Schizophrenia Research 151 (2013) 20–28

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Schizophrenia Research

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Pretreatment of aripiprazole and minocycline, but not haloperidol,suppresses oligodendrocyte damage from interferon-γ-stimulatedmicroglia in co-culture model

Yoshihiro Seki a, Takahiro A. Kato a,b,⁎, Akira Monji a,c,⁎, Yoshito Mizoguchi c, Hideki Horikawa a,Mina Sato-Kasai a, Daigo Yoshiga d, Shigenobu Kanba a

a Department of Neuropsychiatry, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japanb Innovation Center for Medical Redox Navigation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japanc Department of Psychiatry, Faculty of Medicine, Saga University, Saga 849-8501, Japand Department of Oral and Maxillofacial Surgery, Division of Oral and Maxillofacial Reconstructive Surgery, Kyushu Dental College, Kitakyushu 803-8580, Japan

⁎ Corresponding authors at: Department of NeuropsychiSciences, Kyushu University, Maidashi 3-1-1, Higashi-Tel.: +81 92 642 5627; fax: +81 92 642 5644.

E-mail addresses: takahiro@npsych.med.kyushu-u.ac.jamonji@hf.rim.or.jp (A. Monji).

0920-9964/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.schres.2013.09.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 March 2013Accepted 4 September 2013Available online 4 October 2013

Keywords:OligodendrocyteMicrogliaSchizophreniaAntipsychoticsMinocyclineTumor necrosis factor-alpha (TNF-α)Nitric oxideInterferon-gamma (IFN-γ)

Recent imaging studies have indicated that the pathophysiology of schizophrenia is closely related to whitemat-ter abnormalities and microglial activation. Additionally, recent clinical trials have suggested that atypical anti-psychotics may have brain protective properties and that minocycline, an antibiotic with inhibitory effects onmicroglial activation, improves symptoms of schizophrenia. We have reported that not only atypical antipsy-chotics with dopamine D2 receptor (D2R) antagonism but also aripiprazole, a unique antipsychotic drug withD2R partial agonism, inhibit microglial activation in vitro. Thus, atypical antipsychotics may exert a beneficial in-fluence on bothmicroglia and oligodendrocytes, while the underlyingmechanisms have not been clarified. Here,we investigatedwhether antipsychotics suppress oligodendrocyte damage by inhibitingmicroglial activation uti-lizing a co-culture model with microglia and oligodendrocytes. Pretreatment of aripiprazole and minocyclinesuppressed apoptosis of oligodendrocytes in the co-culturemodel with interferon-γ (IFN-γ)-activatedmicroglia,while haloperidol, a traditional antipsychotic drug, did not. Aripiprazole and minocycline inhibited the produc-tion of tumor necrosis factor-alpha (TNF-α) from IFN-γ-activated microglia. Moreover, aripiprazole andminocycline attenuated the phosphorylation of signal transducer and activator of transcription 1 (STAT1) in mi-croglia. Overall, our results suggest that aripiprazole andminocyclinemay have antipsychotic effects through re-ducing oligodendrocyte damage caused by microglial activation. These results put forward a novel therapeutichypothesis in schizophrenia research. Future in vivo studies to confirm the present results should be performed.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Multiple theories have been put forth regarding the pathogenesis ofschizophrenia, while the underlying mechanisms remain to be identi-fied (Fatemi and Folsom, 2009; Jaaro-Peled et al., 2009). Recent imagingstudies have shown that first-episode schizophrenia patients have a sig-nificant volume reduction inwhitematter with abnormal brain connec-tivity (Price et al., 2006; Schlosser et al., 2007). Deviant myelinationof schizophrenia patients has been evident in postmortem studies(Uranova et al., 2004; Uranova et al., 2007; Bernstein et al., 2009) andimaging studies (Miyata et al., 2009; Kubota et al., 2011; Kubota et al.,2013). Combined with evidence of dysregulation of myelination-related

atry, Graduate School of Medicalku, Fukuoka 812-8582, Japan.

p (T.A. Kato),

ghts reserved.

genes, a disruption of oligodendrocyte function in schizophrenia hasbeen strongly implicated (McCullumsmith et al., 2007).

Mittelbronn et al. demonstrated that local distribution of microgliain the normal adult human brain differs by up to one order ofmagnitudeand that there is significantly more microglia in white matter than ingray matter (Mittelbronn et al., 2001). Therefore, microglial activationplays an important role especially in white matter disorders (Schniederand Dwork, 2011). Microglial cytotoxicity of oligodendrocytes is medi-ated through free radical-related molecules (nitric oxide (NO) and/orperoxynitrite) and pro-inflammatory cytokines such as tumor necrosisfactor-alpha (TNF-α) (Buntinx et al., 2004; Steelman and Li, 2011).TNF-α is known to compromise the growth of oligodendrocytes andthe expression of mRNA for myelin basic protein (MBP) in cultures(Cammer and Zhang, 1999). Furthermore, TNF-α is reported to inhibitthe survival and proliferation of oligodendrocyte progenitors and theirsubsequent differentiation into mature myelinating phenotypes(Feldhaus et al., 2004). Recent postmortem and imaging studies havesuggested the microglial activation (Radewicz et al., 2000; Steiner et al.,2006, 2008c; van Berckel et al., 2008; Doorduin et al., 2009; Takano

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et al., 2010) and oligodendrocyte dysfunction (Uranova et al., 2004;Uranova et al., 2007; Bernstein et al., 2009) in schizophrenia patients.Based on the above evidence, microglial activation may be involved inthe pathological process of schizophrenia by damaging oligodendrocytes.

