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Experimental Model of Parkinson's DiseaseInflammation and Oxidative Stress in anDopaminergic Neurons by Inhibiting Brain Paroxetine Prevents Loss of Nigrostriatal

Young C. Chung, Sang R. Kim and Byung K. Jin

http://www.jimmunol.org/content/185/2/1230doi: 10.4049/jimmunol.1000208June 2010;

2010; 185:1230-1237; Prepublished online 21J Immunol 

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8.DC1http://www.jimmunol.org/content/suppl/2010/06/18/jimmunol.100020

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

Paroxetine Prevents Loss of Nigrostriatal DopaminergicNeurons by Inhibiting Brain Inflammation and OxidativeStress in an Experimental Model of Parkinson’s Disease

Young C. Chung,*,†,‡,x,{ Sang R. Kim,*,1 and Byung K. Jin‡,x,{

The present study examined whether the antidepressant paroxetine promotes the survival of nigrostriatal dopaminergic (DA) neu-

rons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. MPTP induced degener-

ation of nigrostriatal DA neurons and glial activation as visualized by tyrosine hydroxylase, macrophage Ag complex-1, and/or

glial fibrillary acidic protein immunoreactivity. Real-time PCR, Western blotting, and immunohistochemistry showed upregula-

tion of proinflammatory cytokines, activation of microglial NADPH oxidase and astroglial myeloperoxidase, and subsequent

reactive oxygen species production and oxidative DNA damage in the MPTP-treated substantia nigra. Treatment with paroxetine

prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This

neuroprotection afforded by paroxetine was associated with the suppression of astroglial myeloperoxidase expression and/or

NADPH oxidase-derived reactive oxygen species production and reduced expression of proinflammatory cytokines, including

IL-1b, TNF-a, and inducible NO synthase, by activated microglia. The present findings show that paroxetine may possess anti-

inflammatory properties and inhibit glial activation-mediated oxidative stress, suggesting that paroxetine and its analogues may

have therapeutic value in the treatment of aspects of Parkinson’s disease related to neuroinflammation. The Journal of Immu-

nology, 2010, 185: 1230–1237.

Parkinson’s disease (PD) is a common neurodegenerativedisease characterized by abnormal motor behavior, char-acterized by resting tremor, rigidity, and bradykinesia (1,

2). The neuropathological features of PD are progressive loss ofdopaminergic (DA) neurons in the substantia nigra (SN) and de-pletion of dopamine in the striatum (STR), the site to which theseneuronal nerve terminals project (3). Although PD is a sporadicdisease of unknown pathogenesis, accumulating evidence suggeststhat glial activation-derived oxidative stress increases the risk ofdeveloping PD (4). In vivo and in vitro 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models of PD show that key enzymes

involved in the production of reactive oxygen species(ROS)/reactive nitrogen species (RNS), such as microglial NAPDHoxidase and inducible NO synthase (iNOS), and astroglial myelo-peroxidase (MPO) are upregulated in damaged areas and contributeto DA neuronal death (5–8). Additionally, proinflammatory cyto-kines, such as TNF-a and IL-1b, are also increased and contributeto DA neuronal death in the MPTP model of PD (9).Paroxetine, a common selective serotonin reuptake inhibitor, is

widely prescribed as an antidepressant because it has fewer sideeffects and lower toxicity than earlier-generation selective seroto-nin reuptake inhibitors (10). The most frequent neuropsychiatricsymptom in PD is depression (11), and several clinical reportshave shown that paroxetine is safe and effective in treatingPD-associated depression (12, 13). Antidepressants such as fluox-etine and imipramine have been shown to possess potent anti-inflammatory effects in the rat cerebral ischemia model of middlecerebral artery occlusion (14) and in a mouse model of Alz-heimer’s disease (15). These studies demonstrated that fluoxetineand/or imipramine attenuated expression of TNF-a and IL-1b.Similarly, paroxetine was found to decrease the release of TNF-ain rat splenocytes (16) and inhibit infiltration of peripheral immunecells and production of the proinflammatory molecule substanceP in an animal model of atopic dermatitis (17). However, little isknown about the effects of paroxetine in the CNS, especially inthe nigrostriatal DA system in the context of PD. Thus, the cur-rent study sought to determine whether paroxetine could preventthe degeneration of nigrostriatal DA neurons by inhibiting glialactivation and, ultimately, reducing oxidative stress in the MPTPmodel of PD.

Materials and MethodsAnimals and treatments

All of the experiments were done in accordance with approved animalprotocol and guidelines established by Kyung Hee University. All of theexperiments were conducted with 8- to 10-wk-old male C57BL/6 mice

*Neuroscience Graduate Program and †Division of Cell Transformation and Restora-tion, School of Medicine, Ajou University, Suwon; and ‡Department of Biochemistryand Molecular Biology, xNeurodegeneration Control Research Center, and {Aged-Related and Brain Disease Research Center, School of Medicine, Kyung Hee Univer-sity, Seoul, South Korea

1Current address: Department of Neurology, College of Physicians and Surgeons,Columbia University, New York, NY.

Received for publication January 21, 2010. Accepted for publication May 6, 2010.

This work was supported by the Basic Science Research Program through the Na-tional Research Foundation of Korea funded by the Ministry of Education, Science,and Technology (Grant 20090063274).

Address correspondence and reprint requests to Dr. Byung K. Jin, Department ofBiochemistry and Molecular Biology, Neurodegeneration Control Research Center,and Age-Related and Brain Disease Research Center, School of Medicine, KyungHee University, Seoul 130-701, South Korea. E-mail address: bkjin@khu.ac.kr

The online version of this article contains supplemental material.

