The Potent PDE10A Inhibitor MP-10 (PF-2545920) Suppresses Microglial Activation in LPS-Induced Neuroinflammation and MPTP-Induced Parkinson’s Disease Mouse Models

Do-Yeon Kim 1 • Jin-Sun Park1 • Yea-Hyun Leem 1 • Jung-Eun Park1 • Hee-Sun Kim 1,2

Received: 2 May 2020 / Accepted: 8 July 2020
Ⓒ Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
MP-10 (PF-2545920) is a selective inhibitor of phosphodiesterase 10A (PDE10A), an enzyme highly enriched in the striatum, nucleus accumbens, olfactory tubercle, and substantia nigra. The therapeutic effect of MP-10 has been reported in psychiatric and neurodegenerative disorders such as schizophrenia, depression, and Huntington’s disease. However, the effect of MP-10 in Parkinson’s disease (PD) has not been reported to date. In this study, we examined the effect of MP-10 in neuroinflammation and PD mouse models. MP-10 inhibited nitric oxide, tumor necrosis factor alpha, and interleukin (IL)-6 production, while it promoted IL-10 production in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Subsequent western blot and reverse transcription polymerase chain reaction analyses showed that MP-10 reduced the mRNA and protein levels of inducible nitric oxide synthase, cyclooxygenase-2, proinflammatory cytokines, and matrix metalloproteinase-3, −8, and − 9 in LPS-stimulated BV2 cells. Further mechanistic studies revealed that MP-10 exerts anti-inflammatory effects by inhibiting the phosphorylation of c-Jun N-terminal kinase and Akt, reducing the activity of nuclear factor-kappa B/activator protein-1, and upregulating the nuclear factor erythroid 2-related factor 2/antioxidant response element and protein kinase A/cAMP response element-binding protein signaling pathways. The anti-inflammatory effect of MP-10 was confirmed in vivo. Specifically, MP-10 inhibited microglial activation and proinflammatory gene expression in the brains of LPS-injected mice. Moreover, MP-10 rescued behavioral deficits and recovered dopaminergic neuronal cell death in the brains of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mice. MP-10 also reduced microglial activation in this PD mouse model. These data collectively suggest that MP-10 may have therapeutic potential in PD and other neuroinflammatory disorders.

Keywords MP-10 . Microglia . Neuroinflammation . Neuroprotection . Parkinson’s disease . Molecular mechanism

Introduction

Microglia are the brain’s resident immune cells. They main- tain brain homeostasis by phagocytosing apoptotic cells and neuronal synapses and support neuronal survival by releasing trophic factors during neurodevelopment (Cronk and Kipnis 2013; Garden and Moller 2006). Microglia are activated by

* Hee-Sun Kim [email protected]

1 Department of Molecular Medicine and Ewha Medical Research Institute, School of Medicine, Ewha Womans University, 808-1 Magok-dong, Gangseo-gu, Seoul 07804, South Korea
2 Department of Brain & Cognitive Sciences, Ewha Womans University, Seoul, South Korea

brain injury, environmental toxins, infection, and aging pro- cess and release inflammatory and neurotoxic factors such as nitric oxide (NO), reactive oxygen species (ROS), cytokines, prostaglandins, and matrix metalloproteinases (MMPs) (Glass et al. 2010; Liu et al. 2019). A prolonged and unresolved inflammatory response results in chronic neuroinflammation, which promotes neuronal cell death and initiates the onset of neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington’s disease (HD) (Cherry et al. 2014; Subramaniam and Federoff 2017).
PD, the second most common neurodegenerative disease, is caused by progressive degeneration of dopaminergic neu- rons in the substantia nigra pars compacta. The main symp- toms of PD include impaired movement, resting tremor, and rigidity (Sveinbornsdottir 2016). Microglial activation is fre- quently found in the post-mortem brains of PD patients, and

