FCPR16, a novel phosphodiesterase 4 inhibitor, produces an antidepressant-like effect in mice exposed to chronic unpredictable mild stress

Accepted Manuscript FCPR16, a novel phosphodiesterase 4 inhibitor, produces an antidepressant-like effect in mice exposed to chronic unpredictable mild stress

Qiuping Zhong, Hui Yu, Chang Huang, Jiahong Zhong, Haitao Wang, Jiangping Xu, Yufang Cheng PII: DOI: Reference:

S0278-5846(18)30028-9 https://doi.org/10.1016/j.pnpbp.2018.10.017 PNP 9526

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date: Revised date: Accepted date:

23 January 2018 28 September 2018 30 October 2018

Please cite this article as: Qiuping Zhong, Hui Yu, Chang Huang, Jiahong Zhong, Haitao Wang, Jiangping Xu, Yufang Cheng , FCPR16, a novel phosphodiesterase 4 inhibitor, produces an antidepressant-like effect in mice exposed to chronic unpredictable mild stress. Pnp (2018), https://doi.org/10.1016/j.pnpbp.2018.10.017

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FCPR16, a novel phosphodiesterase 4 inhibitor, produces an antidepressant-like effect in mice exposed to chronic unpredictable mild stress Qiuping Zhong 1, Hui Yu1, Chang Huang 1 , Jiahong Zhong 1, Haitao Wang 1, Jiangping Xu1,2 *, Yufang

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Cheng 1 *

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1, Neuropharmacology and Drug Discovery Group, School of Pharmaceutical Sciences, Southern Medical University,

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Guangzhou, China

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2, Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, China

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Abstract

The canonical phosphodiesterase 4 (PDE4) inhibitors produce antidepressant-like effects in a variety of animal models. However, severe side effects, particularly vomiting and nausea, limit their clinical

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application. FCPR16 is a novel PDE4 inhibitor with less vomiting potential. However, whether it will

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exert an antidepressant-like effect remains unclear. Here, we aimed to evaluate the effect of FCPR16 in mice subjected to chronic unpredictable mild stress (CUMS). Our results showed that FCPR16 produced

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antidepressant-like effects in multiple behavioral tests, including a forced swimming test, tail suspension

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test, sucrose preference test and novelty suppression feeding test. Simultaneously, data indicated that FCPR16 enhanced the levels of several proteins, including cAMP, brain derived neurotrophic factor, exchange protein directly activated by cAMP 2 (EPAC-2), synapsin1, postsynaptic density protein 95, phosphorylated cAMP response element binding protein and extracellular regulated protein kinases 1/2, which were downregulated by CUMS in both the cerebral cortex and hippocampus. The number of DCX+ cells in the hippocampus of CUMS mice was increased after FCPR16 treatment. Moreover, treatment with FCPR16 resulted in decreased expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β)

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and increased expression of anti-inflammatory cytokines (IL-10) in mice challenged with CUMS. Consistently, the mRNA levels of microglial M1 markers (iNOS and TNF-α) were downregulated, while

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M2 markers (Arginase 1 and CD206) were upregulated in CUMS-exposed mice after FCPR16 treatment. Immunofluorescence analysis showed that FCPR16 inhibited the activation of microglial cells and

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increased the number of CD206+ in CUMS-exposed mice. Collectively, these results suggested that FCPR16 is a potential compound with effects against depressive-like behaviors, and the

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antidepressant-like effect of FCPR16 is possibly mediated through activation of the cAMP -mediated signaling pathways and inhibition of neuroinflammation in both the cerebral cortex and hippocampus.

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Keywords

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Depression, phosphodiesterase 4 inhibitors, FCPR16, neuroinflammation, microglial phenotypes

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1. Introduction Depression is a highly prevalent mental disorder. According to a report from the World Health

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Organization (WHO, 2017), over 300 million individuals are suffering from depression. The prevalence rate has been increasing year by year over the past decade. Approximately 800 thousand people die from

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suicide every year. Depression has become a heavy burden on society and the economy. However, the defects of clinical drugs increase the difficulty for antidepressant therapy. The effects of existing drugs

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usually emerge after 6-8 weeks treatment (Uher et al., 2011). Only less than 50% of patients effectively respond to drugs that are currently on the market (Geddes and Miklowitz, 2013). The rate of adverse

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effects is high (39% suicide, 60% feeling emotionally numb or others) (Read et al., 2014). The selective serotonin reuptake inhibitors (SSRIs), which are commonly used medications for depression treatment,

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have been reported to accelerate the transition from an insulin-resistant state to overt diabetes (Isaac et al., 2013). Searching for new therapeutic targets is particularly urgent.

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Phosphodiesterase 4 (PDE4), an enzyme that selectively hydrolyzes cAMP, is highly expressed in neurons

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and glial cells (Lakics et al., 2010). PDE4 inhibitors exhibit antidepressant-like effects by increasing intracellular cAMP (Garcia et al., 2016). Elevated cAMP triggers a series of signaling cascades, which

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ultimately regulate a variety of pathophysiological processes. The cAMP/protein kinase A (PKA)/cAMP response element binding protein (CREB) signaling pathway is an important mediator in the regulation of

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central nervous system (CNS) functions, including neuronal survival, axonal regeneration and synaptic plasticity (Conti and Beavo, 2007; Plattner et al., 2015). Deficits in these processes are closely linked to the development of depression. Exchange proteins directly activated by cAMP (EPACs), a family of cAMP sensor proteins that include isoforms EPAC-1 and EPAC-2, were first reported in 1998 (Kawasaki et al., 1998). EPAC-2 is highly expressed in the CNS (Wang et al., 2017) and has been implicated in the regulation of neurotransmitter release, neuronal differentiation, neurite growth, memory, learning, and a variety of brain diseases (Schmidt et al., 2013). An in vivo study (Liebenberg et al., 2011) showed that a

