Fluoxetine prevents MPTP-induced loss of dopaminergic neurons by inhibiting microglial activation

Neuropharmacology 60 (2011) 963e974

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Fluoxetine prevents MPTP-induced loss of dopaminergic neurons by inhibiting microglial activation Young C. Chung a, b, c, 2, Sang R. Kim d,1, 2, Ju-Young Park f, 2, Eun S. Chung b, c, 2, Keun W. Park b, So Y. Won a, b, c, Eugene Bok a, b, c, d, e, Minyoung Jin b, Eun S. Park a, b, c, Sung-Hwa Yoon f, Hyuk W. Ko b, Yoon-Seong Kim g, Byung K. Jin a, b, c, * a

Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea Neurodegeneration Control Research Center, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea Aged-related and Brain Disease Research Center, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea d Neuroscience Graduate Program, Ajou University, Suwon 443-479, Republic of Korea e Division of Cell Transformation and Restoration, School of Medicine, Ajou University, Suwon 443-479, Republic of Korea f Department of Molecular Science and Technology, Ajou University, Suwon 443-479, Republic of Korea g Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32827, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 December 2010 Received in revised form 20 January 2011 Accepted 24 January 2011

Parkinson’s disease (PD) is characterized by degeneration of nigrostriatal dopaminergic (DA) neurons. Mice treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) exhibit microglial activationinduced oxidative stress and inflammation, and nigrostriatal DA neuronal damage, and thus serve as an experimental model of PD. Here, we report that fluoxetine, one of the most commonly prescribed antidepressants, prevents MPTP-induced degeneration of nigrostriatal DA neurons and increases striatal dopamine levels with the partial motor recovery. This was accompanied by inhibiting transient expression of proinflammatory cytokines and inducible nitric oxide synthase; and attenuating microglial NADPH oxidase activation, reactive oxygen species/reactive nitrogen species production, and consequent oxidative damage. Interestingly, fluoxetine was found to protect DA neuronal damage from 1-methyl-4phenyl-pyridinium (MPPþ) neurotoxicity in co-cultures of mesencephalic neurons and microglia but not in neuron-enriched mesencephalic cultures devoid of microglia. The present in vivo and in vitro findings show that fluoxetine may possess anti-inflammatory properties and inhibit glial activation-mediated oxidative stress. Therefore, we carefully propose that neuroprotection of fluoxetine might be associated with its anti-inflammatory properties and could be employed as novel therapeutic agents for PD and other disorders associated with neuroinflammation and microglia-derived oxidative damage. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Parkinson’s disease Fluoxetine Microglial activation Oxidative stress Neuroinflammation

1. Introduction Parkinson’s disease (PD) is characterized by the progressive degeneration of nigrostriatal dopaminergic (DA) neurons and is

Abbreviations: DA neurons, dopaminergic neurons; PD, Parkinson’s disease; SN, substantia nigra; STR, striatum; TH, tyrosine hydroxylase; iNOS, inducible NO synthase; ROS, reactive oxygen species; RNS, reactive nitrogen species; 8-OHdG, 8-hydroxy-20 -deoxyguanosine; MDA, malondialdehyde. * Corresponding author. Aged-related and Brain Disease Research Center, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea. Tel.: þ82 2 969 4563; fax: þ82 2 969 4564. E-mail address: [email protected] (B.K. Jin). 1 Present address: Department of Neurology, BB-307, The College of Physicians and Surgeons, Columbia University, 650 West 168th Street, New York, NY 10032, USA. 2 These authors contributed equally to this work. 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.01.043

associated with major clinical symptoms, including resting tremor, rigidity, and bradykinesia (Dauer and Przedborski, 2003; Savitt et al., 2006). The most prominent biochemical change in PD is a reduction in striatal dopamine levels that may result in a characteristic motor dysfunction. Although the etiology of PD is unknown, accumulating evidence suggests that PD is partly caused by glial activation, which may exert a neurotoxic effect through production of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) that, in turn, mediate oxidative stress (Gao et al., 2003; Liberatore et al., 1999; Wu et al., 2003). In the substantia nigra (SN) of PD patients and MPTP models of PD, key enzymes involved in the production ROS/RNS, such as microglial NAPDH oxidase and inducible nitric oxide synthase (iNOS), and astroglial myeloperoxidase (MPO), are upregulated in damaged areas and contribute to DA neuronal death (Choi et al., 2005a; Liberatore et al., 1999; Wu et al., 2003). In addition, proinflammatory cytokines, such as tumor

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necrosis factor-a (TNF-a) and interleukine-1b (IL-1b), are also elevated and are involved in DA neuronal death in MPTP-treated mice (Zhao et al., 2007). Fluoxetine, the selective serotonin reuptake inhibitor most commonly prescribed as an antidepressant, does not worsen parkinsonian motor symptomatology and is thus a safe and effective drug for treating depression in PD patients (Dell’Agnello et al., 2001). Recent studies revealed that fluoxetine inhibits the expression of the proinflammatory cytokines, TNF-a, IL-1b and cyclooxygenase-2 (COX-2), and suppresses nuclear factor kB (NF-kB) activity in the rat middle cerebral artery occlusion (MCAO) model of cerebral ischemia (Lim et al., 2009) and kainic acid (KA)-treated mouse hippocampus (Jin et al., 2009). However, little is known about the effects of fluoxetine, with respect to microglial activation, in the nigrostriatal DA system. Here, we show that in the MPTP mouse model of PD, fluoxetine prevents the degeneration of nigrostriatal DA neurons in a specific dose by inhibiting microglial activation and ultimately decreasing in ROS/RNS generation and oxidative stress. 2. Materials and methods 2.1. Animal and treatment All experiments were done in accordance with approved animal protocol and guidelines established by Kyung Hee University. Eight-ten week old male C57BL6 mice (20e25 g, Charles River Breeding Laboratory) were used. For MPTP intoxication, mice received four intraperitoneal (i.p.) injection of MPTP (20 mg/kg free base; Sigma) dissolved in saline at 2 h interval. The fluoxetine treatment was done through injection of various doses (2.5 mg/kg body weight/single/day, 5 mg/kg/single/day, 5 mg/kg/twice/day, 10 mg/kg/single/day) into the peritoneum at the indicated time points, the first injection being 12 h after the last MPTP injection. Some mice were injected with fluoxetine alone or vehicle as a control. When calculated with mice body weight, 5 mg/kg/single/day fluoxetine is approximately equivalent to 0.1e0.125 mg/day. 2.2. Measurement of MPTP and MPPþ levels in the striatum Striatal MPPþ level were measured by liquid chromatography electrosprayionisation mass spectrometry (LC/ESI-MASS). The LC/ESI-MASS system was composed of three Agilent model G1311A HPLC quaternary pumps (Palo Alto, CA, USA), a G1313A standard autosampler, and G1316A thermostatted column compartment. Dissected striatal tissues were sonicated and centrifuged at 9000 rpm, for 20 min in chilled 400 ml of 0.1 M perchloric acid, and 100 ml of supernatant was isocratically eluted through a 150 mm  1.5 mm i.d., 4 mm, Zorbax Eclipse XDB-C18 (Palo Alto, CA, USA) maintained at 23  C at flow rate of 0.2 ml min1 for the separation of MPTP and MPPþ. The retention times of MPTP and MPPþ were 6.967 and 7.763 min. An isocratic elution profile consist of 70% buffer A containing 0.1% formic acid in H2O (v/v) and 30% buffer B containing 0.1% formic acid in acetonitrile (v/v). All samples were normalized for protein content, which was determined spectrophotometrically using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). 2.3. Tissue preparation and immunostaining Animals were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/ml) and then fixed with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB). Brains were dissected from the skull, postfixed overnight in buffered 4% paraformaldehyde at 4  C, stored in a 30% sucrose solution at 4  C until they sank, were frozen sectioned on a sliding microtome in 30mm-thick coronal sections. All sections were collected in six separate series and processed for immunostaining as described previously (Kim et al., 2006). In brief, brain sections were rinsed in PBS and then incubated overnight at room temperature with primary antibodies. The following day, brain sections were rinsed with PBS and 0.5% bovine serum albumin (BSA), incubated with the appropriate biotinylated secondary antibody, and processed with an avidinebiotin complex kit (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). The bound antiserum was visualized by incubating with 0.05% diaminobenzidine-HCl (DAB) and 0.003% hydrogen peroxide in 0.1 M PB. The DAB reaction was stopped by rinsing tissues in 0.1 M PB. Labeled tissue sections were then mounted on gelatin-coated slides and analyzed under a bright-field microscope (Nikon, Mellville, NY). The primary antibodies were used anti-Mac-1 (1:200; Serotec, Oxford, UK) for microglia, anti-tyrosine hydroxylase (TH, 1:2000; Pel-Freez Biologicals, Rogers, AR, USA) for dopaminergic neurons, anti-neuronal nuclei (NeuN, 1:400; Chemicon, Temecula, CA), anti-CD68 (ED1, 1:1000; Serotec, Oxford, UK) for phagocytic microglia, anti-8-hydroxy-20 -deoxyguanosine (8-OHdG, 1:200; JaICA, Fukuroi, Shizuoka) for oxidative DNA, antinitrotyrosine (1:100; Abcam, Cambridge, UK). For Nissl staining, some of SN tissues