Various antipsychotics, which had classically been regarded tomodu-late solely neurons and synaptic networks, have recently been revealed tohave direct anti-inflammatory properties on activated microglia from aseries of in vitro studies (Kato et al., 2007, 2011a, 2013).Wehave reportedthat not only antipsychotics with dopamine D2 receptor (D2R) antago-nism but also aripiprazole with D2R partial agonism inhibit microglialactivation in vitro (Kato et al., 2008, 2011b). In spite of a different phar-macological profile, aripiprazole is effective against the positive andnegative symptoms of patients with schizophrenia like other antipsy-chotics with lower side effects (Kasper et al., 2003; Potkin et al., 2003;Tandon et al., 2006). In addition, aripiprazole has been proved to be ef-fective not only for schizophrenia but also for other psychiatric disor-ders such as major depressive disorders and bipolar disorders (Kecket al., 2006; Berman et al., 2007). On the other hand, minocycline, asemi-synthetic tetracycline antibiotic, is known to be one of the mostwell-known inhibitors of microglial activation (Yrjanheikki et al., 1998;Du et al., 2001). Minocycline, which can easily cross the blood–brainbarrier, has been reported to provide neuroprotection via suppressingmicroglial activation in a number of neuronal disorders includingamyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington'sdisease and Alzheimer's disease (Kriz et al., 2002; Van Den Boschet al., 2002; Plane et al., 2010). Recent studies using animal models ofschizophrenia have suggested that minocycline can be beneficial forthe treatment of schizophrenia (Zhang et al., 2007; Fujita et al., 2008;Mizoguchi et al., 2008; Levkovitz et al., 2010). Furthermore, therapeuticimprovement in psychotic symptoms has been demonstrated byminocycline in patients with schizophrenia (Ahuja and Carroll, 2007;Miyaoka et al., 2007; Miyaoka, 2008; Chaves et al., 2010; Levkovitzet al., 2010; Kelly et al., 2011; Chaudhry et al., 2012).

To the best of our knowledge, the protective effects of antipsychoticsand minocycline on oligodendrocytes via the modulation of microglialinflammatory responses have never been reported. Our previous inves-tigations have suggested that aripiprazole is the most effective antipsy-chotic, which directly and significantly inhibits microglial activationin vitro (Kato et al., 2008, 2011b). In the present study, we thus investi-gate the underlying mechanism of how aripiprazole, haloperidol andminocycline affect the degenerative process of oligodendrocytes viathe modulation of microglial activation utilizing a co-culture modelwith microglia and oligodendrocytes.

2. Materials and methods

All experimental procedureswere conducted in accordancewith theStandard Guidelines for Animal Experiments of the Graduate School ofMedical Sciences, Kyushu University, Fukuoka, Japan.

2.1. Chemicals and reagents

Aripiprazole was generously provided from Otsuka PharmaceuticalCo., Ltd. (Tokyo, Japan). Recombinant interferon-gamma (IFN-γ) waspurchased from R&D systems (Minneapolis, MN, USA). Minocycline hy-drochloride, haloperidol and other chemical reagents were purchasedfrom Sigma Chemicals (St. Louis, MO, USA). Minocycline was dissolvedwith distilledwater. Aripiprazole andhaloperidolwere dissolved initial-ly into 20 mMwith dimethyl sulfoxide (DMSO) and then were dilutedinto the final concentration for each experiment.

2.2. Cell cultures

Primary oligodendrocyte cultures were prepared as described (Chenet al., 2007) with the following modifications. Using a dissecting mi-croscope, the subventricular zone was removed from the 3-day-old

Sprague–Dawley rats (n = 6). The tissues were mechanically disso-ciated into single cells on 100-mm pore nylon mesh cell strainers(BD Falcon, Franklin Lakes, NJ, USA), and collected in PBS. Cells werethen filtered through 40-mm nylon mesh cell strainers (BD Falcon).The cell suspensionwas centrifuged for 5 min at 800 g, and the cell pel-let was resuspended in cold neurobasal medium (NB; Gibco, Carlsbad,CA, USA)/B27/L-Glu/PSA/EGF/bFGF consisting of NB supplementedwith B27 (Gibco), L-glutamine (L-Glu; Gibco), penicillin/streptomycin/amphotericin B (Gibco), 20 ng/mL EGF (Sigma-Aldrich) and 10 ng/mLbasic fibroblast growth factor (bFGF; Sigma-Aldrich). The cell sus-pension was plated in poly-L-lysine (PLL) coated 12 well plates at1.5 × 105 cells per well. Around day 7, T3 (Sigma-Aldrich) and T4(Sigma-Aldrich) were added into the cultured-media (T3: 30 μg/mL,T4: 40 μg/mL). 14 days after T3 and T4 addition, differentiated oligo-dendrocytes were subjected to further experiments.