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cAMP analogue that inhibits PKA but not EPAC (Rp-8-Br-cAMP) still produced antidepressant-like effects in rats during the forced swimming test (FST) and increased the phosphorylation of CREB. The

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results implied that in addition to activating the cAMP/PKA/CREB signaling pathway, elevated cAMP may also stimulate EPAC and upregulate the proteins downstream of EPAC to mediate antidepressant

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activity. On the other hand, an abundant number of studies have shown that long-term stress is accompanied by neuroinflammation (Howren et al., 2009). Elevated inflammatory cytokines are also a

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critical pathogeny of depression (Kubera et al., 2011), and downregulation of inflammatory levels could exert neuroprotective functions and reverse depressive-like behaviors (Kohler et al., 2014). A previous

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study has shown that the PDE4 inhibitor rolipram reversed amyloid β-induced memory deficits in rats via suppression of neuronal inflammation (Wang et al., 2012). Neuroinflammation suppression is one of the

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ways that PDE4 inhibitors exert antidepressant-like effects. Basic and clinical studies (Price and Drevets, 2010; MacQueen and Frodl, 2011) demonstrated that depression was associated with a reduced size of

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and fewer synapses in the cerebral cortex and hippocampus, the brain regions for regulating mood. It was

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accompanied by a decline in the expression of synapse-related protein, i.e., synapsin 1 and postsynaptic density protein 95 (PSD95). Brain derived neurotropic factor (BDNF), a protein downstream of the

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cAMP/PKA/CREB signaling pathway, promotes neuronal development early in life and maintains synaptic plasticity in the adult brain (Krishnan and Nestler, 2008). PDE4 inhibitors can reverse synaptic

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dysfunction through activation of the cAMP/PKA/CREB signaling pathway. However, rolipram, a first generation PDE4 inhibitor, displayed a strong antidepressant-like effect in preclinical studies as well as in phase II clinical trials (Scott et al., 1991) but was not approved for clinical use

due

to

the

severe

emesis

effect.

FCPR16

(N-(2-chlorophenyl)-3-cyclopropylmethoxy-4-difluoromethoxybenzamide), a structural analogue of the traditional PDE4 inhibitor with 90 nM affinity for PDE4 (Zhou et al., 2016), which was designed and synthesized in our laboratory, did not cause emesis during the 180 min observation period at 3 mg/kg in

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Beagle dogs (Chen et al., 2017). Previous studies (Zhou and Ge et al., 2016) showed that FCPR16 reduced the expression of tumor necrosis factor- (TNF-α) in lipopolysaccharide (LPS)-exposed BV-2

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cells and improved the survival rate of rats in a middle cerebral artery occlusion (MCAO) model by upregulating cAMP-mediated signaling pathways and suppressing the expression of inflammation

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cytokines (Chen and Yu et al., 2017). Nevertheless, whether FCPR16 has an antidepressant potency is unclear. In the current study, we evaluated the antidepressant-like effect of FCPR16 via behavior tests in a

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mouse model of chronic unpredictable mild stress (CUMS) and investigated the mechanism. We hypothesized that FCPR16 would exert an antidepressant-like effect in CUMS-exposed mice by

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regulating the cAMP/PKA/CREB/BDNF and cAMP/EPAC/extracellular signal-regulated kinase (ERK) signal pathways and exerting anti-neuroinflammation. Our results showed that FCPR16 improved the

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depressant-like behaviors of CUMS mice in FST, tail suspension test (TST), sucrose preference test (SPT) and novelty-suppressed feeding test (NSFT), which was implicated in the upregulation of cAMP, BDNF,

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EPAC-2, synapsin 1 and PSD95 levels as well as the phosphorylation of CREB. FCPR16 treatment

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downregulated the expression of the inflammatory cytokines TNF-, interleukin-6 (IL-6) and interleukin-1 (IL-1) and promoted shifting of the microglial phenotype from M1 to M2 in the cerebral

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cortex and hippocampus.

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2. Materials and methods 2.1. Animals

Male C57BL/6 mice (18-22 g) were purchased from the Laboratory Animal Centre of Southern Medical University (Guangzhou, China) and allowed to acclimate for 1 week before the experiments. The mice were housed individually under standard conditions (22 ± 2ºC, 50%-70% humidity, 12-12 h light/dark cycle with lights on at 07:00) with access to food and water ad libitum. All experimental procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH, Eighth

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Edition, 2011) and approved by the Laboratory Animal Ethics Committee of Southern Medical University. Studies from animal experiments showed that behaviors and monoamine transmitter releases in rodents

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could be influenced by estrogen fluctuations (Fink et al., 1996; Okada et al., 1997; Galea et al., 2001; Dalla et al., 2004). To avoid the impact of estrogen on depressive-like behaviors in our experiments, male

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mice were selected for this study. 2.2. Compound

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Escitalopram was purchased from Aladdin (Shanghai, China). FCPR16 (Figure 1(a)) was structurally reconstructed and synthesized by Professor Zhou (Zhou and Ge et al., 2016). These compounds were in

0.5%

sodium

carboxymethylcellulose.

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suspended

carboxymethylcellulose.

vehicle

was

0.5%

sodium

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2.3. Experimental designs

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The experiment included two parts. Part I evaluated the antidepressant-like potency of FCPR16 in the

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behavioral despair model (Wang et al., 2017). Mice were randomly divided into 6 groups (n=8-12 per

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group) using the random number program in SPSS software and received vehicle (0.1 ml/10 g), positive control escitalopram (10 mg/kg, i.g.) or FCPR16 (0.35 mg/kg, 0.7 mg/kg, 1.4 mg/kg and 2.8 mg/kg, i.g.).

