Ca2+ exchange inhibitor, prevents dopaminergic neurotoxicity in an MPTP mouse model of Parkinson’s disease

Neuropharmacology 61 (2011) 1441e1451

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SEA0400, a specific Naþ/Ca2þ exchange inhibitor, prevents dopaminergic neurotoxicity in an MPTP mouse model of Parkinson’s disease Yukio Agoa,1, Toshiyuki Kawasakia, b,1, Tetsuaki Nashidaa, Yuki Otaa, Yana Conga, Mari Kitamotoa, Teisuke Takahashic, Kazuhiro Takumaa, Toshio Matsudaa, d, * a

Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan Functional Probe Research Laboratory, RIKEN, Center for Molecular Imaging Science, Kobe MI R&D Center, C6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan c Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Kita-ku, Saitama, Saitama 331-9530, Japan d United Graduate School of Child Development, Osaka University, Kanazawa University and Hamamatsu University School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 May 2011 Received in revised form 24 August 2011 Accepted 26 August 2011

We have recently shown that the Naþ/Ca2þ exchanger (NCX) is involved in nitric oxide (NO)-induced cytotoxicity in cultured astrocytes and neurons. However, there is no in vivo evidence suggesting the role of NCX in neurodegenerative disorders associated with NO. NO is implicated in the pathogenesis of neurodegenerative disorders such as Parkinson’s disease. This study examined the effect of SEA0400, the specific NCX inhibitor, on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity, a model of Parkinson’s disease, in C57BL/6J mice. MPTP treatment (10 mg/kg, four times at 2-h intervals) decreased dopamine levels in the midbrain and impaired motor coordination, and these effects were counteracted by S-methylthiocitrulline, a selective neuronal NO synthase inhibitor. SEA0400 protected against the dopaminergic neurotoxicity (determined by dopamine levels in the midbrain and striatum, tyrosine hydroxylase immunoreactivity in the substantia nigra and striatum, striatal dopamine release, and motor deficits) in MPTP-treated mice. SEA0400 had no radical-scavenging activity. SEA0400 did not affect MPTP metabolism and MPTP-induced NO production and microglial activation, while it attenuated MPTP-induced increases in extracellular signal-regulated kinase (ERK) phosphorylation and lipid peroxidation product, thiobarbituric acid reactive substance. These findings suggest that SEA0400 protects against MPTP-induced neurotoxicity probably by blocking ERK phosphorylation and lipid peroxidation which are downstream of NCX-mediated Ca2þ influx. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Naþ/Ca2þ exchanger (NCX) Nitric oxide (NO) SEA0400 MPTP Parkinson’s disease Dopamine Mice

1. Introduction The Naþ/Ca2þ exchanger (NCX) plays a key role in the regulation of intracellular Ca2þ concentrations (Annunziato et al., 2004; Lee et al., 2005; Lytton, 2007; Philipson and Nicoll, 2000), and there are three isoforms (NCX1, NCX2 and NCX3) of NCX in the brain. Previous studies have shown that NCX activity is stimulated by nitric oxide (NO) in some cells including astrocytes and neuronal preparations (Asano et al., 1995). These findings suggest that NCX is involved in some effects of NO in the brain. In this relation, we have studied whether NCX is involved in NO-induced cytotoxicity in astrocytes (Kitao et al., 2010), microglia (Nagano et al., 2005) and * Corresponding author. Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: þ81 6 6879 8161; fax: þ81 6 6879 8159. E-mail address: [email protected] (T. Matsuda). 1 Y.A. and T.K. contributed equally to this work. 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.08.041

neuroblastoma SH-SY5Y cells (Nashida et al., 2011), using the specific inhibitor of NCX SEA0400 (Matsuda et al., 2001). SEA0400 inhibits NCX1 more potently than other isoforms (Iwamoto et al., 2004). These studies suggest that NCX-mediated abnormality of intracellular Ca2þ is responsible for the NO-induced cytotoxicity. NO increases phosphorylation of extracellular signal-regulated kinase (ERK), a family of mitogen-activated kinases, and reactive oxygen species (ROS) production, and these effects are attenuated by SEA0400 in astrocytes (Kitao et al., 2010) and SH-SY5Y cells (Nashida et al., 2011). The roles of ERK and Ca2þ homeostasis in NO-induced neurotoxicity were also demonstrated in previous studies (Brorson et al., 1997; Brorson and Zhang, 1997; Harper and Wilkie, 2003; Miloso et al., 2008). These in vitro studies suggest that the NCXmediated Ca2þ influx in plasma membrane triggers the activation of ERK and ROS production, resulting in cytotoxicity. However, little is known about the involvement of NCX in the neurodegenerative disorders associated with NO.