neuroinflammation plays a pivotal role in the pathogenesis of PD (Collins et al. 2012). Activated microglia release neuro- toxic factors that induce dopaminergic neuronal cell death. Moreover, neuroinflammation promotes the spread of α- synuclein aggregates into other brain regions, contributing to the progression of PD (Lee et al. 2014). Thus, the develop- ment of agents that can control neuroinflammation has been suggested as a potential strategy for the treatment of PD (Subramaniam and Federoff 2017).
MP-10 is a selective and potent phosphodiesterase 10A (PDE10A) inhibitor both in vitro and in vivo (Grauer et al. 2009; Schulke et al. 2014). PDE10A is highly expressed in the striatum of the brain and integrates dopaminergic and gluta- matergic signals by modulating cAMP/cGMP levels (Wilson and Brandon 2015). PDE10A affects synaptic transmission, neuronal excitability, and synaptic plasticity. Thus, PDE10A has been suggested to be an important target for the treatment of movement disorders and psychiatric disorders associated with basal ganglia dysfunction (Jankowska et al. 2019). MP- 10, a drug initially developed by Pfizer for the treatment of schizophrenia, has progressed through Phase II clinical trials in humans for the treatment of schizophrenia and HD (Knott et al. 2017; Zagorska et al. 2018). Previous studies have re- ported that MP-10 increases intracellular cAMP/cGMP levels, contributing to striatal dopamine receptor-modulated behav- iors such as sociality and cognition in schizopherenia rodent models (Grauer et al. 2009; Nikiforuk et al. 2016). Given that dopamine receptor agonists are currently used to control PD symptoms, these finding suggest that MP-10 could be an ef- fective PD medication/anti-PD drug. Notably, several studies have demonstrated that D2 receptor activation improves PD pathology in 6-hydroxydopamine-treated rats (Christina et al. 2003; Matsukawa et al. 2007). A recent study reported that MP-10 produces greater induction of c-Fos in dopamine D2 receptor expressing neurons than in D1 neurons in the neostriatum (Wilson et al. 2015). Taken together, these find- ings suggest that MP-10 may have therapeutic potential in PD, HD, and psychiatric disorders.
Although several studies have reported the therapeutic effects of MP-10 in schizophrenia and HD, the effect of MP-10 in PD has not yet been reported. Moreover, the effect of MP-10 on neuroinflammation has not been demonstrated to date. Therefore, in the present study, we investigated whether MP-10 has anti-inflammatory activity in lipopolysaccharide (LPS)-stim- ulated microglia and analyzed the detailed molecular mecha- nisms underlying its anti-inflammatory effect. We confirmed the anti-inflammatory effects of MP-10 in the brains of mice with systemic inflammation. Furthermore, we demonstrated the neu- roprotective and anti-inflammatory effects of MP-10 in a 1-meth- yl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. These data suggest that MP-10 may have thera- peutic potential in PD and other neurodegenerative diseases as- sociated with microglial activation.
Materials and Methods

Reagents and Antibodies

MP-10 (PF-2545920; 2-[[4-(1-methyl-4-pyridin-4-ylpyrazol- 3-yl) phenoxy]methyl] quinolone) was purchased from Selleck Chemicals (Houston, TX, USA). All cell culture re- agents were purchased from Gibco BRL (Grand Island, NY, USA). LPS (Escherichia coli serotype 055:B5) was obtained from Sigma-Aldrich (St. Louis, MO, USA). MPTP was pur- chased from Tokyo Chemical Industry Co. (Tokyo, Japan). All reverse transcription polymerase chain reaction (RT-PCR) reagents and enzymes and electrophoretic mobility shift assay (EMSA) oligonucleotides were purchased from Promega (Madison, WI, USA). Antibodies against the phospho- and total forms of mitogen-activated protein kinases (MAPKs), Akt, and cAMP response element-binding protein (CREB) were purchased from Cell Signaling Technology (Beverley, CA, USA). Antibodies against inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, IL-6, IL-10, tumor necrosis fac- tor alpha (TNF-α), cyclooxygenase-2 (COX-2), and tyrosine hydroxylase (TH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against heme oxygenase-1 (HO-1) and ionized calcium-binding adapter molecule-1 (Iba-1) were purchased from Enzo Life Sciences (Farmingdale, NY, USA) and Wako (Osaka, Japan), respectively. All other chemicals were obtained from Sigma-Aldrich unless otherwise stated.

Animals

Male C57BL/6 mice (22–25 g, 7 weeks old) were purchased from Orient Bio Inc. (Seongnam, Korea). The mice were housed at 21 °C under a 12 h light:12 h dark cycle and had ad libitum access to food and water. Every effort was made to minimize to the suffering of the animals. All experiments were performed in accordance with the National Institutes of Health and Ewha Womans University guidelines for the Care and Use of Laboratory Animals, and the study was approved by the Institutional Animal Care and Use Committee of the Medical School of Ewha Womans University (#EUM 15– 0371, #EUM 19-0429).

BV2 Microglial Cell Culture

The immortalized mouse BV2 microglial cell line (Bocchini et al. 1992) was grown and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, streptomycin (10 μg/mL), and penicillin (10 U/mL) in a 37 °C incuba- tor with 5% CO2.

Measurement of Nitrite, Cytokine, and Intracellular ROS Levels

BV2 cells (1 × 105 cells per well in a 24-well plate) were pretreated with MP-10 for 1 h and stimulated with LPS (100 ng/mL) for 16 h. The supernatants of the cultured cells were collected and nitrite accumulation was measured using Griess reagent (Promega). The concentrations of TNF-α, IL- 6, and IL-10 in the supernatants were measured by enzyme- linked immunosorbent assay with a kit from BD Biosciences (San Jose, CA, USA). Intracellular ROS levels were measured with H2DCF-DA (Sigma-Aldrich), as previously described (Cho et al. 2019).