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Forth-five minutes after a single compound administration, mice were subjected to forced swimming test (FST), open field test (OFT) or TST. OFT was taken as an evaluation of locomotor activity. Part II further

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confirmed the antidepressant-like effect in the CUMS mice and explored the mechanism of the effect. Mice were randomly divided into 4 groups including one control and three experimental groups (n=12-14 per group) using the random number program in SPSS software. Experimental groups were subjected to CUMS as described below for 6 weeks. After CUMS, the experimental groups received FCPR16 (1.5 mg/kg, i.g.), positive control escitalopram (10 mg/kg, i.g.), or vehicle once daily for 3 weeks (0.1 ml/10 g). The control group was not exposed to stressors and received vehicle. The FST and TST were conducted at the baseline (6 weeks after CUMS and prior to drug treatment) and upon completion of the

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3-week drug treatment. The SPT and NSFT were conducted after treatment. After the behavior tests were completed, biochemical tests were conducted (Figure 2).

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2.4. Chronic unpredictable mild stress (CUMS) The CUMS procedure was performed as described previously (Willner, 1997; Thakare et al., 2017) with

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minor modifications. It contained 7 different stressors (Table 1) randomly conducted across 9 weeks. The same stressor was not used on two consecutive days.

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Table 1. Stressors used for CUMS. Stressor

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restriction of behaviors

4h

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water and food deprivation

18 h

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45º cage tilt

18 h

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swimming in water at 4-6ºC

5 min

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tail pinch

5 min

6

water deprivation

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illumination

Duration

24 h overnight

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7 2.5. Behavior test

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2.5.1. Sucrose preference test (SPT)

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The SPT was performed as described previously (Willner et al., 1987; Zhao et al., 2014) with slight modifications. Mice were individually placed in cages with two bottles. On day 1, two bottles contained water. On day 2, one bottle contained water and the other contained 1% sucrose. Two of the bottles’ positions were switched and kept until 12 h later. On day 3, mice were deprived of water and food for 24 h. On day 4, the SPT was conducted and the test session was 4 h. Two bottles contained water and 1% sucrose and the positions were switched 2 h later. The sucrose preference ratio (SPR) was calculated as

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follows: [sucrose intake (g)/ (sucrose intake (g) + water (g))] × 100%. 2.5.2. Forced swimming test (FST) Mice were forced to swim individually in a cylindrical glass container (diameter: 13 cm, height: 24 cm,

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depth: 14 cm) (Castagne et al., 2011) that contained water (22  2ºC). Each session lasted for 6 min and

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was recorded with a video camera. The immobility time was scored for the last 4 min of the session. 2.5.3. Tail suspension test (TST)

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The TST was conducted as described previously (Castagne and Moser et al., 2011). Mice were suspended, and the experimenter made sure that the mice hung with their tails in a straight line. The testing session

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lasted for 6 min, and the immobility time was recorded. 2.5.4. Open-field test (OFT)

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OFT was conducted with minor modifications (Cui et al., 2012). Mice were placed individually in the center of a box (60 cm × 60 cm × 20 cm) divided into 4 quadrants of equal areas with 36 lattices and

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recorded for 6 min. The numbers of rearing (the mice stood on rear limbs) and crossing (the mice crossed

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the lattices with four paws) were scored during the last 5 min. 2.5.5. Novelty-suppressed feeding test (NSFT)

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The NSFT was conducted as described previously (Li et al., 2017). Mice were deprived of food for 24 h and placed in a box (60 cm × 60 cm × 20 cm) with fresh bedding. Food was put in the center of the box,

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and mice were placed in a corner of the box. The latency of first-time feeding was recorded. 2.6. Western blot

Western blot analyses were performed as previously described (Zou et al., 2017). Briefly, proteins were extracted using 1×RIPA lysis buffer containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Sigma, USA). The protein concentration was measured using the BCA method (Thermo Scientific, USA). Proteins were separated by SDS-PAGE, electrotransferred, blocked and then incubated a primary antibody overnight at 4ºC. The following primary antibodies were used: GAPDH

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(ab181602, abcam, UK), ERK1/2 (WL01864, Wanleibio, China), p-ERK1/2 (WLP003, Wanleibio), EPAC-2 (ab193665, abcam), CREB (#9197, CST, USA), p-CREB (#9198, CST), BDNF (SAB2108004,

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Sigma), synapsin 1 (ab64581, abcam), and PSD95 (ab18258, abcam). The blots were then incubated with a secondary antibody for 2 h at room temperature before visualization with an ECL method and analysis

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by ImageJ software. GAPDH was used as an internal control. 2.7. Enzyme-linked immunosorbent assay (ELISA)

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The cerebral cortex and hippocampus of mice were quickly dissected on ice, homogenized and centrifuged at 12000 × rpm for 15 min at 4ºC. Supernatants were used to determine the concentrations of

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IL-6 (EK0411, BOSTER, China), TNF-α (EK0527, BOSTER), IL-10 (EK0417, BOSTER), IL-1β (EK0394, BOSTER) and cAMP (KGE012B, R&D system, USA) by ELISA kits. Data were all

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normalized to total protein.