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Parkinson’s disease is a progressive neurodegenerative disorder characterized by the relatively selective loss of nigrostriatal dopaminergic neurons (Surendran and Rajasankar, 2010). Although the mechanisms underlying Parkinson’s disease are not completely understood, there is considerable evidence showing that the inflammatory processes and cellular oxidative damage occurring in Parkinson’s disease may result from the actions of altered production of NO (Aquilano et al., 2008; Whitton, 2007; Zhang et al., 2006). Indeed, high levels of neuronal NO synthase (nNOS) and inducible NO synthase (iNOS) expressions were observed in the nigrostriatal region and basal ganglia in the post mortem brains with Parkinson’s disease (Eve et al., 1998; Hunot et al., 1996). 1-Methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), which impairs mitochondrial respiration by inhibiting complex I, causes dopaminergic neurotoxicity, leading to behavioral impairment similar to the features of Parkinson’s disease (Sedelis et al., 2001). Thus, the MPTP-induced dopaminergic neurodegeneration model has been used extensively to study the mechanisms underlying Parkinson’s disease. Previous studies show that the NO system plays a key role in MPTP neurotoxicity (Chalimoniuk et al., 2006; Dehmer et al., 2000; Klivenyi et al., 2000; Kurosaki et al., 2002; Liberatore et al., 1999; Matthews et al., 1997; Przedborski et al., 1996; Watanabe et al., 2004). Moreover, Papa et al. (2003) showed that NCX1 is localized in the substantia nigra and NCX1, NCX2 and NCX3 are localized in the striatum of rats. We have also found the mRNA expression of all NCX isoforms in the midbrain regions containing the substantia nigra and striatum of mice (unpublished observation). The present study examined the effect of SEA0400 on MPTP-induced dopaminergic neurotoxicity in mice to study the involvement of NCX in NO-mediated neurodegenerative disorders. We demonstrated that the selective nNOS inhibitor S-methylthiocitrulline (SMTC) blocks MPTP-induced neurotoxicity and SEA0400 attenuates MPTP-induced ERK phosphorylation, lipid peroxidation, dopaminergic neurotoxicity and impairment of motor coordination in mice. 2. Methods 2.1. Animals and drugs The experimental procedures concerning the use of animals in this work were conducted according to the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society. Every effort was made to minimize animal suffering, and to reduce the number of animals used. Six-week-old male C57BL/6J mice (Japan SLC Inc., Hamamatsu, Japan) were housed in cages (24  17  12 cm3) in groups of 5e6 animals under controlled environmental conditions (22  1  C; 12:12-h lightedark cycle, lights on at 08:00 h; food and water ad libitum) for 1 week before use in experiments. We used a total of 480 mice in all the experiments. The following drugs were used: MPTP, SMTC (Sigma, St. Louis, MO), SEA0400 (Taisho Pharmaceutical Co. Ltd, Saitama, Japan) and methamphetamine (Dainippon Pharma Co., Osaka, Japan). The primary antibodies used were as follows: rabbit polyclonal antibodies against tyrosine hydroxylase (Chemicon, Temecula, CA), ionized calcium-binding adapter molecule 1 (Iba-1) (Wako Pure chemical Industries, Osaka, Japan), nitrotyrosine (Millipore Corp., Billerica, MA) and total- and phospho-ERK (Cell Signaling Technology, Beverly, MA). Vectastain ABC standard kit (Vector Laboratories Inc., Burlingame, CA) was used for immunodetection. All other commercially available chemicals used in the experiments were of superfine quality. MPTP, SMTC and methamphetamine were dissolved in saline (0.9% solution of NaCl), and SEA0400 was administered as a lipid emulsion containing 20% soybean oil (Matsuda et al., 2001). These drugs were injected at a fixed volume of 5 ml/kg body weight. 2.2. Experimental design Mice were s.c. administered with MPTP (10 mg/kg) four times at 2-h intervals. SEA0400 (3 or 10 mg/kg) was i.p. administered 30 min before every MPTP dosing. SMTC (10 mg/kg) was also i.p. administered 30 min before every MPTP dosing, referring to the previous study (Matthews et al., 1997). The experiments of microglial activation, ERK phosphorylation, lipid peroxidation and motor functions were performed 1 day after the final MPTP dosing. The experiments for dopamine assay, tyrosine hydroxylase immunostaining and microdialysis were performed 3 days after the final MPTP dosing, because the mice used for behavioral experiments were used for these experiments (Kawasaki et al., 2007, 2008).

2.3. Dopamine levels Concentrations of dopamine in the midbrain regions containing the substantia nigra and striatum were quantified by high-performance liquid chromatography (HPLC) with an electrochemical detector (ECD-100; Eicom Corp., Kyoto, Japan), as previously reported (Kawasaki et al., 2007, 2008). Briefly, the midbrain and striatum were individually isolated, frozen on dry ice, and stored at 80  C until assay. Tissue samples were homogenized in 0.2 M perchloric acid containing 100 mM EDTA and isoproterenol as an internal standard. The homogenate was centrifuged at 15,000g for 15 min at 0  C. The supernatant was filtered through a 0.22 mm membrane filter (Millipore Corp.), and then a 10-ml aliquot of the sample was injected onto the HPLC column for dopamine assay. Values are expressed as ng/g or mg/g tissue (wet weight).

2.4. Tyrosine hydroxylase immunohistochemistry The brains were quickly removed and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 3 days and then transferred to 15% sucrose in 0.1 M phosphate buffer for 2 days. Serial 20-mm-thick coronal sections containing the midstriatum (þ1.2 through 0.1 mm with respect to bregma) and midbrain (3.4 through 3.64 mm with respect to bregma) (Franklin and Paxinos, 1997) were cut using a cryostat microtome at 20  C. The free-floating sections were preincubated for 15 min in 0.3% hydrogen peroxide in 100 mM phosphate-buffered saline containing 0.3% Triton X-100 (PBS-T). The sections were washed in PBS-T and then incubated with the antibody against tyrosine hydroxylase (1:1000) in PBS-T overnight at room temperature. Subsequent primary incubation sections were washed in PBS-T and incubated in a secondary antibody solution containing biotinylated antirabbit IgG (Vector Laboratories Inc.) in PBS-T for 2 h at room temperature. The sections were then incubated with the avidinebiotin peroxidase complex (1:2000) in PBS-T for 1 h at room temperature. Visualization was performed using 50 mM Tris-HCl buffer, pH 7.6, containing 0.02% 3,30 -diaminobenzidine and 0.005% hydrogen peroxide with 0.6% nickel ammonium sulfate. The sections were dehydrated with ethanol (70, 80, 90, and 100%, xylene) and then mounted and coverslipped. Immunostaining images in the dorsomedial regions of the midstriatum sections were collected and analyzed using the NIH Image software package (Bethesda, MD). To quantify the optical density of tyrosine hydroxylase immunostaining, samples from a 1.0-mm2 area of the dorsomedial regions of the midstriatum were counted from six independent sections of the right hemisphere, generating an average count for each treated subject. The optical density of tyrosine hydroxylasepositive dopaminergic neurons in the striatum was expressed as percentage change from basal levels, with 100% defined as the average count for vehicle-treated control mice. The number of tyrosine hydroxylase-immunopositive neurons in the substantia nigra was quantified by counting blindly from six independent sections of the right hemisphere.