Abbreviations used in this paper: 8-OHdG, 8-hydroxy-29-deoxyguanosine; C, control;DA, dopaminergic; ED-1, CD68; GFAP, glial fibrillary acidic protein; Iba-1, ionizedcalcium-binding adaptor molecule 1; iNOS, inducible NO synthase; ip, immunopos-itive; M, MPTP; MAC-1, macrophage Ag complex-1; MPO, myeloperoxidase; MP,MPTP and paroxetine; MPP+, 1-methyl-4-phenyl-pyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; P, paroxetine; PD, Parkinson’s disease; ROS,reactive oxygen species; RNS, reactive nitrogen species; SN, substantia nigra;SNpc, substantia nigra pars compacta; SNr, substantia nigra reticulata; STR,striatum; TH, tyrosine hydroxylase; VTA, ventral tegmental area.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

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(22–24 g; Charles River Breeding Laboratory, Yokohama, Japan) in a roommaintained at 20–22˚C on a 12 h light/dark cycle with food and wateravailable ad libitum. For MPTP intoxication, mice received four i.p. injec-tions of MPTP-HCl (20 mg/kg; Sigma-Aldrich, St. Louis, MO) dissolvedin PBS at 2 h intervals as previously described (18). For paroxetine treat-ment, mice received a single injection per day of paroxetine (10 mg/kgbody weight, equivalent to 0.2–0.25 mg/day) into the peritoneum for 6 d,beginning at 12 h after last MPTP injection. Some mice were injected withparoxetine alone or vehicle as a control.

Immunohistochemistry

Animals were transcardially perfused with a saline solution containing 0.5%sodium nitrate and heparin (10 U/ml) and then fixed with 4% para-formaldehyde dissolved in 0.1 M phosphate buffer. Brains were dissectedfrom the skull, postfixed overnight in buffered 4% paraformaldehyde at 4˚C,stored in a 30% sucrose solution for 24–48 h at 4˚C until they sank, frozen,and sectioned on a sliding microtome in 30-mm-thick coronal sections. Allof the sections were collected in six separate series and processed forimmunohistochemical staining as described previously (19). The primaryAbs included those directed against tyrosine hydroxylase (TH; 1:2000;Pel-Freez, Brown Deer, WI), macrophage Ag complex-1 (MAC-1;1:200; Serotec, Oxford, U.K.), ionized calcium-binding adaptor molecule1 (Iba-1; 1:1000; Wako Chemicals, Osaka, Japan), CD68 (ED-1; 1:1000;Serotec), glial fibrillary acidic protein (GFAP; 1:5000; Neuromics, Edina,MN), MPO (1:500; Thermo Scientific, Fremont, CA), 8-hydroxy-29-deoxyguanosine (8-OHdG; 1:200; JaICA, Shizuoka, Japan), iNOS(1:200, BD Biosciences, San Jose, CA), IL-1b (1:200, R&D Systems,Minneapolis, MN), and p47phox (1:200; Santa Cruz Biotechnology, SantaCruz, CA). Stained cells were viewed and analyzed under a bright-fieldmicroscope (Nikon, Tokyo, Japan) or viewed with a confocal laser-scanning microscope (Olympus Optical, Tokyo, Japan). For Nissl staining,SN tissues immunostained with TH Ab were mounted on gelatin-coatedslide, dried for 1 h at room temperature, and stained with 0.5% cresylviolet (Sigma-Aldrich). To validate immunohistochemical data, we per-formed immunohistochemistry with each isotype-matched control Ab(Supplemental Fig. 3, Supplemental Table I).

Stereological cell counting

The unbiased stereological estimation of the total number of the TH-positivecells and Nissl-stained neurons in the SN was made using the optical frac-tionator method performed on an Olympus Computer Assisted Stereolog-ical Toolbox system, version 2.1.4 (Olympus, Ballerup, Denmark), as wedescribed in detail (19, 20). The sections used for counting covered theentire SN from the rostral tip of the pars compacta back to the caudal endof the pars reticulate (anterioposterior, 22.06 to 24.16 mm from bregma)(21). The SN was delineated at a31.25 objective and generated a countinggrid of 1503 150 mm. An unbiased counting frame of known area (47.87336.19 mm = 1733 mm2) superimposed on the image was placed randomlyon the first counting area and systemically moved through all of the count-ing areas until the entire delineated area was sampled. Actual counting wasperformed using a 3100 oil immersion objective. The estimate of the totalnumber of neurons was calculated according to the optical fractionatorequation (22). More than 300 points over all of the sections of eachspecimen were analyzed.

Densitometric analysis

As previously described (23), an average of 17 coronal sections of thestriatum, starting from the rostral anterioposterior (+1.60 mm) to ante-rioposterior (0.00 mm), according to bregma of the brain atlas (21), wereexamined at a 35 magnification using the Image-Pro Plus system (version4.0; Media Cybernetics, Silver Spring, MD) on a computer attached toa light microscope (Zeiss Axioskop, Oberkochen, Germany), interfacedwith a charge-coupled device video camera (MegaPlus model 1.4i; Kodak,New York, NY). To determine the density of the TH-immunoreactivestaining in the striatum, a square frame of 700 3 700 mm was placed inthe dorsal part of the striatum. A second square frame of 200 3 200 mmwas placed in the region of the corpus callosum to measure backgroundvalues. To control for variations in background illumination, the average ofthe background density readings from the corpus callosum was subtractedfrom the average of density readings of the striatum for each section. Thenthe average of all of the sections of each animal was calculated separatelybefore data were statistically processed.