were mounted on gelatin-coated slide and dried for 1 h at room temperature, stained with 0.5% cresyl violet (SigmaeAldrich). 2.4. Stereological cell counts The unbiased stereological estimation of the total number of the TH-positive cells in the substantia nigra (SN) was made using the optical fractionator (West et al., 1991), as we described in detail (Kim et al., 2005, 2006). The Computer-Assisted Stereological Toolbox system, version 2.1.4 (Olympus, Ballerup, Denmark) equipped with an Olympus BX51 microscope, a motorized microscope stage (Prior Scientific, Rockland, MA) run by an IBM-compatible computer, and a microcator (Heidenhain ND 281B) connected to the stage and feeding the computer with the distance information in the z-axis was used. The borders of the SN at all levels in the rostrocaudal axis were defined. The medial border was defined by a vertical line passing through the medial tip of the cerebral peduncle, by the medial terminal nucleus of the accessory nucleus of the optic tract for excluding the TH-positive cells in the VTA. The ventral border followed the dorsal border of the cerebral peduncle, including the TH-positive cells in the pars reticulata, and the area extended laterally to include the pars lateralis in addition to the pars compacta. The sections used for counting covered the entire SN from the rostral tip of the pars compacta back to the caudal end of the pars reticulate (anterioposterior, 2.06 to 4.16 mm from bregma) (Paxinos and Franklyn, 2001). The SN was delineated at a 1.25 objective and generated counting grid of 150150 mm. An unbiased counting frame of known area (47.87  36.19 mm ¼ 1733 mm2) superimposed on the image was placed randomly on the first counting area and systemically moved through all counting areas until the entire delineated area was sampled. Actual counting was performed using a 100 oil objective. The estimate of the total number of neurons was calculated according to the optical fractionator formula (West et al., 1991). More than total 300 points over all sections of each specimen were analyzed. 2.5. Densitometric analysis As previously described (Ferger et al., 2004), an average of 17 coronal sections of the striatum, starting from the rostral anteroposterior (AP) (þ1.60 mm) to AP (0.00 mm), according to bregma of the brain atlas (Paxinos and Franklyn, 2001), were examined at a 5 magnification using the IMAGE PRO PLUS system (Version 4.0, Media Cybernetics, Silver Spring, Maryland, USA) on a computer attached to a light microscope (Zeiss Axioskop, Oberkochen, Germany), interfaced with a CCD video camera (Kodak Mega Plus model 1.4 I, New York, NY, USA). To determine the density of the TH-immunoreactive staining in the striatum, a square frame of 700  700 mm was placed in the dorsal part of the striatum. A second square frame of 200  200 mm was placed in the region of the corpus callosum to measure background values. To control for variations in background illumination, the average of the background density readings from the corpus callosum was subtracted from the average of density readings of the striatum for each section. Then the average of all sections of each animal was calculated separately before data were statistically processed. 2.6. Rotarod test To determine forelimb and hindlimb motor coordination and balance, we used an accelerating rotarod (UgoBasile, Comerio, Italy) with some modifications (Chung et al., 2010). The rotarod unit consisted of a rotating spindle (diameter, 3 cm) where mice were challenged for speed. To acclimate mice on the rotarod apparatus, animals were given a training session (10 rpm for 20 min), 7 consecutive days before MPTP injection. Animals that stayed on the rod without falling during training were selected and randomly divided into experimental groups. On 7 days from final MPTP injection, mice receiving various treatment regimes were placed on the rotating rod and tested at 20 rpm for 20 min. The latency to fall off the rotarod within this time period was recorded by magnetic trip plates. 2.7. Measurement of dopamine levels in the striatum Levels of dopamine in striatum were measured by reverse-phase high performance liquid chromatography (HPLC) with electrochemical detector as previously described (Ryu et al., 2005). The isolated striata were homogenized and centrifuged at 9000 rpm for 20 min in 400 ml of 0.1 M perchloric acid and 0.1 mM EDTA. The 10 ml of supernatant was injected into an autosampler at 4  C (Waters 717 plus autosampler) and eluted through mBondapak C18 column (3.9  300 mm  10 mm, ESA) with mobile phase for catecholamine analysis (Chromosystems, Munich, Germany). The peaks of dopamine content were analyzed by ESA CoulochemII electrochemical detector and integrated using a commercially available program (Breeze, Waters Corp.). All samples were normalized for protein content as spectrophotometrically determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). 2.8. Immunofluorescence double labeling For double-immunofluorescence staining, tissue sections were processed as described previously (Park et al., 2009). Briefly, free-floating sections were mounted