2.5. Rotarod test The rotarod test is widely used to measure coordinated motor skills (Sedelis et al., 2001). The rotarod (Neuroscience Inc., Tokyo, Japan) consisted of a rotating rod (2.8-cm diameter) and individual compartments for each mouse. Mice were trained for two consecutive days prior to MPTP dosing in an acceleration mode (2e16 rpm) for over 2 min. The training was repeated at a fixed speed (16 rpm) until the mice were able to stay on the rod for at least 600 s. On day 1 after the MPTP dosing, the mice were assessed for their coordination capability on the rod at 16 rpm for a maximum recording time of 600 s (Kawasaki et al., 2007, 2008). Rotarod test was performed twice every 30 min, and the results were averaged to obtain a single value for each mouse.

2.6. In vivo dopamine release Mice were anaesthetized with sodium pentobarbital (40 mg/kg, i.p.) and stereotaxically implanted with a guide cannula for a dialysis probe (Eicom Corp.) unilaterally in the striatum (A þ0.4 mm, L 1.7 mm, V 4.5 mm, from the bregma and skull), as previously reported (Kawasaki et al., 2006). The cannula was cemented in place with dental acrylic, and the animal was kept warm and allowed to recover from anesthesia. Postoperative analgesia was performed with a single injection of buprenorphine (0.1 mg/kg, i.p.). The active probe membranes were 2 mm long. The next day of the surgery, mice were administered with MPTP (10 mg/kg, s.c.) four times at 2-h intervals. SEA0400 (3 or 10 mg/kg, i.p.) was administered 30 min before every MPTP dosing. On day 3 after the MPTP treatment, the probe was perfused with Ringer’s solution [147.2 mM NaCl, 4.0 mM KCl, and 2.2 mM CaCl2 (pH 6.0); Fuso Pharmaceutical Industries, Osaka, Japan] at a constant flow rate of 2 ml/min. A stabilization period of 3 h was established before the onset of the experiments. Microdialysis samples (20 ml) were collected every 10 min and injected immediately onto the HPLC column for dopamine assay, as previously reported (Ago et al., 2008, 2009). After the experiments, Evans Blue dye was microinjected through the cannula to histologically verify the position of the probe. Only results from animals with correct probe placement were used in the analysis.

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Fig. 1. Effects of nNOS inhibitor on MPTP-induced increase in nitrotyrosine levels, decrease in dopamine levels and motor deficit. (A) Mice were administered with 10 mg/kg of MPTP s.c. four times at 2-h intervals, sacrificed at 0, 0.5, 1, 3 and 6 h after the final MPTP dosing, and then the nitrotyrosine levels in the midbrain and striatum were determined by dot-blot analysis. Data are expressed as the mean  SEM from 5 to 10 mice. **P < 0.01, compared with the nitrotyrosine levels at 0 h. Mice were administered with saline or 10 mg/kg of MPTP s.c. four times at 2-h intervals. Vehicle or 10 mg/kg of S-methylthiocitrulline (SMTC) was administered i.p. 30 min before every MPTP dosing. (B) Mice were sacrificed 1 h after the final MPTP dosing and the nitrotyrosine levels in the midbrain were determined. Data are expressed as the mean  SEM from 4 mice. **P < 0.01, compared with the saline-treated mice; ##P < 0.01, compared with the MPTP-treated mice. (C) Mice were sacrificed 3 days after the final MPTP dosing and the dopamine contents in the midbrain and striatum were determined by HPLC/ECD methods. Data are expressed as the mean  SEM from 5 to 12 mice. *P < 0.05, **P < 0.01, compared with the saline-treated mice; ##P < 0.01, compared with the MPTP-treated mice. (D) One day after the final MPTP dosing, motor coordination with the rotarod was assessed. Data are expressed as the mean  SEM from 8 to 14 mice. **P < 0.01, compared with the saline-treated mice; #P < 0.05, compared with the MPTP-treated mice.

2.7. 1-Methyl-4-phenylpyridinium ion levels

2.9. Iba-1-immunoreactivity þ

The 1-methyl-4-phenylpyridinium ion (MPP ) levels were determined according to the HPLC with a UV detection method (Kawasaki et al., 2007, 2008). At 0.5, 1 and 3 h after a single dosing of MPTP (10 mg/kg, s.c.), mice were sacrificed, and the striatum and ventral midbrain were dissected. Tissue samples were homogenized in 0.1 M perchloric acid containing 100 mM EDTA. After centrifugation (15,000g for 15 min at 0  C), 10 ml of the supernatant was injected onto a C18-reverse-phase column (Symmetry C18, 5 mm, 4.6  150 mm: Waters, Milford, MA). The mobile phase [50 mM potassium phosphate and 11% (v/v) acetonitrile] was delivered at a flow rate of 1.0 ml/min. The UV detector was set to 295 nm. Data were calculated by analyzing the peak area of the external standard of MPPþ iodide (Sigma). Values are expressed as ng/mg tissue (wet weight). 2.8. Free radical-scavenging activity The free radical-scavenging activity was determined by the reaction with galvinoxyl radical (Lin et al., 2003). Galvinoxyl radical (5 mM, Sigma) was mixed with SEA0400 and edaravone (Mitsubishi Pharma Co., Osaka, Japan) in ethanol solution. The reaction was carried out at room temperature for 30 min. The concentration of galvinoxyl radical residue at 30 min after the reaction was determined by measuring the absorbance at 428 nm with a Shimadzu UV-1650PC spectrophotometer (Kyoto, Japan). The radical-scavenging activity was expressed as percentage change from the concentration of galvinoxyl radical before the reaction, with 100% defined as the average of three independent experiments.

