Mice deficient for wild-type p53-induced phosphatase 1 display elevated anxiety- and depression-like behaviors

Neuroscience 293 (2015) 12–22

MICE DEFICIENT FOR WILD-TYPE P53-INDUCED PHOSPHATASE 1 DISPLAY ELEVATED ANXIETY- AND DEPRESSION-LIKE BEHAVIORS C. S. RUAN, a,b*  F. H. ZHOU, b,d  Z. Y. HE, a,g S. F. WANG, a C. R. YANG, a,h Y. J. SHEN, a Y. GUO, a H. B. ZHAO, a L. CHEN, a D. LIU, e,f J. LIU, b B. T. BAUNE, e Z. C. XIAO a,c AND X. F. ZHOU a,b

anxiety-like but not depression-like behaviors were further elevated in mice under CUMS. Although limitations like male-alone sampling and multiply behavioral testing exist, the present study suggests a potential protective function of Wip1 in mood stabilization. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

a Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650500, PR China b School of Pharmacy and Medical Sciences, Division of Health Sciences, University of South Australia, Adelaide, SA 5000, Australia

Key words: Wip1, CUMS, exploratory behavior, anxiety-like behavior, depression-like behavior, fluoxetine.

c

Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia d School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, SA 5001, Australia

INTRODUCTION

e

Discipline of Psychiatry, School of Medicine, University of Adelaide, Adelaide, SA 5000, Australia

Mood disorders are a common type of severe mental illness in the world demonstrating co-occurring symptoms of anxiety and depression. A high suicide rate and a significant burden for society and family require effective treatment (Cassano and Fava, 2002). A combination of factors such as environmental, psychological, biological and genetic factors cause mood dysfunction (Ahn et al., 2009; Gillespie et al., 2009). So far, the most promising theory regarding mood dysfunction is related to the dysregulation of neurotransmitters such as serotonin (Young et al., 1994; Young and Leyton, 2002), dopamine (Diehl and Gershon, 1992; Brown and Gershon, 1993), gamma-aminobutyric acid (Petty, 1995; Krystal et al., 2001) and noradrenaline (Asnis et al., 1995; Brunello et al., 2003). Treatments through inhibition of serotonin uptake, such as selective serotonin reuptake inhibitors (SSRIs) have been widely used for depressed human patients (Vaswani et al., 2003). In the recent years, growing evidences have shown that deficiency in neurogenesis (Sablina et al., 2007; Snyder et al., 2011), increase in apoptosis (Eilat et al., 1999; Treusch et al., 2011) and increase in neuroinflammation (Raison et al., 2006; Miller et al., 2009) have also been implicated in the development of mood disorders. However, the molecular mechanisms underlying mood regulation remain poorly understood. Wild-type p53-induced phosphatase 1 (Wip1), encoded by the protein phosphatase, Mg2+/Mn2+-dependent, 1D (PPM1D) gene is an oncogene initially identified in cells treated with gamma radiation (Fiscella et al., 1997). In addition, Wip1 has also shown negative responses to a variety of stresses including ultraviolet radiation (Takekawa et al., 2000), hydrogen peroxide (Oshima et al., 2007), anisomycin (Takekawa et al., 2000) and methyl methane sulfonate (Park et al., 2012). Being a serine/threonine phosphatase, Wip1 negatively

f Mental Health Service, Northern Adelaide Local Health Network, Adelaide, SA 5000, Australia g MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, PR China h

Department of Pathology, Second Hospital of Tianjin Medical University, Tianjin 300000, PR China

Abstract—Mood disorders are a severe health burden but molecular mechanisms underlying mood dysfunction remain poorly understood. Here, we show that wild-type p53-induced phosphatase 1 (Wip1) negatively responds to the stress-induced negative mood-related behaviors. Specifically, we show that Wip1 protein but not its mRNA level was downregulated in the hippocampus but not in the neocortex after 4 weeks of chronic unpredictable mild stress (CUMS) in mice. Moreover, the CUMS-responsive WIP1 downregulation in the hippocampus was restored by chronic treatment of fluoxetine (i.p. 20 mg/kg) along with the CUMS procedure. In addition, Wip1 knockout mice displayed decreased exploratory behaviors as well as increased anxiety-like and depression-like behaviors in mice without impaired motor activities under the non-CUMS condition. Furthermore, the Wip1 deficiency-responsive

*Correspondence to: C. S. Ruan, School of Pharmacy and Medical Sciences, Division of Health Sciences, University of South Australia, Adelaide, SA 5000, Australia. Tel: +618-8302-1521; fax: +6188302-1087. E-mail address: [email protected] (C. S. Ruan).   Co-first author, contributed equally to this study. Abbreviations: CUMS, chronic unpredictable mild stress; EOM, elevated zero maze test; FST, forced swimming test; MAPK, mitogen-activated protein kinase; OFT, open field test; PPM1D, Mg2+/Mn2+-dependent, 1D; SPT, sucrose preference test; SSRIs, selective serotonin reuptake inhibitors; TST, tail suspension test; Wip1, Wild-type p53-induced phosphatase 1; WT, wild-type. http://dx.doi.org/10.1016/j.neuroscience.2015.02.037 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 12

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22

regulates mitogen-activated protein (MAP) kinase p38 by dephosphorylation of a threonine residue essential for p38 MAP kinase activation (Takekawa et al., 2000; Gururajan et al., 2014; Lu et al., 2014). Also as a stress-stimulated molecule (Sun et al., 2013; Zhou et al., 2014), the activation of p38 MAPK is associated with a number of disease-related signaling pathways such as apoptosis (Fukunaga et al., 2004; Van Laethem et al., 2004) and neuroinflammation (Herlaar and Brown, 1999; Zwerina et al., 2006), whereas selective inhibition of p38 MAPK exhibits anti-depressant and anxiolytic-related behaviors in rodents (Bruchas et al., 2007, 2011; Corsi et al., 2011, Peng et al., 2013). The positive stressresponsive p38 MAPK suggests an opposite role of Wip1 in mood regulation. Similarly, increasing evidences have shown that Wip1 may also inhibit the pro-apoptotic and pro-inflammatory pathways respectively through the deactivation of its other dephosphorylating substrates, p53 (Takekawa et al., 2000; Yu et al., 2007; Bachis et al., 2008) and NF-kappaB (Chew et al., 2009; Lowe et al., 2012; Demirtasß et al., 2014). Wip1 have also demonstrated a role in regulating neurogenesis through p53-dependent cell cycle control (Demidov et al., 2007; Zhu et al., 2009). Therefore, we hypothesize that Wip1 may play a regulatory role against stress-induced mood behaviors. To test our hypothesis, we examined the expression profile of Wip1 in the neocortex and hippocampus of mice under chronic unpredictable mild stress (CUMS). We also examined the Wip1 protein expression in the hippocampus of CUMS mice under chronic treatment of fluoxetine. Finally, we measured the exploratory, anxietylike and depression-like behaviors in Wip1 knockout mice under stress-free and stress conditions by the open field test (OFT), elevated zero maze test (EOM), tail suspension test (TST) and forced swimming test (FST).