Y.C. Chung et al. / Neuropharmacology 60 (2011) 963e974 on gelatin-coated slides and dried for 1 h at room temperature. After washing in PBS, sections were incubated in 0.2% TritonX-100 for 30 min and rinsed three times with 0.5% BSA. The sections were incubated in a combination of a goat polyclonal antibody to p47 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a rat monoclonal antibody against MAC-1 (1:200; Serotec, Oxford, UK) overnight at 4  C. After washing in PBS, the sections were incubated simultaneously with a mixture of FITC-conjugated rabbit anti-rat IgG (1:200; Vector Laboratories) and Texas Redconjugated donkey anti-goat IgG (1:200; Molecular probe, Europe, BV) for 1 h at room temperature. Slides were coverslipped with Vectashield medium (Vector Laboratories) and viewed using an IX71 confocal laser scanning microscope (Olympus Optical, Tokyo, Japan). To analyze the localization of different antigens in double-stained samples, images were obtained from the same area and merged using interactive software.  2.9. In situ detection of O 2 and O2 -derived oxidants

Three days after last MPTP injection, hydroethidine (Molecular Probes; 1 mg/ml in PBS containing 1% dimethylsulfoxide) was administered intraperitoneally. After 15 min, the animals were transcardially perfused, postfixed, and the brains were cut into 30 mm. Hydroethidine histochemistry was performed for in situ visualization of  O 2 and O2 -derived oxidants, as previously described (Kim et al., 2007; Wu et al., 2003). The oxidized hydroethidine product, ethidium, was examined by confocal microscopy (Olympus). 2.10. Western blot assay For iNOS and MDA analyses, SN tissues from the animals were dissected at 3 days after injection of MPTP. SN samples were homogenized with ice-cold lysis buffer containing 20 mM TriseHCl, pH 7.5, 1 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma) in a Dounce homogenizer (Wheaton, Millville, NJ, USA). The tissue homogenates were centrifuged at 4  C for 20 min at 14,000  g, and the supernatant was transferred to a fresh tube. The extracts were frozen and kept at 80  C. For NADPH oxidase analyses, brain tissues from the animal were prepared the sample with ProteioExtractÔ Native Membrane Protein Extraction Kit (Calbiochem, La Jola, CA, USA) for separating the cytosolic and membrane fractionation. Equal amounts of protein (30 mg) were mixed with loading buffer (0.125 M TriseHCl, pH 6.8, 20% glycerol, 4% SDS, 10% mercaptoethanol, and 0.002% bromophenol blue), boiled for 5 min, and separated by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidenedifluoride membranes (Millipore, Bedford, MA) using an electrophoretic transfer system (Bio-Rad, Hercules, CA, USA). The membranes were washed with Tris-buffered saline solution (TBS) and then blocked for 1 h in TBS containing 5% skim milk. The membranes were then incubated overnight at 4  C with one of the following the specific primary antibodies: mouse anti-iNOS (1:1000, BD Biosciences, CA, USA), rabbit anti-MDA (1:1000, Cell Biolabs, CA, USA), anti-rac-1 (1:1000, Santa Cruz Biotechnology, CA, USA) and rabbit anti-p47phox (1:500; Santa Cruz Biotechnology, CA, USA). After washing, the membranes were incubated for 1 h at RT with secondary antibodies (1:2000; Amersham Biosciences, Arlington Heights, IL) and washed again. Finally, the blots were developed with enhanced chemiluminescence detection reagents (Amersham Biosciences). The blots were reprobed with antibodies against actin (1:2000; Santa Cruz Biotechnology, CA, USA). To determine the relative degree of membrane purification, the membrane fraction was subjected to immunoblotting for calnexin, a membrane marker, using a rabbit polyclonal antibody against calnexin (1:1000; Stressgen, British Columbia, Canada). For semiquantitative analyses, the densities of bands on immunoblots were measured with the Computer Imaging Device and accompanying software (Fujifilm). 2.11. Detection for protein carbonylation As previously described (Choi et al., 2005c; Park et al., 2009), the extent of protein oxidation was assessed by measuring protein carbonyl levels with an OxyBlot protein oxidation detection kit (Chemicon, Temecula, CA) according to the protocol of the manufacturer. Protein samples were prepared from mice SN tissues harvested 3 days after injection with MPTP in the absence or presence of fluoxetine (5 mg/kg, ip). Subsequently, protein samples (15 mg) were mixed in a microcentrifuge tube with 5 ml of 12% SDS and 10 ml of 1 2,4-dinitrophenylhydrazine (DNPH) solution. Ten microliters of 1 neutralization solution (a kit component) was added instead of the DNPH solution as the negative control. Tubes were incubated at room temperature for 15 min and then mixed with 7.5 ml of neutralization solution. Next, the samples were mixed in equal volumes of SDS sample buffer and separated by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidenedifluoride membranes (Millipore). The membranes were then blocked for 1 h at room temperature in TBS containing 0.1% Tween 20 and 1% BSA. Membranes were incubated overnight at room temperature with the anti-DNPH antibody (1:150) and then incubated at room temperature for 1 h with secondary antibodies (1:300). Blots were developed using enhanced chemiluminescence reagents (Amersham Biosciences). Proteins that underwent oxidative modification (i.e., carbonyl group formation) were identified as a band in the