The number of Iba-1-immunoreactive microglia was determined as a marker of microglial activation. Serial 20-mm-thick coronal sections were cut using a cryostat microtome at 20  C, as described above, and they were washed in PBS-T and then incubated with the antibody against Iba-1 (1:5000) in PBS-T overnight at room temperature. Visualization of the immunostaining was performed as described above. To quantify the number of Iba-1-immunoreactive microglia, samples from a 1.0-mm2 area of the dorsomedial regions of the midstriatum were counted from six independent sections of the right hemisphere, generating an average count for each treated subject. To quantify the microglial activation in the substantia nigra, we used a semi-quantitative scoring system with scores ranging from 1 to 3 to evaluate differences between the treatment groups, as previously reported (Kawasaki et al., 2007, 2008). Briefly, a score of 1 was given when increased immunoreactivity was observed in the absence of morphologically-activated microglia. A score of 2 was given when in addition to the criteria for a score of 1, a number of individually distributed and activated microglia were seen. A score of 3 was given when in addition to the previous mentioned criteria (scores 1 and 2), clusters of activated microglia were observed. A blinded observer performed the scoring. 2.10. Nitrotyrosine levels The levels of total nitrotyrosine were determined by dot-blot analysis (Choi et al., 2005; Liberatore et al., 1999; Wu et al., 2002). Brain tissue was homogenized with 10 mM HEPES containing 137 mM NaCl, 9.6 mM KCl, 1.1 mM KH2PO4, 0.6 mM MgSO4

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Fig. 2. Effects of SEA0400 on MPTP-induced decreases in dopamine levels and tyrosine hydroxylase immunoreactivity. Mice were administered with saline or 10 mg/kg of MPTP s.c. four times at 2-h intervals. Vehicle or SEA0400 (3 or 10 mg/kg) was administered i.p. 30 min before every MPTP dosing. Mice were sacrificed 3 days after the final MPTP dosing. (A) The dopamine contents in the midbrain and striatum were determined by HPLC/ECD methods. Data are expressed as the mean  SEM from 5 to 12 mice. **P < 0.01, compared with the saline/vehicle-treated mice; #P < 0.05, ##P < 0.01, compared with the MPTP/vehicle-treated mice. Representative photomicrographs of tyrosine hydroxylase (TH) staining in the substantia nigra (B) and striatum (C) are shown. Scale bar, 200 mm (B) and 1 mm (C). The number of TH-immunopositive neurons in the substantia nigra was counted and densitometric analysis using NIH Image software package for the TH-positive fibers in the striatum was performed. Data are expressed as the mean  SEM from 5 to 11 mice. **P < 0.01, compared with the saline/vehicle-treated mice; #P < 0.05, ##P < 0.01, compared with the MPTP/vehicle-treated mice.

and a protease inhibitor cocktail (Sigma), and then centrifuged at 17,000g for 15 min. The protein concentration in the supernatant was determined using a BCA protein assay kit (Piece Biotechnology, Inc., Rockford, IL). The samples (100 ng) were loaded onto a nitrocellulose membrane (GE Healthcare UK Ltd., Buckingham, UK). The membrane was blocked with non-fat skim milk for 1 h, and then incubated overnight at 4  C with primary antibodies against nitrotyrosine (1:1000). The membranes were

further incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (1:2000). Specific proteins were detected using Amersham ECL PlusÔ Western Blotting Detection Reagents (GE Healthcare UK Ltd.). In preliminary experiments, the specificity of anti-nitrotyrosine antibody was confirmed by demonstrating that immunostaining was abolished by pre-incubating the membranes with 100 mM sodium hydrosulfite before incubation with the primary antibody.

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2.11. Lipid peroxidation Lipid peroxidation was assessed as a marker of ROS production by determining the concentrations of thiobarbituric acid reactive substances (TBARS) (Kawasaki et al., 2007, 2008). The sample of brain tissue was homogenized with three volumes of KCl [1.15% (w/v)] containing butylated hydroxytoluene (200 mM). An aliquot of the resulting sample was treated with SDS [8% (w/v)] followed by acetic acid (20%), and the mixture was vortexed for 1 min. Thiobarbituric acid (0.8%) was then added, and the resulting mixture was incubated at 95  C for 60 min. After cooling at room temperature, 3 ml of nbutanol was added, and the mixture was shaken vigorously. After centrifugation at 1500g for 5 min, absorbance of the supernatant (organic layer) was measured at 532 nm using a Shimadzu UV-1650PC spectrophotometer (Kyoto, Japan). 2.12. ERK phosphorylation The levels of ERK phosphorylation were determined by Western blot analysis as previously described (Kitao et al., 2010). Briefly, brain tissue was homogenized with lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 1 mg/ml aprotinin, and 5 mg/ml leupeptin). The tissue homogenates were centrifuged at 17,000g for 15 min and the protein concentration in the supernatant was determined using a BCA protein assay kit (Piece Biotechnology, Inc.). The samples were mixed with 0.1% (w/v) bromophenol blue, 3% SDS, 10% glycerol and 5% (v/v) 2-mercaptoethanol, boiled for 5 min, and then loaded (equal amount of protein/lane) on 10% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to polyvinylidenedifluoride membrane (Millipore Corp.). The blotted membranes were blocked with non-fat skim milk for 1 h, and then incubated overnight at 4  C with primary antibodies raised against phospho-ERK (1:1000). The membranes were further incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (1:2000). To quantify the relative amount of proteins, the membranes were stripped at 55  C for 30 min and incubated overnight at 4  C with the primary antibodies raised against total-ERK (1:1000). Protein bands were detected using Amersham ECL PlusÔ Western Blotting Detection Reagents (GE Healthcare UK Ltd.). 2.13. Statistical analysis Data are presented as mean  SEM or median and range. For in vivo microdialysis studies, all data were calculated as percentage change from the dialysate basal concentrations, with 100% defined as the average of three fractions before administration. Analyses were performed using two-way analysis of variance (ANOVA) for treatment as the intersubject factor and repeated measures with time as the intrasubject factor. Other data were analyzed using one- or two-way ANOVA followed by Fisher’s PLSD test or the KruskaleWallis test followed by a MannWhitney U test. Statistical analyses were performed using a software package Statview 5.0J for the Apple Macintosh computer (SAS Institute Inc., Cary, NC). P values of 5% or less were considered statistically significant.