EXPERIMENTAL PROCEDURES Animals C57BL/6 mouse breeders were purchased from the Vital River Laboratories (Beijing, China), and Wip1+/ mouse breeders (Choi et al., 2002) were provided by Dr. L. A. Donehower from the Baylor College of Medicine. All animals were bred in the animal house of the Kunming Medical University (KMU). 810 weeks of C57BL/6 mice were used for experiments 1 (Wip1 expression in CUMS model) and 2 (fluoxetine treatment in CUMS model), and 810 weeks of littermates of Wip1+/+ (wild-type (WT)) and Wip1/ were used for experiment 3 (testing of mood behaviors in Wip1/ mice). Animals for experiments were socially-housed (<five animals per cage) with water and food available ad libitum under standard housing conditions (12-h light/ 12-h dark cycles, 22 ± 1 °C, 52 ± 2% humidity). All procedures involving animals were conducted between 7 a.m. and 7 p.m., in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Department of Laboratory Animal Science of Kunming Medical University.

13

CUMS procedure CUMS was performed as previously described (Ruan et al., 2014; Yang et al., 2014a). Briefly, as control mice were socially housed and undisturbed, mice for CUMS were singly housed and daily experienced a random stimulus of 10 for a specified duration. Stimuli were cold water swimming (13 ± 1 °C, 5 min) (A), warm water swimming (37 ± 2 °C, 5 min) (B), moist bedding (8 h) (C), cage tilt (45°, 8 h) (D), cage shaking (180 rpm, 10 min) (E), tail pinch (1 cm from the tail end, 1 min) (F), food deprivation (12 h) (G), water deprivation (12 h) (H), overnight illumination (12 h) (I) and no stress (24 h) (J). To examine the effect of CUMS on animals, OFT was weekly performed starting from the beginning, while EOM, TST as well as FST were performed at the beginning and end of the CUMS procedure. Drug administration To intervene the progression of mood behaviors in mice under CUMS, fluoxetine (H20110442, Lilly, Fegersheim, France), a clinical antidepressant, was used to treat the CUMS mice daily along with the CUMS procedure by intraperitoneal injection (20 mg/kg, dissolved in saline) (Chouinard, 1985; Dulawa et al., 2004; Ruan et al., 2014). Meanwhile, non-stressed mice with saline, nonstressed mice with fluoxetine and CUMS mice with saline were set as controls. Behavioral tests Sucrose preference test (SPT). SPT was performed as previously described (Ruan et al., 2014). Briefly, animals were trained to drink from two bottles of 1% (w/v) sucrose solution for 24 h, and one bottle of 1% sucrose solution and one bottle of tap water for the next 24 h. After another 24 h of food and water deprivation, one bottle of 1% sucrose solution and one bottle of tap water were given to the animals for 1 h, and the amount of sucrose and water consumptions were measured. The suppression of sucrose consumption indicated the depression-like behavior (Willner et al., 1987; Vollmer et al., 2013). OFT. OFT was performed as previously described (Ruan et al., 2014). Briefly, animals were placed in an open arena and recorded by a video camera for 5 min. Travel distance, turn angle, entries in the peripheral zone, entries in the central zone and percentage of time spent in the central zone were automatically analyzed by ANYmaze video-tracking software (Stoelting, Wood Dale, IL, USA), while rearing numbers were counted manually blinding to the treatment. Travel distance and turn angle (Duffy and Ford, 1997; Spink et al., 2001; Venkitaramani et al., 2011) in the peripheral zone respectively indicated the locomotor and non-locomotor movements of the body, while travel distance and turn angle in the central zone as well as rearing indicated the exploratory behaviors. Entries in the peripheral zone and the central zone, as well as the percentage of time spent in the central zone

14

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22

indicated the anxiety-like behaviors (Corman and Shafer, 1968; Prut and Belzung, 2003). EOM. EOM was performed as previously described (Ruan et al., 2014). Briefly, animals were placed in an annular platform elevated above the floor and recorded by a video camera for 5 min. Data were automatically analyzed by ANY-maze software. The travel distance and turn angle in the closed arms respectively indicated the locomotor and non-locomotor movements of the body, while the travel distance and turn angle in the open arms indicated the exploratory behaviors. Entries in the closed arms and open arms, as well as the percentage of time spent in the open arms indicated the anxiety-like behaviors (Shepherd et al., 1994; Kulkarni et al., 2007). TST. TST was performed as previously described (Ruan et al., 2014). Briefly, animals were suspended above the floor and recorded by a video camera for 6 min. The duration of immobile behavior was manually measured blinding to the treatment. The increase of immobility indicated the depression-like behavior (Steru et al., 1985; Trullas et al., 1989). FST. FST was performed as previously described (Ruan et al., 2014). Briefly, animals were placed in a cylinder containing water and recorded by a video camera for 6 min. The duration of climbing and immobile behaviors were manually measured in the first 2 min and last 4 min respectively blinding to the treatment. The increase of immobility and decrease of climbing both indicated the depression-like behaviors (Velazquez-Moctezuma and Diaz Ruiz, 1992; Castagne´ et al., 2011).

harvested and quantified by the BCA Protein Assay Kit (CWBIO, Beijing, China). 30 ug of denatured proteins was loaded and separated by running 10% SDS–PAGE gel, and transferred on to a PVDF membrane (Millipore, Billerica, MA, USA) at 400 mA for 2 h. After blocking with 5% skimmed milk powder at room temperature for 1 h, rabbit anti-WIP1 (D4F7) monoclonal antibody (#11901; 1:1000; Cell Signaling Technology, Danvers, MA, USA) or mouse anti-b-actin (CW0281A) monoclonal antibody (1:2000; CWBIO, Beijing, China) was incubated on a membrane at 4 °C, overnight. Horseradish peroxidaseconjugated secondary antibodies (1:2000; CWBIO, Beijing, China) were further incubated at room temperature for 1 h. Among incubations, TBST was used for washing after probing with antibody, 10 min  3. Imaging was performed using eECL (CW0049C; CWBIO, Beijing, China), and data were quantified by densitometric analysis using Image J software (NIH, Bethesda, MD, USA). Statistical analyses All data are presented as mean ± standard error of the mean (s.e.m.) and analyzed by IBM SPSS Statistics 21. Unpaired two-tailed Student’s t-test was used for comparison of the difference between the two groups (Control and CUMS in Fig. 1; WT and Wip1/ in Fig. 3). Two-way ANOVA were used to examine the effects of two main factors (CUMS and fluoxetine in Fig. 2) as well as the interaction of these two factors. Two-way repeated measures ANOVA were used to examine the effects of two main factors (CUMS and Wip1 in Fig. 4 and 5) as well as the interaction of these two factors due to within-subjects design. P < 0.05 was considered significant.