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samples derivatized with DNPH. The optical density of the bands was measured using the Computer Imaging Device and accompanying software (Fujifilm). Levels of protein carbonyls were quantified and expressed as the fold increase versus untreated controls. 2.12. Real-time PCR Animals treated with or without fluoxetine (5 mg/kg, i.p.) were decapitated 48 h after injection of MPTP, and the bilateral SN regions were immediately isolated. Total RNA was prepared with RNAzol B (Tel-Test, Friendwood, TX, USA) and RT was carried out using the Superscript II reverse transcriptase (Life Technologies, Rockville, MD, USA) according to the manufacturer’s instructions. The primer sequences used in this study were as follows: 50 -CTGCTGGTGGTGACAAGCACATTT-30 (forward) and 50 ATGTCATGAGCAAAGGCGCAGAAC-30 (reverse) for iNOS; 50 -GCGACGTGGAACTGGCAGAAGAG-30 (forward) and 50 -TGAGAGGGAGGCCATTTGGGAAC-30 (reverse) for TNF-a; 50 -GCAACTGTTCCTGAACTCAACT-30 (forward) and 50 -ATCTTTTGGGGTCCGTCAACT-30 (reverse) for IL-1b; and 50 -TCAACAGCAACTCCCACTCTTCCA-30 (forward) and 50 -ACCCTGTTGCTGTAGCCGTATTCA-30 (reverse) for glyceraldehyde-3-phosphate dehydrogenase. Real-time PCRs were performed in a reaction volume of 10 ml including 2 ml 1/50 diluted RT product as a template, 5 ml of SYBR Green PCR master mix (Takara, Japan) and 10 pmol of each primer described above. The PCR amplifications were performed with 50 cycles of denatureation at 95  C for 5 s, annealing at 60  C for 10 s and extension at 72  C for 20 s using Light Cycler (Roche applied science, Indianapolis, USA). The DCT value was determined by subtracting average CT values of glyceraldehyde-3-phosphate dehydrogenase from average CT values of IL1b, TNF-a and iNOS from PCR reactions. To express the relative amount of IL-1b, TNFa and iNOS, DDCT value was calculated by subtracting DCT value of control group from DCT value of each group. The ratios of expression levels of IL-1b, TNF-a and iNOS were calculated as 2ðmeanDDCT Þ . 2.13. Measurement of proinflammatory cytokines At 3 days from final MPTP treatment, mice treated with or without fluoxetine (5 mg/kg, ip) were sacrificed and the SN tissues were isolated. The amount of IL-1b and TNF-a from SN was measured with sandwich ELISA techniques. Tissues were homogenized in 200 ml of ice-cold RIPA buffer (60 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholic acid, and 50 mM Tris, pH 8.0) and centrifuged at 14,000  g at 4  C for 20 min. Equal amounts of protein (100 mg) from each sample were placed in ELISA kit strips coated with the appropriate antibody. Sandwich ELISA was then performed according to the manufacturer’s instructions (BioSource, Camarillo, CA, USA). The detection limit of IL-1b and TNF-a were 5 pg/ml and 25 pg/ml. 2.14. Neuron-enriched mesencephalic cultures and drug treatment SD rat ventral mesencephalon were isolated from embryonic day 14 (E14) fetal brain and dissected as described previously (Kim et al., 2005). Tissues were cut into small segments and incubated in Ca2þ-, Mg2þ-free Hanks’ balanced salt solution (CMF-HBSS) for 10 min at 37  C. Cultures were replaced with a 0.01% trypsin solution in CMF-HBSS, incubated for an additional 9 min, rinsed twice in RF [Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Rockville, MD, USA) supplemented with 10% fetal bovine serum, 6 mg/ml glucose, 204 mg/ml L-glutamine, 100 U/ml penicillin/streptomycin (P/S)] and mechanically triturated. Dissociated cells were plated on 12-mm round aclar plastic coverslips pre-coated with 0.1 mg/ml poly-D-lysine and 4 mg/ml laminin and housed in 24-well culture plates at a density of 1.0  105 cells/coverslip. Cells were incubated in a humidified incubator at 37  C, 5% CO2 for 24 h. The media of two day-old in vitro cultures (DIV 2) incubated in the absence of serum were replaced with chemically defined serum-free medium (DM) composed of Ham’s nutrient mixture (F12-DMEM) and supplemented with 1% ITS (insulin, transferrin, selenium), glucose, L-glutamine and P/S. At DIV 4, cultures were transferred to DM without ITS, treated with MPPþ for 48 h and processed for further studies. 2.15. Mesencephalic microglia cultures Mesencephalic microglia cultures were prepared from the ventral mesencephalons of E14 SD rat brain as previously described with some modifications (Kim et al., 2000). Tissues were triturated and plated in 75-cm2 T-flasks pre-coated with poly-Dlysine at a density of 1  107 cells/flask, and then maintained in DMEM supplemented with 10% FBS. After 2e3 weeks, microglia were detached from the flasks, applied to a nylon mesh to remove astrocytes and then plated into neuron-enriched mesencephalic cultures at a density of 5  104 cells/well. 2.16. Statistical analysis All values are expressed as mean  SEM. Statistical significance (P < 0.05 for all analyses) was assessed by ANOVA using Instat 3.05 (GraphPad, San Diego, CA), followed by StudenteNewmaneKeuls analyses.

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MPTP only (P < 0.001; Fig. 1N). Fluoxetine alone had no effects on motor behavior or striatal dopamine level.

3. Results 3.1. Neuroprotective effect of fluoxetine in MPTP-treated mice We first measured striatal MPTP and MPPþ levels at the indicated time points after the final injection of MPTP by liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MASS; Table 1). MPTP was rapidly eliminated and almost undetectable at 6 h. Striatal MPPþ levels peaked within 30 min (14.89  2.4 mg/mg protein) and then gradually declined, becoming almost negligible after 12 h (0.19  0.1 mg/mg protein). We then evaluated the effects of fluoxetine on MPTP-induced neurotoxicity. Mice administered MPTP (20 mg/kg body weight) and received a single daily intraperitoneal injection of fluoxetine for 6 days commencing 12 h after the final MPTP injection. On day 7, brains were removed and sections were immunostained with a tyrosine hydroxylase (TH) antibody to detect DA neurons (Fig. 1AeI). Compared to the PBS-treated control (Fig. 1AeC), MPTP treatment induced damage in both the SN (Fig. 1D and E) and striatum (Fig. 1F). Treatment with fluoxetine significantly attenuated the loss of DA neurons in the SN (Fig. 1GeH) and DA nerve terminals in the striatum (Fig. 1I). Quantification of nigral THpositive neurons by stereological count revealed that fluoxetine increased the percentage of TH-positive neurons in the SN of MPTPtreated mice by 28% compared to mice administered MPTP only (P < 0.001), and induced 39% increase in the optical density of THpositive fibers (P < 0.001; Fig. 1J). Fluoxetine alone had no effects on the loss of DA neurons in the SN or their fibers in the striatum (Fig. 1M). To verify neuroprotective effects of fluoxetine on general nigral neurons, SN tissues were immunostaining with NeuN antibody. PBS-treated SN (Fig. 1J) had NeuN positive neurons when compared with MPTP-treated SN (Fig. 1K), showing marked loss of NeuN positive neurons. Consistent with upper results, fluoxetine was found to increase the NeuN positive neurons in the SN (Fig. 1L). 3.2. Recovery of behavioral dysfunction and improvement of striatal dopamine levels by fluoxetine We next determined the effects of fluoxetine on MPTP-induced motor deficits by testing rotarod performance (Chung et al., 2010). Seven days after the final MPTP injection, rotarod performance results showed that fluoxetine significantly improved MPTP-induced behavioral dysfunction, increasing the average latency to fall from 12.2  0.6 min recorded in animals treated with MPTP only to 16.4  0.5 min in MPTP-treated animals post-treated with fluoxetine (P < 0.001; Fig. 1N). After the rotarod performance test, mice were sacrificed and striatal tissues were prepared for biochemical assessments. Consistent with rotarod performance results, HPLC analyses showed that MPTP treatment depleted dopamine levels by 37% in the striatum (P < 0.001 compared with PBS-treated controls) and fluoxetine attenuated this depletion, increasing dopamine levels in the striatum by 47% compared to those in animals treated with

Table 1 MPTP and MPPþ levels (mg/mg protein) in the striata of C57 BL/6 mice.

MPTP MPPþ

Control (0 h)

30 min

2h

6h

12 h

0 0

2.57  0.4 14.89  2.4

2.28  0.5 5.80  1.2

0 2.91  0.7

0 0.19  0.1

Mice received four intraperitoneal injections of MPTP (20 mg/kg body weight) at 2 h intervals. Striatal tissues were removed at various time points after the final MPTP injection, and MPTP and MPPþ levels measured by Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MASS).