Western Blot Analysis

Whole cell protein lysates were prepared in RIPA buffer consisting of 10 mM Tris (pH 7.4), 300 mM NaCl, 1%
Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM EDTA, and protease inhibitor cocktail. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis, transferred to nitrocellulose membranes, and in- cubated with primary antibodies diluted according to the man- ufacturers’ instructions. After the membranes were thoroughly washed with TBST, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted to 1:2000 in 5% skim milk), and the blots were developed using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA, USA). For quanti- fication, the density of specific target bands was normalized against β-actin using ImageJ software, version 1.37 (National Institutes of Health, Bethesda, MD, USA).

RT-PCR

Total RNA was isolated from BV2 cells and mouse brain tissue using TRIzol reagent (Invitrogen, CA, USA). For RT- PCR, total RNA (1 μg) was reverse transcribed in a reaction mixture containing 500 ng random primers, 3 mM MgCl2,
0.5 mM dNTP, 1× RT buffer, and 10 U reverse transcriptase (Promega). The synthesized cDNA was used as a template for PCR using GoTaq polymerase (Promega) and primers. RT- PCR was carried out in a Bio-Rad T100 thermal cycler (Bio- Rad, Richmond, CA, USA). Quantitative RT-PCR was per- formed using a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Green PCR Master Mix (Bioline, Taunton, MA, USA). The expression levels of target genes were normalized against that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the following formula: 2(Ct, test gene – Ct, GAPDH). The sequences of the PCR primers used in this study are shown in Table 1.
EMSA

BV2 cells were pretreated with MP-10 for 1 h and stim- ulated with LPS for 1 or 3 h. Nuclear extracts were pre- pared from BV2 cells as previously described (Lee et al. 2015). Oligonucleotides containing the nuclear factor- kappa B (NF-κB) consensus sequence (Promega) were end-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA) in the presence of [γ-32P]ATP. Nuclear proteins (5 μg) were incubated with a 32P-labeled NF-κB probe on ice for 30 min, resolved on a 5% acrylamide gel, and visualized by autoradiography.

Transient Transfection and Luciferase Assay

BV2 cells (2 × 105 cells per well in a 12-well plate) were transfected with 1 μg of reporter plasmid DNA using Metafectene transfection reagent (Biontex, Martinsried/ Planegg, Germany). After 36 h of transfection, the cells were treated with LPS (100 ng/mL) for 6 h in the presence or ab- sence of MP-10. Then, the luciferase assay was performed to determine the effect of MP-10 on reporter gene activity.

Drug Administration

To study the systemic inflammation mouse model, C57/ BL6 mice were randomly divided into four groups (con- trol, LPS, LPS + MP-10 (10 mg/kg), and LPS + MP-10 (30 mg/kg); each group, n = 8). MP-10 was dissolved in a vehicle solution (1% dimethyl sulfoxide, 0.5% Cremophor EL, and 0 .9% sodium chloride) and administered via a daily intraperitoneal (i.p.) injection for 4 days before LPS injection (5 mg/kg, i.p.). To study the MPTP mouse model, C57/BL6 mice were randomly divided into five groups (control, MPTP, MPTP+MP-10 (10 mg/kg), MPTP+MP-10 (30 mg/kg), and MP-10 (30 mg/kg); each group, n = 7). MP-10 (10 or 30 mg/kg/ day, i.p.) was administered for three consecutive days. One day after the final MP-10 treatment, MPTP (20 mg/kg, i.p) was injected four times at 2 h intervals (Park et al. 2019).

Behavioral Tests

To assess the animals’ motor coordination, the rotarod test was performed 1, 3, and 6 days after MPTP injection, the protocol of which was modified from a previous study (Rai et al. 2016). During the test, the speed of the rotarod was accelerated from 4 to 40 rpm in 300 s. Before the main test, all mice underwent training until they were able to remain on the rotarod (4–20 rpm) for 300 s. To evaluate dyskinesia, the pole test (50 cm in height, 1 cm in diameter) was performed 7 days after MPTP injection. As before, prior to the main test,

Table 1 Primers used in PCR
reactions Gene Forward primer (5′ → 3′) Reverse primer (5′ → 3′) Size
iNOS CATTGGAAGTGAAGCGTTTCG CAGCTGGGCTGTACAAACCTT 95 bp COX-2 AGCAACCCGGCCAGCAATCT CCTGCTGCCCGACACCTTCA 139 bp TNF-α TGGGAGTAGACAAGGTACAACCC CATCTTCTCAAAATTCGAGTGACAA 175 bp IL-6 CACGATTTCCCAGAGAACATGTG ACAACCACGGCCTTCCCTACTT 129 bp

IL-1β GATCCACACTCTCCAGCTGCA CAACCAACAAGTGATATTCT
CCATG
152 bp

IL-10 AGCAAGGCAGTGGAGCAGGT GGTTGCCAAGCCTTATCGGA 131 bp HO-1 GGCTGTCGATGTTCGGGAAGG CACGCCAGCCACACAGCACTA 139 bp MMP-3 CTCCAGTATTTGTCCTCTAC ATTCAGTCCCTCTATGGA 245 bp MMP-8 CCAAGGAGTGTCCAAGCCAT CCTGCAGGAAAACTGCATCG 180 bp

MMP-9 GAAGCCATACAGTTTATCCT
GGTC
GTGATCCCCACTTACTATGGAAAC 353 bp

GAPDH GGCATGGACTGTGGTCATGA TTCACCACCATGGAGAAGGC 236 bp

all mice were trained three times to successfully descend from the top to the bottom of the pole.