Rotarod test

An accelerating rotarod (five-lane accelerating rotarod; Ugo Basile,Comerio, Italy) was used to measure forelimb and hindlimb motor

dysfunction (9), with some modifications. The rotarod unit consisted ofa rotating spindle (diameter, 3 cm) where mice were challenged for speed.For training, mice were placed on the rotarod at 10 rpm for 20 min, sevenconsecutive days before MPTP treatment. Mice that stayed on the rodwithout falling during training were selected and randomly divided intoexperimental groups. After 7 d from last MPTP treatment, animals receiv-ing various treatment regimes were placed in a separate compartment onthe rotating rod and tested at 20 rpm for 20 min. The latency to fall wasautomatically recorded by magnetic trip plates.

HPLC analysis

To measure contents of dopamine in STR, we performed reverse-phaseHPLC with electrochemical detector as previously described (24). Dis-sected striatal tissues were homogenized with 0.1 M perchloric acid and0.1 mM EDTA buffer and centrifuged at 9000 rpm for 20 min. The super-natant was injected into an autosampler at 4˚C (Waters 717 Plus Autosam-pler, Waters Division, Milford, MA) and eluted through mBondapak C18column (3.9 3 300 mm 310 mm; ESA, Chelmsford, MA) with mobilephase for catecholamine analysis (Chromosystems, Munich, Germany).The peaks of dopamine content were analyzed by ESA Coulochem IIelectrochemical detector and integrated using a commercially availableprogram (Breeze; Waters). All of the samples were normalized for proteincontent as spectrophotometrically determined using the Bio-Rad ProteinAssay Kit (Bio-Rad, Hercules, CA).

Determination of O22 and O2

2-derived oxidants

Hydroethidine histochemistry was performed for in situ visualization ofO2

2 and O22-derived oxidants (7). Three days after MPTP injection,

hydroethidine (1 mg/ml in PBS containing 1% DMSO; Molecular Probes,Eugene, OR) was administered i.p. After 15 min, the animals were trans-cardially perfused with a saline solution containing 0.5% sodium nitrateand heparin (10 U/ml) and then fixed with 4% paraformaldehyde in 0.1 Mphosphate buffer. After fixation, the brains were cut into 30-mm slicesusing a sliding microtome. Sections were mounted on gelatin-coatedslides, and the oxidized hydroethidine product, ethidium, was examinedby IX71 confocal microscopy (Olympus Optical, Tokyo, Japan). To de-termine specific cell type related to oxidant production, we performeddouble-fluorescence staining with HE and MAC-1 or HE and TH Ab(Supplemental Fig. 2).

Quantitative real time-PCR

Animals treated with or without paroxetine (10 mg/kg, i.p.) were humanelykilled 24 h after injection of MPTP, and the bilateral SN regions were im-mediately isolated. In brief, tissues were homogenized and total RNA wasextracting with RNAzol B (Tel-Test, Friendwood, TX). Reverse transcriptionwas performed with SuperScript II Reverse Transcriptase (Life Technolo-gies, Rockville, MD). The primer sequences used in this study were asfollows: 59-CTGCTGGTGGTGACAAGCACATTT-39 (forward) and 59-ATGTCATGAGCAAAGGCGCAGAAC-39 (reverse) for iNOS; 59-GCGA-CGTGGAACTGGCAGAAGAG-39 (forward) and 59-TGAGAGGGAGGC-CATTTGGGAAC-39 (reverse) for TNF-a; 59-GCAACTGTTCCTGAACT-CAACT-39 (forward) and 59-ATCTTTTGGGGTCCGTCAACT-39 (reverse)for IL-1b; and 59-TCAACAGCAACTCCCACTCTTCCA-39 (forward) and59-ACCCTGTTGCTGTAGCCGTATTCA-39 (reverse) for G3PDH. Real-time PCR was performed in a reaction volume of 10 ml including 2 ml1:50 diluted reverse transcription product as a template, 5 ml SYBR PremixEX Taq (Takara, Shiga, Japan) and 10 pmol of each primer described above.The PCR amplifications were performed with 50 cycles of denaturation at95˚C for 5 s, annealing at 60˚C for 10 s, and extension at 72˚C for 20 s usingLightCycler (Roche Applied Science, Indianapolis, IN). The DCT value wasdetermined by subtracting average CT values of G3PDH from average CT

values of IL-1b, TNF-a, and iNOS from PCR reactions (Supplemental TableII). To express the relative amounts of IL-1b, TNF-a, and iNOS, DDCT

values were calculated by subtracting the DCT value of the control groupfrom the DCT values of each group. The ratios of expression levels of IL-1b,TNF-a, and iNOS were calculated as 22(meanΔΔCt).

Measurement of TNF-a and IL-1b

Animals treated with or without paroxetine (10 mg/kg, i.p.) were humanelykilled 72 h after injection of MPTP, and the bilateral SN regions wereimmediately isolated. The production of TNF-a and IL-1b from mice SNtissues was determined by sandwich ELISA techniques. Tissues werehomogenized in 200 ml ice-cold radioimmunoprecipitation assay buffer(60 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholicacid, and 50 mM Tris [pH 8]) and centrifuged at 14,000 3 g at 4˚C for20 min. Equal amounts of samples (100 mg) were placed in ELISA kit

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strips coated with the appropriate Ab. Sandwich ELISA was then per-formed according to the manufacturer’s instructions (BioSource Interna-tional, Camarillo, CA). The detection limits of TNF-a and IL-1b were 5and 25 pg/ml, respectively. We diluted TNF-a samples and IL-1b samplesas 1:10, respectively.