3.3. Blockade of microglial activation and ROS production by fluoxetine Recent studies additionally report the presence of reactive microglia in MPTP-treated SN exhibiting nigral DA neuronal degeneration (Block et al., 2007; Wu et al., 2003). Accordingly, we investigated whether neuroprotection by fluoxetine is due to inhibition of MPTP-induced microglial activation in the SN in vivo. Three days after the final MPTP treatment, with or without fluoxetine, brain tissues were processed for immunostaining using an antibody against Mac-1 to detect microglial activation. Consistent with earlier reports (Wu et al., 2003), numerous Mac-1-positive (activated) microglia were observed in MPTP-treated SN (Fig. 2D), whereas such cells were largely absent in the PBS-treated control SN (Fig. 2A). Fluoxetine treatment mitigated these effects of MPTP, dramatically decreasing the number of Mac-1-positive cells in the MPTP-treated SN (Fig. 2G). In idiopathic PD patients, a number of ED-1-positive cells, identical to phagocytic microglia were observed in the SN (Croisier et al., 2005). Similarly, ED-1-positive cells are much more numerous in the SN of MPTP-treated mice (Fig. 2E) than in the SN of PBS-treated control mice (Fig. 2B). Fluoxetine treatment almost completely eliminated ED-1-positive cells in the MPTP-treated SN (Fig. 2H). Fluoxetine alone had no effects on microglial activation or phagocytosis (data not shown). In PD patients and MPTP-treated mice, activated microglia  produce O 2 and O2 -derived oxidants that contribute to the degeneration of DA neurons in the SN (Gao et al., 2003; Hald and Lotharius, 2005; Wu et al., 2003). Accordingly, we examined whether fluoxetine exerts its neuroprotective action by inhibiting oxidant production. MPTP-induced oxidant production was visualized in situ by hydroethidine histochemistry as previously described (Kim et al., 2007; Wu et al., 2003) using sections adjacent to those used for Mac-1 and ED-1 immunostaining (Fig. 2C, F and I). Compared with PBS-treated control SN (Fig. 2C), there was a significant increase in the fluorescent products of oxidized hydroethidine (i.e., ethidium accumulation) in the SN of MPTPtreated mice 3 days after the final MPTP injection (Fig. 2F). Fluoxetine dramatically attenuated the MPTP-induced increase in oxidant levels in the SN (Fig. 2I), but had no effect alone (data not shown). 3.4. Ablation of MPTP toxicity by fluoxetine via inhibition of NADPH oxidase NADPH oxidase is composed of cytosolic subunits, including p47phox and Rac 1, and membrane subunit gp91phox (Cross and Segal, 2004; Miller et al., 2009). Upon activation, NADPH oxidase subunits translocate from the cytosol to the plasma membrane (Cross and Segal, 2004; Miller et al., 2009), stimulating enzymatic activity and triggering ROS production in microglia (Choi et al., 2005c; Gao et al., 2003; Wu et al., 2003). The resulting O 2 and O 2 -derived oxidants eventually lead to neurodegeneration, as observed in the MPTP mouse model of PD (Wu et al., 2003). In our experiments, SN tissue samples were separated into membrane and cytosolic components, and examined by Western blotting. Three days after the final MPTP injection, the levels of cytosolic NADPH oxidase subunits (p47phox and Rac 1) were significantly increased in the membrane fraction (Fig. 3AeC; P < 0.01), indicating translocation and activation of the complex. In contrast, MPTP-induced translocation of p47phox (Fig. 3A and C; P < 0.05) and Rac 1 (Fig. 3B and C; P < 0.05) were dramatically decreased in the

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Fig. 1. Fluoxetine attenuates MPTP-induced neurotoxicity in the nigrostriatal pathway of mouse brains. At 7 days after last MPTP injection, animals were sacrificed and brains were prepared for immunohistochemistry. Tyrosine hydroxylase (TH) immunostaining in the substantia nigra (SN) (A,B,D,E,G,H) and striatum (C,F,I) treated with PBS as a control (AeC), MPTP and vehicle (DeF) or MPTP and fluoxetine (GeI). SN tissues immunostained with TH were counterstained with cresyl violet (A,B,D,E,G,H). Note absence of TH expression in some Nissl-stained neurons, indicated by arrow. (B,E,H), Higher magnification of (A), (D) and (G), respectively. SN tissues obtained from the same animals as used in A,D,G were prepared for neuronal nuclei (NeuN) histochemistry to detect neurons. PBS as controls (J), MPTP (K) and MPTP and fluoxetine (L). Insets show higher magnifications of JeL, respectively. (M) Number of TH-positive neurons in the SN (black bars) and optical density of TH-ip fibers in the striatum (white bars) after treatment with MPTP in the absence or presence of fluoxetine. Data are presented as means  SEM of eight to nine animals per group. #P < 0.001, significantly different from control; ##P < 0.001, significantly different from MPTP only. C, PBS-treated control; F, fluoxetine; M, MPTP; M þ F, MPTP and fluoxetine. SNpc, substantia nigra pars compacta; VTA, ventral tegmental area; Scale bars: A,C,D,F,G,I, 300 mm; B,E,H, 50 mm. (N) Rotarod performance (black bars) and dopamine levels in the striatum (white bars) at 7 days post-MPTP lesion. Data are presented as means  SEM of eight to ten animals per group. C, PBStreated control; F, fluoxetine; M, MPTP; M þ F, MPTP and fluoxetine. #P < 0.001, significantly different from control or fluoxetine. ##P < 0.001, significantly different from MPTP only.

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Fig. 2. Fluoxetine inhibits microglial activation and O 2 production in the MPTP-treated SN in vivo. (A,D,G) Mac-1 immunostaining in mouse SN treated with PBS (A), MPTP (D), or MPTP and fluoxetine (G). (B,E,H) ED-1 immunostaining in SN tissue treated with PBS (B), MPTP (E), or MPTP and fluoxetine (H). The inset represents higher magnification. (C,F,I) In  situ visualization of MPTP-induced O 2 and O2 -derived oxidant production in mouse SN treated with PBS (C), MPTP (F), or MPTP and fluoxetine (I). The dotted lines indicate the SNpc, which displays degeneration of dopaminergic neurons after MPTP treatment. Data are representative of 6e8 animals per group. Scale bars: AeI, 500 mm.

SN of mice treated with fluoxetine. Co-localization of the p47phox subunit in activated (Mac-1-positive) microglia was confirmed by double-immunofluorescence staining (Fig. 3D).

significantly attenuated by fluoxetine (P < 0.01; Fig. 4G). Fluoxetine alone had no effect.

