European Journal of Pharmacology 746 (2015) 301–307
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Zonisamide suppresses endoplasmic reticulum stress-induced neuronal cell damage in vitro and in vivo Saori Tsujii a, Mitsue Ishisaka a, Masamitsu Shimazawa a, Takanori Hashizume b, Hideaki Hara a,n a b
Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Japan Laboratory of Drug Metabolism & Pharmacokinetics, Faculty of Pharmacy, Osaka Ohtani University, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 25 July 2014 Received in revised form 16 September 2014 Accepted 16 September 2014 Available online 23 September 2014
Zonisamide has been reported to have protective effects on epilepsy and Parkinson's disease and to work via various mechanisms of action, such as inhibition of monoamine oxidase-B and enhancement of tyrosine hydroxylase. Recently, it has been suggested that zonisamide itself shows neuroprotective actions. Therefore, in the present study we investigated the neuroprotective effects of zonisamide against endoplasmic reticulum (ER) stress. We used human neuroblastoma (SH-SY5Y) cells and investigated the protective effects of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death. In addition, we investigated the effect of zonisamide against 1-methyl-4phenylpyridinium (MPP þ )-induced cell death and the mechanism of protection against ER stress. In vivo, we investigated the effect of zonisamide (20 mg/kg, p.o.) in the 1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease. Zonisamide not only suppressed MPP þ -induced cell death, but also inhibited ER stress-induced cell death and suppressed the expression of ER stress-related factors such as C/EBO homologous protein (CHOP) in vivo. Furthermore, zonisamide inhibited the activation of caspase-3 in vitro. These results suggest that zonisamide affected ER stress via caspase-3. We think that ER stress, particularly the mechanism via caspase-3, is involved in part of the neuroprotective effect of zonisamide against the experimental models of Parkinson's disease. & 2014 Elsevier B.V. All rights reserved.
Keywords: Endoplasmic reticulum stress Parkinson's disease Zonisamide
1. Introduction Parkinson's disease is a neurodegenerative disease characterized by four major symptoms; akinesia, rigidity, balance impairment, and tremors (Savitt et al., 2006). Dopaminergic neurons are reduced in the midbrain substantia nigra in the brains of patients with Parkinson's disease, and the activity of acetylcholine is high due to the dopamine deficiency, which leads to an imbalance of functions (Zhu et al., 2008). However, the cause of the reduction in dopaminergic neurons remains unelucidated. Recently, it has been suggested that cell death induced by oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction are involved in Parkinson's disease (Beal, 2003; Gotz et al., 1990; Ryu et al., 2002; Schapira and Gegg, 2011). The protein associated with ER stress such as Hrd1p/Der3p (HRD1), which promotes ubiquitination, is localized in the brain neurons of patients with Parkinson's disease, and ER stress is involved other
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Corresponding author. Tel.: þ 81 58 230 8100. E-mail address: [email protected] (H. Hara).
http://dx.doi.org/10.1016/j.ejphar.2014.09.023 0014-2999/& 2014 Elsevier B.V. All rights reserved.
neurodegenerative diseases such as Alzheimer's disease and Huntington's disease (Kaneko, 2012). Several therapeutic medicines such as L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine receptor agonists, drugs promoting free dopamine, and anticholinergic drugs are used for treating Parkinson's disease (Quinn, 1995; Robertson et al., 1989). Among them, zonisamide works by inhibiting monoamine oxidase-B (MAO-B) activity and boosting tyrosine hydroxylase activity. Zonisamide has been used as an anti-epileptic drug at high doses of 200–400 mg/day, originally defined as inhibiting of T-type Ca2 þ channels and as a Na þ channel blocker (Suzuki et al., 1992). However, it has been revealed that zonisamide shows a therapeutic effect in Parkinson's disease at a low dose of 25 mg/day (Murata et al., 2007). In addition, it has been reported that zonisamide itself shows neuroprotective effects by increasing protein levels of a yeast homolog, HRD1 (Omura et al., 2012). Here, we investigated whether zonisamide shows a protective effect against ER stress-induced neuronal cell death. Furthermore, we clarified the mechanism of the protective effects of zonisamide against ER stress in in vitro and in vivo Parkinson's disease models.
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2. Material and methods
2.5. Drug treatment
2.1. Cell culture
2.5.1. Measurement of plasma concentration of zonisamide Zonisamide (20 mg/kg) was suspended in a 40% propylene glycol (Nacalai Tesque, Kyoto, Japan) solution and orally administered (p.o.). Blood collection was carried out from vena cava at 0, 2, 8, or 24 h after drug treatment. Blood samples were centrifuged at 1500g for 25 min at 4 1C and the separated plasma was stored at 80 1C until analysis. Plasma concentrations of zonisamide were measured by high performance liquid chromatography equipped with UV detector (UV-HPLC).
Human neuroblastoma (SH-SY5Y) cells were purchased from the European Collection of Cell Culture (Wiltshire, UK) and maintained in Dulbecco's Modified Eagle's medium (DMEM) (Nacalai Tesque, Kyoto, Japan) containing 10% Fetal Bovine Serum (FBS) (VALEANT, Costa Mesa, CA, USA), 100 units/ml penicillin (Meiji Seika, Tokyo, Japan), and 100 μg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37 1C. Cells were passaged by trypsinization every 3–5 days.
2.2. Cell death assay SH-SY5Y cells was seeded at 1 104 cells per well into a 96well plate (number 3072, Falcons, Becton Dickinson and Company, Franklin Lakes, NJ, USA) and incubated for 24 h at 37 1C in a humidified atmosphere containing 5% CO2. The entire medium was replaced with fresh medium containing 1% FBS, and cells were pretreated with zonisamide at 0.03–3 μM (gifted by Sumitomo Dainippon Pharma Co. Ltd., Osaka, Japan) for 1 h, followed by the addition of 2.0 μg/ml tunicamycin, 1 μM thapsigargin, or 0.5 mM 1-methyl-4-phenylpyridinium (MPP þ ) (Sigma-Aldrich). Nuclear staining assay was carried out after a further 27 h of incubation. Cell death was assessed on the basis of combination staining with Hoechst 33342 (Molecular Probes, Eugene, OR, USA) and propidium iodide (PI; Molecular Probes). At 27 h of incubation, Hoechst 33342 and PI dyes were added to the culture medium (at 8 and 0.2 μM, respectively) for 15 min. Images were collected using an Olympus IX70 inverted epifluorescence microscope (Olympus, Tokyo, Japan). At least 300 cells per condition were counted in a blind manner by a single observer (S.T).
2.3. Western blot analysis SH-SY5Y cells were lysed using a cell-lysis buffer with protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor cocktails (SigmaAldrich). Equal amounts of protein were separated on a 5–20% SDSPAGE gradient gel and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). After blocking with Block Ace (Snow Brand Milk Products Co. Ltd., Tokyo, Japan) for 1 h, membranes were incubated with primary antibody. The primary antibodies used were as follows: mouse anti-KDEL antibody (1:1000 dilution) (Stressgen Bioreagents Limited Partnership, Victoria, B.C., Canada), Cleaved Caspase-3 (Asp175) antibody (1:1000 dilution) or monoclonal anti-β-actin antibody (1:4000 dilution) (Sigma-Aldrich). Subsequently, the membrane was incubated with secondary antibody [goat anti-mouse (Pierce Biotechnology, Rockford, IL, USA)]. The immunoreactive bands were visualized using Multi Gauge Ver. 3.0 (Fujifilm, Tokyo, Japan) and measured using a LAS-4000 mini (Fujifilm).