Immunohistochemistry

For histological analysis, mice were anesthetized with sodium pentobarbital (80 mg/kg body weight, i.p. injection) and then perfused transcardially with 0.9% saline followed by 4% para- formaldehyde for tissue fixation. The animals’ brains were then isolated and stored in 30% sucrose solution at 4 °C for cryoprotection. Serial coronal brain sections (40 μm thick) were cut using a cryotome (CM1860; Leica, Mannheim, Germany). For immunohistochemical staining, the sections were treated with 3% H2O2 and 4% bovine serum albumin to inactivate endogenous peroxidation and block non-specific binding, respectively. The sections were then incubated with a primary antibody against TH or Iba-1 (1:1000) overnight. Following this, they were incubated with biotinylated second- ary antibodies for 1 h at 25 °C room temperature, followed by an avidin-biotin-HRP complex reagent solution (Vector Laboratories, Burlingame, CA, USA). Subsequently, a perox- idase reaction was performed using diaminobenzidine tetrahy- drochloride (Vector Laboratories). Finally, the sections were dehydrated and coverslipped for light microscopy.

Statistical Analysis

Differences between experimental groups were analyzed by one-way analysis of variance, and post-hoc comparisons were made using least significant difference tests. All statistical analyses were conducted using SPSS for Windows, version
18.0 (SPSS Inc., Chicago, IL, USA). All values are reported as mean ± standard error of the mean. A p value <0.05 was con- sidered statistically significant.
Results

MP-10 Suppressed the Production of NO, TNF-α, and IL-6 and Promoted the Production of IL-10 in LPS- Stimulated BV2 Microglial Cells

To investigate whether MP-10 has anti-inflammatory effects in microglia, we examined the effect of MP-10 on the LPS- induced production of NO and pro- and anti-inflammatory cytokines in BV2 cells. We found that MP-10 dramatically decreased LPS-induced NO release. In addition, MP-10 inhibited the production of proinflammatory cytokines such as TNF-α and IL-6 (Fig. 1a). In contrast, MP-10 increased the production of the anti-inflammatory cytokine IL-10 in LPS- stimulated BV2 cells. Western blot analysis revealed that MP- 10 downregulated the protein expression of iNOS, COX-2, and proinflammatory cytokines and upregulated IL-10 expres- sion (Fig. 1b). These data indicate that MP-10 has strong anti- inflammatory effects in LPS-stimulated microglia. In addition, we observed that MP-10 inhibited the activity of PDE10A, but not of PDE4, at the concentrations used in this study (5– 20 μM), suggesting that the effect of MP-10 is PDE10- specific (data not shown).

MP-10 Regulated the mRNA Expression of pro- and Anti-Inflammatory Molecules in LPS-Stimulated BV2 Cells

We next performed quantitative RT-PCR analyses to examine the effect of MP-10 on the mRNA expression of pro- and anti- inflammatory molecules. MP-10 suppressed the expression of iNOS, COX-2, TNF-α, IL-1β, and IL-6 and increased IL-10 mRNA levels in LPS-stimulated BV2 cells (Fig. 2a). In addi- tion, we examined the effect of MP-10 on the expression of MMPs, which act as proinflammatory mediators in activated microglia (Lee et al. 2010, 2015). We found that MP-10

a
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COX-2 TNF-α IL-1β IL-6
IL-10

β -actin

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+ LPS

Fig. 1 Effect of MP-10 on NO and cytokine release in LPS-stimulated BV2 microglial cells. (a) Cells were pretreated with MP-10 for 1 h and incubated with LPS (100 ng/mL) for 16 h. After the incubation period, the levels of nitrite, TNF-α, IL-6, and IL-10 released into the media were measured using Griess reagent or enzyme-linked immunosorbent assay.
(b) Western blot analysis was performed to examine the effect of MP-10
on the expression of iNOS, COX-2, TNF-α, IL-1β, IL-6, and IL-10 using cell lysates prepared from BV2 cells. Data represent the mean ± standard error of the mean of three independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. LPS-treated group. COX-2 cyclooxygenase-2, IL interleukin, iNOS inducible nitric oxide synthase, LPS lipopolysaccha- ride, NO nitric oxide, TNF-α tumor necrosis factor alpha

suppressed the LPS-induced mRNA expression of MMP-3,
−8, and −9 (Fig. 2b). These data collectively indicate that MP-10 modulates the transcription of iNOS, COX-2, cyto- kines, and MMPs.