Western blotting

For separating the cytosolic andmembrane fraction, we dissected SN tissuesfrom the animals at 3 d after injection of MPTP. As previously described(25, 26), SN samples were homogenized with using a glass homogenizer inice-cold buffer consisting of the following: 20 mM HEPES, 250 mMsucrose, 10 mM KCl, 1.5 mM MgCl2, 2 mM EDTA, and protease inhibitormixture (Sigma-Aldrich). Homogenates were centrifuged for 5 min at500 3 g at 4˚C, and supernatants were collected and centrifuged for20 min at 13,000 3 g at 4˚C. The pellets were further centrifuged for1 h at 100,000 3 g at 4˚C, and the resulting supernatants and pellets weredesignated as the cytosolic and membrane fractions, respectively. Equalamounts of proteins (30 mg) were separated by 12% SDS-PAGE gels andtransferred to polyvinylidene difluoride membranes (Millipore, Bedford,MA) using an electrophoretic transfer system (Bio-Rad). The membraneswere immunoblotted with goat anti-actin (1:1000; Santa Cruz Biotechnol-ogy), rabbit anti-p47phox (1:1000; BD Biosciences), and mouse anti-Rac1(1:500; Santa Cruz Biotechnology). To determine the relative degree ofmembrane purification, the membrane fraction was subjected to immuno-blotting for calnexin, a membrane marker, using a rabbit anti-calnexin(1:1000; Stressgen, Victoria, BC, Canada). For semiquantitative analyses,the densities of bands on immunoblots were measured with a computerimaging device and accompanying software (Fujifilm, Tokyo, Japan).

Statistical analysis

All of the values are expressed as mean 6 SEM. Statistical significance(p , 0.05 for all analyses) was assessed by ANOVA using Instat 3.05(GraphPad, SanDiego, CA), followed by Student–Newman–Keuls analyses.

ResultsParoxetine protects nigrostriatal DA neurons fromMPTP-induced neurotoxicity in vivo

In the brain, MPTP is converted to 1-methyl-4-phenyl-pyridinium(MPP+), an active toxic metabolite that is primarily responsible forMPTP neurotoxicity (27). Consistent with this, striatal MPP+ con-tent correlates linearly with MPTP toxicity (28). Striatal levels ofMPTP and MPP+ at specific time points after the last MPTP in-jection were measured and quantified by liquid chromatographywith electrospray ionization mass spectrometry. MPP+ levelspeaked within 30 min at 14.9 6 2.4 mg/mg protein and then grad-ually declined, becoming almost negligible (0.2 6 0.1mg/mg protein) after 12 h. These data indicate that MPTP is com-pletely converted to MPP+ and largely cleared within 12 h afterinjection. Because paroxetine was administered 12 h after the finalinjection of MPTP for all in vivo experiments, its effects could notbe attributed to reduced metabolism of MPTP to MPP+ or uptakeof MPP+ into DA neurons. At this time point, the extent of damageof the nigrostriatal DA system was assessed by TH immunostain-ing and the results are shown in Supplemental Fig. 1.The mice in each group received four i.p. injections of MPTP

(20 mg/kg) or PBS as a control at 2 h intervals. Seven days later,brains were removed and sections were immunostained for TH tospecifically detect DA neurons, and then SN tissues were processedfor Nissl staining. Similar to a recent report (18), there was aconsiderable loss of TH-immunopositive (ip) cell bodies in the SN(Fig. 1E, 1F) and fibers in the STR (Fig. 2C) at 7 d in the MPTP-injected mice compared with those of the PBS-treated controlmice (Figs. 1A, 1B, 2A). TH-ip cells in the SN and their nerveterminals in the STR were quantified by stereological counts anddensitometric analyses, respectively. MPTP treatment decreasedthe number of TH-ip neurons by 63% in the SN (Fig. 1I) andreduced the OD of TH-ip fibers by 62% in the STR (Fig. 2E)compared with those of the PBS-treated mice. Moreover, PBS-

treated SN had prominent Nissl substances (Fig. 1B) when com-pared with MPTP-treated SN, showing marked loss of Nissl sub-stances with gliosis (Fig. 1F). The stereological counts of thesesubstances revealed that MPTP decreased the number of Nissl-positive neurons by 61% in the SN compared with those of thePBS-treated SN (Fig. 1I).To investigate whether paroxetine altered MPTP-induced neuro-

toxicity of nigrostriatal DA neurons, we administered paroxetine(10 mg/kg body weight) for 6 d, starting 12 h after the last MPTPinjection. The results of TH immunohistochemistry showed thatparoxetine treatment effectively reduced MPTP-induced loss ofDA neurons in the SN (Fig. 1G, 1H) and their nerve terminalsin the STR (Fig. 2D). When quantified and expressed as a percent-age of TH-ip neurons or fibers in the control SN and STR, re-spectively, paroxetine was found to increase the number of TH-ipneurons by 26% (Fig. 1I; p , 0.01) and the OD of TH-ip fibers by21% (Fig. 2E; p , 0.001). Consistent with these results, parox-etine was found to increase the number of Nissl-stained neuronsby 25% in the SN (Fig. 1I; p , 0.01). Paroxetine alone had noeffects on the number of neurons in the SN (Fig. 1C, 1D, 1I) orDA fibers in the STR (Fig. 2B, 2E).