2.4. Animals 9-week-old male C57BL/6J mice were used in these experiments. Mice were housed at 2472 1C under a 12 h light-dark cycle (lights on from 8:00 to 20:00) and had ad libitum access to food and water. All procedures relating to animal care and treatment conformed to animal care guidelines of the Animal Experiment Committee of Gifu Pharmaceutical University. All efforts were made to minimize both suffering and the number of animals used.
2.5.2. MPTP model in mice The method used to generate a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridin (MPTP) (Sigma-Aldrich) model in mice was performed as previously described (Oida et al., 2006). Briefly, mice were administered orally with zonisamide (20 mg/kg) 1 h before a peritoneal injection of MPTP (20 mg/kg, i.p.) or vehicle, and these injections were continued once a day for 5 days. Six days after the last administration of MPTP, mice were anesthetized with sodium pentobarbital (nembutal, 50 mg/kg, i.p.) and brains were perfusion fixed with 4% paraformaldehyde in 0.1 M phosphate-buffer (pH 7.4). The brains were extracted after a 10 min perfusion fixation at 4 1C and then immersed in the same fixative solution over night. Brain were immersed in 25% sucrose, and embedded in Optimal Cutting Temperature (O.C.T.) compound. 2.6. Immunohistochemistry Frosen sections (10 μm thick) of the substantia nigra were washed for 5 min in 0.01 M phosphate-buffered saline (PBS), and then treated with 0.3% hydrogen peroxidase in 10% methanol. Sections were washed three times with 0.01 M PBS, followed by a 30 min pre-incubation with 1.5% normal goat serum. They were incubated with anti-tyrosine hydroxylase (TH) antibody (1:1000) including 0.3% Triton X-100 overnight at 4 1C. After rinsing twice with 0.01 M PBS, the sections were incubated with biotinylated anti-rabbit secondary antibody for 2 h, and then with an avidin– biotin peroxidase complex for 30 min (both at room temperature). The number of TH-positive neurons in two sections of the substantia nigra was counted under the light microscope at a magnification of 400, and the mean number of TH-positive neurons was calculated. The count of positive cells was performed in a blind manner by a single observer (S.T). 2.7. Double fluorescence immunostaining Frosen sections (10 μm thick) of the substantia nigra were washed for 5 min in 0.01 M PBS, followed by a 1 h pre-incubation with 1.5% normal goat serum. Sections were incubated with antiTH antibody (1:1000) overnight at 4 1C. After rinsing twice with 0.01 M PBS, the sections were incubated with Alexa Fluors 546 donkey anti-rabbit second antibody for 1 h at room temperature. They were then washed with 0.01 M PBS, followed by 1 h incubation with mouse on mouse (M.O.M) Ig blocking reagent. Sections were incubated with mouse anti-CHOP antibody (Santa Cruz Biotechnology, Inc., Texas, USA; 1:1000) including M.O.M protein concentrate overnight at 4 1C. After rinsing twice with 0.01 M PBS, sections were incubated with Alexa Fluors 488 rabbit anti-mouse secondary antibody including M.O.M protein concentrate for 1 h at room temperature. They were then washed with 0.01 M PBS and stained with DNA-specific fluorescent Hoechst 33342. Images were photographed using a laser confocal microscope, and the fluorescence intensity of CHOP was measured by ImageJ (National
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Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih. gov/ij/, 1997–2014.).
Cell seeding (1×104 cells/well)
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Data are presented as mean 7standard error of mean (S.E.M.). Statistical comparisons were made using Student's t-test, Dunnett's test, or Bonferroni, with P o0.05 being considered statistically significant.
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3. Results
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3.1. Effect of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death in vitro To investigate the protective effect of zonisamide against ER stress in SH-SY5Y cells, we examined the effects of zonisamide on tunicamycin- and thapsigargin-induced neuronal cell death. Tunicamycin inhibits the glycosylation of glycoproteins (Behar-Bannelier et al., 1980), and thapsigargin inhibits Ca2 þ -ATPase at the ER membrane (Parra et al., 2013); therefore, they both induce ER stress via different pathways. Typical photographs of Hoechst 33342 and PI staining are shown in Fig. 1B and D. When compared with the control group, tunicamycin at 2.0 μg/ml or thapsigargin at 1 μM significantly increased the number of dead cells (stained with PI). Pretreatment with zonisamide at 0.1–3 μM protected against this cell death in a concentration-dependent manner, and the effect for tunicamycin was significant at 0.3–3 μM (Fig. 1C) and it for thapsigargin was significant at 0.1–3 μM (Fig. 1E). In addition, we performed similar trials using glutamate treatment at 80 mM or H2O2 treatment at 100 mM. Equally, pretreatment with zonisamide at 0.1–3 μM protected against this cell death in a concentration-dependent manner and the effect for glutamate was significant at 0.1–3 μM (Fig. S1B) and it for H2O2 was significant at 1 and 3 μM (Fig. S1E). These results suggest that zonisamide is neuroprotective against neuronal cell death induced by ER stress, glutamate, and oxidative stress. þ
3.2. Effect of zonisamide against MPP -induced ER stress in vitro To investigate the involvement of ER stress in Parkinson's disease, we examined changes in ER stress markers with MPP þ treatment in SH-SY5Y cells. It has been reported that the expression of GRP78 is increased by ER stress (Li et al., 2014). The expression of GRP78 was significantly increased by MPP þ exposure (Fig. 2C). Therefore, this suggested that ER stress is induced by MPP þ exposure. Next, we investigated the effects of zonisamide against MPP þ -induced neuronal cell death. Typical photographs of Hoechst 33342 and PI staining are shown in Fig. 2D. When compared with the control group, MPP þ treatment significantly increased the number of dead cells. Pretreatment with zonisamide protected against this cell death (Fig. 2E). 3.3. Effect of zonisamide against MPTP-induced ER stress in vivo Next, we evaluated the effect of zonisamide by using experimental animal models. The number of TH-positive neuronal cells in the substantia nigra was decreased after MPTP treatment in mice, but rescued by zonisamide treatment (Fig. 3A and B). In addition, to confirm the involvement of ER stress in Parkinson's disease in vivo, we examined changes in CHOP expression in MPTP-treated mice compared with control mice. The expression of CHOP in the striatum is shown in Fig. 3C. The expression of CHOP was increased in MPTP-treated mice, and pretreatment with zonisamide attenuated this increase (Fig. 3D).
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Fig. 1. Effects of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death. (A) The protocol performed in this study. SH-SY5Y cells were treated with zonisamide for 1 h followed by the addition of 2.0 μg/ml tunicamycin or 1 μM thapsigargin. A nuclear staining assay was carried out after 27 h of incubation. (B and D) Typical photographs of Hoechst 33342 (blue: viable cells) and propidium iodide (PI) staining (red: dead cells). Scale bar¼ 50 μm. (C, E) Measurement of PI positive cells. Each column and bar represent the mean7 S.E.M., n¼ 6. ##; Po 0.01 vs. Control (t-test), n; P o0.05, nn; P o0.01 vs. Vehicle (Dunnett's test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Mechanism of protection of zonisamide against tunicamycininduced cell death in vitro To examine in the mechanism of action of zonisamide protecting against ER stress, we examined changes in factors induced by apoptosis: GRP78, unfolded protein response (UPR), and caspase-3 activity under tunicamycin treatment in SH-SY5Y cells. Zonisamide did not affect to the expression of GRP78 (data not shown).