3.5. Effects of fluoxetine on the MPTP-induced oxidative damages

3.6. Effects of fluoxetine on the MPTP-induced expression of proinflammatory cytokines and iNOS

The levels of 8-hydroxy-2-deoxy guanosine (8-OHdG), a measure of oxidative nucleic acid damage, are increased in the cerebrospinal fluid of PD patients and in the striatum of MPTPtreated mice (Kikuchi et al., 2002; Oyagi et al., 2008). Thus, we examined the effects of fluoxetine on MPTP-induced oxidative damage in nucleic acids by immunostaining with an anti-8-OHdG antibody. Substantial increases of 8-OHdG content were evident in the SN 3 days after the final MPTP injection (Fig. 4B) compared with the SN of PBS-treated controls (Fig. 4A). Remarkably, this MPTPinduced increase in 8-OHdG levels was completely abrogated in the SN of animals treated with fluoxetine (Fig. 4C). Fluoxetine alone had no effect (data not shown). There is a significant elevation of oxidative protein damage in PD patients (Floor and Wetzel, 1998) and in MPTP model of PD (Wu et al., 2003). To examine the effects of fluoxetine on MPTPinduced oxidative damage to proteins, we assessed protein carbonyl levels in the SN by Western blotting (Fig. 4D and E). MPTP alone significantly increased the levels of protein carbonyls in the SN 3 days after the final MPTP injection (P < 0.01 compared to PBStreated controls). This increase was significantly reduced by treatment with fluoxetine (P < 0.01; Fig. 4E), which had no effects alone. The levels of MDA, a marker for lipid oxidation products, are elevated in PD patients and in the MPTP model of PD (Han and Zhao, 2010; Kikuchi et al., 2002). To assess the effects of fluoxetine on MPTP-induced lipid peroxidation in the SN, we analyzed SN samples for the presence of MDA by Western blotting. Consistent with observed changes in 8-OHdG and protein carbonyls, MDA levels were significantly increased in the SN of MPTP-treated mice (P < 0.01 compared to PBS-treated controls), an increase that was

It has been shown that transgenic mice expressing a dominantnegative inhibitor of IL-1b converting enzyme, or those deficient in TNF-a or iNOS are resistant to MPTP-induced neurotoxicity (Klevenyi et al., 1999; Liberatore et al., 1999; Sriram et al., 2002). Thus, we investigated whether MPTP-induced expression of IL-1b, TNF-a and/ or iNOS in the SN were affected by fluoxetine. Two days after the final MPTP injection, SN tissues were dissected and prepared for real-time PCR analysis. The results of real-time PCR showed that fluoxetine attenuated MPTP-induced expression of IL-1b, TNF-a and iNOS mRNA in the SN, reducing IL-1b by 45% (P < 0.05), TNF-a by 58% (P < 0.01) and iNOS by 78% (P < 0.01; Fig. 5A). To confirm that these changes at the mRNA level are reflected in changes at the protein level, we analyzed tissue lysates by ELISA and Western blotting 3 days after the final MPTP injection (Fig. 5BeD). Similar to real-time PCR results, ELISAs and Western blot analyses showed that the levels of IL-1b, TNF-a and iNOS protein were significantly increased in the SN of MPTP-treated mice compared with the SN of PBS-treated mice (Fig. 5BeD). Treatment with fluoxetine inhibited these MPTP-induced increases, reducing expression of IL-1b by 39% (P < 0.01; Fig. 4B), TNF-a by 29% (P < 0.01; Fig. 4B) and iNOS by 53% (P < 0.01; Fig. 4C and D) in the SN. Fluoxetine alone had no effects on the SN levels of IL-1b, TNFa or iNOS. Nitration of protein tyrosine residues, a well-known marker for oxidative stress in PD patients (Dawson and Dawson, 2003), is mediated by iNOS-derived nitric oxide (Liang et al., 2007). To measure the extent of oxidative damage to proteins by iNOS, we performed immunostaining with nitrotyrosine in the SN after MPTP injection, with or without fluoxetine (Fig. 5EeG). Our results

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Fig. 3. Fluoxetine inhibits NADPH oxidase activation in the MPTP-treated SN in vivo. (AeD) At 3 days after the final MPTP injection, translocation of NADPH oxidase subunits [(A) 47phox, (B) Rac 1] was detected by Western blotting in the SN. Data are representative of five to seven animals per group. (C) The histogram represents quantitation of p47phox and Rac 1 levels as a ratio of the membrane fraction to total, respectively. Data are representative as means  SEM of five to seven animals per group. *P < 0.01, significantly different from control; #P < 0.05 and &P < 0.01, significantly different from MPTP only. (D) Co-localization of p47phox (red) within Mac-1-positive activated microglia (green) in SN treated with MPTP. Two images are merged (yellow).

revealed that the levels of nitrotyrosine were dramatically increased in the SN 3 days after MPTP injection (Fig. 5F) compared with the PBS-treated SN (Fig. 5E). This MPTP-induced nitration of protein was dramatically inhibited by fluoxetine (Fig. 5G), which had no effect alone (data not shown). 3.7. Effectiveness of fluoxetine against microglia-derived neurotoxicity Our results showed that the in vivo neuroprotective effects of fluoxetine are not attributable to reduced metabolism of MPTP to MPPþ or MPPþ uptake into DA neurons (Table 1). However, the possibility remained that fluoxetine might promote neuronal survival by preventing MPPþ-induced blockade of mitochondrial respiration in neurons. To test this hypothesis, we performed additional experiments with mesencephalic neurons cultured alone or cocultured with microglia. In microglia-free, neuron-enriched mesencephalic cultures, pre-treatment with 0.5e1.0 mM fluoxetine (30 min or 2 h before MPPþ treatment) had no protective effect, and 10 mM fluoxetine alone was neurotoxic (Fig. 6AeC and G). By contrast, in co-cultures of mesencephalic neurons and microglia, fluoxetine (0.5e1.0 mM) blocked MPPþ-induced death of DA neurons (Fig. 6DeG). 4. Discussion The conversion of MPTP to MPPþ, mediated by MAO-B in brain astrocytes, is the key process leading to MPTP-induced neurotoxicity

(Przedborski et al., 2000). In keeping with this observation, striatal MPPþ content correlates linearly with MPTP toxicity (Giovanni et al., 1991). Theoretically, fluoxetine should have prevented neurotoxicity by blocking MPPþ formation due to its MAO-B inhibition function (Brooks et al.,1989). However, it was administered 12 h after the final injection of MPTP which would have been long after the complete conversion of MPTP to MPPþ (6 h) and the MPPþ levels in the DA neurons had peaked. This means that our experimental conditions did not allow fluoxetine to take its neuroprotective role in reducing the metabolism of MPTP to MPPþ nor prevent MPPþ uptake into DA neurons. Microglia, resident immunocompetent and phagocytic cells in the central nervous system (CNS), play a critical role in innate defense (Kim and de Vellis, 2005). It has been generally believed that these intrinsic immune cells serve neuron-protective and -supportive functions in normal CNS. Under neuropathological conditions, microglia are rapidly activated in response to neuronal damages (Block and Hong, 2005) and produce various potentially neurotoxic compounds, including ROS/RNS and/or proinflammatory cytokines (Block et al., 2007). Numerous studies have provided evidence for oxidative stress in PD patients and in MPTP-treated mice, including high levels of oxidative nucleic acid damage (Oyagi et al., 2008; Zhang et al., 1999), protein oxidation (Alam et al., 1997; Wu et al., 2003) and lipid peroxidation (Dexter et al., 1989; Han and Zhao, 2010). One such pathogenic condition is PD, where oxidative damage may account for the degeneration of DA neurons in the SN of PD brain

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Fig. 4. Fluoxetine prevents oxidative damages on nucleic acids, proteins and lipids in MPTP-treated SN in vivo. (AeC) At 3 days after the final MPTP injection, 8-OHdG immunostaining and then performed Nissl staining in mouse SN treated with PBS (A), MPTP (B), or MPTP and fluoxetine (C). The inset represents higher magnification. The dotted lines indicate the SNpc. Data are representative of six to eight animals per group. Scale bars: 250 mm. (D,E) The levels of protein carbonyls were analyzed by Western blotting. (E) Bars represent the means  SEM of four to five samples. #P < 0.01 compared with control, ##P < 0.01 compared with MPTP only. (F,G) Samples used in (D) were analyzed by Western blotting for MDA as markers of oxidatively modified lipids. (F) Bars represent the means  SEM of four to five samples. #P < 0.01 compared with control, &P < 0.01 compared with MPTP only.