3. Results 3.1. Effects of nNOS inhibitor on MPTP-induced increase in nitrotyrosine levels, decrease in dopamine levels and impairment of motor coordination To confirm that MPTP-induced neurotoxicity is associated with NO, we examined the effects of the nNOS inhibitor, SMTC (Furfine et al., 1994), in MPTP-treated mice. Repeated MPTP administration (10 mg/kg, four times at 2-h intervals) caused a rapid increase in the levels of nitrotyrosine, a marker of NO production, in the midbrain, but not in the striatum (Fig. 1A). One-way ANOVA revealed the significant main effect of the time in the midbrain [F4,25 ¼ 16.412, P < 0.0001], but not in the striatum [F4,30 ¼ 0.360, not significant; n.s.]. Pretreatment with SMTC at doses of 10 mg/kg blocked the increase in nitrotyrosine levels (Fig. 1B). Two-way ANOVA revealed the significant main effects of the treatment with MPTP [F1,12 ¼ 5.456, P ¼ 0.0377] and SMTC [F1,12 ¼ 11.731, P ¼ 0.0050], and the significant interaction between the MPTP and SMTC treatments [F1,12 ¼ 9.643, P ¼ 0.0091]. In agreement with this effect, SMTC protected against MPTP-induced decrease in dopamine levels in the midbrain, but not in the striatum (Fig. 1C). For midbrain, two-way ANOVA revealed the significant main effect of the MPTP treatment [F1,38 ¼ 61.203, P < 0.0001], but not of SMTC treatment [F1,38 ¼ 2.570, n.s.], and the significant interaction between the MPTP and SMTC treatments [F1,38 ¼ 9.237, P ¼ 0.0043]. For striatum, there was a main

Fig. 3. Effects of SEA0400 on motor deficit and methamphetamine-induced striatal dopamine release in MPTP-treated mice. Mice were administered with saline or 10 mg/ kg of MPTP s.c. four times at 2-h intervals. Vehicle or SEA0400 (3 or 10 mg/kg) was administered i.p. 30 min before every MPTP dosing. (A) One day after the final MPTP dosing, motor coordination with the rotarod was assessed. Data are expressed as the mean  SEM from 6 to 12 mice. **P < 0.01, compared with the saline/vehicle-treated mice; ##P < 0.01, compared with the MPTP/vehicle-treated mice. (B) Three days after the final MPTP dosing, striatal dopamine release was assessed by microdialysis. Methamphetamine (1 mg/kg, i.p.) was administered at 0 min (arrow). Data are expressed as the mean  SEM from 3 to 6 mice.

significant effect of the MPTP treatment [F1,21 ¼ 1985.182, P < 0.0001], but not of SMTC treatment [F1,21 ¼ 1.805, n.s.], and no significant interaction between the MPTP and SMTC treatments [F1,21 ¼ 0.633, n.s.]. Moreover, SMTC blocked almost completely MPTP-induced loss of motor coordination (Fig. 1D). Two-way ANOVA revealed the significant main effect of the treatment with MPTP [F1,45 ¼ 6.355, P ¼ 0.0153], but not with SMTC [F1,45 ¼ 2.836, n.s.], and no significant interaction between the MPTP and SMTC treatments [F1,45 ¼ 3.523, n.s.].

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Fig. 4. Effects of SEA0400 on free radical-scavenging activity and MPTP-induced increases in nitrotyrosine and MPPþ levels. (A) The MPPþ contents in the midbrain and striatum were measured at 0.5, 1 and 3 h after a single injection of MPTP (10 mg/kg, s.c.). Vehicle or 10 mg/kg of SEA0400 was administered i.p. 30 min before the MPTP injection. Data are expressed as the mean  SEM from 3 to 4 mice. (B) The free radical-scavenging activity was determined by the reaction with galvinoxyl radical and expressed as percentage change from the concentration before the reaction. Data are expressed as the mean  SEM from 3 to 4 independent experiments. (C) Mice were administered with saline or 10 mg/kg of MPTP s.c. four times at 2-h intervals. Vehicle or 10 mg/kg of SEA0400 was administered i.p. 30 min before every MPTP dosing. Mice were sacrificed 1 h after the final MPTP dosing and the nitrotyrosine levels in the midbrain were determined. Data are expressed as the mean  SEM from 5 mice. *P < 0.05, compared with the saline-treated mice.

3.2. Effects of SEA0400 on MPTP-induced decreases in dopamine levels and tyrosine hydroxylase immunoreactivity Pretreatment with SEA0400 (3, 10 mg/kg) attenuated MPTPinduced reduction in dopamine levels in the midbrain, although SEA0400 alone did not affect the dopamine levels (Fig. 2A). Oneway ANOVA revealed the significant main effect of the treatment [F4,27 ¼ 37.756, P < 0.0001]. Repeated MPTP administration also decreased dopamine levels in the striatum, and SEA0400 (3, 10 mg/ kg) slightly but significantly attenuated MPTP-induced decrease in striatal dopamine levels. One-way ANOVA revealed the significant main effect of the treatment [F4,43 ¼ 229.397, P < 0.0001]. Tyrosine hydroxylase immunoreactivity is a marker of dopaminergic neurons. Fig. 2B and C show representative photomicrographs of tyrosine hydroxylase staining in the substantia nigra and striatum 3 days after the final MPTP dosing, respectively. Repeated MPTP administration caused a marked reduction in the number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra, and this effect was attenuated dose-dependently by pretreatment

with SEA0400 (3, 10 mg/kg). One-way ANOVA revealed the significant main effect of the treatment [F4,29 ¼ 29.247, P < 0.0001]. MPTP treatment also reduced the tyrosine hydroxylase immunostaining in dopaminergic terminals in the striatum, and this effect was slightly but significantly attenuated by pretreatment with SEA0400 (10 mg/kg). One-way ANOVA revealed the significant main effect of the treatment [F4,32 ¼ 39.679, P < 0.0001]. 3.3. Effects of SEA0400 on impairment of motor coordination and methamphetamine-induced striatal dopamine release in MPTP-treated mice SEA0400 (3, 10 mg/kg) improved MPTP-induced loss of motor coordination, although it alone did not affect the rotarod performance (Fig. 3A). One-way ANOVA revealed the significant main effect of the treatment [F4,43 ¼ 6.656, P ¼ 0.0003]. To determine the ability of the residual population of dopamine terminals to increase dopamine release as a function of lesion size (Castañeda et al., 1990; Imai et al., 2007), mice were given a challenge injection of