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of zonisamide in mice following a single oral administration. Fig. 5 shows the change in zonisamide plasma concentration after the oral administration. The concentrations showed approximately 11, 4, and 0.005 μg/ml at 2, 8, and 24 h after administration, respectively. The elimination phase half-life (t1/2b) was 1.7 h. The C2h concentration was approximately 11 μg/ml, which is equivalent to approximately 50 μM. The neuroprotective effects of zonisamide were seen in the range of 0.1–3 μM in vitro. This concentration range is lower than 50 μM; therefore, it was considered that the concentrations used in in vitro were reasonable.
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4. Discussion
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Fig. 2. Involvement of ER stress in MPP-induced stress and effect of zonisamide against MPP þ -induced stress in vitro. (A) The protocol performed in this study. SH-SY5Y cells were treated with zonisamide for 1 h followed by the addition of 0.1–1.0 mM MPP þ . A nuclear staining assay was carried out after 48 h of incubation. (B) Representative band images show immunoreactivity against GRP78 and β-actin. (C) GRP78 expression was significantly increased by 0.5 mM MPP þ exposure. Data represent means and S.E.M., n¼ 3 or 4. ##; P o0.01 vs. Control (Bonferroni-test). (D) Typical photograph of Hoechst 33342 (blue: viable cells) and propidium iodide (PI) staining (red: dead cells). Scale bar¼ 50 μm. (E) Measurement of PI positive cells. Each column and bar represent the mean 7 S.E.M., n ¼6. ##; Po 0.01 vs. Control (t-test), nn; Po 0.01 vs. Vehicle (t-test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
We investigated caspase-3, downstream signal of ER stress. The cleavage of caspase-3 was increased by tunicamycin and this effect was prevented by pretreatment with zonisamide (Fig. 4A and B). 3.5. Plasma concentrations of zonisamide after single oral administration in mice To clarify whether the concentrations of zonisamide used in vitro were physiologically relevant, we measured the plasma concentration
In the present study, we examined the protective effects of zonisamide on cell death induced by ER stress, via thapsigargin and tunicamycin treatment, in vitro. It is known that zonisamide increases a amount of dopamine in the brain by inhibition of MAO-B, which metabolizes dopamine (Yabe et al., 2009). Recently, it has been reported that zonisamide itself has a neuroprotective effect by increasing the levels of manganese superoxide dismutase (MnSOD) (Kawajiri et al., 2010), glutathione (Murata, 2010), and inhibiting caspase-3 activation (Omura et al., 2012). These findings indicate that zonisamide has multifunctional actions. However, a clear mechanism of action for zonisamide has not yet been revealed. We showed here that zonisamide suppressed neuronal cell death from ER stress induced by tunicamycin or thapsigargin. Furthermore, zonisamide was protective against oxidative stress. However, previous reports suggest that antioxidants do not protect against ER stress induced by tunicamycin and thapsigargin (Yamauchi et al., 2011). Therefore, it is suggested that, in this study, the protective effect of zonisamide may be mainly due to the protection of the ER stress process, rather than an antioxidative effect. MPP þ -treatment is often used as a Parkinson's disease model in vitro. While there are some reports that show the involvement of ER stress in Parkinson's disease (Quinn, 1995; Yamauchi et al., 2011), it is not clear whether MPP þ induces ER stress. In this study, we investigated the change in expression of ER stress markers in MPP þ -treated SH-SY5Y cells. As a result, it was confirmed that the expression level of GRP78 was increased under MPP þ -treatment, suggesting that MPP þ -treatment induces ER stress. This phenomenon was also observed in vivo as there was an increase in the optical density of CHOP-positive fibers in striatum following MPTP-treatment. Zonisamide suppressed the expression of CHOP; therefore, zonisamide may have some effect on ER stress. We examined the change in caspase-3 activation with tunicamycin-treatment to assess the mechanism of action of zonisamide protecting against ER stress. Caspase-3 is a structural factor in the apoptotic pathway activated by ER stress, and induces apoptosis by activating itself (Rutkowski and Kaufman, 2004). The results of this study suggest that zonisamide protects against ER stress by affecting caspase-3. It has been reported that zonisamide dose not affect expression of mRNA of GRP78, CHOP, and Poly (ADPribose) polymerase-2 (PARK2), and that zonisamide inhibits activation of caspase-3 by inducing HRD1 (Omura et al., 2012). Even in our present study, zonisamide did not affect GRP78 induced by MPP þ (data not shown). Therefore, the protective effect of zonisamide against ER stress may modulate the factor upstream of caspase-3 except for GRP78, IRE, and Bax. Also, there are reports that cannabidiol confers oligoprotective effects during inflammation, which are accompanied by a decrease in the expression of ER apoptotic effectors (CHOP, Bax, and caspase 12) and cleaved caspase-3 (Mecha et al., 2012), and that lipopolysaccharide suppresses ER stress-induced CHOP via toll-like receptor activation (Woo et al., 2012). Inflammation relates to the pathogenesis of
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Zonisamide MPTP Fig. 3. Effect of zonisamide against MPTP-induced ER stress in vivo. Zonisamide was administered orally 5 times to MPTP-treated mice. (A and C) Immunochemical staining of tyrosine hydroxylase (TH) or double immunostaining fluorescence TH (red) and CHOP (green) in substantia nigra. Hoechst 33342 (blue) stained nucleus. (B) Optical density of TH-positive fibers in substantia nigra. (D) Proportional stained area of CHOP in substantia nigra cells. Values are expressed as the mean 7 S.E.M., n¼ 3–5. #; P o 0.05, ##; P o0.01 vs. Control (t-test), n; Po 0.05, nn; Po 0.01 vs. Vehicle (ttest). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Parkinson's disease (Gyoneva et al., 2014); therefore, it is possible that zonisamide effects not only caspase-3 but also other ER apoptotic factors such as Bax and caspase-12, or toll-like receptors. Further investigations will be necessary regarding this point.
We investigated whether the concentrations of zonisamide used in vitro in this study were physiologically relevant by measuring plasma concentrations of zonisamide after a single oral dose. C2h of zonisamide in this study was approximately 11 μg/ml.
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C2h, approximately 5.5 μg/ml in this study and 0.57 μg/ml in humans. From this study, it has been revealed that there was a 10–times difference in the plasma level of zonisamide between mice and humans. In addition, the effective concentrations used in vitro were 0.1–3 μM, which are approximately 0.02–0.64 μg/ml. Therefore, the concentration in vitro is less than the effective plasma concentration in mice. These findings indicate that the concentration of zonisamide used in the in vitro experiments is reasonable, and that zonisamide would show these protective effects in the central nervous system in human.
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These findings indicate that ER stress may play a role in vitro and in vivo in experimental models of Parkinson's disease, and that zonisamide possesses neuroprotective effects against ER stress via caspase-3.