MP-10 Inhibited the Phosphorylation of c-Jun N- Terminal Kinase and Akt and Reduced the Activity of NF-κB and Activator Protein-1 in LPS-Stimulated BV2 Cells

To investigate the molecular mechanism underlying the anti- inflammatory effects of MP-10, we examined the effect of MP-10 on inflammatory signaling molecules such as MAPKs, Akt, and the transcription factors NF-κB and activa- tor protein-1 (AP-1). As shown in Fig. 3a, MP-10 modestly decreased the phosphorylation of c-Jun N-terminal kinase (JNK) without affecting the phosphorylation of extracellular signal-regulated kinase (ERK) or p38. In addition, MP-10 significantly inhibited LPS-induced Akt phosphorylation. The EMSA results showed that MP-10 decreased the DNA
binding activity of NF-κB in a dose-dependent manner (Fig. 3b). Furthermore, MP-10 inhibited the reporter gene activity of NF-κB and AP-1 (Fig. 3c). These data suggest that MP-10 exerts anti-inflammatory effects by inhibiting the phosphory- lation of JNK and Akt and reducing the activity of the down- stream transcription factors NF-κB and AP-1.

MP-10 Promoted the Nuclear Factor Erythroid 2- Related Factor 2/Antioxidant Response Element and Protein Kinase a/CREB Signaling Pathways

ROS are known to be early signaling inducers of inflammato- ry reactions (Bedard and Krause 2007). We found that MP-10 decreased intracellular ROS levels in a dose-dependent man- ner (Fig. 4a). Next, we examined the effect of MP-10 on the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) and protein kinase A (PKA)/CREB signaling pathways, both of which have antioxidant and anti- inflammatory functions in microglia (Lee et al. 2009; Jung et al. 2010; Park et al. 2019). MP-10 increased the mRNA

iNOS/GAPDH
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Fig. 2 Effect of MP-10 on the mRNA expression of proinflammatory molecules in LPS-stimulated BV2 cells. (a) BV2 cells were pretreated with MP-10 for 1 h and incubated with LPS (100 ng/mL) for 6 h. The mRNA levels of iNOS, COX-2, TNF-α, IL-1β, IL-6, and IL-10 were determined by qRT-PCR analysis. (b) The effect of MP-10 on the mRNA expression of MMP-3, −8, and −9 was also determined by traditional RT- PCR analysis. Representative gels and quantifications of the data are
shown. Data represent the mean ± standard error of the mean of three independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. LPS- treated group. COX-2 cyclooxygenase-2, GAPDH glyceraldehyde 3- phosphate dehydrogenase, IL interleukin, iNOS inducible nitric oxide synthase, LPS lipopolysaccharide, MMP matrix metalloproteinase, qRT- PCR quantitative reverse transcription polymerase chain reaction, TNF-α tumor necrosis factor alpha

and protein levels of HO-1, the expression of which is under the control of Nrf2/ARE signaling (Fig. 4b, c). Consistent with this, MP-10 enhanced the reporter gene activity of ARE-luc, HO-1 E1-luc, and HO-1 E2-luc, all of which have ARE motifs bound by Nrf2 (Fig. 4d–f). Furthermore, MP-10 increased the phosphorylation and nuclear translocation of CREB and enhanced CRE-luc reporter gene activity (Fig. 4g–i).

MP-10 Inhibited Microglial Activation and Proinflammatory Gene Expression in LPS-Injected Mouse Brains

To confirm the anti-inflammatory effect of MP-10 in vivo, mice were administered MP-10 (10 or 30 mg/kg) before LPS injection. After 3 days, the mice were sacrificed and microglial activation was assessed by staining their brain
tissue with an antibody against Iba-1, a marker of microglial activation. LPS treatment increased the number of Iba-1- positive activated microglial cells in the cerebral cortex, hip- pocampus, and substantia nigra (Fig. 5a, b). However, MP-10 treatment decreased the number of Iba-1-positive activated microglia. When we examined the effect of MP-10 on gene expression in LPS-injected mouse brains, MP-10 reduced the expression of iNOS, proinflammatory cytokines, and MMP- 3,-8, and −9 and increased the expression of the anti- inflammatory cytokine IL-10 (Fig. 5c, d).