Paroxetine increases striatal dopamine levels and improvesmotor behavior in MPTP mice

We next determined whether paroxetine improves MPTP-inducedmotor deficits by testing performance on a rotarod apparatus withsome modifications (9). Animals receiving various treatment

FIGURE 1. Paroxetineprevents MPTP-induced neurotoxicity in the SN

of mouse brains. Animals receiving PBS as a control (A, B), paroxetine

alone (C, D), MPTP (E, F), or MPTP and paroxetine (G, H) were sacrificed

7 d after last MPTP administration. The brain tissues were cut using

a sliding microtome in 30-mm-thick coronal sections and immunostained

with Ab to TH (brown) for DA neurons, and then Nissl staining (blue) was

performed. Note the absence of TH expression in some Nissl-stained neu-

rons, indicated by arrows. B, D, F, and H show higher magnifications of A,

C, E, and G, respectively. I, Numbers of TH-ip and Nissl-stained neurons

in the SN were counted. Four to 10 animals were used for each experi-

mental group. pp , 0.001, significantly different from controls; ppp ,0.01, significantly different from MPTP only. A–D, Scale bars, 200 mm. C,

control; M, MPTP; MP, MPTP and paroxetine; P, paroxetine; SNpc, sub-

stantia nigra pars compacta; SNr, substantia nigra reticulata; VTA, ventral

tegmental area.

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regimes were evaluated 7 d after the last MPTP injection by mea-suring the latency to falling. MPTP treatment decreased sustainedrotarod time to 11.29 6 0.49 min, a 56% decrease compared withthat of PBS treatment (Fig. 2F; p , 0.001). The behavioral dys-function in MPTP-treated mice was partially recovered by parox-etine treatment, which increased the latency to falling to 14.07 60.35 min (Fig. 2F; p , 0.05). Paroxetine alone had no effects onmotor behavior.After rotarod performance tests, the mice were sacrificed for

biochemical assessments. In parallel with behavioral dysfunction,HPLC analysis revealed that MPTP reduced STR dopamine levelsto 75% of those in PBS controls (Fig. 2F). By contrast, treatment ofMPTP-injected mice with paroxetine increased dopamine levelsby 22% compared with MPTP only (Fig. 2F; p , 0.05). Parox-etine alone had no effect on STR dopamine levels.

Paroxetine inhibits microglial activation in the SN in vivo

Several lines of evidence suggest that activated microglia playa critical role in DA neuronal cell death in the MPTP model (29).Thus, we next examined whether the neuroprotective effect ofparoxetine resulted from inhibition of microglial activation in theSN. At 3 d after the last MPTP injection, brain tissues from micetreated with or without paroxetine were processed for immunos-taining with MAC-1, Iba-1, and ED-1 Abs to detect activatedmicroglia. In contrast with PBS controls, where relatively fewpositive microglia were observed in the SN (Fig. 3A), there werenumerous robustly immunoreactive MAC-1–positive (activated)microglia in the MPTP-treated SN (Fig. 3B), consistent with aprevious report (30). Paroxetine treatment dramatically reducedthe number of MAC-1-ip microglia in the MPTP-treated SN(Fig. 3C), indicating that paroxetine suppressed MPTP-inducedmicroglial activation.Consistent with MAC-1 staining, Iba-1–positive microglia

exhibited the typical ramified morphology of resting microgliain the PBS-treated control SN (Fig. 3D). In contrast, the majorityof Iba-1–positive microglia displayed an activated morphology,including larger cell bodies with short, thick, or no processes inMPTP-treated SN (Fig. 3E). Treatment with paroxetine mitigatedthese effects of MPTP, dramatically decreasing the number ofactivated microglia in the MPTP-treated SN (Fig. 3F).Microglia in the MPTP-treated SN appeared to reach a state

similar to that of active phagocytes (Fig. 3H), as determined byED-1 immunohistochemical staining, which labels phagocyticmicroglia, in particular, the presence of injured cells or debris(31). Few such ED-1-ip cells were observed in the paroxetine-treated SN (Fig. 3I), similar to the results of MAC-1 immu-

nostaining. ED-1-ip cells were not detectable in the PBS-treated SN (Fig. 3G). Paroxetine alone had no effect on micro-glial activation (data not shown).

Paroxetine inhibits MPTP-induced expression of IL-1b,TNF-a, and iNOS

It has been shown that mice expressing a dominant-negative inhib-itor of IL-1b–converting enzyme or those deficient in TNF-a oriNOS are resistant to MPTP-induced neurotoxicity (6, 32, 33).Thus, we examined whether paroxetine might modulate DA neu-ronal survival by affecting MPTP-induced expression of IL-1b,TNF-a, and/or iNOS in the SN. For this purpose, paroxetine wasadministered 12 h after the last MPTP injection and animals weresacrificed 12 h later. An analysis of dissected brain tissues by real-time PCR revealed that paroxetine administered after MPTP sig-nificantly inhibited MPTP-induced expression of proinflammatorycytokines, reducing IL-1b, TNF-a, and iNOS expression by57, 60, and 76%, respectively (Fig. 4A), but had no effect whenadministered alone. Consistent with real-time PCR analyses,ELISA results showed that the levels of IL-1b and TNF-a pro-tein were significantly increased at 3 d in the MPTP-treated SNcompared with those of the PBS-treated SN (Fig. 4B). TheseMPTP-induced increases were significantly attenuated by treat-ment with paroxetine, which had no effect alone. To identify thecell types expressing IL-1b and iNOS protein, we performed

FIGURE 2. Paroxetine protects neurotoxicity

induced by MPTP in the STR in vivo. The STR

tissues obtained from the same animals as used

in Fig. 1 were immunostained with TH Ab for

dopaminergic fibers: PBS (A), paroxetine only

(B), MPTP (C), or MPTP and paroxetine (D). E,

OD of TH-ip fiber in the STR. Four to six ani-

mals were used for each experimental group.

pp, 0.001, significantly different from controls;

ppp , 0.001, significantly different from MPTP

only. A–D, Scale bars, 250 mm. F, After the

rotarod test, striatal tissues were dissected from

animals to determine the dopamine level in the

striatum. Five to six animals were used for each

experimental group. pp , 0.001, significantly

different from controls; ppp, 0.05, significantly

different fromMPTP only. C, control; M,MPTP;

MP, MPTP and paroxetine; P, paroxetine.