2.8. RNA extraction and real-time PCR (RT-PCR)

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Total RNA was extracted using Trizol reagent (9109, TaKaRa, Japan) and reverse transcribed (RR036A)

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and amplified (RR420A) using a kit from TaKaRa. The RT-PCR conditions were as follows: initial 95ºC for 30 s follow by 40 cycles at 95ºC for 5 s and extension at 60ºC for 31 s. Data were normalized against

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GAPDH and are presented as fold changes. Primer sequences are listed in Table 2. Table 2. Primer sequences used in RT-PCR experiments. Reverse (5’ to 3’)

AGGTCGGTGTGAAACGGATTTG

TGTAGACCATGTAGTTGAGGTCA

GTTCTCAGCCCAACAATACAAGA

GTGGACGGGTCGATGTCAC

CCCTCACACTCAGATCATCTTCT

GCTACGACGTGGGCTACAG

Arg1

CTCCAAGCCAAAGTCCTTAGAG

AGGAGCTGTCATTAGGGACATC

CD206

CTCTGTTCAGCTATTGGACGC

CGGAATTTCTGGGATTCAGCTTC

GAPDH iNOS TNF-α

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Forward (5’ to 3’)

Gene

Arg1: Arginase 1

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2.9. Immunohistochemistry Mice were perfused with ice-cold phosphate-buffered saline and 4% paraformaldehyde. Brains were

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postfixed in 4% paraformaldehyde and dehydrated in graded sucrose solutions. Brains sections were prepared using a freezing microtome (Leica, Germany). Sections were washed and permeated by 0.3%

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Triton for 15 min. After blocking at room temperature for 1 h, sections were incubated with rat anti-CD206 (GTX42264, GeneTex, USA), rabbit anti-Iba1 (019-19741, Wako, Japan) or guinea pig

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anti-doublecortin (ab2253, Millipore, USA) overnight at 4ºC. Subsequently, sections were incubated with an appropriate secondary antibody at room temperature for 2 h and DAPI for 10 min. The secondary

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antibodies included Alexa Fluor® 555 goat anti-rat IgG (405420, Biolegend, USA), Dylight 488 goat anti-rat IgG (A23220, Abbkine, USA) and Alexa Fluor 488 AffiniPure goat anti-guinea pig IgG (H+L)

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(106-545-003, Jackson, USA). Image analysis was performed by a confocal microscope (Nikon, Japan). For quantification of immunofluorescence, 6 fields from each group were analyzed by Nikon Imaging

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Elements Software. For each field, the total number of Iba1-positive cells and CD206-positive cells were

2.10. Statistical analysis

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counted.

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All data are presented as the mean ± S.E.M. All statistical comparisons were computed using SPSS 19.0 software. When normality and equal variance were achieved, potential differences between the mean

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values were analyzed with a one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. In CUMS model, at week 6, TST and FST data between the control and CUMS group were analyzed with one-way ANOVA. At week 9, data of TST and FST were analyzed with repeated ANOVA(week as a within factor (repeated factor)) (Li et al., 2009). For evaluation of the NSFT results, Kaplan-Meier survival analysis was used followed by the Mantel-Cox log-rank test. P-value of less than 0.05 was reported as statistically significant. All experiments were conducted in a blinded manner, experimenters did not know the treatment conditions.

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3. Results 3.1. FCPR16 ameliorated depressant-like behaviors in mice

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To evaluate whether the compound FCPR16 has an antidepressant-like effect, we conducted FST and TST 45 min after a single administration. In the behavioral despair model, compared with the control group,

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FCPR16 (1.4 mg/kg and 2.8 mg/kg) and the positive control escitalopram both decreased the immobility time in the FST (F (5, 60) = 6.998, p < 0.001, Figure 1(b)) and the TST (F (5, 60) = 9.521, p < 0.001,

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Figure 1(c)). Neither agent altered the number of crossing (F (5, 60) = 1.105, p > 0.05, Figure 1(d)) or rearing (F (5, 60) = 2.008, p > 0.05, Figure 1(e)) in the OFT. These findings suggested that the effect of

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FCPR16 to decrease the immobility time in FST and TST was not a result of psychostimulant disturbances. FCPR16 may have the potential to improve depressant-like behaviors in mice.

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To further investigate the antidepressant-like effect of FCPR16, mice were exposed to CUMS for 6 weeks and assessed with the TST, FST, SPT and NSFT after treatment for another 3 weeks (Figure 2). At week 6,

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in the FST (F (3, 50) = 8.362, p < 0.001, Figure 3(a)) and TST (F (3, 48) = 6.775, p < 0.001, Figure 3(c)),

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the immobility time in CUMS-exposed groups showed significant increases compared with that in the control group without CUMS. A one-way ANOVA revealed that there was no significant difference (p >

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0.05) between the CUMS (CUMS + vehicle), FCPR16 (CUMS + FCPR16) and escitalopram (CUMS + Escitalopram) groups on immobility times before treatments.

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At week 9, for the FST, a repeated ANOVA showed significant effects for week (F (1, 50) = 15.613, p < 0.001), group (F (3, 50) = 13.239, p < 0.001) and group × week interaction (F (3, 50) = 3.966, p < 0.05) on immobility time (Figure 3(b)). A post hoc test showed that immobility time was significantly decreased after FCPR16 (p < 0.01) or escitalopram (p < 0.05) treatment compared with the CUMS group. In the TST, a repeated ANOVA showed significant effects for week (F (1, 48) = 23.605, p < 0.001), group (F (3, 48) = 7.213, p < 0.001) and group × week interaction (F (3, 48) = 3.989, p < 0.05) on immobility time (Figure 3(d)). Post hoc test results showed that, compared with the CUMS group, the immobility

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times for mice were significantly decreased after FCPR16 (p < 0.05) or escitalopram (p < 0.05) treatment. In the SPT, the SPR of CUMS-exposed mice showed a significant decrease compared with the control

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group. However, FCPR16 (p < 0.05) and escitalopram (p < 0.05) treatment attenuated the effect of CUMS (F (3, 50) = 4.295, p < 0.01, Figure 3(e)) and increased the SPR of mice after 3 weeks treatment

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compared with the CUMS group. In NSFT, the mean latency in CUMS-exposed mice (166.3 s) was significantly longer than that of the controls (96.9 s) as well as the FCPR16 (108.7 s) and escitalopram

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(120.9 s) treatment groups (Figure 3(f)). In summary, FCPR16 produced an antidepressant-like effect in

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CUMS-exposed mice.