Real-time PCR RNA isolation and real-time PCR were performed as previously described (Ruan et al., 2014). Briefly, total RNA from the neocortex and hippocampus was isolated by TRIzol reagent (Sigma, St. Louis, MO, USA). RNA was quantified and reverse transcribed into cDNA by a PrimeScript RT reagent Kit (DRR037A; Takara, Otsu, Shiga, Japan). Using all Faststart Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland), all the realtime reactions were performed on the ABI 7300 platform (Applied Biosystems, Waltham, MA, USA). PPM1D forward primer: 50 -CAGAAAGGCTTCACCTCGTC-30 , reverse primer: 50 -CACCTCCACAGCTCTCACAA-30 ; b-actin forward primer: 50 - TGA GAC CTT CAA CAC CCC AG-30 , reverse primer: 50 - GCC ATC TCT TGC TCG AAG TC-30 . PPM1D mRNA was normalized to b-actin and the relative expression was calculated by the standard 2DDCT method. Western blot Western blot was performed as previously described (Ruan et al., 2014). Briefly, neocortex and hippocampus were collected and homogenized in RIPA buffer containing protease inhibitor cocktail (Roche, Basel, Switzerland). After incubation at 4 °C for 1 h, the lysates were centrifuged at 12,000 rpm, 4 °C for 15 min. Supernatant was

RESULTS WIP1 is downregulated in hippocampus under stress Growing evidences have implicated that Wip1 negatively responds to a range of stresses (Takekawa et al., 2000; Oshima et al., 2007; Park et al., 2012), together with its potential function in facilitating neurogenesis as well as inhibiting apoptosis and neuroinflammation through deactivating p53, p38 MAPK and NF-kappaB signaling (Herlaar and Brown, 1999; Fukunaga et al., 2004; Van Laethem et al., 2004; Zwerina et al., 2006; Demidov et al., 2007; Zhu et al., 2009). We hereby hypothesize that Wip1 may also respond to environmental stresses. To test this hypothesis, we duplicated a depression model in mice using a modified CUMS protocol (Willner et al., 1987; Ruan et al., 2014) as shown in Fig. 1a and examined the expression profile of Wip1 in the neocortex and hippocampus, which is associated with mood activities (Sheline, 2003; Hastings et al., 2004). Real-time PCR analyses showed no significant differences of Wip1 mRNA expression in both the neocortex (t(12) = 0.23, P = 0.82; Fig. 1b) and the hippocampus (t(12) = 0.01, P = 0.99; Fig. 1c) between control and CUMS groups. However, western blot analyses showed an over 20 percent reduction of Wip1 protein in the hippocampus (t(12) = 2.54, P < 0.05; Fig. 1e) but not neocortex

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22

15

Fig. 1. Expression of Wip1 in the brain after chronic unpredictable mild stress (CUMS). (a) Diagram of CUMS protocol. A, cold water swimming (13 ± 1 °C, 5 min); B, warm water swimming (37 ± 2 °C, 5 min); C, moist bedding (8 h); D, cage tilt (45°, 8 h); E, cage shaking (180 rpm, 10 min); F, tail pinch (1 cm from the tail end, 1 min); G, food deprivation (12 h); H, water deprivation (12 h); I, overnight illumination (12 h), and J, no stress (24 h). (b–c) Wip1 mRNA in the neocortex (b) and hippocampus (c) were analyzed by real-time PCR between CUMS and control mice, relative expression of Wip1 was quantified. b-actin was detected as loading control. n = 7 each. (d–e) WIP1 protein in neocortex (d) and hippocampus (e) were analyzed by western blot between CUMS and control mice, relative expression of WIP1 was quantified by densitometry, b-actin was detected as loading control. ⁄, P < 0.05 (unpaired Student’s t-tests); n = 7 each. All results are presented as mean ± s.e.m.

Fig. 2. Wip1 protein expression in the hippocampus after chronic treatment with fluoxetine along with CUMS. (a) Diagram of CUMS protocol and drug administration. Fluoxetine (20 mg/kg) or saline was daily i.p. injected along with the CUMS stimuli for 28 days. (b–d) Percentage of sucrose consumption in the sucrose preference test (b), percentages of immobility in the forced swimming test (c) and tail suspension test (d) were compared among control + saline, control + FLX, CUMS + saline and CUMS + FLX groups. (e) Expressions of Wip1 protein in the hippocampus after treating with saline or fluoxetine with and without CUMS were analyzed by western blot and quantified by densitometry. b-actin was detected as loading control. ns, non-significant; ⁄, P < 0.05; ⁄⁄, P < 0.01; ⁄⁄⁄, P < 0.001 (two-way ANOVA); n = 6 each. FLX, fluoxetine; SPT, sucrose preference test; FST, forced swimming test; TST, tail suspension test. All results are presented as mean ± s.e.m.

16

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22

Fig. 3. Exploratory behaviors in Wip1-deficient mice before CUMS. (a–b) Travel distance (a) and turn angle (b) in peripheral and central zones in the open field test were measured in Wip1/ (n = 8) versus wild-type (n = 10) mice. (c) Rearing numbers in the open field test were measured in Wip1/ versus wild-type mice. (d–e) Travel distance (d) and turn angle (e) in closed and open arms in the elevated zero maze test were measured in Wip1/ (n = 8) versus wild-type (n = 10) mice. (f) Diagram of the CUMS protocol and behavioral testing. ⁄, P < 0.05; ⁄⁄, P < 0.01 (unpaired Student’s t-tests). OFT, open field test; EOM, elevated zero maze test. All results are presented as mean ± s.e.m.

(t(12) = 0.78, P = 0.45; Fig. 1d). Together, these findings suggest that Wip1 protein in the hippocampus also negatively responds to the environmental stress. Stress-responsive downregulation of WIP1 is restored by fluoxetine Given that Wip1 protein is responsive to the repeated environmental stresses which are one of the main causes for human mood dysfunction (Ahn et al., 2009; Gillespie et al., 2009), we propose that this negative stress-response of Wip1 might be involved in the neurotransmitter dysregulation, a central mechanism for mood regulation. To test our hypothesis, fluoxetine or Prozac, the first SSRIs-type antidepressant for human depression treatment (Wong et al., 1974) was used to examine the function of WIP1 in the hippocampus by treating the mice along with the CUMS procedure (see Fig. 2a). SPT (Willner et al., 1987; Vollmer et al., 2013), FST (Velazquez-Moctezuma and Diaz Ruiz, 1992; Castagne´ et al., 2011) and TST (Steru et al., 1985; Trullas et al., 1989) were performed after stress in order to assess the stress-induced depression-like behaviors. The percentage of sucrose consumption in SPT was plotted in Fig. 2b. A

two-way ANOVA analysis revealed significant main effects of stress (F1,22 = 5.50, P < 0.05) and treatment (F1,22 = 12.59, P < 0.01), as well as a significant interaction between stress and treatment (F1,22 = 10.82, P < 0.01). Subsequent pairwise comparisons on the percentage of sucrose consumption showed a decrease in the CUMS-saline group versus the control-saline group (P < 0.001), an increase in the CUMS-fluoxetine versus the CUMS-saline group (P < 0.001), no significant difference between the control-saline group and the control-fluoxetine group (P = 0.86) as well as the control-fluoxetine group and the CUMS-fluoxetine group (P = 0.51). Moreover, the percentages of immobility in FST and TST were respectively plotted in Fig. 2c and Fig. 2d. A two-way ANOVA analysis revealed significant main effects of stress (F1,22 = 36.17, P < 0.001; Fig. 2c; F1,22 = 43.28, P < 0.001; Fig. 2d) and treatment (F1,22 = 2.61, P < 0.05; Fig. 2c; F1,22 = 3.36, P < 0.05; Fig. 2d), as well as a significant interaction between stress and treatment (F1,22 = 3.47, P < 0.05; Fig. 2c; F1,22 = 9.18, P < 0.01; Fig. 2d). Further pairwise comparisons on the percentage of immobility showed increases in the CUMS-saline group versus the control-saline group (P < 0.001; Fig. 2c; P < 0.001; Fig. 2d) and the

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22

17

Fig. 4. Anxiety-like behaviors in Wip1 deficient mice before and after CUMS. (a–c) Entries in the peripheral zone (a), central zone (b) as well as percentage of time spent in the central zone (c) in the open field test were repeatedly measured before CUMS (pre-CUMS) and after 1, 2 and 3 week(s) of CUMS in Wip1/ (n = 8) versus wild-type (n = 10) mice. (d–f) Entries in closed arms (d), open arms (e) as well as percentage of time spent in open arms (f) in the elevated zero maze test were repeatedly measured before CUMS and after 3 weeks of CUMS in Wip1/ (n = 8) versus wild-type (n = 10) mice. ⁄, P < 0.05; ⁄⁄, P < 0.01; ⁄⁄⁄, P < 0.001 (repeated measures two-way ANOVA). All results are presented as mean ± s.e.m.