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Fig. 5. Effects of SEA0400 on MPTP-induced activation of microglia. Mice were administered with saline or 10 mg/kg of MPTP s.c. four times at 2-h intervals. Vehicle or 10 mg/kg of SEA0400 was administered i.p. 30 min before every MPTP dosing. Mice were sacrificed 1 day after the final MPTP dosing. (A) Representative photomicrographs of Iba-1 immunostaining in the substantia nigra and striatum are shown. Scale bar, 200 mm (inset, 20 mm) in the substantia nigra and 100 mm in the striatum. (B) Qualitative results of Iba-1 immunostaining in the substantia nigra are shown. The microglial activation was assessed using microglia score (see in the Materials and Methods). Horizontal lines indicate the median values. Data are expressed as the mean  SEM from 6 mice. **P < 0.01 compared with the saline-treated mice using Mann-Whitney U post hoc test, following KruskaleWallis test [P ¼ 0.0007]. (C) Quantitative results of Iba-1 immunostaining in the striatum are shown. Data are expressed as the mean  SEM from 6 mice. **P < 0.01, compared with the saline-treated mice.

methamphetamine, a dopamine-releasing agent (Fig. 3B). The basal extracellular levels of dopamine in the striatum (not corrected for in vitro probe recovery) were significantly reduced in the MPTPtreated mice [1.67  0.34 pg], compared with the vehicle-treated

mice [4.03  0.55 pg]. Moreover, methamphetamine (1 mg/kg)induced dopamine release was also reduced in the MPTP-treated mice [repeated measures two-way ANOVA (treatment  time interaction): F11,110 ¼ 11.130, P < 0.0001]. Pretreatment with

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SEA0400 (3, 10 mg/kg) significantly improved the basal extracellular levels of dopamine [3.42  0.48 pg for 3 mg/kg; 3.66  0.60 pg for 10 mg/kg] and the response to methamphetamine challenge [repeated measures two-way ANOVA (treatment  time interaction): F11,99 ¼ 6.957, P < 0.0001 for 3 mg/kg; F11,88 ¼ 13.259, P < 0.0001 for 10 mg/kg]. 3.4. Effects of SEA0400 on free radical-scavenging activity, MPTP-induced increases in nitrotyrosine, and MPPþ levels SEA0400 (10 mg/kg) did not affect the MPPþ levels in the midbrain and striatum at 0.5, 1 or 3 h after a single administration of MPTP (10 mg/kg) (Fig. 4A). Two-way ANOVA revealed the significant main effect of the time [F2,18 ¼ 46.046, P < 0.0001 for midbrain; F2,17 ¼ 77.647, P < 0.0001 for striatum], but not of SEA0400 treatment [F1,18 ¼ 0.003, n.s. for midbrain; F1,17 ¼ 0.070, n.s. for striatum], and no significant interaction between the time and SEA0400 treatment [F2,18 ¼ 0.154, n.s. for midbrain; F2,17 ¼ 1.633, n.s. for striatum]. Galvinoxyl is a stable phenoxy radical that exhibits characteristic UV absorption at 428 nm in ethanol solution. This allows easy measurement of the depletion of galvinoxyl radicals in the presence of antioxidants (Lin et al., 2003). When the radical scavenger edaravone, a positive control, was added to the ethanol solution of galvinoxyl, the UV spectrum of galvinoxyl was diminished in a dose-dependent manner (Fig. 4B). The ED50 value of edaravone was 0.76  0.10 mM. Under this condition, SEA0400 up to 100 mM did not show any scavenging activity toward galvinoxyl radicals. Moreover, SEA0400 did not affect MPTP-induced increase in nitrotyrosine levels in the midbrain (Fig. 4C). Two-way ANOVA revealed the significant main effect of the treatment with MPTP [F1,16 ¼ 15.785, P ¼ 0.0011], but not with SEA0400 [F1,16 ¼ 0.007, n.s.], and no significant interaction between the MPTP and SEA0400 treatments [F1,16 ¼ 0.054, n.s.]. 3.5. Effects of SEA0400 on MPTP-induced activation of microglia Microglial activation by MPTP treatment was assessed by staining fixed brain sections with an antibody against Iba-1, a marker of activated microglia (Fig. 5). Representative photomicrographs of Iba-1 immunostaining 1 day after the final MPTP dosing are shown in Fig. 5A. Microglia in the substantia nigra showed changes in cellular morphology, such as enlarged cell bodies and thickening of their processes (Stence et al., 2001), and were evaluated by a semi-quantitative scoring system because they were difficult to count (Fig. 5B). Few morphologically-activated microglia were expressed in the substantia nigra of vehicletreated mice (median, 1; range, 1e2). Repeated MPTP administration induced morphological changes of microglia into macrophagelike round cells and Iba-1 positive microglial cells were mainly observed by forming a cluster in both the medial and lateral portion of the substantia nigra (median, 2.5; range, 2e3). Pretreatment with SEA0400 (10 mg/kg) did not affect the MPTP-induced activation of microglia in the substantia nigra (median, 3; range, 2e3), and it alone did not have any effect (median, 1; range, 1e2). Fig. 5C shows the quantitative results of Iba-1 immunostaining in the striatum. Repeated MPTP administration caused a robust increase in the number of Iba-1-immunoreactive microglia in the striatum. Pretreatment with SEA0400 (10 mg/kg) did not affect MPTPinduced activation of microglia in the striatum. Two-way ANOVA revealed the significant main effect of the treatment with MPTP [F1,20 ¼ 190.568, P < 0.0001], but not with SEA0400 [F1,20 ¼ 1.375, n.s.], and no significant interaction between the MPTP and SEA0400 treatments [F1,20 ¼ 0.012, n.s.].

Fig. 6. Effects of SEA0400 on MPTP-induced ERK phosphorylation and lipid peroxidation in the midbrain. Mice were administered with saline or 10 mg/kg of MPTP s.c. four times at 2-h intervals. Vehicle or 10 mg/kg of SEA0400 was administered i.p. 30 min before every MPTP dosing. Mice were sacrificed 1 day after the final MPTP dosing. (A) The levels of ERK phosphorylation were determined by Western blot analysis. Typical immunoblot images were detected by antibodies against phosphoERK (upper) and total-ERK (middle). Phospho-ERK levels, which were normalized by the respective total-ERK levels, were expressed as % of the control (lower). Data are expressed as the mean  SEM from 6 to 9 mice. **P < 0.01, compared with the salinetreated mice; #P < 0.05, compared with the MPTP-treated mice. (B) Lipid peroxidation was determined by a spectrophotometrical assay of TBARS. Data are expressed as the mean  SEM from 7 to 11 mice. **P < 0.01, compared with saline-treated mice; #P < 0.05, compared with MPTP-treated mice.