FIGURE 3. Paroxetine prevents microglial activation in the SN in vivo.

Animals receiving PBS as a control (A, D, G), MPTP (B, E, H), or MPTP

and paroxetine (C, F, I) were sacrificed 3 d after the last MPTP injection.

The mouse brain tissues were removed and immunostained with Abs to

MAC-1 (A–C), Iba-1 (D–F), and ED-1 (G–I) to identify microglia. Insets

show higher magnifications of A–I, respectively. Dotted lines indicate

SNpc. A–I, Scale bars, 100 mm.

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double-immunofluorescent staining in SN sections obtained 3 dafter MPTP injection using a combination of Abs against MAC-1and IL-1b or iNOS. Simultaneous imaging of immunofluorescencein the same tissue sections revealed that IL-1b (Fig. 4C) andiNOS (Fig. 4D) immunoreactivity was localized within MAC-1-ip microglia.

Paroxetine attenuates MPTP-induced oxidant production andoxidative stress via microglial NADPH oxidase

The production of ROS, such as hydrogen peroxide and superoxide,are increased in the midbrain of MPTP-treated mice, and oxidantoriginating from microglia is thought to mediate the loss of DAneurons in the SN (34). Thus, we examined whether paroxetinerescued nigral DA neurons by inhibiting MPTP-induced oxidantproduction. The fluorescent product of oxidized hydroethidine(i.e., ethidium) was significantly increased in the MPTP-injectedSN 72 h after administration (Fig. 5B) compared with that of thePBS-injected SN (Fig. 5A). Paroxetine dramatically decreasedMPTP-induced oxidant production in the SN in vivo (Fig. 5C)but had no effect alone (data not shown).NADPH oxidase is composed of the cytosolic components

p47phox, p67phox, and Rac1 and the membrane componentsgp91phox and p22phox (35, 36). Translocation of NADPH oxidasesubunits from the cytosol to the plasma membrane in activatedmicroglia produces ROS, which ultimately contribute to DAneuronal death in the MPTP mouse model of PD (7). To inves-tigate whether paroxetine modulates the activity of NADPH oxi-dase, we performed Western blotting after separating cell lysatesinto membrane and cytosolic components. Western blot analysesshowed that the levels of p47phox and Rac1 in the MPTP-treatedSN were significantly increased in membrane fractions 3 d after

MPTP injection compared with those of the PBS-treated SN con-trols (Fig. 5D, 5E), indicating increased translocation of thesesubunits. Administration of paroxetine after MPTP injection sig-nificantly decreased translocation of p47phox and Rac1 from thecytosol to the membrane in the SN (Fig. 5D, 5E). Paroxetine alonehad no effects on the level of p47phox or Rac1 in either cytosolic ormembrane fractions. These results confirm that MPTP-inducedactivation of NADPH oxidase is inhibited by paroxetine. More-over, the p47phox-ip cells present in the SN 3 d after MPTP in-jection corresponded to activated microglia (Fig. 5F) but notastrocytes or neurons (data not shown).

FIGURE 4. Paroxetine reduces the MPTP-induced inflammation in the

SN of mouse brains. Animals were administered MPTP or vehicle in the

presence or absence of paroxetine and sacrificed 1 d later for real-time

PCR (A) or 3 d later for a sandwich ELISA in the SN (B). Paroxetine

dramatically attenuated MPTP-induced expression of proinflammatory

cytokines including IL-1b, TNF-a, and iNOS. Each bar represents the

mean 6 SEM of four to five animals per group. pp , 0.05, significantly

different from control; ppp , 0.05, significantly different from MPTP.

Colocalization of IL-1b (E) or iNOS (I) immunoreactivity within

activated microglia in the SN in vivo. The sections of mouse SN were

prepared 3 d after MPTP injection and then immunostained simultaneously

with MAC-1 (C, G; green) as a marker of microglia and IL-1b (D; red)

or iNOS (H; red). To verify the colocalized images, double-

immunofluorescence staining was performed with each isotype-matched

control (F, J). Images from the same double-labeled tissue were merged

(E, I). C–J, Scale bars, 50 mm. C, control; M, MPTP; MP, MPTP and

paroxetine; P, paroxetine.

FIGURE 5. Paroxetine inhibits MPTP-induced oxidant production, ac-

tivation of NADPH oxidase, and oxidative DNA damage in the SN in vivo.

A–C, Animals receiving PBS as a control (A), MPTP (B), or MPTP and

paroxetine (C) were sacrificed 3 d after the last MPTP injection. SN tissues

were prepared for hydroethidine histochemistry to detect oxidant produc-

tion. Dotted lines indicate SNpc. D, Translocation of cytosolic subunits

(p47phox and Rac1) from the cytosol to the plasma membrane after MPTP

injection, indicating activation of NADPH oxidase in the SN. Animals

were decapitated after injection of MPTP, and SN tissues were isolated.

Tissue lysates were fractionated and analyzed by immunoblot analysis

with p47phox or Rac1 Ab. The membrane protein calnexin was used to

normalize the data. E, The histogram shows quantification of p47phox and

Rac1 levels expressed as the ratio of membrane fraction to total. The

results represent the mean 6 SEM of three to four separate experiments.

pp , 0.01, compared with control; pp p , 0.05, compared with MPTP

only. F, Localization of p47phox immunoreactivity in activated microglia in

the SN treated with MPTP. The sections of SN were prepared 3 d after

MPTP injection and then simultaneously stained with an Ab against

p47phox and MAC-1 as a marker for microglia. G–I, The SN tissues

obtained from the same animals as used in A–C were prepared for

8-OHdG histochemistry to detect oxidative DNA damage in the SN. Dot-

ted lines indicate SNpc. Insets show higher magnifications of G–I, respec-

tively. Scale bars: A–C, 150 mm; F, 50 mm; G–I, 100 mm. C, control;

M, MPTP; MP, MPTP and paroxetine; P, paroxetine.