Primary microglia are known to be a mixture of heterogeneous mi-croglia. To avoid contamination of heterogeneous microglia population,immortalized microglia are frequently used especially in co-culture ex-periments (Bi et al., 2011; Cui et al., 2012; Dentesano et al., 2012; Gresa-Arribas et al., 2012). We thus decided to use a single microglial cell linein our co-culture system. The immortalized rat microglial cell line HAPI(Cheepsunthorn et al., 2001; Zhou et al., 2008) was kindly provided byDrs. Morales NP. and Hyodo F. (Kyushu University). The cells were cul-tured in DMEM (low glucose; Invitrogen), 5% FBS (Hyclone), 4 mMglu-tamine (Invitrogen), 100,000 U/L penicillin G, 100 mg/L streptomycin(Mediatech), and maintained in 5% CO2 at 37 °C.

2.3. Co-culture experiment with microglial cell line and primaryoligodendrocyte

The HAPI microglial cells were plated on tissue culture inserts for12-well plates (Greiner Bio One GmbH, Frickenhausen, Germany) at adensity of 5.0 × 105 cells. The microglial cells were incubated for 12 hin the presence or absence of aripiprazole (20 μM), haloperidol (20 μM)or minocycline (10 μM). In order to minimize oligodendrocyte damagedue to pretreatment of IFN-γ challenge,wehave chosen to treat the targetdrugs (aripiprazole, haloperidol andminocycline) before IFN-γ challenge.This experimental design is represented in Fig. 1B. Each tissue cultureinsert was placed on the primary oligodendrocytes in 12-well plates(Fig. 1A). 100 U/mL IFN-γ (R&D) was added immediately to the culturedmedium of HAPI cells and primary oligodendrocytes. 24 h after the incu-bation, the tissue culture inserts were removed from the 12-well plates.Morphological changes in the primary oligodendrocytes were observedunder a phase-contrast microscope. This experiment was independentlyconducted three times.

2.4. Immunofluorescence

Co-cultured primary oligodendrocytes were rinsed twice in 0.1 mol/LHEPES/KOH and were fixed with 4% paraformaldehyde for 10 min andthen rinsedwith 0.1 mol/L HEPES/KOH for 10 min. Indirect immunofluo-rescence was performed using the following antibodies: rabbit anti-cleaved caspase-3 polyclonal antibody (1:200 dilution, Cell Signaling,Danvers, MA) and mouse anti-PLP monoclonal antibody (1:100 dilution,Abcam, Cambridge, MA). Cells were incubated in primary antibodiesdiluted in 0.1% Triton-X 100 in PBS containing 5% normal goat serum at4 °C overnight. After rinsing twice with PBS for 5 min, FITC- or Texasred-conjugated secondary antibodies (Southern Biotech, Birmingham,AL, USA) were used for detection. Fluorescent images were capturedwith a fluorescence microscope (OLYMPUS BX50; Olympus Co. Ltd,Tokyo, Japan).

For phosphorylated STAT1 (pSTAT1) immunofluorescence, theHAPI cells were plated on 24-well tissue culture plates (Greiner BioOne GmbH) at 2 × 104 cells per 500 μL medium. The cells werepre-incubated in the presence or absence of minocycline (10 μM)or aripiprazole (10 μM) for 12 h followed by the addition of 100 U/mL

Fig. 1. Experimental design of co-culture experiment and themorphological changes of primary oligodendrocytes. (A) The co-culture system of primary oligodendrocytes/HAPI microglialcells. HAPI cells are pre-incubated with haloperidol, aripiprazole or minocycline for 12 h followed by a co-culture with primary oligodendrocytes in IFN-γ-containing medium for 24 h.(B) The time course of applying each drug and grouping into five as follows: Group 1 (no treatment HAPI cells + primary oligodendrocyte), Group 2 (IFN-γ-alone stimulated HAPIcells + primary oligodendrocyte), Group 3 (minocycline/IFN-γ stimulated HAPI cells + primary oligodendrocyte), Group 4 (aripiprazole/IFN-γ stimulated HAPI cells + primary oligo-dendrocyte) andGroup5 (haloperidol/IFN-γ stimulatedHAPI cells + primary oligodendrocyte). Abbreviations inB:M,minocycline;A, aripiprazole; H, haloperidol. (C) Themorphologicalchanges of oligodendrocytes after co-culture experiment. Low (a) and high (b–e) magnification of each condition is shown. In Group 2, although some normal shapes were observed(b), degenerative changes such as retraction of their processes, (c) swollen cytoplasm and large vacuoles (d), and roundish morphology (e) were mainly observed. Degenerative changeswere not evident in Group 3 and Group 4, but fragmented shapes were observed in Group 5. Abbreviations in B: MINO, minocycline; ARI, aripiprazole; HAL, haloperidol.