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Figure 1. Effect of FCPR16 in mice in the behavioral despair model. Chemical structure of FCPR16 (N-(2-chlorophenyl)-3-cyclopropylmethoxy-4-difluoromethoxybenzamide) (a). Immobility time in the FST (b) and TST (c). The number of crossing (d) and rearing (e) in the OFT. Data are shown as the mean ± S.E.M. *P<0.05, ***P<0.001 vs the control group. n = 8-12.

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Figure 2. The experimental schematic. Mice were divided into 4 groups: one control group and three

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experimental groups. Experimental groups were subjected to CUMS as described below for 6 weeks. TST and FST were conducted at week 6 to demonstrate the successful construction of the depression model in

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mice. Then, mice in the experimental groups received FCPR16, positive control escitalopram and vehicle once daily for 3 weeks. The control group also received the same volume vehicle once daily for 3 weeks. 3

weeks

treatment,

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After

the

mice

performed

the

TST,

FST,

SPT

and

NSFT.

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Figure 3. Antidepressant-like effect of FCPR16 in CUMS-exposed mice. The immobility time of mice in FST after 6 weeks CUMS and prior to treatment (a) and after 3 weeks treatment (b). The immobility

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time of mice in TST after 6 weeks CUMS and prior to treatment (c) and after 3 weeks treatment (d). The sucrose preference ratio in the SPT (e) and the result in the NSFT (f) after 3 weeks treatment. Data are

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CUMS group. n = 12-14. FCPR16: 1.5 mg/kg. Escitalopram: 10 mg/kg.

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shown as the mean ± S.E.M. *p<0.05, **p<0.01, *** p<0.001 vs the control group. #p<0.05, ##p<0.01 vs the

3.2. FCPR16 upregulated cAMP-mediated signaling pathways and synapse-related protein

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expression in the cerebral cortex

To evaluate whether FCPR16 has a regulatory role in the cAMP canonical pathways, cAMP/PKA/CREB

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and cAMP/EPAC/ERK expression levels were measured by Western blotting. In the cerebral cortex of mice, the results showed that FCPR16 treatment reversed the CUMS-induced reduction in cAMP levels

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(F (3, 8) = 15.900, p<0.01, Figure 4(a)) and upregulated the protein expression of cAMP-mediated signaling pathways, including phosphorylation of CREB (F (3, 8) = 12.130, p<0.01, Figures 4(b) and (e)),

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BDNF (F (3, 8) = 15.740, p<0.01, Figures 4(b) and (f)) and EPAC-2 (F (3, 8) = 19.390, p<0.001, Figures

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4(c) and (g)). However, the phosphorylation of ERK1/2 (F (3, 12) = 2.634, p>0.05, Figures 4(c) and (h)) was increased slightly without statistical significance in both the FCPR16 and escitalopram groups

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compared with the CUMS group. The above data suggested that FCPR16 exerted an antidepressant-like effect accompanied by upregulation of the cAMP/CREB/BDNF and cAMP/EPAC-2 pathways.

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To examine the protective effect of FCPR16 on synapse function in the cerebral cortex of CUMS-exposed mice, we measured the expression levels of synapse-associated proteins synapsin 1 and PSD95. The results showed decreased expression of synapsin 1 and PSD95 in the cerebral cortex of mice upon CUMS. However, FCPR16 treatment reversed the alteration and increased the expression levels of synapsin 1 (F (3, 8) = 19.330, p<0.001, Figure 4(d) and (i)) and PSD95 (F (3, 8) = 13.820, p<0.01, Figure 4(d) and (j)) compared with the CUMS group.

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3.3. FCPR16 upregulated cAMP-mediated signaling pathways and synapse-related protein expression in the hippocampus

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In addition to the cerebral cortex, the hippocampus is one of the brain regions associated with depression. Thus, we also measured the expression levels of the above proteins in the hippocampus. The results

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showed that the changes in protein expression levels in the hippocampus were similar to those of the cerebral cortex. On one hand, FCPR16 treatment upregulated the level of cAMP (F (3, 8) = 16.140,

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p<0.001, Figure 5(a)) and activated cAMP-mediated signaling pathways, increasing the protein expression of BDNF (F (3, 8) = 11.690, p<0.01, Figure 5(b) and (f)), EPAC-2 (F (3, 8) = 9.081, p<0.01,

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Figure 5(c) and (g)) and phosphorylated CREB (F (3, 8) = 6.057, p<0.05, Figure 5(b) and (e)) compared with the CUMS group. Similarly, there was no significant difference in the changes in phosphorylation of

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ERK1/2 (F (3, 12) = 3.138, p>0.05, Figure 5(c) and (h)) between the CUMS and FCPR16 group. On the other hand, FCPR16 treatment reversed CUMS-induced decreases in the expression of synapse-related

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protein synapsin 1 (F (3, 12) = 11.920, p<0.001, Figure 5(d) and (i)) and PSD95 (F (3, 12) = 5.567,

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p<0.05, Figure 5(d) and (j)).