CUMS-fluoxetine group versus the control-fluoxetine group and (P < 0.5; Fig. 2c; P < 0.05; Fig. 2d), a decrease in the CUMS-fluoxetine group versus the CUMS-saline group (P < 0.05; Fig. 2c; P < 0.05; Fig. 2d), as well as no significant difference between the control-saline group and the control-fluoxetine group (P = 0.25; Fig. 2c; P = 0.08; Fig. 2d). These data indicate that CUMS-induced depression-like behaviors were attenuated by the treatment of fluoxetine, while no similar effect was observed in control animals. Next, WIP1 expression in the hippocampus was further analyzed by western blot (see Fig. 2d), and a two-way ANOVA analysis revealed main effects of stress (F1,22 = 12.38, P < 0.01) and treatment (F1,22 = 2.09, P < 0.05), as well as an interaction between stress and treatment (F1,22 = 4.02, P < 0.05). Subsequent pairwise comparisons showed a decrease of WIP1 level in the CUMS-saline group versus the control-saline group (P < 0.001), an increase in the CUMS-fluoxetine group versus the CUMS-saline group (P < 0.05), and no significant difference between the control-saline group and the control-fluoxetine group

(P = 0.86) as well as the control-fluoxetine group and the CUMS-fluoxetine group (P = 0.43). Collectively, the stress-responsive downregulation of WIP1 in the hippocampus is restorable by the chronic treatment of fluoxetine, suggesting a potential signaling of Wip1 involves the regulation of serotonin transmission. Loss of Wip1 impairs exploratory behaviors in mice As it is known that Wip1 negatively responds to the environmental stresses and is involved in the serotonin regulation in the hippocampus, it suggests a potential role of Wip1 in mood stabilization. To determine the function of Wip1 in the regulation of mood, 8 of the adult male Wip1 knockout mice (Choi et al., 2002) were generated for characterizing the mood-related phenotypes through performing OFT (Corman and Shafer, 1968; Prut and Belzung, 2003), EOM (Shepherd et al., 1994; Kulkarni et al., 2007), FST and TST as shown in Fig. 3f, meanwhile 10 of the adult male WT littermates were tested as control. Before CUMS, OFT showed a reduction

18

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22

Fig. 5. Depression-like behaviors in Wip1-deficient mice before and after CUMS. Percentage of immobility was measured in the tail suspension test (a) and percentages of immobility (b) and climbing (c) were measured in the forced swimming test before and after 3 weeks of CUMS in Wip1/ (n = 8) versus wild-type (n = 10) mice. ns, non-significant; ⁄, P < 0.05; ⁄⁄, P < 0.01; ⁄⁄⁄, P < 0.001 (repeated measures two-way ANOVA). All results are presented as mean ± s.e.m.

of the traveling distance in the central zone in Wip1/ versus WT mice (t(16) = 2.70, P < 0.05; Fig. 3a), whereas no significant difference was measured in the peripheral zone between them (t(16) = 0.12, P = 0.91; Fig 3a). Similarly, the traveling distance in EOM was decreased in the open arms (t(16) = 3.25, P < 0.01; Fig. 3d), and no significant difference in the closed arms (t(16) = 0.93, P = 0.38; Fig. 3d) was detected. Moreover, turn angles (Duffy and Ford, 1997; Spink et al., 2001; Venkitaramani et al., 2011) of the body were also reduced in the central zone (t(16) = 2.52, P < 0.05; Fig. 3b) and the open arms (t(16) = 2.04, P < 0.05; Fig. 3e) in Wip1/ compared with WT mice, and also no significant difference was found in the peripheral zone (t(16) = 1.33, P = 0.20; Fig. 3b) and closed arms (t(16) = 1.35, P = 0.20; Fig. 3e). Decreased traveling distances and turn angles in Wip1/ vs WT mice in the central zone and open arms indicate an impaired exploratory behavior in mice without WIP1, while no significant alteration of these two parameters in the peripheral zone and closed arms suggests no locomotor defect in Wip1/ mice. In addition, the rearing behavior in OFT was also reduced in Wip1/ relative to WT mice (t(16) = 2.40, P < 0.05; Fig. 3c), which also suggest an impaired exploratory behavior in Wip1/ mice. Taken together, mice deficient for WIP1 show decreased exploratory behaviors without impairment of the basic movement. Loss of Wip1 displays anxiety-like behaviors in mice In order to further assess the anxiety-like behavior of Wip1 knockout mice with and without stress, all animals were performed with OFT before (pre-CUMS) and after (CUMS-1w, 2w, and 3w) CUMS. Entries in the peripheral zone, entries in the central zone and percentage of time spent in the central zone were respectively plotted in Fig. 4a–c. Repeated measures two-way ANOVA analyses showed main effects of stress (F3,14 = 14.85, P < 0.001; Fig. 4a; F3,14 = 13.68,

P < 0.001; Fig. 4b; F3,14 = 8.78, P < 0.001; Fig. 4c) and genotype (F3,14 = 30.06, P < 0.001; Fig. 4a; F3,14 = 30.11, P < 0.001; Fig. 4b; F3,14 = 41.54, P < 0.001; Fig. 4c) without significant interaction between stress and genotype (F3,14 = 2.12, P = 0.12; Fig. 4a; F3,14 = 2.13, P = 0.12; Fig. 4b; F3,14 = 0.29, P = 0.83; Fig. 4c). Pairwise comparisons revealed reductions of entries in the peripheral zone (WT: P < 0.001; Wip1/: P < 0.001; Fig. 4a) and the central zone (WT: P < 0.001; Wip1/: P < 0.001; Fig. 4b) as well as percentage of time spent in the central zone (WT: P < 0.001; Wip1/: P < 0.001; Fig. 4c) in both WT and Wip1/ mice between preCUMS and CUMS-3w, suggesting that the stressinduced anxiety-like behaviors were successfully developed; also, decreases of entries in the peripheral zone and the central zone in Wip1/ versus WT mice at pre-CUMS (P < 0.05; Fig. 4a; P < 0.05; Fig. 4b), CUMS-2w (P < 0.001; Fig. 4a; P < 0.001; Fig. 4b) and CUMS-3w (P < 0.001; Fig. 4a; P < 0.001; Fig. 4b), as well as a decrease of percentage of time in the central zone at pre-CUMS (P < 0.05; Fig. 4c), CUMS-1w (P < 0.05; Fig. 4c), CUMS-2w (P < 0.01; Fig. 4c) and CUMS-3w (P < 0.001; Fig. 4c). Taken together, these data indicated that mice without WIP1 were more anxious in the open field than WT mice with and without stress. In addition, EOM was also performed before (pre-CUMS) and after 3 weeks of CUMS (CUMS) to confirm the findings in open field (see Fig. 4d–f). Repeated measures two-way ANOVA analyses showed main effects of stress (F1,16 = 12.87, P < 0.01; Fig. 4d; F1,16 = 14.68, P < 0.01; Fig. 4e; F1, 16 = 25.51, P < 0.001; Fig. 4f) and genotype (F1,16 = 21.88, P < 0.01; Fig. 4d; F1,16 = 24.86, P < 0.01; Fig. 4e; F1,16 = 20.66, P < 0.01; Fig. 4f) without significant interaction between stress and genotype (F1,16 = 0.26, P = 0.62; Fig. 4d; F1,16 = 0.22, P = 0.65; Fig. 4e; F1,16 = 1.42, P = 0.26; Fig. 4f). Further pair comparisons revealed reductions of entries in closed arms (WT: P < 0.01; Wip1/: P < 0.05; Fig. 4d) and