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IFN-γ to cultured medium for 30 min (n = 4, respectively). Then, thecells were rinsed twice in 0.1 mol/L HEPES/KOH and were fixed with4% paraformaldehyde for 10 min and rinsed with 0.1 mol/L HEPES/KOH for 10 min. They were blocked with 1.0% bovine serum albuminin PBS containing 0.3% Triton X-100 and 0.05% sodium azide for 1 h at20 °C. After blocking, they were incubated in rabbit phospho-STAT1 anti-body (Ser 727) (1:1000; Cell Signaling Technology, Danvers, MA) for4 days at 20 °C. After rinsing three times with PBS for 5 min, a mixtureof Cy3-conjugated secondary antibody (1:300; Jackson ImmunoResearchLaboratories) and YOYO-1 (1:10,000; Invitrogen, Carlsbad, CA) was usedfor detection. Fluorescent images were captured with a fluorescence mi-croscope (Axio ScopeA1, Carl Zeiss, Oberkochen,Germany) andanopticalsectioning system (Apotome.2, Carl Zeiss).

2.5. Digital image analysis

Four cultureswere selected from each condition, and two specimensper culture were processed for pSTAT1 and YOYO-1 staining. Eight-bitblack and white images were captured with a digital camera (RetigaEX, Roper Scientific, San Diego, CA) using a dry objective lens (×20,NA). The same capturing conditions of digital camera were used for allspecimens.

The intensity of pSTAT1wasmeasured using ImageJ 1.42 (NIMH). Theoutlines of nucleus were traced manually, and then the mean score ofpSTAT1 intensity was measured for each. To avoid double counting, weused the Measure and Label plugin. All data were presented as the

mean ± SEM and were analyzed by a one-way ANOVA, followed byTukey's HSD test. Statistical significance was established at a level ofp b 0.05.

For preparing illustrations, selected images were processed usingAdobe Photoshop CS4 (Adobe Systems,Mountain View, CA). Only bright-ness and contrast were adjusted for the whole frame, and no part of aframe was enhanced or modified in any way.

2.6. Real time-polymerase chain reaction (real time-PCR)

The HAPI microglial cells were incubated for 12 h in the presence orabsence of minocycline (1 μM). 100 U/mL IFN-γ (R&D)was then addedto the culturedmedium of HAPI microglial cells. Total RNAwas isolated6 h after the incubation by the RNeasy Mini Kit (Qiagen). The reversetranscription (RT) reaction was performed by the superscript II(Invitrogen, Carlsbad, CA). The expression of iNOS and TNF-α wasquantified using SYBR Green master mix (TOYOBO, Osaka, Japan).The gene-specific primers are used as follows: iNOS (forward: 5′-TGTCTTGGTTCTATGGAATCACTC-3′ and reverse: 5′-CGTTCACAGACCTAATCTAAACCT-3′), TNF-α (5′-ATGATCCGAGATGTGGAACTGGCA-3′ and reverse: 5′-AATGAGAAGAGGCTGAGGCACAGA-3′), β-actin(forward: 5′-TGTCTTGGTTCTATGGAATCACTC-3′ and reverse: 5′-CGTTCACAGACCTAATCTAAACCT-3′). The linearity of the amplificationsas a function of cycle number was tested in preliminary experi-ments, and the mRNA expression levels were normalized to the ex-pression levels of the house keeping gene β-actin.

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2.7. Nitrite production assessment

The accumulation of NO2−, a stable end-product, extensively used as

an indicator of NO production by cultured cells, was assayed using theGriess reaction. The HAPI cells were plated on 96-well tissue cultureplates (Greiner Bio One GmbH) at 1 × 105 cells per 200 μL medium.The cells were pre-incubated in the presence or absence of minocyclinefor 12 h followed by the addition of 100 U/mL IFN-γ to the culturedme-dium. After 24 h of incubation, the cell-free supernatants were assayedfor NO accumulation using a Griess kit (Dojindo Molecular Technolo-gies, Kumamoto, Japan) and a plate reader (Labsystems Multiscan MS,Frankfurt, Germany).

2.8. Enzyme-linked immunosorbent assay

TNF-α concentrations in the conditionedmedia fromHAPI cells thatwere pre-incubated with minocycline (1 μM) and subsequently stimu-latedwith 100 U/mL IFN-γweremeasured by enzyme-linked immuno-sorbent assay (ELISA) with Quantikine kits for Rat TNF-α (Invitrogen)according to the manufacturer's instructions. Optical densities weredetermined by measurement of indicator color shift at 450 nm on amicroplate reader (Multiscan MS, Labsystems, Finland).