MP-10 Showed Neuroprotective and Anti- Inflammatory Effects in the MPTP-Induced PD Mouse Model

To investigate whether MP-10 has anti-inflammatory and neu- roprotective effects in an MPTP-induced PD model, mice

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Fig. 3 MP-10 inhibits the phosphorylation of JNK and Akt and reduces the activity of NF-κB and AP-1 in LPS-stimulated BV2 cells (a) Cell extracts were prepared from BV2 cells pretreated with MP-10 and incu- bated with LPS (100 ng/mL) for 1 h. These extracts were subjected to western blot analysis using antibodies against the phospho- or total forms of JNK, ERK, p38 MAPK, and Akt. The autoradiograms are representa- tive of three independent experiments. The levels of the phosphorylated forms of MAPKs and Akt were normalized with respect to the level of each total form and expressed as fold changes relative to the control
group. (b) Electrophoretic mobility shift assay for NF-κB was performed using nuclear extracts prepared from BV2 cells pretreated with MP-10 and incubated with LPS for 3 h. (c) Transient transfection analysis of [κB]3-luc and AP-1-luc reporter gene activity. Data represent the mean
± standard error of the mean of three independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. LPS-treated group. AP-1 activator pro- tein-1, ERK extracellular signal-regulated kinase, JNK c-Jun N-terminal kinase, LPS lipopolysaccharide, MAPK mitogen-activated protein kinase, NF-κB nuclear factor-kappa B

were administered MP-10 for 3 days before MPTP injection, and behavioral and immunohistochemical analyses were sub- sequently performed. A schematic of the experimental proce- dure is shown in Fig. 6a. In the rotarod test, on days 1 and 3, MPTP administration significantly decreased the time the mice spent on the rod, and MP-10 treatment attenuated this decrease (Fig. 6b). On day 6, MP-10 administration slightly
increased the riding time of MPTP-treated mice, although this difference was not significant. Additionally, in the pole test, the descending time of MPTP-treated mice was longer than that of control mice. In contrast, MP-10 (30 mg/kg) adminis- tration reduced the descending time of MPTP-treated mice (Fig. 6c). These results suggest that MP-10 administration improved PD-like symptoms such as motor impairments and

a b c

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ROS production
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(μM)

+ LPS
Fig. 4 MP-10 suppresses ROS production and enhances HO-1 expres- sion by upregulating Nrf2/ARE and PKA/CREB signaling. (a) BV2 cells were treated with MP-10 for 1 h and incubated with LPS for 16 h. Intracellular ROS levels were measured by the DCF-DA method. (b, c) BV2 cells were treated with MP-10 for 1 h and incubated with LPS for 6 h. Reverse transcription polymerase chain reaction (b) and western blot analysis (c) were performed to determine the effect of MP-10 on the mRNA and protein expression of HO-1. (d–f) Transient transfection analysis of ARE-luc (d), HO-1 E1-luc (e), and HO-1 E2-luc (f) reporter gene activity. (g) Western blot analysis was performed to assess the levels of the phosphorylated and total forms of CREB using cell lysates
+ LPS
prepared from BV2 cells treated with LPS for 30 min. (h) Western blot analysis was performed to detect the nuclear translocation of CREB. (i) Transient transfection analysis of CRE-luc reporter gene activity. Data represent the mean ± standard error of the mean of three independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. LPS-treated group. ARE antioxidant response element, CREB cAMP response element-binding protein, GAPDH glyceraldehyde 3-phosphate dehydro- genase, HO-1 heme oxygenase-1, LPS lipopolysaccharide, Nrf2 nuclear factor erythroid 2–related factor 2, PKA protein kinase A, ROS reactive oxygen species

dyskinesia. Following this, dopaminergic neuronal cell death was assessed by TH staining. The results showed that MP-10 recovered striatal dopaminergic fibers, as demonstrated by the observed increase in the optical density of striatal TH+ fibers. Moreover, MP-10 recovered nigral dopaminergic cells, as demonstrated by the increased number of nigral TH+ cells in MPTP-injected mouse brains (Fig. 6d). In addition, MP-10 significantly decreased the number of Iba-1-positive
microglial cells in the striatum and substantia nigra of MPTP-injected mice (Fig. 7).

Discussion

In the present study, we demonstrated that MP-10 suppressed microglial activation under in vitro and in vivo

Hippocampus
Cortex
a Control LPS LPS+MP-10 (10mg/kg) LPS+MP-10 (30mg/kg)
b
#
400
Iba-1+ activated microglia (cells/mm2)
#
300
200
100
0
HIPP
400
#
300
200
100
0
400
#
#
300
200
100
0
LPS - + + +

c Control LPS LPS+MP-10 MP-10
Scale bar : 200 μm; insert : 100 μm

Substantia nigra
d
MP-10 - -
10 30 (mg/kg)

iNOS TNF-α COX-2 IL-1β IL-6
IL-10
15
10 *
5
Fold induction
0
8
6 *
4
2
0
iNOS 20
# 15
10
5
0
IL-1β 6
# 4
2
0
3
* TNF-α
# 2
1
0
IL-6 15
* 10
#
5
0

MMP-3 MMP-8
20
15 *
10

5
15
MMP-3
10
0
# 5
4
* MMP-8 3
# 2
1

MMP-9
0
LPS -
+ + -
- + + - 0

GAPDH
MP-10 - -
30 30
- - 30 30
- - 30 30 (mg/kg)