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Acknowledgment
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Appendix A. Supporting information
Fig. 4. Effect of zonisamide against the cleavage of caspase-3 under tunicamycin stress. SH-SY5Y cells were treated with zonisamide for 1 h followed by the addition of 2.0 μg/ml tunicamycin. Sampling was carried out after 27 h of incubation. (A) Representative band images show immunoreactivity against cleaved caspase3 and β-actin. (B) Cleaved caspase-3 expression was significantly increased by tunicamycin exposure. Data represent means 7 S.E.M., n¼ 3. ##; P o 0.01 vs. Control (t-test), n; Po 0.05 vs. Vehicle (t-test).
Plasma concentration of zonisamide (ng/ml)
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We received a financial support of Sumitomo Dainippon Pharma Co., Ltd (Osaka, Japan) as a collaborative research.
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Fig. 5. Plasma concentrations of zonisamide after single oral administration. Zonisamide was administered orally at a dose of 20 mg/kg. Collecting of blood samples was carried out after 2, 8, and 24 h. n¼5 or 6.
On the other hand, trough levels of following repeated administration of 25 mg zonisamide in humans is reported to be 1.14 μg/ml (Sumitomo Dainippon Pharma Co., Ltd., 2014). However, the plasma protein binding rate of zonisamide in humans or rats is approximately 50% (Sumitomo Dainippon Pharma Co., Ltd., 2014), therefore the effective plasma concentration is half the
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2014.09.023. References Beal, M.F., 2003. Mitochondria, oxidative damage, and inflammation in Parkinson's disease. Ann. NY Acad. Sci. 991, 120–131. Behar-Bannelier, M., Pinteric, L., Murray, R.K., 1980. Effects of acute hepatic ischemia on the electrophoretic patterns of the polypeptides and phosphopolypeptides of the microsomal membrane fraction of rat liver. Can. J. Biochem. 58, 1039–1050. Gotz, M.E., Freyberger, A., Riederer, P., 1990. Oxidative stress: a role in the pathogenesis of Parkinson's disease. J Neural Transm. 29, 241–249. Gyoneva, S., Shapiro, L., Lazo, C., Garnier-Amblard, E., Smith, Y., Miller, G.W., Traynelis, S.F., 2014. Adenosine A2A receptor antagonism reverses inflammation-induced impairment of microglial process extension in a model of Parkinson's disease. Neurobiol. Dis. 67, 191–202. Kaneko, M., 2012. [Molecular pharmacological studies on the protection mechanism against endoplasmic reticulum stress-induced neurodegenerative disease]. Yakugaku Zasshi 132, 1437–1442. Kawajiri, S., Machida, Y., Saiki, S., Sato, S., Hattori, N., 2010. Zonisamide reduces cell death in SH-SY5Y cells via an anti-apoptotic effect and by upregulating MnSOD. Neurosci. Lett. 481, 88–91. Li, H., Zhu, X., Fang, F., Jiang, D., Tang, L., 2014. Down-regulation of GRP78 enhances apoptosis via CHOP pathway in retinal ischemia-reperfusion injury. Neurosci. Lett. 575, 68–73. Mecha, M., Torrao, A.S., Mestre, L., Carrillo-Salinas, F.J., Mechoulam, R., Guaza, C., 2012. Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress. Cell Death Dis. 3, e331. Murata, M., 2010. [Discovery of an antiparkinsonian drug: zonisamide]. Rinsho Shinkeigaku 50, 780–782 ( ¼ Clinical neurology). Murata, M., Hasegawa, K., Kanazawa, I., The Japan Zonisamide on PD Study Group, 2007. Zonisamide improves motor function in Parkinson disease; a randomized, double-blined study. Neurology 68, 45–50. Oida, Y., Kitaichi, K., Nakayama, H., Ito, Y., Fujimoto, Y., Shimazawa, M., Nagai, H., Hara, H., 2006. Rifampicin attenuates the MPTP-induced neurotoxicity in mouse brain. Brain Res. 1082, 196–204. Omura, T., Asari, M., Yamamoto, J., Kamiyama, N., Oka, K., Hoshina, C., Maseda, C., Awaya, T., Tasaki, Y., Shiono, H., Shimizu, K., Matsubara, K., 2012. HRD1 levels increased by zonisamide prevented cell death and caspase-3 activation caused by endoplasmic reticulum stress in SH-SY5Y cells. J Mol. Neurosci. 46, 527–535. Parra, V., Moraga, F., Kuzmicic, J., Lopez-Crisosto, C., Troncoso, R., Torrealba, N., Criollo, A., Diaz-Elizondo, J., Rothermel, B.A., Quest, A.F., Lavandero, S., 2013. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death. Biochim. Biophys. Acta 1832, 1334 (1344). Quinn, N., 1995. Drug treatment of Parkinson's disease. BMJ 310, 575–579.
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Robertson, H.A., Peterson, M.R., Murphy, K., Robertson, G.S., 1989. D1-dopamine receptor agonists selectively activate striatal c-fos independent of rotational behaviour. Brain Res. 503, 346–349. Rutkowski, D.T., Kaufman, R.J., 2004. A trip to the ER: coping with stress. Trends Cell Biol. 14, 20–28. Ryu, E.J., Harding, H.P., Angelastro, J.M., Vitolo, O.V., Ron, D., Greene, L.A., 2002. Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease. J. Neurosci. 22, 10690–10698. Sumitomo Dainippon Pharma Co., Ltd., 8th edition revision. Pharmaceuticals interview form, Levodopa activation type therapeutic agent for Parkinson's disease TRERIEFs Tablet 25 mg, Item VII Pharmacokinetics, 2014. August. Savitt, J.M., Dawson, V.L., Dawson, T.M., 2006. Diagnosis and treatment of Parkinson disease: molecules to medicine. J. Clin. Invest. 116, 1744–1754. Schapira, A.H., Gegg, M., 2011. Mitochondrial contribution to Parkinson's disease pathogenesis. Parkinson Dis. 2011, 159160. Suzuki, S., Kawakami, K., Nishimura, S., Watanabe, Y., Yagi, K., Seino, M., Miyamoto, K., 1992. Zonisamide blocks T-type calcium channel in cultured neurons of rat cerebral cortex. Epilepsy Res. 12, 21–27.