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Figure 4. Effect of FCPR16 on the regulation of signaling pathways in the cerebral cortex of mice. The levels of cAMP (a), p-CREB (b, e), BDNF (b, f), EPAC-2 (c, g) p-ERK1/2 (c, h), synapsin 1 (d, i) and PSD95 (d, j). Data are shown as the mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001 vs the control group. p<0.05,

##

p<0.01,

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#

p<0.001 vs the CUMS group. n = 3-4 samples per group (repeated 3 times per

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Figure 5. Effect of FCPR16 on the regulation of signaling pathways in the hippocampus of mice. The level of cAMP (a), p-CREB (b, e), BDNF (b, f), EPAC-2 (c, g), p-ERK1/2 (c, h), synapsin 1 (d, i) and

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PSD95 (d, j). Data are shown as the mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001 vs the control group. #

p<0.05,

##

p<0.01,

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p<0.001 vs the CUMS group. n = 3-4 samples per group (repeated 3 times per

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3.4. FCPR16 promoted neurogenesis in the hippocampus of mice exposed to CUMS FCPR16 treatment upregulated the cAMP signaling pathways and the expression levels of

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synaptic-related proteins. To evaluate whether FCPR16 improves neurogenesis in the hippocampus of CUMS-treated mice, we measured the number of DCX+ cells in the hippocampus by immunofluorescence

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and detected the expression of DCX by Western blotting. The number of DCX+ cells in the hippocampus of mice, indicative of the numbers of neuronal progenitor cells, was increased after FCPR16 treatment

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compared with the CUMS group (Figure 6 (a)). The Western blot results suggested that the expression level of DCX was higher in the hippocampus of mice treated with FCPR16 (F (3, 12) = 16.75, p<0.001,

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Figure 6 (b) and (c)) than that of the CUMS group.

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Figure 6. Effect of FCPR16 on Doublecortin (DCX) in the hippocampus of mice. DCX (green) and DAPI

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(blue) were measured by immunofluorescence (a). White arrows point to DCX+ cells. Scale bar = 100 μm. The expression of DCX was measured by Western blotting (b, c). Data are shown as the mean ± S.E.M. ***

p<0.001 vs the control group. #p<0.05, ##p<0.01 vs the CUMS group. n = 4 per group (repeated 3 times

per sample).

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3.5. FCPR16 suppressed inflammatory cytokine expression in the cerebral cortex of CUMS mice To examine whether FCPR16 regulates inflammatory cytokine expression, inflammatory cytokines were measured by ELISA kits. In the cerebral cortex, as shown in the results, CUMS increased the expression

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of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β compared with the control group, while it

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decreased the anti-inflammatory cytokine IL-10. With FCPR16 treatment, the expression levels of TNF-α (F (3, 16) = 8.711, p<0.01, Figure 7(a)), IL-6 (F (3, 16) = 4.137, p<0.05, Figure 7(b)) and IL-1β (F (3, 16)

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= 7.333, p<0.01, Figure 7(c)) were significantly lower than those of CUMS-exposed mice. The level of the anti-inflammatory cytokine IL-10 in FCPR16-treated mice was higher than that in mice exposed to

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CUMS (F (3, 16) = 2.733, p>0.05, Figure 7(d)), but there was no significant difference.

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3.6. FCPR16 suppressed inflammatory cytokine expression in the hippocampus of CUMS mice In the hippocampus, CUMS also upregulated the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β,

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while it downregulated the anti-inflammatory cytokine IL-10. However, after FCPR16 treatment, the

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levels of pro-inflammatory cytokines TNF-α (F (3, 16) = 4.005, p<0.05, Figure 7(e)), IL-6 (F (3, 16) = 20.200, p<0.001, Figure 7(f)) and IL-1β (F (3, 16) = 62.440, p<0.001, Figure 7(g)) were decreased, while

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the level of anti-inflammatory cytokine IL-10 (F (3, 16) = 37.900, p<0.001, Figure 7(h)) was increased compared with the CUMS group. Our results demonstrated that FCPR16 protected mice against

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CUMS-induced neuroinflammation in both the cerebral cortex and hippocampus.

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Figure 7. Effect of FCPR16 on the release of inflammatory cytokines. The expression of pro-inflammatory cytokines TNF-α (a, e) and IL-6 (b, f), IL-1β (c, g) and anti-inflammatory cytokine IL-10 (d, h) in the cerebral cortex and hippocampus, respectively. Data are shown as the mean ± S.E.M. p<0.05,

***

p<0.001 vs the control group. # p<0.05,

##

p<0.01,

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*

p<0.001 vs the CUMS group. n = 5

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samples per group (each sample was measured in triplicates).

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3.7. FCPR16 improved the shift in microglial phenotype from M1 to M2 in the cerebral cortex The above results suggested that FCPR16 suppressed the expression of pro-inflammatory cytokines in the

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cerebral cortex and hippocampus of CUMS-exposed mice. Thus, to further confirm whether FCPR16 had effects on regulating the microglial phenotypes, we examined the mRNA levels of microglial M1 (TNF-

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and iNOS) and M2 (Arg1 and CD206) markers by RT-PCR. In the cerebral cortex, the results showed that FCPR16 decreased the CUMS-induced increases in the mRNA levels of the M1 markers TNF-α (F (3, 12)

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= 14.230, p<0.001, Figure 8(a)) and iNOS (F (3, 12) = 17.450, p<0.001, Figure 8(b)). The mRNA levels

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of the M2 markers Arg1 (F (3, 8) = 64.390, p<0.001, Figure 8(c)) and CD206 (F (3, 8) = 12.610, p<0.01, Figure 8(d)) in the FCPR16 group were higher than those in the CUMS group.

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To further verify the results, immunofluorescence double labeling was performed using microglial marker Iba1 and microglial M2 marker CD206 (Figure 9 (a)). The ratio of CD206 + cells to Iba1 + cells (F (3, 20) =

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6.249, p<0.01, Figure 9(a) and (c)) was increased in the cerebral cortex of the FCPR16-treated group compared with the CUMS group.