Fig. 5 Effects of MP-10 on microglial activation and the mRNA expres- sion of proinflammatory molecules in the brains of LPS-injected mice. (a, b) Immunohistochemical staining for Iba-1 and quantification of the number of Iba-1-positive activated microglia 3 days after LPS injection (each group: n = 8). MP-10 treatment reduced microglial activation in the cortex, hippocampus, and substantia nigra of LPS-injected mice in a dose-dependent manner (cerebral cortex: F3, 28 = 239.55, p < 0.01; hippo- campus: F3, 28 = 174.29, p < 0.01; substantia nigra: F3, 28 = 85.02, p < 0.01). Representative images (a) and the quantification of the
obtained data (b) are shown. (c, d) Effects of MP-10 on the mRNA levels of iNOS, cytokines, and MMPs in the cortices of LPS-injected mice. Representative gels (c) and quantification of the obtained data (d) are shown. *P < 0.05 vs. control group; #P < 0.05 vs. LPS-treated group. COX-2 cyclooxygenase-2, CTX cortex, HIPP hippocampus, GAPDH glyceraldehyde 3-phosphate dehydrogenase, Iba-1 ionized calcium- binding adapter molecule 1, IL interleukin, iNOS inducible nitric oxide synthase, LPS lipopolysaccharide, MMP matrix metalloproteinase, SN substantia nigra, TNF-α tumor necrosis factor alpha

neuroinflammatory conditions. MP-10 reduced the expression of iNOS, proinflammatory cytokines, and MMPs in LPS- stimulated BV2 microglial cells and LPS-injected mouse brains. Moreover, MP-10 exerted anti-inflammatory effects by inhibiting the phosphorylation of JNK and Akt and reduc- ing the activity of NF-κB and AP-1 in LPS-stimulated microg- lia. In addition, MP-10 exerted antioxidant effects by reducing intracellular ROS levels and upregulating Nrf2/ARE signal- ing. Moreover, MP-10 promoted PKA/CREB signaling in
LPS-stimulated microglia, which appears to play a pivotal role in mediating anti-inflammatory and antioxidant effects of MP- 10 in microglia.
PDE inhibitors increase intracellular cAMP levels by inhibiting the breakdown of cAMP by PDE. cAMP is known to regulate microglial function and activation, and a recent study reported that cAMP is a critical determinant of M1/M2 microglial polarization (Ghosh et al. 2016; Pearse and Hughes 2016). In this regard, previous studies have reported that PDE

a MP-10

MPTP

Sacrifice and Analysis

-3d -2d -1d 0d 1d 3d 6d 7d

b +1d
Latency time (sec.)
150

100

50

0

+3d
Rotarod Test

+6d
Pole Test

c
Descending time (sec.)
20

15

10

5

0

MPTP - + + + - - + + + - - + + + -
MP-10 - - 10 30 30 - - 10 30 30 - - 10 30 30 (mg/kg)
MPTP - +
MP-10 - -
+ +
10 30
-
30 (mg/kg)

d
Striatum
CON MPTP

MPTP+
MP-10 (10mg/kg)

MPTP+
MP-10 (30mg/kg)

MP-10 (30mg/kg)

Substantia Nigra
TH+ optical density (%)
150

100

50

150

TH+ cells/mm2 (%)
100

50

Scale bar : 500 μm

0
MPTP -
MP-10 -
+ + + -
- 10 30 30
0
MPTP -
MP-10 -

+ + +
- 10 30
-
30 (mg/kg)

Fig. 6 Effect of MP-10 on locomotor activity and dopaminergic neuronal cell death in the brains of MPTP-injected mice. (a) Schematic of the experimental procedure. Mice were intraperitoneally injected with MP- 10 (10 or 30 mg/kg) every day for 3 days before MPTP injection. Mice were sacrificed 7 days after MPTP injection, and histological analysis was performed (each group: n = 7). (b) The rotarod test was performed 1, 3, and 6 days after MPTP injection (1 day: F4, 19 = 9.83, p < 0.01; 3 days: F4,
19 = 5.98, p < 0.01; 6 days: F4, 19 = 1.52, p > 0.05). (c) The pole test was performed 7 days after MPTP injection (pole test: F4, 19 = 6.26, p < 0.01).
(d) Representative images of TH-positive neuronal cells in the striatum and substantia nigra. Quantitative analysis was performed by measuring the optical density of TH-positive fibers in the striatum (top panel: F4, 20 = 22.66, p < 0.01) and the number of TH-positive cells in the substantia nigra (bottom panel: F4, 20 = 20.15, p < 0.01). *P < 0.05 vs. control group; #P < 0.05 vs. MPTP-treated group. CON control, MPTP 1-methyl-4-phe- nyl-1,2,3,6-tetrahydropyridine, SN substantia nigra, TH tyrosine hydroxylase