(Beal, 2002; Fahn and Cohen, 1992) and in MPTP-treated mice (Miller et al., 2009). The ROS responsible for these molecular modifications can be generated by microglial NADPH oxidase and play an important role in development of oxidative stress in the MPTP model of PD (Block et al., 2007; Hirsch and Hunot, 2009). Several studies, including our work, have implicated activation of microglial NADPH oxidase in degeneration of DA neurons, both in the SN of MPTP-treated mice (Gao et al., 2003; Wu et al., 2003) and in thrombin-treated SN (Choi et al., 2005b). Fluoxetine was found to inhibit microglial NADPH oxidase activation and reduce ROS production and oxidation of nucleic acid, protein, and lipids. These data support the hypothesis that the observed neuroprotective effects of fluoxetine are associated with an ability of the drug to inhibit microglial NADPH oxidase-derived ROS production and oxidative damage to DA neurons. RNS, another major contributor to oxidative stress, are implicated in the pathogenesis and progression of PD (Aquilano et al., 2008). Moreover, NO, which is synthesized through a reaction catalyzed by iNOS, reacts with O 2 to form peroxynitrite which subsequently causes oxidative damage to proteins by modifying tyrosine residues (Kavya et al., 2006). Several studies have demonstrated that reactive glia expressing iNOS and/or increased

levels of nitrotyrosine (a marker for RNS) are present in the midbrains of PD patients (Giasson et al., 2000; Hunot et al., 1996). In the MPTP mice model of PD, iNOS contributes to DA neuronal death through MPTP-induced RNS and increases in nitrotyrosine levels (Liberatore et al., 1999), although the existence of iNOS in human microglia remains controversial (Heneka et al., 2001). Treatment with fluoxetine not only reduced iNOS expression, but also decreased the levels of nitrotyrosine in the SN of MPTP-treated mice. Collectively, these results suggest that fluoxetine decreases MPTP-induced NO production and expression of iNOS in activated microglia, reducing oxidative damage and leading to increased neuronal survival. In addition to ROS/RNS, microglia-derived proinflammatory cytokines may be involved in nigrostriatal DA neuronal death. Several lines of evidence point to the presence of activated glial cells expressing the proinflammatory cytokines IL-1b and TNF-a in the SN of PD patients (Nagatsu et al., 2000) and MPTP-treated mice (Moon et al., 2009; Zhao et al., 2007). TNF-a and IL-1b, originating from activated glia, may trigger intracellular death-related signaling pathways or participate in the induction of iNOS expression in the MPTP model (Teismann et al., 2003). In the present study, both real-time PCR and sandwich ELISA assays

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Fig. 5. Fluoxetine attenuates the expression of proinflammatory cytokines and iNOS in the MPTP-treated SN in vivo. (A) At 2 days after the final MPTP injection, transcriptional levels of proinflammatory cytokines and iNOS in the SN were determined using real-time PCR. The results represent the means  SEM of four to five separate experiments. # P < 0.01, significantly different from control; &P < 0.05, $P < 0.01 significantly different from MPTP only. (B) At 3 days after the final MPTP injection, the amounts of IL-1b and TNFa were measured using the sandwich ELISA technique. Each bar represents the mean  SEM of four to five animals per group; #P < 0.01, significantly different from control; & P < 0.01, significantly different from MPTP only. (C,D) Western blot analysis of iNOS expression. (D) The histogram shows quantitation of iNOS, respectively. The results represent the means  SEM of four to five separate experiments. #P < 0.01, significantly different from control; &P < 0.01, significantly different from MPTP only. (E,G) The SN tissues obtained from the same animals as used in Fig. 4A were prepared for nitrotyrosine histochemistry to detect oxidative damage to proteins by modification of tyrosine residues and then counterstained with cresyl violet in the SN. PBS as a control (E), MPTP (F), or MPTP and fluoxetine (G). Dotted lines indicate SNpc. Scale bars: EeG, 300 mm.

showed that MPTP induced an increase in the expression of IL-1b and TNF-a in the SN. These increases were attenuated by fluoxetine suggesting that the anti-inflammatory actions of fluoxetine contribute to its neuroprotective effects. This is in line with our recent studies showing that fluoxetine inhibits the expression of TNF-a and IL-1b in cultured microglia treated with lipopolysaccharide (Lim et al., 2009) and in hippocampus treated with kainate (Jin et al., 2009). The most prominent biochemical changes in the striatum of PD patients and MPTP-treated mice are decreased levels of dopamine (Jackson-Lewis and Przedborski, 2007; Savitt et al., 2006). Such deficits in striatal dopamine in MPTP-treated mice led to a decreased latency to fall on an accelerating rotarod apparatus, reflecting diminished coordination and balance (Moon et al., 2009). Fluoxetine was found to increase striatal dopamine levels and ameliorate motor deficits in MPTP-treated mice. These behavioral and in vivo biochemical effects of fluoxetine on the lesioned nigrostriatal DA system together with the present finding that fluoxetine inhibits microglial activation-mediated oxidative stress suggest that fluoxetine and its analogs may be useful pharmacological tools for treating PD and other disorders associated with neuroinflammation and microglia-derived oxidative damage. It is worthy to note whether neuroprotection afforded by fluoxetine results in the partial restoration or prevention of further

decrease of DA neurons in nigrostriatal pathway. Our TH immunostaining revealed that MPTP reduced optical density of striatal TH-positive fibers by 15% after 12 h from last MPTP injection, indicating that majority of striatal TH-positive fibers seem to be alive (data not shown). Therefore, it is likely that the observed neuroprotective effects may be due to the prevention of further decrease of nigrostriatal DA neurons. Notably, we found that fluoxetine failed to protect DA neurons from MPPþ neurotoxicity in cultures enriched for mesencephalic neurons, as assessed by TH immunostaining. In contrast, the neuroprotective actions of fluoxetine were clearly evident in cocultures of mesencephalic microglia and neurons, providing further evidence that fluoxetine acts through microglia to mediate its neuroprotective effects and confirming that fluoxetine does not act by blocking MPPþ entry into DA neurons. Additionally, fluoxetine was unable to mitigate the neurotoxicity of 6-OHDA or rotenone, other mitochondrial inhibitors of DA neurons (Bolin et al., 2002; Bove et al., 2005), in neuron-enriched mesencephalic cultures devoid of microglia (data not shown). Although not tested directly, these results collectively provide indirect evidence that the neuroprotective effects of fluoxetine are unrelated to the prevention of MPPþ-induced inhibition of mitochondrial activity. To verify the effects of fluoxetine on serotonin system under our experimental conditions, we measured the striatal serotonin level

