E., 1994. p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature 371, 419–423. Viani, P., Giussani, P., Riboni, L., Bassi, R., Tettamanti, G., 1997. Behaviour of nitric oxide synthase in rat cerebellar granule cells differentiating in culture. FEBS Lett. 408, 131–134. Wang, K.K.W., 1999. Calpain substrates, assay methods, regulation and its inhibitory agents. In: Wang, K.K.W., Yuen, P. (Eds.), CALPAIN: Pharmacology and Toxicology of CalciumDependent Protease. Taylor & Francis, Philadelphia, pp. 77–101. Yoshimura, N., Kikuchi, T., Sasaki, T., Kitahara, A., Hatanaka, M., Murachi, T., 1983. Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney. J. Biol. Chem. 258, 8883–8889. Zhang, J., Miyamoto, K., Hashioka, S., Hao, H.P., Murao, K., Saido, T.C., Nakanishi, H., 2003. Activation of mu-calpain in developing cortical neurons following methylmercury treatment. Dev. Brain Res. 142, 105–110.