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Woo, C.W., Kutzler, L., Kimball, S.R., Tabas, I., 2012. Toll-like receptor activation suppresses ER stress factor CHOP and translation inhibition through activation of eIF2B. Nat. Cell. Biol. 14, 192–200. Yabe, H., Choudhury, M.E., Kubo, M., Nishikawa, N., Nagai, M., Nomoto, M., 2009. Zonisamide increases dopamine turnover in the striatum of mice and common marmosets treated with MPTP. J. Pharmacol. Sci. 110 (1), 64–68. Yamauchi, M., Tsuruma, K., Imai, S., Nakanishi, T., Umigai, N., Shimazawa, M., Hara, H., 2011. Crocetin prevents retinal degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase activity. Eur. J. Pharmacol. 650, 110–119. Zhu, W., Wang, D., Zheng, J., An, Y., Wang, Q., Zhang, W., Jin, L., Gao, H., Lin, L., 2008. Effect of (R)-salsolinol and N-methyl-(R)-salsolinol on the balance impairment between dopamine and acetylcholine in rat brain: involvement in pathogenesis of Parkinson disease. Clin. Chem. 54 (4), 705–712.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Zonisamide suppresses endoplasmic reticulum stress-induced neuronal cell damage in vitro and in vivo Saori Tsujii a, Mitsue Ishisaka a, Masamitsu Shimazawa a, Takanori Hashizume b, Hideaki Hara a,n a b
Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Japan Laboratory of Drug Metabolism & Pharmacokinetics, Faculty of Pharmacy, Osaka Ohtani University, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 25 July 2014 Received in revised form 16 September 2014 Accepted 16 September 2014 Available online 23 September 2014
Zonisamide has been reported to have protective effects on epilepsy and Parkinson's disease and to work via various mechanisms of action, such as inhibition of monoamine oxidase-B and enhancement of tyrosine hydroxylase. Recently, it has been suggested that zonisamide itself shows neuroprotective actions. Therefore, in the present study we investigated the neuroprotective effects of zonisamide against endoplasmic reticulum (ER) stress. We used human neuroblastoma (SH-SY5Y) cells and investigated the protective effects of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death. In addition, we investigated the effect of zonisamide against 1-methyl-4phenylpyridinium (MPP þ )-induced cell death and the mechanism of protection against ER stress. In vivo, we investigated the effect of zonisamide (20 mg/kg, p.o.) in the 1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease. Zonisamide not only suppressed MPP þ -induced cell death, but also inhibited ER stress-induced cell death and suppressed the expression of ER stress-related factors such as C/EBO homologous protein (CHOP) in vivo. Furthermore, zonisamide inhibited the activation of caspase-3 in vitro. These results suggest that zonisamide affected ER stress via caspase-3. We think that ER stress, particularly the mechanism via caspase-3, is involved in part of the neuroprotective effect of zonisamide against the experimental models of Parkinson's disease. & 2014 Elsevier B.V. All rights reserved.
Keywords: Endoplasmic reticulum stress Parkinson's disease Zonisamide
1. Introduction Parkinson's disease is a neurodegenerative disease characterized by four major symptoms; akinesia, rigidity, balance impairment, and tremors (Savitt et al., 2006). Dopaminergic neurons are reduced in the midbrain substantia nigra in the brains of patients with Parkinson's disease, and the activity of acetylcholine is high due to the dopamine deficiency, which leads to an imbalance of functions (Zhu et al., 2008). However, the cause of the reduction in dopaminergic neurons remains unelucidated. Recently, it has been suggested that cell death induced by oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction are involved in Parkinson's disease (Beal, 2003; Gotz et al., 1990; Ryu et al., 2002; Schapira and Gegg, 2011). The protein associated with ER stress such as Hrd1p/Der3p (HRD1), which promotes ubiquitination, is localized in the brain neurons of patients with Parkinson's disease, and ER stress is involved other
n
Corresponding author. Tel.: þ 81 58 230 8100. E-mail address: [email protected] (H. Hara).
http://dx.doi.org/10.1016/j.ejphar.2014.09.023 0014-2999/& 2014 Elsevier B.V. All rights reserved.
neurodegenerative diseases such as Alzheimer's disease and Huntington's disease (Kaneko, 2012). Several therapeutic medicines such as L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine receptor agonists, drugs promoting free dopamine, and anticholinergic drugs are used for treating Parkinson's disease (Quinn, 1995; Robertson et al., 1989). Among them, zonisamide works by inhibiting monoamine oxidase-B (MAO-B) activity and boosting tyrosine hydroxylase activity. Zonisamide has been used as an anti-epileptic drug at high doses of 200–400 mg/day, originally defined as inhibiting of T-type Ca2 þ channels and as a Na þ channel blocker (Suzuki et al., 1992). However, it has been revealed that zonisamide shows a therapeutic effect in Parkinson's disease at a low dose of 25 mg/day (Murata et al., 2007). In addition, it has been reported that zonisamide itself shows neuroprotective effects by increasing protein levels of a yeast homolog, HRD1 (Omura et al., 2012). Here, we investigated whether zonisamide shows a protective effect against ER stress-induced neuronal cell death. Furthermore, we clarified the mechanism of the protective effects of zonisamide against ER stress in in vitro and in vivo Parkinson's disease models.
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2. Material and methods
2.5. Drug treatment
2.1. Cell culture
2.5.1. Measurement of plasma concentration of zonisamide Zonisamide (20 mg/kg) was suspended in a 40% propylene glycol (Nacalai Tesque, Kyoto, Japan) solution and orally administered (p.o.). Blood collection was carried out from vena cava at 0, 2, 8, or 24 h after drug treatment. Blood samples were centrifuged at 1500g for 25 min at 4 1C and the separated plasma was stored at 80 1C until analysis. Plasma concentrations of zonisamide were measured by high performance liquid chromatography equipped with UV detector (UV-HPLC).
Human neuroblastoma (SH-SY5Y) cells were purchased from the European Collection of Cell Culture (Wiltshire, UK) and maintained in Dulbecco's Modified Eagle's medium (DMEM) (Nacalai Tesque, Kyoto, Japan) containing 10% Fetal Bovine Serum (FBS) (VALEANT, Costa Mesa, CA, USA), 100 units/ml penicillin (Meiji Seika, Tokyo, Japan), and 100 μg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37 1C. Cells were passaged by trypsinization every 3–5 days.
2.2. Cell death assay SH-SY5Y cells was seeded at 1 104 cells per well into a 96well plate (number 3072, Falcons, Becton Dickinson and Company, Franklin Lakes, NJ, USA) and incubated for 24 h at 37 1C in a humidified atmosphere containing 5% CO2. The entire medium was replaced with fresh medium containing 1% FBS, and cells were pretreated with zonisamide at 0.03–3 μM (gifted by Sumitomo Dainippon Pharma Co. Ltd., Osaka, Japan) for 1 h, followed by the addition of 2.0 μg/ml tunicamycin, 1 μM thapsigargin, or 0.5 mM 1-methyl-4-phenylpyridinium (MPP þ ) (Sigma-Aldrich). Nuclear staining assay was carried out after a further 27 h of incubation. Cell death was assessed on the basis of combination staining with Hoechst 33342 (Molecular Probes, Eugene, OR, USA) and propidium iodide (PI; Molecular Probes). At 27 h of incubation, Hoechst 33342 and PI dyes were added to the culture medium (at 8 and 0.2 μM, respectively) for 15 min. Images were collected using an Olympus IX70 inverted epifluorescence microscope (Olympus, Tokyo, Japan). At least 300 cells per condition were counted in a blind manner by a single observer (S.T).
2.3. Western blot analysis SH-SY5Y cells were lysed using a cell-lysis buffer with protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor cocktails (SigmaAldrich). Equal amounts of protein were separated on a 5–20% SDSPAGE gradient gel and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). After blocking with Block Ace (Snow Brand Milk Products Co. Ltd., Tokyo, Japan) for 1 h, membranes were incubated with primary antibody. The primary antibodies used were as follows: mouse anti-KDEL antibody (1:1000 dilution) (Stressgen Bioreagents Limited Partnership, Victoria, B.C., Canada), Cleaved Caspase-3 (Asp175) antibody (1:1000 dilution) or monoclonal anti-β-actin antibody (1:4000 dilution) (Sigma-Aldrich). Subsequently, the membrane was incubated with secondary antibody [goat anti-mouse (Pierce Biotechnology, Rockford, IL, USA)]. The immunoreactive bands were visualized using Multi Gauge Ver. 3.0 (Fujifilm, Tokyo, Japan) and measured using a LAS-4000 mini (Fujifilm).