19

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22

open arms (WT: P < 0.01; Wip1/: P < 0.05; Fig. 4e) as well as percentage of time spent in open arms (WT: P < 0.001; Wip1/: P < 0.05; Fig. 4f) in WT or Wip1/ mice between pre-CUMS and CUMS, suggesting the stress-induced anxiety-like behaviors were successfully developed. Moreover, the pair comparisons also showed decreases of entries in closed arms, entries in open arms and percentage of time spent in open arms in Wip1/ versus WT mice at preCUMS (P < 0.05; Fig. 4d; P < 0.05; Fig. 4e; P < 0.01; Fig. 4f) and CUMS (P < 0.001; Fig. 4d; P < 0.001; Fig. 4e; P < 0.001; Fig. 4f). In addition, significant reductions of these behaviors were also observed between stressed Wip1/ mice and baseline stressfree WT mice (P < 0.001; Fig. 4d; P < 0.001; Fig. 4e; P < 0.001; Fig. 4f). Thus collectively, both OFT and EOM indicate that mice without WIP1 display increased anxiety-like behaviors under both non-stressed and stressed conditions. Loss of Wip1 displays depression-like behaviors in mice To further assess the depressive behaviors, the same batch of mice were performed with TST and FST before (pre-CUMS) and after 3 weeks of CUMS (CUMS). Percentages of immobility in these two tests were plotted in Fig. 5a-b. Repeated measures two-way ANOVA analyses showed a main effect of stress (F1,16 = 16.00, P < 0.01; Fig. 5a; F1,16 = 12.49, P < 0.01; Fig. 5b) but not genotype (F1,16 = 0.18, P = 0.68; Fig. 5a; F1,16 = 0.18, P = 0.14; Fig. 5b), however, a significant interaction between stress and genotype was detected in TST (F1,16 = 33.71, P < 0.001; Fig. 5a) but not FST (F1,16 = 1.03, P = 0.34; Fig. 5b). Subsequent pair comparisons on WT mice showed that percentages of immobility were significantly increased in both TST (P < 0.001) and FST (P < 0.01). These suggest that depression-like behaviors are successfully induced by CUMS. However, further comparisons showed no significant difference in Wip1/ mice between pre-CUMS and CUMS groups (P = 0.33; Fig. 5a; P = 0.17; Fig. 5b). Furthermore, an increase of immobility was detected in Wip1/ versus WT mice before CUMS (P < 0.01; Fig. 5a) by TST, while a trend of increase was also detected by FST (P = 0.10; Fig. 5b). Although the Wip1/ mice did not show higher immobility than WT mice in both of these two tests after CUMS (significant lower, P < 0.05; Fig. 5a; P = 0.97; Fig. 5b), they still constantly maintained an elevated immobility compared to the baseline stress-free WT mice in both of these two tests (P < 0.05; Fig. 5a; P < 0.01; Fig. 5b). These indicate that Wip1-deficiency-induced depression-like behaviors cannot be further elevated by CUMS. In addition, the percentage of climbing in FST was plotted in Fig. 5c. Repeated measures two-way ANOVA analysis showed a main effect of stress (F1,16 = 7.64, P < 0.05; Fig. 5c) and genotype (F1,16 = 31.93, P < 0.001; Fig. 5c) without a significant interaction between stress and genotype (F1,16 = 1.14, P = 0.31; Fig. 5c). Further pair comparisons revealed a significant reduction in climbing

in Wip1/ (P < 0.05) but not WT mice (P = 0.32) between pre-CUMS and CUMS, decreases of climbing in Wip1/ versus WT mice before (P < 0.001) and after CUMS (P < 0.001), as well as a significant reduction in Wip1/ with CUMS relative to stress-free WT (P < 0.001). Collectively, the above data suggest that mice deficient for Wip1 display increased depression-like behaviors with and without stress compared to non-stressed WT mice, however, this Wip1 deficiency caused depression-like phenotype may not be further elevated by CUMS.

DISCUSSION In the present study, we present the novel role of Wip1 in the regulation of mood in mice. Our data demonstrate that CUMS reduces Wip1 protein but not its mRNA level in the hippocampus but not in the neocortex in mice, and this CUMS-responsive downregulation of WIP1 is restored by chronic treatment of fluoxetine along with the fluoxetine-revoked attenuation of depression-like behaviors. Furthermore, Wip1 deficiency causes reduced exploratory behaviors but not motor activities in mice as demonstrated by OFT and EOM. Finally, mice deficient for Wip1 also display anxiety-like and depression-like phenotypes under both CUMS-free and CUMS conditions as revealed by OFT and EOM for anxious behaviors, and TST and FST for depressive behaviors. Although limitations exist, these results suggest that Wip1 plays a critical function in mood stabilization through the suppression of stress response which is consistent with the current literature. Wip1 is a protein that regulates stressful events such as physical and chemical stimuli. Previous reports have demonstrated a common role of Wip1 in suppressing response to various stresses including ionizing radiation (Fiscella et al., 1997), ultraviolet radiation (Takekawa et al., 2000), hydrogen peroxide (Oshima et al., 2007), anisomycin (Takekawa et al., 2000) and methyl methane sulfonate (Park et al., 2012). We found that Wip1 protein but not its mRNA in the hippocampus is down-regulated in response to CUMS. Our result is consistent with the previous studies in which Wip1 protein was downregulated in response to DNA damage response (Macurek et al., 2012; Vaz and Ramadan, 2013). Although a rapid (several hours) upregulation of Wip1 mRNA was consistently observed in response to a number of stresses such as ultraviolet radiation and ionizing radiation (Takekawa et al., 2000; Demidov et al., 2006; Dayaram et al., 2013), no change of Wip1 mRNA is detected after 28 days of CUMS. The inconsistency could be due to a different time course of stress, i.e., the rapid response of Wip1 is generated as a negative feedback in suppressing stress-responsive p38 MAPK/p53-mediated proapoptotic signaling (Takekawa et al., 2000; Yu et al., 2007; Bachis et al., 2008), or NF-kappaB-mediated proinflammatory reaction (Chew et al., 2009; Lowe et al., 2012; Demirtasß et al., 2014) by dephosphorylation of these substrates. Chronic stressful stimulation would result in the repression of the Wip1 activity. As nonconcomitant mRNA and protein expression commonly occurs (Pierce et al., 2012), the reduction of Wip1 protein