2.9. Cell viability

Cell viability of the present experimental conditionswas determinedby colorimetric measurements of the reduction product of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT). Theoriginal medium was removed from the 96-well plates, and the cellswere incubated for 2 h at 37 °C in the presence of phenol red-free min-imum essential medium (Invitrogen) containing 0.5 mg/mL MTT. A100 μL MTT lysis buffer (5% sodium dodecyl sulfate (SDS) and 5 mMHCl) was then added to each well, and the plates were incubated at37 °C overnight to dissolve the formazan that had formed in the wells.MTT is reduced to formazan in themitochondria of living cells. ReducedMTT was measured by means of a plate reader (Labsystems MultiscanMS, Frankfurt, Germany) at a wavelength of 570 nm.

2.10. Quantitative analyses and statistics

To determine the expression ratio of cleaved caspase-3 in oligoden-drocytes, the number of DAPI positive nuclei and the cleaved caspase-3positive oligodendrocytes was counted from 4 optic fields for each con-dition.Next, the intensity of pSTAT1 inHAPI cellswasmeasured for eachcondition. All data are represented as the means ± standard error ofmeans and they were analyzed by a one-way analysis of variance(ANOVA). The Tukeymethodwasused for post hoc comparisons. Signif-icancewas established at a level of p b 0.05. All statistical analyses wereperformed with SPSS version 13.0 for Windows.

3. Results

3.1. Cell viability

In the present experiments, the cell viability of HAPI microglialcells was not affected by aripiprazole (20 μM), haloperidol (20 μM)and minocycline (10 μM) (data not shown). Therefore, we used thesedrugs under this concentration in the present study.

3.2. Protective effects of minocycline and antipsychotics on oligodendrocytedamage

IFN-γ is a typical Th1 cytokine and a major immuno-activator re-leased by infiltrating T cells as well as activated microglia in the CNS(Kawanokuchi et al., 2006). Elevated mRNA levels of IFN-γ have beenreported in the brain of schizophrenia patients (Freudenreich et al.,

2010), and we thus applied IFN-γ as a stimuli for microglial cells. Toinvestigate direct interactions between microglial activation and thedegeneration of oligodendrocytes, a co-culture experiment with HAPImicroglial cells and primary oligodendrocytes was performed utilizingthe tissue culture inserts (Fig. 1A). In order to evaluate the protective ef-fects of aripiprazole, haloperidol and minocycline, we compared fivedifferent groups with various drugs. In order to minimize oligodendro-cyte damage due to pretreatment of IFN-γ challenge, we have chosen totreat the target drugs (aripiprazole, haloperidol and minocycline)before IFN-γ challenge. Our experimental design is represented asFig. 1B. Group 1 [Negative Control] showed a differentiated oligo-dendrocyte and exhibited the morphology of multipolar mature oli-godendrocytes rather than bipolar early precursor cells (Woodruffet al., 2001; Jakovcevski and Zecevic, 2005) (Fig. 1C; Group 1; aand b). Although Group 2 [Positive Control; INF-γ] showed a typicalmorphology of oligodendrocyte (Fig. 1C; Group 2; a and b) partly, de-generative changes of the oligodendrocyte were frequently observedsuch as retraction of their processes (Fig. 1C; Group 2; c) swollen cyto-plasm/large vacuoles (Fig. 1C; Group 2; d) and roundish morphology(Fig. 1C; Group 2; e) compared to Group 1. In contrast, Group 3[INF-γ + minocycline] (Fig. 1C; Group 3; a and b) and Group 4[INF-γ + aripiprazole] (Fig. 1C; Group 4; a and b) displayed no obviousdegenerative changes of oligodendrocytes. Group 5 [INF-γ + haloperi-dol] showed the fragmentation of oligodendrocytes processes (Fig. 1C;Group 5; a and b), indicative of cytoskeletal breakdown.

A striking degeneration of oligodendrocytes was observed in thetreatment of IFN-γ (Fig. 1C;Group 2).We then performed immunocyto-chemistry for proteolipid protein (PLP), a mature oligodendrocytemarker, and cleaved caspase-3 to confirm whether the apoptosis wascaused bymicroglial activation. PLPpositive cells in Group 2weremark-edly immunoreactive for cleaved caspase-3 (Fig. 2A; Group 2). The ex-pression of cleaved caspase-3 was inhibited by aripiprazole andminocycline (Fig. 2A; Group 3 andGroup 4). In the haloperidol pretreat-ment group the immunoreactivity of cleaved caspase-3 was observedslightly (Fig. 2A; Group 5). A quantitative analysis showed that theexpression ratio of the cleaved caspase-3 was significantly lower inGroup 3 and Group 4 (Fig. 2B) than in Group 2. Group 5 tended to belower than Group 2 (Fig. 2B). These results suggest that aripiprazoleand minocycline are equally efficacious drugs in the inhibition of oligo-dendrocyte damage.