3.6. Effects of SEA0400 on MPTP-induced ERK phosphorylation and lipid peroxidation in the midbrain We have previously observed that NO-induced Ca2þ influx via NCX activates the ERK signal, which plays a key role in NO-induced ROS production and cytotoxicity in astrocytes (Kitao et al., 2010) and SH-SY5Y cells (Nashida et al., 2011). Repeated MPTP administration increased the phosphorylation of ERK in the midbrain 1 day

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after the final MPTP dosing, and this effect was blocked by pretreatment with SEA0400 (10 mg/kg) (Fig. 6A). Two-way ANOVA revealed the significant main effect of the treatment with SEA0400 [F1,25 ¼ 7.916, P ¼ 0.0094], but not with MPTP [F1,25 ¼ 1.545, n.s.], and the significant interaction between the MPTP and SEA0400 treatments [F1,25 ¼ 10.531, P ¼ 0.0033]. MPTP increased the level of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation and a marker of oxidative stress status, in the midbrain, and this effect was significantly attenuated by SEA0400 (10 mg/kg) (Fig. 6B). Two-way ANOVA revealed the significant main effect of the treatment with MPTP [F1,34 ¼ 8.803, P ¼ 0.0055], but not with SEA0400 [F1,34 ¼ 2.971, n.s.], and no significant interaction between the MPTP and SEA0400 treatments [F1,34 ¼ 2.944, n.s.]. 4. Discussion The present study addresses whether the plasma membrane NCX is involved in NO-mediated dopaminergic neurodegeneration in vivo. Previous studies show that MPTP neurotoxicity is dependent on NOS activities (Chalimoniuk et al., 2006; Liberatore et al., 1999; Przedborski et al., 1996). In this study, we showed that repeated MPTP administration caused a rapid but transient increase in nitrotyrosine levels in the midbrain, but not the striatum, and the increase was blocked by SMTC, a selective nNOS inhibitor. Furthermore, we observed that SMTC attenuated MPTP-induced impairment of motor coordination and decreases in dopamine levels in the midbrain, but not the striatum. The less effect of the nNOS inhibitor on MPTP-induced striatal dopaminergic neurotoxicity is in agreement with the previous studies (Klivenyi et al., 2000; Matthews et al., 1997). It should be noted that the dopaminergic function in the substantia nigra is responsible for the improvement of MPTP-induced impairment of motor coordination (Crocker et al., 2003; Hayley et al., 2004; Kawasaki et al., 2007, 2008). Taken together, this study first confirms that MPTP-induced neurotoxicity in the midbrain is associated with NO. In this neurotoxicity model, the present study examined the involvement of NCX in neurodegenerative disorders, and we found that the specific NCX inhibitor SEA0400 improved MPTP-induced impairment of motor coordination and attenuated the decreases in dopamine levels and tyrosine hydroxylase immunoreactivity in the substantia nigra (midbrain) and striatum. We have found in the separate experiment using [3H]SEA0400 (999 GBq/mmol) that mouse brain penetration of SEA0400 was excellent: the peak concentration of the radioactivity in the brain was observed at 30 min when it was measured at 10, 30 and 60 min after its i.p. injection, and the maximal concentrations of this inhibitor in the brain were calculated to be about 4 and 15 mM, when it was i.p. injected at 3 and 10 mg/kg, respectively. The excellent brain penetration of SEA0400 was observed in the rats (Matsuda et al., 2001). These findings suggest that i.p administration of SEA0400 may cause almost complete inhibition of brain NCX. Generally, dopaminergic neuronal impairment is assessed by measuring dopamine levels, and immunoreactivity of dopaminergic neuron markers such as tyrosine hydroxylase and dopamine transporters. Besides these strategies, this study challenged to determine in vivo dopamine release that reflects the functional activity of dopaminergic neurons. As expected, the microdialysis study showed that MPTP treatment decreased the striatal dopamine release and this effect was attenuated by SEA0400. In this experiment, the basal release and methamphetamine-induced increase in the release showed similar changes in the responses to MPTP and SEA0400. This neurochemical study supports the fact that SEA0400 protects against MPTP-induced dopaminergic neurotoxicity and impairment of motor coordination. In vivo MPTP-induced neurotoxicity is considered to be composed of three steps, namely, MPPþ production, MPPþ-induced

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signal cascades including NO production, and NO-induced cytotoxicity (Fig. 7). The effect of SEA0400 could not be explained by the pharmacokinetics of MPTP, because the levels of MPPþ after MPTP administration were not altered by the drug. SEA0400 did not have any radical-scavenging activity, unlike edaravone which could protect MPTP-induced dopaminergic neurotoxicity (Kawasaki et al., 2007). In addition, SEA0400 did not affect MPTP-induced increases in Iba-1-immunoreactivity and nitrotyrosine levels. This observation suggests that SEA0400 does not affect MPTP-induced microglial activation, although microglial activation is involved in MPTPinduced dopaminergic neurotoxicity (Du et al., 2001; Gao et al., 2002, 2003; Wu et al., 2002). Moreover, the lack of the effect of SEA0400 on NO production suggests that NCX-mediated Ca2þ influx is not coupled with NOS activation. It is likely that the neuroprotective effect of SEA0400 is due to attenuation of NO-induced cytotoxicity in which NCX-mediated Ca2þ influx is involved (Kitao et al., 2010; Nashida et al., 2011). To support this idea, we examined the effect of SEA0400 on downstream signals of NO production, such as ERK phosphorylation and lipid peroxidation, as a marker of oxidative stress. We have shown that repeated MPTP administration increases the production of TBARS in the midbrain, but not the striatum and this change in the midbrain is closely related to motor dysfunction (Kawasaki et al., 2007, 2008). In this study, we found that SEA0400 inhibits MPTP-induced increases in TBARS production and ERK phosphorylation in the midbrain. Moreover, we have found in the separate experiment that MPTP treatment did not alter the levels of NCX1 (data not shown). These findings suggest that MPTP increases NO production which stimulates the activity of NCX, and the resulting increase in intracellular Ca2þ causes ERK phosphorylation and ROS production (lipid peroxidation), leading to dopaminergic neurotoxicity. It should be