20

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22

but not mRNA in the hippocampus suggests CUMS affects either the protein translation or stabilization/degradation process. Similar with our previous finding (Ruan et al., 2014), a specific brain region, hippocampus, is identified in which Wip1 protein is altered under CUMS, suggesting a link between stress and Wip1 suppression in the hippocampus. As fluoxetine, the most common used anti-depressant, can alleviate the depression symptoms in a subpopulation of depression subjects, we used fluoxetine in these stressed mice to see whether the reduction in Wip1 expression is reversed. Fluoxetine has long been implicated to attenuate CUMS-responsive impairment of hippocampal neurogenesis and elevated apoptosis and inflammation (Lee et al., 2001; Dulawa et al., 2004; Huang and Herbert, 2006; Roumestan et al., 2007). Chronic treatment with fluoxetine in the present study restores the CUMS-responsive depression-like behaviors such as increased sucrose consumption in SPT as well as decreased immobility in both TST and FST. As expected, the reduction of Wip1 protein level in the hippocampus is also significantly reversed by fluoxetine. Our result is consistent with the recent studies showing that fluoxetine reduces inflammation through deactivation of the NF-kappaB or/and p38 MAPK signaling (Yao et al., 2006; Kale et al., 2007; Yang et al., 2014b) while it is known that Wip1 selectively deactivates NF-kappaB (Chew et al., 2009) and p38 MAPK (Takekawa et al., 2000; Yu et al., 2007). Together with the data showing that the mice with Wip1 deficiency display anxiety- and depression-like behaviors without any stress, the reduction of Wip1 under stress and restoration of its level after the treatment with fluoxetine suggests that Wip1 is an important protein that regulates mood behaviors. Wip1 knockout mouse is a good model to address the role of Wip1 in the regulation of mood behaviors. Our data demonstrate the exploratory behaviors, the motivation/tendency to explore a novel environment (Stapley and Keogh, 2004), are impaired as displayed by the decreased traveling distances and decreased turn angles in both the central zone of the open field and open arms of the elevated zero maze, as well as the reduced rearing behavior in the open field. Meanwhile, Wip1 deficiency has no effect on motor activities as no differences in traveling distances and turn angles were observed. Our data are consistent with the findings that mice treated with p38 MAPK inhibitor SB203580 did not show motor deficit (Bruchas et al., 2007; Galeotti and Ghelardini, 2012). Our data suggest that the impairment of exploratory behaviors in Wip1-deficient mice is not due to basal motor activities. Moreover, mice deficient for Wip1 consistently show anxiety-like phenotypes under both CUMS-free and CUMS condition. The Wip1 deficiency-induced anxietyrelated behaviors are further elevated under CUMS exposure. In addition, mice deficient for Wip1 also display depression-like phenotypes with and without CUMS relative to the baseline stress-free WT mice, as displayed by the increased immobility in TST and FST and the decreased climbing behavior in FST. These Wip1 deficiency-induced depression-like behaviors seem not to be further increased under CUMS exposure as no consistent

and significant alteration is detected. Taken together, our data suggest that Wip1 deficiency results in an anxiety-like and a depression-like phenotype in mice under stressed or non-stressed conditions compared to stress-free WT mice. However, these mutant mice were more anxious but not more depressive under stress. Our findings are consistent with studies which reported that selective deletion of p38a MAPK attenuated the depression-like behavior as displayed by reduced immobility in FST (Bruchas et al., 2011). In addition, other studies also showed that inhibiting p38 MAPK with inhibitors attenuated the anxiety-like behavior as demonstrated by reduced time spent in central zone in OFT and open arms in the elevated plus-maze test (Corsi et al., 2011; Peng et al., 2013), as well as depression-like behavior as shown by reduced immobility in FST or TST in mice (Bruchas et al., 2007; Galeotti and Ghelardini, 2012). A number of limitations still exist in this study. For example, the results were achieved based only on the males, which leave an unsolved question for the other sex. In addition, the other stress-responsive limbic areas were not examined. Furthermore, within-subject design using a small sample-size, multiple behavioral testing might affect the statistical significance examination. At last, the basal immobility of WT mice in FST is considerably higher than normal basal immobile behavior and our previous results (Ruan et al., 2014), which may be caused by repeated testing as we know FST and TST have been used as acute depression models. In conclusion, we are the first to define the potential function of Wip1 in stabilization of mood. The protein level of Wip1 in the hippocampus is reduced in response to the CUMS while fluoxetine restored the Wip1 downregulation. Moreover, loss of Wip1 in mice results in an exploratory deficit as well as an anxiety- and depression-like phenotype. Sustained environmental stresses further render the Wip1-deficient mice more anxious but not more depressive. This study highlights the possible link between stress-induced Wip1 responses and mood dysfunction and suggests a drug target for mental illness. Acknowledgments—We are grateful to Dr. L. A. Donehower from the Baylor College of Medicine for providing Wip1/ mice. This work was supported by grants from the Chinese MST 2011CB944200 and NHMRC grants (APP1021408 and APP1021409) to X.F.Z., the Talent Program of Yunnan Province, China, and the Professorial Fellowship of Monash University, Australia, to Z.C.X., as well as Chinese NSFC grant (31460314) to S.F.W. We wish to thank Ms. Diana Jaeger from University of South Australia for critical reading of this manuscript. X.F.Z. is a visiting professor of KMU.

REFERENCES Ahn WK, Proctor CC, Flanagan EH (2009) Mental health clinicians’ beliefs about the biological, psychological, and environmental bases of mental disorders. Cognit Sci 33:147–182. Asnis GM, McGinn LK, Sanderson WC (1995) Atypical depression: clinical aspects and noradrenergic function. Am J Psychiatry 152:31–36.