3.3. Minocycline inhibits TNF-α released by IFN-γ-activated microglia, butnot nitric oxide

The above results strongly indicate that IFN-γ-activated microglialcells secrete inflammatorymolecules at high enough levels to be degen-erative to primary oligodendrocytes in their local environment. Microg-lia are reported to be a major source of locally produced NO and TNF-α(Li et al., 2005, 2008). In addition, previous studies have demonstratedthat oligodendrocyte cell death is dependent on TNF-α production (Liet al., 2008). Therefore, we hypothesize that the protective effect ofaripiprazole and minocycline on oligodendrocyte damage may be in-duced by inhibiting TNF-α from IFN-γ-activated microglial cells. Previ-ously, we have shown that aripiprazole significantly inhibits TNF-αgeneration from IFN-γ-activated microglial cells (Kato et al., 2008),however the effect of minocycline has not been reported. To investigatewhether the detrimental effects of minocycline were attributable toTNF-α production on IFN-γ-activated microglial cells, we performedquantitative PCR for two targets of iNOS and TNF-α. These resultsshowed that the iNOSmRNA level in minocycline-pretreated microglialcells was not altered compared to that of IFN-γ alone-treatedmicroglialcells (Fig. 3A). In contrast, the expression of TNF-α mRNA was signifi-cantly reduced in minocycline-pretreated microglial cells (Fig. 3B).Therefore, we then measured the NO and TNF-α releases. The releasedNO was unaltered in minocycline pretreated microglial cells (Fig. 3C). Incontrast, the secreted TNF-α was significantly inhibited by minocycline

Fig. 2.Double immunofluorescence for PLP and cleaved caspase-3. (A) Numerous cleaved caspase-3-positive cells were observed in Group 2 compared to Group 1. In contrast, in Group 3and Group 4, the cleaved caspase-3 expression was inhibited. In Group 5, the expression of cleaved caspase-3 was slightly observed, but there is no significance compared to Group 2.(B) The results based on three independent experiments were expressed as the ratio of the values obtained in cleaved caspase-3 positive nuclei or DAPI positive nuclei. Each resultwas shown as the mean ± SEM (##p b 0.001 in comparison to the control group. ***p b 0.001 in comparison to the IFN-γ stimulated group). Scale bar = 50 μm.

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(Fig. 3D). These results suggest that minocycline protects againstoligodendrocyte degeneration through inhibiting at least the pro-duction of TNF-α from microglial cells as well as aripiprazole.

3.4. Aripiprazole and minocycline inhibit phosphorylation of STAT1

Previous reports have suggested that increased transcription of TNF-α mRNA by IFN-γ uses the JAK-STAT1 pathway (Takagi et al., 2005). Todetermine whether minocycline or aripiprazole inhibit an IFN-γ-mediated increase in the expression of pSTAT1, we performed immuno-cytochemistry for pSTAT1 in these drugs-pretreated HAPI cells. pSTAT1was strongly stained in the nucleus of positive control cells (IFN-γ-treat-ed group) compared to control cells. In contrast, inhibition of phosphor-ylated STAT1 was observed in minocycline- or aripiprazole-pretreatedcells (Fig. 4A). Additionally, the intensity of pSTAT1 was significantly at-tenuated to control levels with pretreatment by minocycline oraripiprazole (Fig. 4B).

4. Discussion

This is the first report to demonstrate that pretreatment ofaripiprazole and minocycline inhibits oligodendrocyte damage bysuppressing IFN-γ-activated microglial cells.

We have already demonstrated that aripiprazole inhibits TNF-α pro-duction from IFN-γ-activatedmicroglial cells via suppressing the eleva-tion of intracellular calcium signaling, not via D2R receptor-relatedsignaling (Kato et al., 2008). In the present study, we have proved thataripiprazole andminocycline have the potential to inhibit phosphoryla-tion of STAT1 and lead to the attenuation of TNF-α production in

Fig. 3. Anti-inflammatory effects of minocycline onmicroglial cells. Real time PCR results showdid not inhibit the induction of iNOS expression (A). TNF-αwas induced by IFN-γ-stimulationreleasewas induced by IFN-γ in HAPI cells, andminocycline did not inhibit NO release (C).Minowas shown as the mean ± SEM (*p b 0.05; **p b 0.01 in comparison to the IFN-γ or control g

microglial cells. The JAK1/STAT1 pathway is activated by IFN-γ and isan essential regulator of diverse cellular functions including cytokineproduction (Gough et al., 2008). The plasma concentration of IFN-γwas reported to be significantly higher in patients with schizophreniathan in controls (Kim et al., 2004), and minocycline has been reportedto be effective in the treatment of schizophrenia (Ahuja and Carroll,2007; Miyaoka et al., 2007; Miyaoka, 2008; Chaves et al., 2010;Levkovitz et al., 2010; Kelly et al., 2011; Chaudhry et al., 2012). Togeth-er, it is suggested that abnormal constitutive activation of JAK1-STAT1pathway may be related to the pathophysiology of schizophrenia. Fur-ther investigations are required regarding these molecules.