2.4. Animals 9-week-old male C57BL/6J mice were used in these experiments. Mice were housed at 2472 1C under a 12 h light-dark cycle (lights on from 8:00 to 20:00) and had ad libitum access to food and water. All procedures relating to animal care and treatment conformed to animal care guidelines of the Animal Experiment Committee of Gifu Pharmaceutical University. All efforts were made to minimize both suffering and the number of animals used.
2.5.2. MPTP model in mice The method used to generate a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridin (MPTP) (Sigma-Aldrich) model in mice was performed as previously described (Oida et al., 2006). Briefly, mice were administered orally with zonisamide (20 mg/kg) 1 h before a peritoneal injection of MPTP (20 mg/kg, i.p.) or vehicle, and these injections were continued once a day for 5 days. Six days after the last administration of MPTP, mice were anesthetized with sodium pentobarbital (nembutal, 50 mg/kg, i.p.) and brains were perfusion fixed with 4% paraformaldehyde in 0.1 M phosphate-buffer (pH 7.4). The brains were extracted after a 10 min perfusion fixation at 4 1C and then immersed in the same fixative solution over night. Brain were immersed in 25% sucrose, and embedded in Optimal Cutting Temperature (O.C.T.) compound. 2.6. Immunohistochemistry Frosen sections (10 μm thick) of the substantia nigra were washed for 5 min in 0.01 M phosphate-buffered saline (PBS), and then treated with 0.3% hydrogen peroxidase in 10% methanol. Sections were washed three times with 0.01 M PBS, followed by a 30 min pre-incubation with 1.5% normal goat serum. They were incubated with anti-tyrosine hydroxylase (TH) antibody (1:1000) including 0.3% Triton X-100 overnight at 4 1C. After rinsing twice with 0.01 M PBS, the sections were incubated with biotinylated anti-rabbit secondary antibody for 2 h, and then with an avidin– biotin peroxidase complex for 30 min (both at room temperature). The number of TH-positive neurons in two sections of the substantia nigra was counted under the light microscope at a magnification of 400, and the mean number of TH-positive neurons was calculated. The count of positive cells was performed in a blind manner by a single observer (S.T). 2.7. Double fluorescence immunostaining Frosen sections (10 μm thick) of the substantia nigra were washed for 5 min in 0.01 M PBS, followed by a 1 h pre-incubation with 1.5% normal goat serum. Sections were incubated with antiTH antibody (1:1000) overnight at 4 1C. After rinsing twice with 0.01 M PBS, the sections were incubated with Alexa Fluors 546 donkey anti-rabbit second antibody for 1 h at room temperature. They were then washed with 0.01 M PBS, followed by 1 h incubation with mouse on mouse (M.O.M) Ig blocking reagent. Sections were incubated with mouse anti-CHOP antibody (Santa Cruz Biotechnology, Inc., Texas, USA; 1:1000) including M.O.M protein concentrate overnight at 4 1C. After rinsing twice with 0.01 M PBS, sections were incubated with Alexa Fluors 488 rabbit anti-mouse secondary antibody including M.O.M protein concentrate for 1 h at room temperature. They were then washed with 0.01 M PBS and stained with DNA-specific fluorescent Hoechst 33342. Images were photographed using a laser confocal microscope, and the fluorescence intensity of CHOP was measured by ImageJ (National
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Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih. gov/ij/, 1997–2014.).
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Data are presented as mean 7standard error of mean (S.E.M.). Statistical comparisons were made using Student's t-test, Dunnett's test, or Bonferroni, with P o0.05 being considered statistically significant.
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3.1. Effect of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death in vitro To investigate the protective effect of zonisamide against ER stress in SH-SY5Y cells, we examined the effects of zonisamide on tunicamycin- and thapsigargin-induced neuronal cell death. Tunicamycin inhibits the glycosylation of glycoproteins (Behar-Bannelier et al., 1980), and thapsigargin inhibits Ca2 þ -ATPase at the ER membrane (Parra et al., 2013); therefore, they both induce ER stress via different pathways. Typical photographs of Hoechst 33342 and PI staining are shown in Fig. 1B and D. When compared with the control group, tunicamycin at 2.0 μg/ml or thapsigargin at 1 μM significantly increased the number of dead cells (stained with PI). Pretreatment with zonisamide at 0.1–3 μM protected against this cell death in a concentration-dependent manner, and the effect for tunicamycin was significant at 0.3–3 μM (Fig. 1C) and it for thapsigargin was significant at 0.1–3 μM (Fig. 1E). In addition, we performed similar trials using glutamate treatment at 80 mM or H2O2 treatment at 100 mM. Equally, pretreatment with zonisamide at 0.1–3 μM protected against this cell death in a concentration-dependent manner and the effect for glutamate was significant at 0.1–3 μM (Fig. S1B) and it for H2O2 was significant at 1 and 3 μM (Fig. S1E). These results suggest that zonisamide is neuroprotective against neuronal cell death induced by ER stress, glutamate, and oxidative stress. þ
3.2. Effect of zonisamide against MPP -induced ER stress in vitro To investigate the involvement of ER stress in Parkinson's disease, we examined changes in ER stress markers with MPP þ treatment in SH-SY5Y cells. It has been reported that the expression of GRP78 is increased by ER stress (Li et al., 2014). The expression of GRP78 was significantly increased by MPP þ exposure (Fig. 2C). Therefore, this suggested that ER stress is induced by MPP þ exposure. Next, we investigated the effects of zonisamide against MPP þ -induced neuronal cell death. Typical photographs of Hoechst 33342 and PI staining are shown in Fig. 2D. When compared with the control group, MPP þ treatment significantly increased the number of dead cells. Pretreatment with zonisamide protected against this cell death (Fig. 2E). 3.3. Effect of zonisamide against MPTP-induced ER stress in vivo Next, we evaluated the effect of zonisamide by using experimental animal models. The number of TH-positive neuronal cells in the substantia nigra was decreased after MPTP treatment in mice, but rescued by zonisamide treatment (Fig. 3A and B). In addition, to confirm the involvement of ER stress in Parkinson's disease in vivo, we examined changes in CHOP expression in MPTP-treated mice compared with control mice. The expression of CHOP in the striatum is shown in Fig. 3C. The expression of CHOP was increased in MPTP-treated mice, and pretreatment with zonisamide attenuated this increase (Fig. 3D).
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Fig. 1. Effects of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death. (A) The protocol performed in this study. SH-SY5Y cells were treated with zonisamide for 1 h followed by the addition of 2.0 μg/ml tunicamycin or 1 μM thapsigargin. A nuclear staining assay was carried out after 27 h of incubation. (B and D) Typical photographs of Hoechst 33342 (blue: viable cells) and propidium iodide (PI) staining (red: dead cells). Scale bar¼ 50 μm. (C, E) Measurement of PI positive cells. Each column and bar represent the mean7 S.E.M., n¼ 6. ##; Po 0.01 vs. Control (t-test), n; P o0.05, nn; P o0.01 vs. Vehicle (Dunnett's test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Mechanism of protection of zonisamide against tunicamycininduced cell death in vitro To examine in the mechanism of action of zonisamide protecting against ER stress, we examined changes in factors induced by apoptosis: GRP78, unfolded protein response (UPR), and caspase-3 activity under tunicamycin treatment in SH-SY5Y cells. Zonisamide did not affect to the expression of GRP78 (data not shown).