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22 Bachis A, Cruz MI, Nosheny RL, Mocchetti I (2008) Chronic unpredictable stress promotes neuronal apoptosis in the cerebral cortex. Neurosci Lett 442:104–108. Brown A, Gershon S (1993) Dopamine and depression. J Neural Transm/Gen Sect JNT 91:75–109. Bruchas MR, Land BB, Aita M, Xu M, Barot SK, Li S, Chavkin C (2007) Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria. J Neurosci 27:11614–11623. Bruchas MR, Schindler AG, Shankar H, Messinger DI, Miyatake M, Land BB, Lemos JC, Hagan CE, Neumaier JF, Quintana A (2011) Selective p38a MAPK deletion in serotonergic neurons produces stress resilience in models of depression and addiction. Neuron 71:498–511. Brunello N, Blier P, Judd LL, Mendlewicz J, Nelson CJ, Souery D, Zohar J, Racagni G (2003) Noradrenaline in mood and anxiety disorders: basic and clinical studies. Int Clin Psychopharmacol 18:191–202. Cassano P, Fava M (2002) Depression and public health: an overview. J Psychosom Res 53:849–857. Castagne´ V, Moser P, Roux S, Porsolt RD (2011) Rodent models of depression: forced swim and tail suspension behavioral despair tests in rats and mice. Curr Protoc Neurosci 8:8.10 A. Chew J, Biswas S, Shreeram S, Humaidi M, Wong ET, Dhillion MK, Teo H, Hazra A, Fang CC, Lopez-Collazo E, Bulavin DV, Tergaonkar V (2009) WIP1 phosphatase is a negative regulator of NF-kappaB signalling. Nat Cell Biol 11:659–666. Choi J, Nannenga B, Demidov ON, Bulavin DV, Cooney A, Brayton C, Zhang Y, Mbawuike IN, Bradley A, Appella E, Donehower LA (2002) Mice deficient for the wild-type p53-induced phosphatase gene (Wip1) exhibit defects in reproductive organs, immune function, and cell cycle control. Mol Cell Biol 22:1094–1105. Chouinard G (1985) A double-blind controlled clinical trial of fluoxetine and amitriptyline in the treatment of outpatients with major depressive disorder. J Clin Psychiatry 46:32–37. Corman CD, Shafer JN (1968) Open-field activity and exploratory behavior. Psychonomic Sci 13:55–56. Corsi M, Faiferman I, Pich EM, Ratti E, Wren PB (2011) Use of a p38 kinase inhibitor for treating psychiatric disorders. U.S. Patent No. 7,989,479. Dayaram T, Lemoine FJ, Donehower LA, Marriott SJ (2013) Activation of WIP1 phosphatase by HTLV-1 Tax mitigates the cellular response to DNA damage. PLoS One 8:e55989. Demidov O, Kek C, Shreeram S, Timofeev O, Fornace A, Appella E, Bulavin D (2006) The role of the MKK6/p38 MAPK pathway in Wip1-dependent regulation of ErbB2-driven mammary gland tumorigenesis. Oncogene 26:2502–2506. Demidov ON, Timofeev O, Lwin HN, Kek C, Appella E, Bulavin DV (2007) Wip1 phosphatase regulates p53-dependent apoptosis of stem cells and tumorigenesis in the mouse intestine. Cell Stem Cell 1:180–190. Demirtasß T, Utkan T, Karson A, Yazır Y, Bayramgu¨rler D, Gacar N (2014) The link between unpredictable chronic mild stress model for depression and vascular inflammation? Inflammation 37(5):1432–1438. Diehl DJ, Gershon S (1992) The role of dopamine in mood disorders. Compr Psychiatry 33:115–120. Duffy KJ, Ford RM (1997) Turn angle and run time distributions characterize swimming behavior for Pseudomonas putida. J Bacteriol 179:1428–1430. Dulawa SC, Holick KA, Gundersen B, Hen R (2004) Effects of chronic fluoxetine in animal models of anxiety and depression. Neuropsychopharmacology 29:1321–1330. Eilat E, Mendlovic S, Doron A, Zakuth V, Spirer Z (1999) Increased apoptosis in patients with major depression: a preliminary study. J Immunol 163:533–534. Fiscella M, Zhang H, Fan S, Sakaguchi K, Shen S, Mercer WE, Vande Woude GF, O’Connor PM, Appella E (1997) Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. Proc Natl Acad Sci U S A 94:6048–6053.

21

Fukunaga M, Miyata S, Liu BF, Miyazaki H, Hirota Y, Higo S, Hamada Y, Ueyama S, Kasuga M (2004) Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells. Biochem Biophys Res Commun 320:689–695. Galeotti N, Ghelardini C (2012) Regionally selective activation and differential regulation of ERK, JNK and p38 MAP kinase signalling pathway by protein kinase C in mood modulation. Int J Neuropsychopharmacol 15:781–793. Gillespie CF, Phifer J, Bradley B, Ressler KJ (2009) Risk and resilience: genetic and environmental influences on development of the stress response. Depression Anxiety 26:984–992. Gururajan A, Hill R, van den Buuse M (2014) Long-term differential effects of chronic young-adult corticosterone exposure on anxiety and depression-like behaviour in BDNF heterozygous rats depend on the experimental paradigm used. Neurosci Lett 576:6–10. Hastings RS, Parsey RV, Oquendo MA, Arango V, Mann JJ (2004) Volumetric analysis of the prefrontal cortex, amygdala, and hippocampus in major depression. Neuropsychopharmacology 29:952–959. Herlaar E, Brown Z (1999) P38 MAPK signalling cascades in inflammatory disease. Mol Med Today 5:439–447. Huang GJ, Herbert J (2006) Stimulation of neurogenesis in the hippocampus of the adult rat by fluoxetine requires rhythmic change in corticosterone. Biol Psychiatry 59:619–624. Kale AY, Paranjape SA, Briski KP (2007) Site-specific habituation of insulin-induced hypoglycemic induction of fos immunoreactivity in glucocorticoid receptor: immunopositive neurons in the male rat brain. Exp Brain Res 176:260–266. Krystal J, Sanacora G, Blumberg H, Anand A, Charney D, Marek G, Epperson C, Goddard A, Mason G (2001) Glutamate and GABA systems as targets for novel antidepressant and mood-stabilizing treatments. Mol Psychiatry 7:S71–80. Kulkarni S, Singh K, Bishnoi M (2007) Elevated zero maze: a paradigm to evaluate antianxiety effects of drugs. Methods Find Exp Clin Pharmacol 29:343–348. Lee H, Kim J, Yim S, Kim M, Kim S, Kim Y, Kim C, Chung J (2001) Fluoxetine enhances cell proliferation and prevents apoptosis in dentate gyrus of maternally separated rats. Mol Psychiatry 6(610). 725-618. Lowe J, Cha H, Lee MO, Mazur SJ, Appella E, Fornace Jr AJ (2012) Regulation of the Wip1 phosphatase and its effects on the stress response. Front Biosci 17:1480. Lu J, Xu Y, Hu W, Gao Y, Ni X, Sheng H, Liu Y (2014) Exercise ameliorates depression-like behavior and increases hippocampal BDNF level in ovariectomized rats. Neurosci Lett 573:13–18. Macurek L, Benada J, Mu¨llers E, Halim VA, Krejcˇı´ kova´ K, Burdova´ K, Pecha´cˇkova´ S, Hodny´ Z, Lindqvist A, Medema RH (2012) Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis. Cell Cycle 12:251–262. Miller AH, Maletic V, Raison CL (2009) Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression. Biol Psychiatry 65:732–741. Oshima T, Sasaki M, Kataoka H, Miwa H, Takeuchi T, Joh T (2007) Wip1 protects hydrogen peroxide-induced colonic epithelial barrier dysfunction. Cell Mol Life Sci: CMLS 64:3139–3147. Park JY, Song JY, Kim HM, Han HS, Seol HS, Jang SJ, Choi J (2012) P53-Independent expression of wild-type p53-induced phosphatase 1 (Wip1) in methylmethane sulfonate-treated cancer cell lines and human tumors. Int J Biochem Cell Biol 44:896–904. Peng Z, Wang H, Zhang R, Chen Y, Xue F, Nie H, Chen Y, Wu D, Wang Y, Wang H, Tan Q (2013) Gastrodin ameliorates anxietylike behaviors and inhibits IL-1beta level and p38 MAPK phosphorylation of hippocampus in the rat model of posttraumatic stress disorder. Physiol Res 62(5):537–545. Petty F (1995) GABA and mood disorders: a brief review and hypothesis. J Affect Disord 34:275–281. Pierce A, Williamson A, Jaworska E, Griffiths JR, Taylor S, Walker M, O’Dea MA, Spooncer E, Unwin RD, Poolman T (2012)