Fig. 7. Proposed mechanism for the involvement of NCX in MPTP-induced dopaminergic neurotoxicity and motor deficit. MPTP increases NO production and microglial activation which stimulates the activity of NCX, and the resulting increase in intracellular Ca2þ causes ERK phosphorylation and ROS production (lipid peroxidation), leading to dopaminergic neurotoxicity and motor deficit. SEA0400 protects against MPTP-induced neurotoxicity by blocking ERK phosphorylation and lipid peroxidation, which are downstream of NCX-mediated Ca2þ influx, without affecting NO production and microglial activation.

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noted that interferon-g secreted by activated microglia also stimulates the activity of NCX (Nagano et al., 2004). Taken together, it is likely that SEA0400 prevents MPTP-induced dopaminergic degeneration and impairment of motor coordination via inhibition of NCX-mediated Ca2þ signal cascades in the substantia nigra (Fig. 7). In conclusion, the present study shows that SEA0400 protects against MPTP-induced neurotoxicity probably by inhibiting NCXmediated Ca2þ influx, leading to neurotoxicity signals such as ERK phosphorylation and lipid peroxidation. NO is emerging as one of the predominant effector of neurodegeneration and is implicated in the pathogenesis of Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and brain ischemia (Calabrese et al., 2009; Love, 1999; Moreno-López et al., 2011). Thus, this finding implies that NCX may be a novel target molecule for treating Parkinson’s disease and other neurodegenerative disorders associated with NO. Conflicts of interest The authors state no conflict of interest. Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and “Molecular Imaging Research Program” of the Ministry of Education, Culture, Sports, Science and Technology of Japan. A part of this work was also supported by grants from the Uehara Memorial Foundation (Japan) and Taisho Pharmaceutical Co. Ltd. (Japan). References Ago, Y., Arikawa, S., Yata, M., Yano, K., Abe, M., Takuma, K., Matsuda, T., 2009. Role of prefrontal dopaminergic neurotransmission in glucocorticoid receptor-mediated modulation of methamphetamine-induced hyperactivity. Synapse 63, 7e14. Ago, Y., Arikawa, S., Yata, M., Yano, K., Abe, M., Takuma, K., Matsuda, T., 2008. Antidepressant-like effects of the glucocorticoid receptor antagonist RU-43044 are associated with changes in prefrontal dopamine in mouse models of depression. Neuropharmacology 55, 1355e1363. Annunziato, L., Pignataro, G., Di Renzo, G.F., 2004. Pharmacology of brain Naþ/Ca2þ exchanger: from molecular biology to therapeutic perspectives. Pharmacol. Rev. 56, 633e654. Aquilano, K., Baldelli, S., Rotilio, G., Ciriolo, M.R., 2008. Role of nitric oxide synthases in Parkinson’s disease: a review on the antioxidant and anti-inflammatory activity of polyphenols. Neurochem. Res. 33, 2416e2426. Asano, S., Matsuda, T., Takuma, K., Kim, H.S., Sato, T., Nishikawa, T., Baba, A., 1995. Nitroprusside and cyclic GMP stimulate NaþeCa2þ exchange activity in neuronal preparations and cultured rat astrocytes. J. Neurochem. 64, 2437e2441. Brorson, J.R., Sulit, R.A., Zhang, H., 1997. Nitric oxide disrupts Ca2þ homeostasis in hippocampal neurons. J. Neurochem. 68, 95e105. Brorson, J.R., Zhang, H., 1997. Disrupted [Ca2þ]i homeostasis contributes to the toxicity of nitric oxide in cultured hippocampal neurons. J. Neurochem. 69, 1882e1889. Calabrese, V., Cornelius, C., Rizzarelli, E., Owen, J.B., Dinkova-Kostova, A.T., Butterfield, D.A., 2009. Nitric oxide in cell survival: a janus molecule. Antioxid. Redox Signal. 11, 2717e2739. Castañeda, E., Whishaw, I.Q., Robinson, T.E., 1990. Changes in striatal dopamine neurotransmission assessed with microdialysis following recovery from a bilateral 6-OHDA lesion: variation as a function of lesion size. J. Neurosci. 10, 1847e1854. Chalimoniuk, M., Lukacova, N., Marsala, J., Langfort, J., 2006. Alterations of the expression and activity of midbrain nitric oxide synthase and soluble guanylyl cyclase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice. Neuroscience 141, 1033e1046. Choi, J.Y., Jang, E.H., Park, C.S., Kang, J.H., 2005. Enhanced susceptibility to 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity in high-fat diet-induced obesity. Free Radic. Biol. Med. 38, 806e816. Crocker, S.J., Smith, P.D., Jackson-Lewis, V., Lamba, W.R., Hayley, S.P., Grimm, E., Callaghan, S.M., Slack, R.S., Melloni, E., Przedborski, S., Robertson, G.S., Anisman, H., Merali, Z., Park, D.S., 2003. Inhibition of calpains prevents neuronal and behavioral deficits in an MPTP mouse model of Parkinson’s disease. J. Neurosci. 23, 4081e4091. Dehmer, T., Lindenau, J., Haid, S., Dichgans, J., Schulz, J.B., 2000. Deficiency of inducible nitric oxide synthase protects against MPTP toxicity in vivo. J. Neurochem. 74, 2213e2216. Du, Y., Ma, Z., Lin, S., Dodel, R.C., Gao, F., Bales, K.R., Triarhou, L.C., Chernet, E., Perry, K.W., Nelson, D.L., Luecke, S., Phebus, L.A., Bymaster, F.P., Paul, S.M., 2001.

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