The primary target of minocycline and aripiprazole on microglialcells has not beenwell clarified. Giuliani et al. have shown the direct ac-tion ofminocycline on activated T cells and onmicroglia, which resultedin the decreased ability of T cells to contact microglia (Giuliani et al.,2005). Nikodemova et al. have shown that the inhibitory effect ofminocycline on microglial activation is stimulus-specific and that it alsodownregulates MHC class II expression in microglia (Nikodemova et al.,2007). Horvath et al. have shown the inhibitory effect of minocycline onmicroglial migration (Horvath et al., 2008). Further evidence of other cy-tokine pathways interacting with minocycline is required to confirm theprecise target ofminocyclinewith a particular focus onmicroglia. The ex-pression of TNF-α mRNA has been reported to be ablated by inhibitingphosphorylation of ERK and p38 (Rutault et al., 2001; Fan et al., 2007).Furthermore, it has been reported that minocycline attenuates the phos-phorylation both of P38 and ERK (Nikodemova et al., 2006), and also weconfirmed the same results (data not shown). This may indicate that thesuppression of phosphorylated ERK and p38 by minocycline results inthe attenuation of TNF-α release from activated HAPI microglial cells.

ed that the relative expression of iNOSwas upregulated by IFN-γ stimulation, minocyclineat mRNA level, and minocycline significantly attenuated the expression of TNF-α (B). NOcycline significantly inhibited TNF-α release by IFN-γ-activated HAPI cells (D). Each resultroup). Abbreviations in A, B, C and D: MINO, minocycline.

Fig. 4. Immunofluorescence for pSTAT1. pSTAT1 immunoreactivity in the presence or ab-sence of aripiprazole or minocycline followed by the addition of IFN-γ (A). pSTAT1 inten-sity is significantly attenuated inMINO- or ARI-pretreated cells compared to IFN-γ-treatedHAPI cells (B). Each result was shown as the mean ± SEM (#p b 0.05 in comparison to thecontrol group. *p b 0.05 in comparison to the IFN-γ or control group). Scale bar = 20 μm.Abbreviations in A and B: MINO, minocycline; ARI, aripiprazole.

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Aripiprazole may have similar intracellular mechanisms as report-ed in a previous in vitro study (Kato et al., 2008). Further investiga-tions should be conducted to clarify deeper underlying molecularmechanisms.

Traditionally, most studies on schizophrenia have focused on al-terations in gray matter, however, recent evidence suggests thatneurodevelopmental problems also occur in white matter, especiallyin oligodendrocytes and in the myelin that these cells produce. Xiaoet al. reported that demyelination in the brains of mice which hadbeen administered a copper chelator, cuprizone, led to abnormal behav-iors such as spatial workingmemory impairment and hyperlocomotion(Xiao et al., 2008). Because the former is regarded as an endophenotypeof schizophrenia, they proposed that cuprizone-fedmicemay representan animal model of schizophrenia. This study and their following study

(Zhang et al., 2008) also revealed that quetiapine, an atypical antipsy-chotic, ameliorated both the demyelination and the abnormal behav-iors, underscoring the causal relationship of the demyelination withsymptoms of schizophrenia. In addition to the above reports, it hasbeen reported that the numbers of CD68+,MHC class II+, and allograftinflammatory factor-1 (AIF-1)+ microglia, which are identified asmicroglial activation (Renaud et al., 2005), are higher in white matterthan in gray matter (Mittelbronn et al., 2001). Therefore, microglial ac-tivation may cause more damage in white matter than in gray matter,and our in vitro co-culture method would help to reenact the in vivomechanisms of schizophrenia by investigating interaction betweenmi-croglia and oligodendrocytes. Recent MRI imaging studies suggest thatantipsychotics impact on intracortical myelination in schizophrenia pa-tients (Bartzokis et al., 2007, 2009, 2012). Bartzokis et al. have shownthe protective properties of risperidone, an atypical antipsychotic,compared with fluphenazine, a typical antipsychotic, on intracorticalmyelination in schizophrenia (Bartzokis et al., 2009). When comparedwith the healthy controls and fluphenazine-treated schizophrenia pa-tients, risperidone-treated schizophrenia patients showed significantlyhigher intracortical myelin volume. In the present study, we have notinvestigated the effects of risperidone, while our previous studieshave shown that risperidone and quetiapine, but not haloperidol, inhib-it IFN-γ-activated microglial activation in vitro (Kato et al., 2007; Bianet al., 2008). Therefore, based on the present study, we hypothesizethat risperidone and quetiapine may have protective properties onintracortical myelination and white matter protection via suppressingmicroglial activation, and further in vitro and in vivo investigationsshould be conducted.