22

C. S. Ruan et al. / Neuroscience 293 (2015) 12–22

Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation. PLoS One 7:e38928. Prut L, Belzung C (2003) The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 463:3–33. Raison CL, Capuron L, Miller AH (2006) Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends Immunol 27:24–31. Roumestan C, Michel A, Bichon F, Portet K, Detoc M, Henriquet C, Jaffuel D, Mathieu M (2007) Anti-inflammatory properties of desipramine and fluoxetine. Respir Res 8:35. Ruan CS, Wang SF, Shen YJ, Guo Y, Yang CR, Zhou FH, Tan LT, Zhou L, Liu JJ, Wang WY, Xiao ZC, Zhou XF (2014) Deletion of TRIM32 protects mice from anxiety- and depression-like behaviors under mild stress. Eur J Neurosci 40:2680–2690. Sablina AA, Chen W, Arroyo JD, Corral L, Hector M, Bulmer SE, DeCaprio JA, Hahn WC (2007) The tumor suppressor PP2A Abeta regulates the RalA GTPase. Cell 129:969–982. Sheline YI (2003) Neuroimaging studies of mood disorder effects on the brain. Biol Psychiatry 54:338–352. Shepherd JK, Grewal SS, Fletcher A, Bill DJ, Dourish CT (1994) Behavioural and pharmacological characterisation of the elevated ‘‘zero-maze’’ as an animal model of anxiety. Psychopharmacology 116:56–64. Snyder JS, Soumier A, Brewer M, Pickel J, Cameron HA (2011) Adult hippocampal neurogenesis buffers stress responses and depressive behaviour. Nature 476:458–461. Spink AJ, Tegelenbosch RA, Buma MO, Noldus LP (2001) The EthoVision video tracking system–a tool for behavioral phenotyping of transgenic mice. Physiol Behav 73:731–744. Stapley J, Keogh JS (2004) Exploratory and antipredator behaviours differ between territorial and nonterritorial male lizards. Anim Behav 68:841–846. Steru L, Chermat R, Thierry B, Simon P (1985) The tail suspension test: a new method for screening antidepressants in mice. Psychopharmacology 85:367–370. Sun ZG, Huang QZ, Xu CY, Chen LP (2013) [Effects of shuyu ningxln recipe on the praxiology and the expressions of hippocampal BDNF and trkB of model rats with chronic stress-induced depression]. Zhongguo Zhong xi yi jie he za zhi Zhongguo Zhongxiyi jiehe zazhi = Chinese journal of integrated traditional and Western medicine / Zhongguo Zhong xi yi jie he xue hui. Zhongguo Zhong yi yan jiu yuan zhu ban 33:370–375. Takekawa M, Adachi M, Nakahata A, Nakayama I, Itoh F, Tsukuda H, Taya Y, Imai K (2000) P53-inducible wip1 phosphatase mediates a negative feedback regulation of p38 MAPK-p53 signaling in response to UV radiation. EMBO J 19:6517–6526. Treusch S, Hamamichi S, Goodman JL, Matlack KE, Chung CY, Baru V, Shulman JM, Parrado A, Bevis BJ, Valastyan JS, Han H, Lindhagen-Persson M, Reiman EM, Evans DA, Bennett DA, Olofsson A, DeJager PL, Tanzi RE, Caldwell KA, Caldwell GA, Lindquist S (2011) Functional links between Abeta toxicity, endocytic trafficking, and Alzheimer’s disease risk factors in yeast. Science 334:1241–1245. Trullas R, Jackson B, Skolnick P (1989) Genetic differences in a tail suspension test for evaluating antidepressant activity. Psychopharmacology 99:287–288. Van Laethem A, Van Kelst S, Lippens S, Declercq W, Vandenabeele P, Janssens S, Vandenheede JR, Garmyn M, Agostinis P (2004) Activation of p38 MAPK is required for Bax translocation to mitochondria, cytochrome c release and apoptosis induced by UVB irradiation in human keratinocytes. FASEB J 18:1946–1948.

Vaswani M, Linda FK, Ramesh S (2003) Role of selective serotonin reuptake inhibitors in psychiatric disorders: a comprehensive review. Prog Neuropsychopharmacol Biol Psychiatry 27:85–102. Vaz B, Ramadan K (2013) Wip1 downregulation conserves truncated DNA damage response (DDR) in mitosis. Cell Cycle 12:391. Velazquez-Moctezuma J, Diaz Ruiz O (1992) Neonatal treatment with clomipramine increased immobility in the forced swim test: an attribute of animal models of depression. Pharmacol Biochem Behav 42:737–739. Venkitaramani DV, Moura PJ, Picciotto MR, Lombroso PJ (2011) Striatal-enriched protein tyrosine phosphatase (STEP) knockout mice have enhanced hippocampal memory. Eur J Neurosci 33:2288–2298. Vollmer LE, Ghosal S, Rush JA, Sallee FR, Herman JP, Weinert M, Sah R (2013) Attenuated stress-evoked anxiety, increased sucrose preference and delayed spatial learning in glucocorticoid-induced receptor-deficient mice. Genes Brain Behav 12:241–249. Willner P, Towell A, Sampson D, Sophokleous S, Muscat R (1987) Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology 93:358–364. Wong DT, Horng JS, Bymaster FP, Hauser KL, Molloy BB (1974) A selective inhibitor of serotonin uptake: Lilly 110140, 3-(ptrifluoromethylphenoxy)-N-methyl-3-phenylpropylamine. Life Sci 15:471–479. Yang CR, Zhang ZG, Bai YY, Zhou HF, Zhou L, Ruan CS, Li F, Li CQ, Zheng HY, Shen LJ, Zhou XF (2014a) Foraging activity is reduced in a mouse model of depression. Neurotox Res 25: 235–247. Yang JM, Rui BB, Chen C, Chen H, Xu TJ, Xu WP, Wei W (2014b) Acetylsalicylic acid enhances the anti-inflammatory effect of fluoxetine through inhibition of NF-kappaB, p38-MAPK and ERK1/2 activation in lipopolysaccharide-induced BV-2 microglia cells. Neuroscience 275:296–304. Yao Z, DuBois DC, Almon RR, Jusko WJ (2006) Modeling circadian rhythms of glucocorticoid receptor and glutamine synthetase expression in rat skeletal muscle. Pharm Res 23:670–679. Young LT, Warsh JJ, Kish SJ, Shannak K, Hornykeiwicz O (1994) Reduced brain 5-HT and elevated NE turnover and metabolites in bipolar affective disorder. Biol Psychiatry 35:121–127. Young SN, Leyton M (2002) The role of serotonin in human mood and social interaction: insight from altered tryptophan levels. Pharmacol Biochem Behav 71:857–865. Yu E, Ahn YS, Jang SJ, Kim MJ, Yoon HS, Gong G, Choi J (2007) Overexpression of the wip1 gene abrogates the p38 MAPK/p53/ Wip1 pathway and silences p16 expression in human breast cancers. Breast Cancer Res Treat 101:269–278. Zhou H, Wang J, Jiang J, Stavrovskaya IG, Li M, Li W, Wu Q, Zhang X, Luo C, Zhou S, Sirianni AC, Sarkar S, Kristal BS, Friedlander RM, Wang X (2014) N-acetyl-serotonin offers neuroprotection through inhibiting mitochondrial death pathways and autophagic activation in experimental models of ischemic injury. J Neurosci 34:2967–2978. Zhu YH, Zhang CW, Lu L, Demidov ON, Sun L, Yang L, Bulavin DV, Xiao ZC (2009) Wip1 regulates the generation of new neural cells in the adult olfactory bulb through p53-dependent cell cycle control. Stem cells 27:1433–1442. Zwerina J, Hayer S, Redlich K, Bobacz K, Kollias G, Smolen JS, Schett G (2006) Activation of p38 MAPK is a key step in tumor necrosis factor–mediated inflammatory bone destruction. Arthritis Rheum 54:463–472.

(Accepted 19 February 2015) (Available online 27 February 2015)