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In MPTP mice, DA neuronal cell death is accompanied by in-creased levels of 8-OHdG, a marker of oxidative nucleic aciddamage (37). To determine whether paroxetine prevented MPTP-induced oxidative damage to DNA in the SN, we immunostainedfor 8-OHdG. Our results revealed that the levels of 8-OHdG weredramatically increased in the SN 3 d after MPTP injection (Fig.5H) compared with those of the PBS-treated SN (Fig. 5G). ThisMPTP-induced oxidative DNA damage was dramatically inhibitedby paroxetine (Fig. 5I), which had no effect alone (data not shown).

Paroxetine inhibits MPTP-induced astroglial activation andMPO expression in the SN in vivo

It has been shown that MPO is upregulated in astrocytes in theSN of PD patients and MPTP-treated mice; furthermore, mice de-ficient in MPO are resistant to MPTP neurotoxicity (5). Thus, wenext examined whether paroxetine inhibited MPTP-inducedastroglial activation and expression of MPO in the SN, whichwould be predicted to enhance neuronal survival. To ascertain this,we immunostained sections adjacent to those used for MAC-1immunostaining for GFAP and MPO 3 d after MPTP injection.In agreement with previous studies (6, 30), resting astrocytes(Fig. 6A; small somas and thin dendrites) were transformed intocells with enlarged bodies and thick dendrites in MPTP-treatedmice (Fig. 6B). Treatment with paroxetine (10 mg/kg, i.p.) afterMPTP injection dramatically decreased the number of activatedastrocytes in the MPTP-treated mice (Fig. 6C). There was also anincrease in immunostaining for MPO-ip cells in the MPTP-treatedSN (Fig. 6E) compared with that of the control SN (Fig. 6D), anincrease that was significantly attenuated by paroxetine (Fig. 6F).Quantification of MPO-ip cells in the SN by stereological cell

counts confirmed these results, showing that the number MPO-ipcells was 263-fold higher in the MPTP-treated SN than that inthe SN of PBS-treated controls (Fig. 6G). Paroxetine reduced thenumber of MPTP-induced MPO-ip cells by 75% (Fig. 6G). Ascontrols, vehicle (PBS) and paroxetine alone had no effects. Ad-ditional immunofluorescence staining revealed that MPO immu-noreactivity was localized within astrocytes 3 d after MPTPtreatment (Fig. 6H).

DiscussionIn this study, we demonstrated that paroxetine protects nigros-triatal DA neurons from MPTP neurotoxicity in vivo by inhibitingbrain inflammation and the resultant oxidative stress. We showedthat paroxetine suppressed MPTP-induced ROS generation andreduced oxidative damage to nucleic acids by inhibiting micro-glia-derived NADPH oxidase and astrocyte-derived MPO, leadingto survival of nigrostriatal DA neurons, recovery of striatal dopa-mine depletion in vivo, and reversal of motor dysfunction. Addi-tionally, paroxetine attenuated the expression of proinflammatorycytokines and iNOS within activated microglia. To our knowl-edge, this is the first study to show that paroxetine preventsnigrostriatal DA neuronal death through blockade of glial activa-tion in the MPTP model of PD.Glial cells play an important role in supporting neurons in the

CNS. However, in the presence of adverse stimuli, they maycontribute to chronic, damaging inflammation and, ultimately, toneuronal cell death. Microglia are intrinsic immune effector cellsthat are dramatically activated in response to neuronal damage (38).Activated microglia produce several potentially neurotoxic sub-stances, including ROS and/or proinflammatory cytokines (29).ROS, such as O2

2 and O22-derived oxidant molecules, may

impose an oxidative stress on DA neurons and induceand/or exacerbate neurotoxicity (39). Several studies have demon-strated evidence of oxidative stress in PD patients and in theMPTP model of PD, including oxidative modifications to nucleicacids (40, 41). Importantly, these ROS can be produced bymicroglia-derived NADPH oxidase and are capable of causingoxidative stress in the MPTP model of PD (29). Several studies,including ours, have demonstrated that activation of microglialNADPH oxidase participates in DA neuronal death in vivo andin vitro (7, 8, 25). The results of the current study show that MPTPactivated NADPH oxidase, as demonstrated by the translocation ofcytosolic components of NADPH oxidase, p47phox and Rac1.NADPH oxidase activation resulted in increased O2

2 and O22

-derived oxidants and DNA damage, as visualized by hydroethi-dine staining and 8-OHdG immunostaining in the SN, respec-tively. Treatment with paroxetine not only inhibited microglialNADPH oxidase activation but also mitigated ROS productionand nucleic acid oxidation. These results verify that paroxetineinhibited MPTP-induced activation of NADPH oxidase and oxi-dative damage, thereby resulting in neuroprotection in theMPTP model.Accompanying oxidative stress, other microglial-derived proin-

flammatory cytokines or cytotoxic factors may be involved innigrostriatal DA neuronal death. It has been shown that iNOS(42) and proinflammatory cytokines, such as TNF-a and IL-1b(43), are increased in the brains of PD patients. Moreover, NO,generated by iNOS, may also be involved in the pathogenesis ofPD (27), although the presence of iNOS in human microgliaremains controversial (44). The neurotoxic effects of NO areattributed to its reaction with O2