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of zonisamide in mice following a single oral administration. Fig. 5 shows the change in zonisamide plasma concentration after the oral administration. The concentrations showed approximately 11, 4, and 0.005 μg/ml at 2, 8, and 24 h after administration, respectively. The elimination phase half-life (t1/2b) was 1.7 h. The C2h concentration was approximately 11 μg/ml, which is equivalent to approximately 50 μM. The neuroprotective effects of zonisamide were seen in the range of 0.1–3 μM in vitro. This concentration range is lower than 50 μM; therefore, it was considered that the concentrations used in in vitro were reasonable.
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Fig. 2. Involvement of ER stress in MPP-induced stress and effect of zonisamide against MPP þ -induced stress in vitro. (A) The protocol performed in this study. SH-SY5Y cells were treated with zonisamide for 1 h followed by the addition of 0.1–1.0 mM MPP þ . A nuclear staining assay was carried out after 48 h of incubation. (B) Representative band images show immunoreactivity against GRP78 and β-actin. (C) GRP78 expression was significantly increased by 0.5 mM MPP þ exposure. Data represent means and S.E.M., n¼ 3 or 4. ##; P o0.01 vs. Control (Bonferroni-test). (D) Typical photograph of Hoechst 33342 (blue: viable cells) and propidium iodide (PI) staining (red: dead cells). Scale bar¼ 50 μm. (E) Measurement of PI positive cells. Each column and bar represent the mean 7 S.E.M., n ¼6. ##; Po 0.01 vs. Control (t-test), nn; Po 0.01 vs. Vehicle (t-test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
We investigated caspase-3, downstream signal of ER stress. The cleavage of caspase-3 was increased by tunicamycin and this effect was prevented by pretreatment with zonisamide (Fig. 4A and B). 3.5. Plasma concentrations of zonisamide after single oral administration in mice To clarify whether the concentrations of zonisamide used in vitro were physiologically relevant, we measured the plasma concentration
In the present study, we examined the protective effects of zonisamide on cell death induced by ER stress, via thapsigargin and tunicamycin treatment, in vitro. It is known that zonisamide increases a amount of dopamine in the brain by inhibition of MAO-B, which metabolizes dopamine (Yabe et al., 2009). Recently, it has been reported that zonisamide itself has a neuroprotective effect by increasing the levels of manganese superoxide dismutase (MnSOD) (Kawajiri et al., 2010), glutathione (Murata, 2010), and inhibiting caspase-3 activation (Omura et al., 2012). These findings indicate that zonisamide has multifunctional actions. However, a clear mechanism of action for zonisamide has not yet been revealed. We showed here that zonisamide suppressed neuronal cell death from ER stress induced by tunicamycin or thapsigargin. Furthermore, zonisamide was protective against oxidative stress. However, previous reports suggest that antioxidants do not protect against ER stress induced by tunicamycin and thapsigargin (Yamauchi et al., 2011). Therefore, it is suggested that, in this study, the protective effect of zonisamide may be mainly due to the protection of the ER stress process, rather than an antioxidative effect. MPP þ -treatment is often used as a Parkinson's disease model in vitro. While there are some reports that show the involvement of ER stress in Parkinson's disease (Quinn, 1995; Yamauchi et al., 2011), it is not clear whether MPP þ induces ER stress. In this study, we investigated the change in expression of ER stress markers in MPP þ -treated SH-SY5Y cells. As a result, it was confirmed that the expression level of GRP78 was increased under MPP þ -treatment, suggesting that MPP þ -treatment induces ER stress. This phenomenon was also observed in vivo as there was an increase in the optical density of CHOP-positive fibers in striatum following MPTP-treatment. Zonisamide suppressed the expression of CHOP; therefore, zonisamide may have some effect on ER stress. We examined the change in caspase-3 activation with tunicamycin-treatment to assess the mechanism of action of zonisamide protecting against ER stress. Caspase-3 is a structural factor in the apoptotic pathway activated by ER stress, and induces apoptosis by activating itself (Rutkowski and Kaufman, 2004). The results of this study suggest that zonisamide protects against ER stress by affecting caspase-3. It has been reported that zonisamide dose not affect expression of mRNA of GRP78, CHOP, and Poly (ADPribose) polymerase-2 (PARK2), and that zonisamide inhibits activation of caspase-3 by inducing HRD1 (Omura et al., 2012). Even in our present study, zonisamide did not affect GRP78 induced by MPP þ (data not shown). Therefore, the protective effect of zonisamide against ER stress may modulate the factor upstream of caspase-3 except for GRP78, IRE, and Bax. Also, there are reports that cannabidiol confers oligoprotective effects during inflammation, which are accompanied by a decrease in the expression of ER apoptotic effectors (CHOP, Bax, and caspase 12) and cleaved caspase-3 (Mecha et al., 2012), and that lipopolysaccharide suppresses ER stress-induced CHOP via toll-like receptor activation (Woo et al., 2012). Inflammation relates to the pathogenesis of
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Zonisamide MPTP Fig. 3. Effect of zonisamide against MPTP-induced ER stress in vivo. Zonisamide was administered orally 5 times to MPTP-treated mice. (A and C) Immunochemical staining of tyrosine hydroxylase (TH) or double immunostaining fluorescence TH (red) and CHOP (green) in substantia nigra. Hoechst 33342 (blue) stained nucleus. (B) Optical density of TH-positive fibers in substantia nigra. (D) Proportional stained area of CHOP in substantia nigra cells. Values are expressed as the mean 7 S.E.M., n¼ 3–5. #; P o 0.05, ##; P o0.01 vs. Control (t-test), n; Po 0.05, nn; Po 0.01 vs. Vehicle (ttest). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Parkinson's disease (Gyoneva et al., 2014); therefore, it is possible that zonisamide effects not only caspase-3 but also other ER apoptotic factors such as Bax and caspase-12, or toll-like receptors. Further investigations will be necessary regarding this point.
We investigated whether the concentrations of zonisamide used in vitro in this study were physiologically relevant by measuring plasma concentrations of zonisamide after a single oral dose. C2h of zonisamide in this study was approximately 11 μg/ml.
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C2h, approximately 5.5 μg/ml in this study and 0.57 μg/ml in humans. From this study, it has been revealed that there was a 10–times difference in the plasma level of zonisamide between mice and humans. In addition, the effective concentrations used in vitro were 0.1–3 μM, which are approximately 0.02–0.64 μg/ml. Therefore, the concentration in vitro is less than the effective plasma concentration in mice. These findings indicate that the concentration of zonisamide used in the in vitro experiments is reasonable, and that zonisamide would show these protective effects in the central nervous system in human.
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These findings indicate that ER stress may play a role in vitro and in vivo in experimental models of Parkinson's disease, and that zonisamide possesses neuroprotective effects against ER stress via caspase-3.
*
Acknowledgment
Control
Vehicle
3
3
μM
Zonisamide Tunicamycin (2 μg/ml)
Appendix A. Supporting information
Fig. 4. Effect of zonisamide against the cleavage of caspase-3 under tunicamycin stress. SH-SY5Y cells were treated with zonisamide for 1 h followed by the addition of 2.0 μg/ml tunicamycin. Sampling was carried out after 27 h of incubation. (A) Representative band images show immunoreactivity against cleaved caspase3 and β-actin. (B) Cleaved caspase-3 expression was significantly increased by tunicamycin exposure. Data represent means 7 S.E.M., n¼ 3. ##; P o 0.01 vs. Control (t-test), n; Po 0.05 vs. Vehicle (t-test).