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Fig. 6. Effects of fluoxetine on MPPþ-induced neurotoxicity in mesencephalic cultures. AeC, TH-positive neurons in neuron-enriched mesencephalic cultures treated with vehicle as a control (A), and 20 mM MPPþ for 24 h in the absence (B) or presence (C) of fluoxetine (0.5 mM) pre-treatment for 30 min. Note that following either MPPþ or MPPþ þ fluoxetine, several of the remaining TH-positive neurons displayed short processes (white arrow) and rounded and shrunken cell bodies (black arrows), compared to the vehicle-treated control. DeF, TH-positive neurons in co-cultures of mesencephalic neurons and microglia treated with vehicle as a control (D), and 20 mM MPPþ for 24 h in the absence (E) or presence (F) of fluoxetine (0.5 mM) pre-treatment for 30 min. Similar to those observed in neuron-enriched cultures (AeC), following MPPþ treatment, many of the remaining THpositive neurons had short processes (white arrow) and rounded and shrunken cell bodies (black arrows), compared to vehicle-treated control. By contrast, following fluoxetine treatment, TH-positive neurons had long and branched neuritic processes. Scale bar: AeF, 25 mM. G, TH-positive neurons were counted. All values are expressed as means  SEM of triplicate cultures from three separate plates. *P < 0.001, compared with each control values. #P < 0.01, compared with MPPþ only-treated co-cultures. ##P < 0.05, compared with MPPþ only-treated co-cultures. F, fluoxetine; MPPþ, 1-methyl-4-phenyl-pyridinium.

with LC-MASS. As a result, fluoxetine did not increase striatal serotonin level (25.4  2.1 mg/mg protein, n ¼ 6) in mice treated with fluoxetine (5 mg/kg body weight, equivalent to w0.1e0.125 mg/day) for 6 days, compared with PBS-treated STR (23.8  1.2 mg/mg protein, n ¼ 4). We also found that fluoxetine improved the motor behavior without affecting striatal serotonin level in MPTP-treated mice (MPTP: 14.6  2.5 mg/mg protein, n ¼ 6; MPTP þ Fluoxetine: 15.3  1.3 mg/mg protein, n ¼ 6). Additionally, fluoxetine-induced behavioral abnormality and weight loss was not detected under our experimental conditions. These results indicate that fluoxetine affects neither serotonin level nor behavioral changes. This observation is line with recent report that fluoxetine (8 mg/kg/body

weight/day over 4 week) neither causes behavior abnormality or elevates serotonin level in prefrontal cortex and hippocampus of adolescent mice (3e7 weeks of age) (Norcross et al., 2008). However, Ansorge and colleagues showed that transient exposure to fluoxetine or other antidepressants from postnatal 4 days (P4) to P21 produced anxiety- or depression-related behavioral response in adult mice through blockade of serotonin transporter (Ansorge et al., 2008). The apparent discrepancy between these studies may be explained by differences in the animal ages, route of drug delivery and dose. The clinical reports relating to the effects of fluoxetine discovered so far concern both alleviating depression and the inconsistent

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capability to aggravate and/or to mitigate PD symptoms (Montastruc et al., 1995; Simons, 1996). However, the current study uncovers the anti-inflammatory effects of fluoxetine on PD only, showing that fluoxetine contributes to the survival of nigrostriatal DA neurons by inhibiting microglial activation in the MPTP model of PD. In conclusion, in view of its clinical safety and ability to effectively penetrate the bloodebrain barrier, we propose that fluoxetine could be beneficial in the treatment of aspects of PD and other disorders associated with neuroinflammation and microgliaderived oxidative damage. Supplementary material related to this article can be found online at doi:10.1016/j.neuropharm.2011.01.043. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20090063274) and partly a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A080781). Appendix. Supplementary material Supplementary material related to this article can be found online at doi:10.1016/j.neuropharm.2011.01.043. References Alam, Z.I., Daniel, S.E., Lees, A.J., Marsden, D.C., Jenner, P., Halliwell, B., 1997. A generalised increase in protein carbonyls in the brain in Parkinson’s but not incidental Lewy body disease. J. Neurochem. 69, 1326e1329. Ansorge, M.S., Morelli, E., Gingrich, J.A., 2008. Inhibition of serotonin but not norepinephrine transport during development produces delayed, persistent perturbations of emotional behaviors in mice. J. Neurosci. 28, 199e207. 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. Beal, M.F., 2002. Oxidatively modified proteins in aging and disease. Free Radic. Biol. Med. 32, 797e803. Block, M.L., Hong, J.S., 2005. Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Prog. Neurobiol. 76, 77e98. Block, M.L., Zecca, L., Hong, J.S., 2007. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat. Rev. Neurosci. 8, 57e69. Bolin, L.M., Strycharska-Orczyk, I., Murray, R., Langston, J.W., Di Monte, D., 2002. Increased vulnerability of dopaminergic neurons in MPTP-lesioned interleukin6 deficient mice. J. Neurochem. 83, 167e175. Bove, J., Prou, D., Perier, C., Przedborski, S., 2005. Toxin-induced models of Parkinson’s disease. NeuroRx 2, 484e494. Brooks, W.J., Jarvis, M.F., Wagner, G.C., 1989. Astrocytes as a primary locus for the conversion MPTP into MPPþ. J. Neural Transm. 76, 1e12. Choi, D.K., Pennathur, S., Perier, C., Tieu, K., Teismann, P., Wu, D.C., Jackson-Lewis, V., Vila, M., Vonsattel, J.P., Heinecke, J.W., Przedborski, S., 2005a. Ablation of the inflammatory enzyme myeloperoxidase mitigates features of Parkinson’s disease in mice. J. Neurosci. 25, 6594e6600. Choi, S.H., Lee, D.Y., Chung, E.S., Hong, Y.B., Kim, S.U., Jin, B.K., 2005b. Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo. J. Neurochem. 95, 1755e1765. Choi, S.H., Lee, D.Y., Kim, S.U., Jin, B.K., 2005c. Thrombin-induced oxidative stress contributes to the death of hippocampal neurons in vivo: role of microglial NADPH oxidase. J. Neurosci. 25, 4082e4090. Chung, Y.C., Kim, S.R., Jin, B.K., 2010. Paroxetine prevents loss of nigrostriatal dopaminergic neurons by inhibiting brain inflammation and oxidative stress in an experimental model of Parkinson’s disease. J. Immunol. 185, 1230e1237. Croisier, E., Moran, L.B., Dexter, D.T., Pearce, R.K., Graeber, M.B., 2005. Microglial inflammation in the parkinsonian substantia nigra: relationship to alpha-synuclein deposition. J. Neuroinflammation 2, 14. Cross, A.R., Segal, A.W., 2004. The NADPH oxidase of professional phagocyteseprototype of the NOX electron transport chain systems. Biochim. Biophys. Acta 1657, 1e22. Dauer, W., Przedborski, S., 2003. Parkinson’s disease: mechanisms and models. Neuron 39, 889e909. Dawson, T.M., Dawson, V.L., 2003. Molecular pathways of neurodegeneration in Parkinson’s disease. Science 302, 819e822.

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