2 to form peroxynitrite, whichcan cause oxidative damage to proteins in the MPTP model (6).TNF-a and IL-1b, originating from activated glia, may triggerintracellular death-related signaling pathways or participate in

FIGURE 6. Paroxetine suppresses MPTP-induced astroglial activation

and expression of MPO in the SN in vivo. The SN tissues obtained from

the same animals as used in Fig. 5 were immunostained with GFAPAb for

astrocyte (A–C) and MPO Ab for MPO immunoreactivity (D–F). Animals

receiving PBS as a control (A, D), MPTP (B, E), or MPTP and paroxetine

(C, F) were sacrificed 3 d after the last MPTP injection. Insets show higher

magnifications of A–C, respectively. Dotted lines indicate SNpc. G, Num-

ber of MPO-ip cells in the SN were counted. Four to five animals were

used for each experimental group. pp , 0.01, significantly different from

controls; ppp , 0.05, significantly different from MPTP only. H, Locali-

zation of MPO immunoreactivity in activated astrocytes in the SN treated

with MPTP. The SN tissues obtained from the same animals as used in B

were simultaneously stained with an Ab against MPO and GFAP as a maker

for astrocyte. Scale bars: A–F, 200 mm; G, 50 mm. C, control; M, MPTP;

MP, MPTP and paroxetine; P, paroxetine.

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the induction of iNOS expression in the MPTP model (45). Thepresent data showed that MPTP increased the expression of iNOS,TNF-a, and IL-1b mRNA in the SN. Treatment with paroxetineinhibited the expression of these three molecules within activatedmicroglia after MPTP injection, an effect that likely accounts, atleast in part, for the observed paroxetine-induced neuroprotection.These results collectively suggest that paroxetine has anti-inflammatory properties that contribute to its neuroprotectiveeffects. Similar to our results, a recent report showed that mino-cycline prevents MPTP neurotoxicity through relatively smallchanges in protein levels of IL-1b and TNF-a (46). Althoughgenetic ablation of TNF-a decreased microglial activation andprevented disruption of blood–brain barriers, survival of DAneurons was not increased in the SN. Thus, biological relevanceof these molecules may be varied depending on experimentalconditions. Although we did not provide direct evidence, thepresent results combined with other observations carefullysuggest that the biological relevance of these two molecules isdue to each molecule and/or synergic effects combined witheach other.Although the role of astroglial activation in PD is disputed,

several studies using the MPTP model support the idea thatastroglial activation can contribute to the degeneration of nigros-triatal DA neurons via generation of neurotoxic substances (47, 48).One such substance expressed in activated astrocytes is MPO,which can use NO2

2 to generate RNS and cause DA neuronaldeath in the MPTP model (5). This increased expression ofastroglial MPO mediates neurotoxicity on DA neurons under twopossible mechanisms. First, MPO is known as the key enzyme forproduction of cytotoxic ROS/RNS (49). These astroglial MPO-derived oxidants can be released and then give deleterious effectson adjacent neurons (5). This was supported by evidence that in-creased levels of 3-chlorotyrosine– and HOCl-modified proteinsand MPO-derived oxidative stress markers were observed inMPTP-treated SN (5). Another possible mechanism is the crosstalk with other immune cells through MPO secretion (50). MPOcan be secreted and then activate neutrophils through bindingwith CD11b/CD18 integrins. Because microglia also expressCD11b/CD18 integrins, MPO secretion may participate in DAneuronal death in the MPTP model of PD via microglial activation.Although the cytokine-like effect of MPO is still unknown, thisenzyme may play an important role in the signaling pathway ofmicroglial activation, resulting in neuronal death. This is consis-tent with our present data showing that MPO was expressed inactivated astrocytes in the MPTP-treated SN, as assessed bydouble-label immunostaining. Additional experiments showed thatparoxetine attenuated MPO immunoreactivity in the SN. Takentogether, our results suggest that paroxetine rescues DA neuronaldeath via suppression of MPO expression in activated astrocytes.The most prominent biochemical change in the denervated

STR caused by MPTP injection is a reduction in the levels ofstriatal dopamine (18). This biochemical deficit in mice yieldsa characteristic motor dysfunction that leads to increased latencyto fall (deteriorated balance) on the accelerating rotarod (9). Thisnotion is consistent with the present demonstration that motorperformance on a rotarod is decreased with the loss of striatal THterminals and, by extension, the consequent reduction of DAlevels in the MPTP-treated STR (51). Paroxetine amelioratedthese MPTP-induced motor deficits; accompanying this behavioralrecovery was an increase in DA levels in the denervated STR.These behavioral and in vivo biochemical effects of paroxetineon the lesioned nigrostriatal DA system, taken together withour demonstration of inhibitory effects of paroxetine on glialactivation-mediated oxidative stress, suggest that paroxetine and

its analogues may have therapeutic value in the treatment PDsymptoms related to neuroinflammation.Finally, the most frequent neuropsychiatric symptom in PD is

depression, which is observed in up to 46% of patients with PD(11). In this context, several clinical reports have concluded thatparoxetine is a safe and effective drug for use in treating the de-pression associated with PD (12, 13). However, paroxetine hasalso been found to exacerbate parkinsonism symptoms associatedwith depression (52). Although our results suggest that paroxetineis beneficial in the treatment of aspects of PD related to glialactivation, these conflicting clinical data suggest that the use ofparoxetine in PD patients warrants caution (53).

AcknowledgmentsWe thank Dr. Noh and Dr. Yoon in Ajou University for providing paroxetine

and valuable comments on this manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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