Plasma concentration of zonisamide (ng/ml)
14000 12000 10000 8000 6000 4000 2000 0 0
2
4
8 12 16 20 Time after the administration (h)
We received a financial support of Sumitomo Dainippon Pharma Co., Ltd (Osaka, Japan) as a collaborative research.
24
Fig. 5. Plasma concentrations of zonisamide after single oral administration. Zonisamide was administered orally at a dose of 20 mg/kg. Collecting of blood samples was carried out after 2, 8, and 24 h. n¼5 or 6.
On the other hand, trough levels of following repeated administration of 25 mg zonisamide in humans is reported to be 1.14 μg/ml (Sumitomo Dainippon Pharma Co., Ltd., 2014). However, the plasma protein binding rate of zonisamide in humans or rats is approximately 50% (Sumitomo Dainippon Pharma Co., Ltd., 2014), therefore the effective plasma concentration is half the
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2014.09.023. References Beal, M.F., 2003. Mitochondria, oxidative damage, and inflammation in Parkinson's disease. Ann. NY Acad. Sci. 991, 120–131. Behar-Bannelier, M., Pinteric, L., Murray, R.K., 1980. Effects of acute hepatic ischemia on the electrophoretic patterns of the polypeptides and phosphopolypeptides of the microsomal membrane fraction of rat liver. Can. J. Biochem. 58, 1039–1050. Gotz, M.E., Freyberger, A., Riederer, P., 1990. Oxidative stress: a role in the pathogenesis of Parkinson's disease. J Neural Transm. 29, 241–249. Gyoneva, S., Shapiro, L., Lazo, C., Garnier-Amblard, E., Smith, Y., Miller, G.W., Traynelis, S.F., 2014. Adenosine A2A receptor antagonism reverses inflammation-induced impairment of microglial process extension in a model of Parkinson's disease. Neurobiol. Dis. 67, 191–202. Kaneko, M., 2012. [Molecular pharmacological studies on the protection mechanism against endoplasmic reticulum stress-induced neurodegenerative disease]. Yakugaku Zasshi 132, 1437–1442. Kawajiri, S., Machida, Y., Saiki, S., Sato, S., Hattori, N., 2010. Zonisamide reduces cell death in SH-SY5Y cells via an anti-apoptotic effect and by upregulating MnSOD. Neurosci. Lett. 481, 88–91. Li, H., Zhu, X., Fang, F., Jiang, D., Tang, L., 2014. Down-regulation of GRP78 enhances apoptosis via CHOP pathway in retinal ischemia-reperfusion injury. Neurosci. Lett. 575, 68–73. Mecha, M., Torrao, A.S., Mestre, L., Carrillo-Salinas, F.J., Mechoulam, R., Guaza, C., 2012. Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress. Cell Death Dis. 3, e331. Murata, M., 2010. [Discovery of an antiparkinsonian drug: zonisamide]. Rinsho Shinkeigaku 50, 780–782 ( ¼ Clinical neurology). Murata, M., Hasegawa, K., Kanazawa, I., The Japan Zonisamide on PD Study Group, 2007. Zonisamide improves motor function in Parkinson disease; a randomized, double-blined study. Neurology 68, 45–50. Oida, Y., Kitaichi, K., Nakayama, H., Ito, Y., Fujimoto, Y., Shimazawa, M., Nagai, H., Hara, H., 2006. Rifampicin attenuates the MPTP-induced neurotoxicity in mouse brain. Brain Res. 1082, 196–204. Omura, T., Asari, M., Yamamoto, J., Kamiyama, N., Oka, K., Hoshina, C., Maseda, C., Awaya, T., Tasaki, Y., Shiono, H., Shimizu, K., Matsubara, K., 2012. HRD1 levels increased by zonisamide prevented cell death and caspase-3 activation caused by endoplasmic reticulum stress in SH-SY5Y cells. J Mol. Neurosci. 46, 527–535. Parra, V., Moraga, F., Kuzmicic, J., Lopez-Crisosto, C., Troncoso, R., Torrealba, N., Criollo, A., Diaz-Elizondo, J., Rothermel, B.A., Quest, A.F., Lavandero, S., 2013. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death. Biochim. Biophys. Acta 1832, 1334 (1344). Quinn, N., 1995. Drug treatment of Parkinson's disease. BMJ 310, 575–579.
S. Tsujii et al. / European Journal of Pharmacology 746 (2015) 301–307
Robertson, H.A., Peterson, M.R., Murphy, K., Robertson, G.S., 1989. D1-dopamine receptor agonists selectively activate striatal c-fos independent of rotational behaviour. Brain Res. 503, 346–349. Rutkowski, D.T., Kaufman, R.J., 2004. A trip to the ER: coping with stress. Trends Cell Biol. 14, 20–28. Ryu, E.J., Harding, H.P., Angelastro, J.M., Vitolo, O.V., Ron, D., Greene, L.A., 2002. Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease. J. Neurosci. 22, 10690–10698. Sumitomo Dainippon Pharma Co., Ltd., 8th edition revision. Pharmaceuticals interview form, Levodopa activation type therapeutic agent for Parkinson's disease TRERIEFs Tablet 25 mg, Item VII Pharmacokinetics, 2014. August. Savitt, J.M., Dawson, V.L., Dawson, T.M., 2006. Diagnosis and treatment of Parkinson disease: molecules to medicine. J. Clin. Invest. 116, 1744–1754. Schapira, A.H., Gegg, M., 2011. Mitochondrial contribution to Parkinson's disease pathogenesis. Parkinson Dis. 2011, 159160. Suzuki, S., Kawakami, K., Nishimura, S., Watanabe, Y., Yagi, K., Seino, M., Miyamoto, K., 1992. Zonisamide blocks T-type calcium channel in cultured neurons of rat cerebral cortex. Epilepsy Res. 12, 21–27.
307
Woo, C.W., Kutzler, L., Kimball, S.R., Tabas, I., 2012. Toll-like receptor activation suppresses ER stress factor CHOP and translation inhibition through activation of eIF2B. Nat. Cell. Biol. 14, 192–200. Yabe, H., Choudhury, M.E., Kubo, M., Nishikawa, N., Nagai, M., Nomoto, M., 2009. Zonisamide increases dopamine turnover in the striatum of mice and common marmosets treated with MPTP. J. Pharmacol. Sci. 110 (1), 64–68. Yamauchi, M., Tsuruma, K., Imai, S., Nakanishi, T., Umigai, N., Shimazawa, M., Hara, H., 2011. Crocetin prevents retinal degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase activity. Eur. J. Pharmacol. 650, 110–119. Zhu, W., Wang, D., Zheng, J., An, Y., Wang, Q., Zhang, W., Jin, L., Gao, H., Lin, L., 2008. Effect of (R)-salsolinol and N-methyl-(R)-salsolinol on the balance impairment between dopamine and acetylcholine in rat brain: involvement in pathogenesis of Parkinson disease. Clin. Chem. 54 (4), 705–712.