Striatum
a
CON
MPTP
MPTP+MP-10 (10mg/kg) MPTP+MP-10 (30mg/kg)
MP-10 (30mg/kg)

Relative change of Iba-1+ cells (%)
b 500
400
300
200
100
0

800

Relative change of Iba-1+ cells (%)
600

400

200

0

Substantia nigra
Scale bar: 200 μm; insert: 100 μm

MPTP - +
MP-10 - -
+ + -
10 30 30
MPTP - +
MP-10 - -
+ +
10 30
-
30 (mg/kg)

Fig. 7 Effect of MP-10 on microglial activation in the brains of MPTP- injected mice. Immunohistochemical staining for Iba-1 and quantification of the number of Iba-1-positive microglia in the striatum (F4, 20 = 120.28, p < 0.01) and substantia nigra (F4, 20 = 51.67, p < 0.01) of MPTP-injected mice. Representative images (a) and the quantification of the obtained
data (b) are shown. *P < 0.05 vs. control group; #P < 0.05 vs. MPTP- treated group. CON control, Iba-1 ionized calcium-binding adapter pro- tein 1, MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, SN substantia nigra

inhibitors exert an anti- inflammatory effect in neuroinflammatory conditions. PDE4 inhibitors, including rolipram, have neuroprotective, anti-inflammatory, and mem- ory enhancing effects in aged mice and primates (Knott et al. 2017; Song and Suk 2017). Moreover, the PDE5 inhibitor sildenafil induced M2 microglial polarization, promoted β- amyloid clearance, and prevented axonal loss in ischemia, AD, and experimental autoimmune encephalomyelitis mouse models (Peixoto et al. 2015; Pifarre et al. 2011). Although several PDE inhibitors have reached clinical trials, they have all subsequently failed owing to a lack of brain penetration or significant side effects (Bertolino et al. 1988; Richter et al. 2013). Therefore, the development of a blood-brain barrier (BBB)-permeable PDE inhibitor with minimal side effects is required. Previous studies have reported that MP-10 can pen- etrate the BBB and demonstrated that [11C]MP-10 is a potent and specific radioligand suitable for positron emission tomog- raphy imaging and PDE10 quantification in the brains of ro- dents and non-human primates (Plisson et al. 2011; Lin et al. 2015). Given that MP-10 has a strong anti-inflammatory ef- fect and can cross the BBB, it may be a promising drug can- didate for the treatment of neuroinflammatory disorders.
The present study showed that MP-10 has neuroprotective effects in an MPTP-induced PD mouse model. In the rotarod and pole tests, MP-10 was shown to improve MPTP-induced motor deficits. In addition, MP-10 recovered TH-positive do- paminergic cells/fibers in the substantia nigra and striatum. Moreover, MP-10 suppressed microglial activation in both regions. Recently, our group reported that another PDE10 inhibitor, papaverine (PAP), also has neuroprotective and anti-inflammatory effects in MPTP-induced PD mice (Lee et al. 2019). PAP inhibited dopaminergic neuronal cell death by increasing the expression of neurotrophic factors such as brain-derived neurotrophic factor, glial cell line-derived neu- rotrophic factor, and B cell lymphoma 2, all of which are under the control of PKA signaling. PAP also suppressed microglial activation and inflammatory gene expression. These results collectively suggest that PDE10 inhibition has neuroprotective and anti-inflammatory effects in PD mouse models.
For several decades, the gold standard therapy for PD has been levodopa. However, after years of use, this drug loses its efficacy and causes dyskinesia (Niccolini et al. 2015). A sig- nificant reduction in the levels of cAMP/cGMP in the cortico-

striatal-pallidal loop has been observed at the peak of levodopa-induced dyskinesia in hemiparkinsonian rats (Giorgi et al. 2008). Thus, PDE inhibition has been suggested to be beneficial for the restoration of cyclic nucleotide signal- ing (Zagorska et al. 2018). In support of this, co-treatment of levodopa with the selective PDE10A inhibitor TP-10 reduced the severity of dyskinesia in 6-hydroxydopamine-treated rats (Heckman et al. 2016). Since PDE10 is the predominant reg- ulator of cAMP/cGMP levels in the striatum, administration of PDE10A inhibitors may not only rescue the decrease in cAMP/cGMP levels but also allow for the prolonged use of levodopa in combination with PDE10A inhibitors (Wilson and Brandon 2015). In this regard, MP-10 may be a potential candidate for potentiating the effect of levodopa by reducing its side effects. Further studies are necessary to address this possibility.
In conclusion, to the best of our knowledge, the present study is the first to report the anti-inflammatory and neuropro- tective effects of MP-10 in neuroinflammation and PD mouse models. Given that MP-10 is a highly selective PDE10 inhib- itor that is able to cross the BBB, MP-10 may be a promising therapeutic agent for the treatment of PD and other neuroinflammatory disorders.

Acknowledgments This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea govern- ment (MSIT) (2018R1A2B6003074).

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflict of interest.

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