3.8. FCPR16 improved the shift in microglial phenotype from M1 to M2 in the hippocampus In the hippocampus, FCPR16 decreased the mRNA levels of the M1 markers TNF- (F (3, 16) = 9.213, p<0.001, Figure 8(e)) and iNOS (F (3, 12) = 15.44, p<0.001, Figure 8(f)) and increased the mRNA levels of the M2 markers Arg1 (F (3, 8) = 4.724, p<0.05, Figure 8(g)) and CD206 (F (3, 8) = 32.600, p<0.001,

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Figure 8(h)) compared with those in the CUMS group. The results of immunofluorescence showed that the ratio of CD206 + cells to Iba1 + cells (F (3, 20) = 56.670, p<0.001, Figure 9(b) and (d)) was increased in

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mice treated with FCPR16 compared with the CUMS group. Taken together, FCPR16 produced an anti-neuroinflammation effect and promoted a shift in microglial

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phenotype from M1 to M2.

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Figure 8. Effects of FCPR16 on microglial polarization. The mRNA level of the microglial M1 markers TNF-α (a, e) and iNOS (b, f) and the microglial M2 markers Arginase 1 (Arg1) (c, g) and CD206 (d, h) in the cerebral cortex and hippocampus, respectively. Data are shown as the mean ± S.E.M. * p<0.05, p<0.01,

***

p<0.001 vs the control group. #p<0.05, ##p<0.01,

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**

p<0.001 vs the CUMS group. n = 3-5

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samples per group (each sample was measured in triplicate).

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Figure 9. Effect of FCPR16 on the microglial immunofluorescence marker (Iba1) and the microglial M2 marker (CD206) in the cerebral cortex (a) and hippocampus (b), and their quantification, respectively (c, d). Scale bar = 100 μm. White arrows point out Iba+ cells. Green arrows point out CD206 + cells. Data are

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shown as the mean ± S.E.M. ##p<0.01, ###p<0.001 vs the CUMS group. n = 6 fields per group.

4. Discussion

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The current data showed that FCPR16 reversed CUMS-induced depressant-like behaviors in mice, accompanied by an upregulation of cAMP/CREB/BDNF and cAMP/EPAC signaling pathways as well as

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the expression of synapsin 1 and PSD95, an improvement in neurogenesis, and downregulation of neuroinflammation. To our knowledge, this is the first report that the PDE4 inhibitor FCPR16 exerted an

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antidepressant-like effect in CUMS-exposed mice. The idea is supported by the following results: (1) FCPR16 treatment ameliorated depressant-like behaviors in mice models of both acute stress and CUMS

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without influencing the locomotor activity; (2) treatment with FCPR16 enhanced the activation of

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cAMP/CREB/BDNF and cAMP/EPAC signaling pathways; (3) FCPR16 improved CUMS-induced decreases in the expression of synapsin 1 and PSD95; (4) FCPR16 promoted neurogenesis in the

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hippocampus of CUMS mice; (5) pro-inflammatory cytokines were decreased while anti-inflammatory cytokines were increased with FCPR16 treatment; and (6) FCPR16 promoted a shift of microglial

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phenotypes from M1 to M2.

In the behavior tests, OFT was used to evaluate locomotor activity in rodents (Wang et al., 2017). The durations were different among different labs, such as 60 min (Zanos et al., 2016), 20 min (Plattner et al., 2015) or 10 min (Zhang et al., 2016). However, we also noticed that 5-6 min of OFT was also commonly used in most labs (Cunha et al., 2017; Han et al., 2018; Zhao et al., 2018). In the present study, 6 min of OFT was used to rule out false positive results in the TST and FST due to hyperlocomotion (Siteneski et al., 2018), which was similar to our method for the OFT. In the TST, we occasionally found that a few

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mice grabbed their tails during the testing process. Under this condition, mice usually showed no intent to struggle, and this behavior is different than the immobility measured in the TST. Hence, mice showing

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tail-grabbing behaviors were not included in the final analysis (Can et al., 2012). Substantial evidence has revealed that PDE4 inhibitors exerted antidepressant-like effects via the

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cAMP/CREB/BDNF signaling pathway (Li et al., 2009; Bollen and Prickaerts, 2012). Using multiple biochemical and pharmacologic techniques, we observed increased cAMP concentrations upon FCPR16

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treatment in both the cerebral cortex and hippocampus and subsequent activation of CREB and EPAC-2. Based on these findings, we speculated that in addition to the cAMP/CREB/BDNF pathway, the

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cAMP/EPAC signaling pathway also contributed to the antidepressant-like effect of FCPR16. Due to the selective distribution of EPAC proteins, the effects of EPAC-2 on the CNS systems are

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foreseeable (Zhou et al., 2016). Previous studies (Srivastava et al., 2012; Yang et al., 2012) suggested that dendritic spine motility and cortical neuron density were reduced and the learning, memory and social

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interaction functions were influenced in EPAC-2-deficient mice. In the current study, CUMS reduced the

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concentration of cAMP and decreased the expression level of EPAC-2 in the cerebral cortex and hippocampus. FCPR16 reversed these changes and improved the phosphorylation of ERK1/2. Summarily,

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these findings suggested that FCPR16 produced an antidepressant-like effect by upregulating the cAMP/CREB/BDNF and cAMP/EPAC signaling pathways.