Kato et al. reported that aripiprazole inhibits the release of both NOand TNF-α from activatedmicroglia, while haloperidol inhibits only NOrelease (Kato et al., 2007, 2008). Additionally, one recent report suggeststhat NO production does not appear to correlate with oligodendrocyteprocess retraction and cell death, while oligodendrocytes are highlysensitive to TNF-α (Li et al., 2008). Upregulation of TNF-α productionhas been reported in the prefrontal cortex of schizophrenia patients(Paterson et al., 2006). In our experiments,minocyclinewas found to in-hibit at least TNF-α release from HAPI microglial cells and resulted in theprotection of oligodendrocyte degeneration. In addition, aripiprazole wasfound to have a similar effect asminocycline on oligodendrocyte degener-ation induced bymicroglial activationwhile in the haloperidol-pretreatedgroup, oligodendrocytes exhibited focal bead-like swellings at their pro-cesses, and this change is one of the characteristics observed in oligoden-drocyte degeneration (Li et al., 2008). These results thus suggest thatminocycline as well as aripiprazole suppress oligodendrocyte degenera-tion through the inhibition of TNF-α release fromactivatedmicroglia. Fur-ther studies are required to develop deeper understandings of inhibitorymechanisms of NO and TNF-α release utilizing other activators ofmicrog-lia such as LPS and PMA. To investigate the direct influence of TNF-α, in-hibitory experiments with TNF-α inhibitors are required. In addition, ourprevious report suggests that haloperidol does not suppress the release ofIL-6 or IL-1β from interferon-γ-activated microglia (Kato et al., 2007).Therefore, in our co-culture experiment, these cytokines may also be dif-ferently affected by aripiprazole and haloperidol. Further experimentsshould be conducted.

On the other hand, one recent report suggests that minocycline hasthe potential to promote favor remyelination directly on oligodendro-cytes (Defaux et al., 2011). In our experiments, only HAPI microglialcells were pre-treated with minocycline, but there is the possibilitythat a small amount of minocycline adhered to the tissue culture insertmay be acting directly on oligodendrocytes. Further research is thus re-quired to confirm the effects of minocycline and antipsychotics on oli-godendrocyte remyelination directly or indirectly via microglial cells.Contrary to our results, Miller et al. have demonstrated that activatedmicroglia increase survival and reduce apoptosis of mature oligoden-drocytes (Miller et al., 2007). Their study used LPS for the stimulationof microglia instead of IFN-γ. This may explain the difference.

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On the other hand, in our experimental design, the target drugs(aripiprazole, haloperidol and minocycline) were administered as pre-treatment to IFN-γ activation, and not after IFN-γ challenge. Ideally, tomake a therapeuticmodel of diseases (first inducing pathology, then in-tervening therapy), we should have added IFN-γ before the target drugtreatment. However, IFN-γ challenge before the target drugs seems toinduce significant oligodendrocyte damage in all the experimental con-ditions. Thus, in order tominimize oligodendrocyte damage due to pre-treatment of IFN-γ challenge, we have chosen to treat the target drugsbefore IFN-γ challenge. While, in vivo it is expected that microglia acti-vation will occur before the administration of minocycline and antipsy-chotics. Thus, the present experimental design may have not reflectedactual process in vivo. Therefore, further detailed mechanisms shouldbe investigated in the future in vivo experiments.

Recently, several studies have reported elevated S100B serum levels inschizophrenia (Steiner et al., 2009). S100B is synthesized in and releasedfromastrocytes andoligodendrocytes, acting as a dose-dependent growthfactor for neurons and glial cells (Steiner et al., 2008b). Nanomolar levelsof S100B stimulate neurite growth and promote neuronal survival.However, micromolar levels of S100B result in opposite effects andcan induce neuronal apoptosis (Van Eldik and Wainwright, 2003). Inaddition, Steiner et al. have reported that schizophrenia may be causedby white matter oligodendrocyte damage of dysfunction, associatedwith a release of S100B into body fluid (Steiner et al., 2008a). In our ex-periments, pretreatment of aripiprazole and minocycline inhibited oli-godendrocyte damage by suppressing IFN-γ-activated microglial cells.Thus, S100B may be modulated by aripiprazole and/or minocycline inour co-culture model. Further studies are required.

Our results may lead to a deeper understanding of the patho-physiology of schizophrenia and establish novel therapeutic strate-gies for schizophrenia with anti-inflammatory/immunosuppressiveagents. Further molecular mechanisms of the inhibitory effect ofaripiprazole and minocycline on microglial activation should be clarified.In vivo studies to confirm the present results should also be performedusing animal models of schizophrenia such as the cuprizone model.

Role of funding sourceThis work was financially supported by Grant-in-Aids from the Japan Society for the

Promotion of Science (JSPS) [to YS, TAK, AM, YM and SK], and from theMitsubishi PharmaResearch Foundation [to TAK and HH].

ContributorsAll authors contributed substantially to the scientific process leading up to thewriting

of the present paper. TAK and AM, the principal investigators of the present research, andYS, thefirst author,made the conception and design of the project andwrote the protocol.The performance of experiments and the data analysis/interpretation were done by YS,TAK, AM, YM, HH and MSK. YS wrote the first draft of the manuscript. Critical revisionsof the manuscript were made by TAK, AM, DY and SK. All authors contributed to andhave approved the final manuscript.

Conflict of interestAll authors declare that they have no financial conflict of interest.

AcknowledgmentsThe authorswould like to thankDr. Masahiro Ohgidani, Dr. Noriaki Sagata, Dr. Yusuke

Yamauchi, and Dr. Kohei Hayakawa (Molecular & Translational Psychiatry Unit, Depart-ment of Neuropsychiatry, Kyushu University) and Dr. Daichi Muratsu (Division of OralandMaxillofacial Oncology, Faculty of Dental Science, KyushuUniversity) for fruitful com-ments and technical supports.

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