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In keeping with previous studies, synaptic damage and malfunction were accompanied by depression, which exhibited a decline in expression levels of synapsin 1 and PSD95 (Qiao et al., 2017). Our results showed that the expression levels of synapsin 1 and PSD95 were decreased in the cerebral cortex and hippocampus of CUMS-exposed mice and were significantly increased after FCPR16 treatment. With FCPR16 treatment, the number of DCX+ cells was increased in the hippocampus, which indicated that FCPR16 potentially improved neurogenesis in the hippocampus of mice exposed to CUMS. Depression and a variety of other psychiatric diseases are typically accompanied by neuroinflammation

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(Maes et al., 2009; Blandini, 2013; Heneka et al., 2014). Inhibiting inflammation and decreasing the levels of pro-inflammatory cytokines could relieve depressive symptoms (Kohler and Benros et al., 2014).

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In this study, we found that FCPR16 had an anti-neuroinflammation effect under chronic stress conditions, as reflected by decreased pro-inflammatory cytokines and increased transcription levels of M2 microglial

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markers.

Escitalopram produces antidepressant effects by increasing extracellular 5-hydroxytryptamine (5-HT).

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Elevated extracellular 5-HT activates G protein-coupled receptors and activates adenylyl cyclase. Escitalopram promotes the cAMP signaling pathway by accelerating generation instead of inhibiting the

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metabolism of cAMP. A previous study showed that simulation of the 5-HT7 receptor could suppress the release of pro-inflammatory cytokines (de Las et al., 2013). However, the results showed that

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escitalopram did not have a clear advantage in shifting M1/M2 microglial phenotypes. The expressions of pro-inflammatory cytokines and the mRNA levels of microglial M1 markers were decreased after

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escitalopram treatment, but the levels of microglial M2 markers did not change significantly. We

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suspected that these results may be related to several issues. First, the regulation of microglial phenotypes is a dynamic process. Different drugs have different latent periods for regulating microglial polarizations

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(Zhang et al., 2017). The peak effect of escitalopram on microglial polarizations may not occur at the third week. Second, the role of escitalopram in cAMP is mainly to promote generation. Whether

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increasing the cAMP level activates PDE4 is unknown. Most importantly, extracellular 5 -HT activates both 5-HT1A and 5-HT2A receptors, which will produce distinct or even opposing effects (Weisstaub et al., 2006; Isom et al., 2013; Hiroaki-Sato et al., 2014; Amidfar et al., 2017). The 5-HT1A receptor is expressed on activated T cells and regulates T cells proliferation (Aune et al., 1993). In contrast, activating the 5-HT2A receptor aggravates the inflammatory response both in peripheral tissues and CNS systems (Ito et al., 2000; Savignac et al., 2016). The latency periods of activating 5-HT1A and 5-HT2A receptors by escitalopram may be discrepant. This may be one of the reasons that less than 50% patients respond to

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SSRIs. However, there are also several insufficiencies in our experiment. Although FCPR16 has an

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antidepressant-like effect in mice, whether it influences the neurotransmitter involved in the pathology of depression is uncertain. It is deficient to evaluate synapsis function only by measuring the expression of

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synapse-associated proteins without measuring synaptic function by electrophysiology. Our studies have demonstrated that FCPR16 could protect neurons against apoptosis in MCAO rats and reverse

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depressant-like behaviors in CUMS mice. We investigated the changes in expression of related proteins in the cerebral cortex and hippocampus, brain regions closely associated with depression. However, we did

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not measure the FCPR16 distribution in specific brain regions. The above deficiencies remain to be

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further studied.

5. Conclusion

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In summary, the current study demonstrated that the novel PDE4 inhibitor FCPR16 possessed an

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antidepressant-like effect in CUMS-exposed mice. The observed behavioral effects were apparently associated with the upregulation of cAMP/CREB/BDNF and cAMP/EPAC signaling pathways, as well as

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the expression of synapsin 1 and PSD95, improvement of neurogenesis, suppression of pro-inflammatory cytokine release and a shift in microglial M1/M2 polarization. Taken together, because of the

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antidepressant-like effect of FCPR16 without an emesis side effect, FCPR16 could be a leading compound for new possible antidepressant research and development.

Disclosures

The authors declare that there is no conflict of interest.

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Ethical Statement All animal experiments performed in studies were in accordance the NIH Guide for the Care and Use of

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Laboratory Animals (NIH, Eighth Edition, 2011) and approved by the Laboratory Animal Ethics Committee of Southern Medical University (number 2016-0041). This article did not contain any studies

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with human performed by any of the authors.

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Acknowledgements

This work was supported by the National Nature Science Foundation of China (grant No: 81503043,

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81373384) and the Funding from Guangzhou Science and Technology Department (grant No.

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2015B020211007).

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Zhou Z.Z., Ge B.C., Zhong Q.P., et al. 2016. Development of highly potent phosphodiesterase 4 inhibitors with anti-neuroinflammation potential: Design, synthesis, and structure-activity relationship study of catecholamides bearing aromatic rings. Eur J Med Chem. 124: 372-379. Zou Z.Q., Chen J.J., Feng H.F., et al. 2017. Novel Phosphodiesterase 4 Inhibitor FCPR03 Alleviates

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Lipopolysaccharide-Induced Neuroinflammation by Regulation of the cAMP/PKA/CREB Signaling

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Pathway and NF-kappaB Inhibition. J Pharmacol Exp Ther. 362(1): 67-77.

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Ethical Statement The authors declare there is no conflict of interest. All animal experiments performed in studies were in

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accordance the NIH Guide for the Care and Use of Laboratory Animals (NIH, Eighth Edition, 2011) and approved by the Laboratory Animal Ethics Committee of Southern Medical University (number

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2016-0041). This article did not contain any studies with human performed by any of the authors.

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Highlights FCPR16 ameliorates depressant-like behaviors in CUMS-exposed mice.

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Treatment of FCPR16 up-regulates cAMP-mediated signaling pathways in mice.

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FCPR16 increases the expression of synapsin1 and PSD95 in mice.

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FCPR16 treatment suppresses the neuroinflammation in mice.

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