CREB signaling pathway

CREB signaling pathway

Pharmacology, Biochemistry and Behavior 120 (2014) 140–148 Contents lists available at ScienceDirect Pharmacology, Biochemistry and Behavior journal...

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Pharmacology, Biochemistry and Behavior 120 (2014) 140–148

Contents lists available at ScienceDirect

Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh

The VGF-derived peptide TLQP62 produces antidepressant-like effects in mice via the BDNF/TrkB/CREB signaling pathway Peipei Lin a,b,1, Chuang Wang a,b,⁎,1, Bing Xu c,1, Siyun Gao a,b,1, Jiejie Guo a,b,1, Xin Zhao a,b, Huihui Huang a,b, Junfang Zhang a,b, Xiaowei Chen a,b, Qinwen Wang a,b, Wenhua Zhou a,b a b c

Ningbo University School of Medicine, Ningbo, Zhejiang 315211, PR China Zhejiang Provincial Key Laboratory of Pathophysiology of Ningbo University School of Medicine, Ningbo, Zhejiang 315211, PR China No. 97 Hospital, Xuzhou, Jiangsu 221000, PR China

a r t i c l e

i n f o

Article history: Received 9 December 2013 Received in revised form 28 February 2014 Accepted 6 March 2014 Available online 11 March 2014 Keywords: VGF-derived peptide Tropomyosin-related kinase B Brain-derived neurotrophic factor (BDNF) cAMP response element-binding protein (CREB) TLQP62 Fluoxetine

a b s t r a c t Recent studies demonstrate that the neuropeptide VGF (nonacronymic)-derived peptide is regulated in the hippocampus by antidepressant therapies. Brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB), cAMP response element-binding protein (CREB) signaling, and monoamine transmitter pathways mediate the behavioral effects of antidepressants, but it is not known if these pathways also contribute to the antidepressant-like effects of VGF-derived peptide TLQP62. Here the antidepressant-like effects of TLQP62 were evaluated by measuring immobility time in the forced swimming and tail suspension tests (FST and TST) following acute microinjection of the TLQP62 (0.25, 0.5 and 1 nmol/side) into the hippocampal CA1 regions. This treatment dose-dependently reduced immobility in the FST and TST compared to phosphate-buffered saline (PBS) infusion without affecting locomotor activity in the open field test (OFT). In addition, daily intrahippocampal microinfusion of TLQP62 (1 nmol/side/day; 21 days) also upregulated the expression of BDNF and the phosphorylation of CREB (pCREB) and TrkB (pTrkB) without altering CREB or TrkB. Blocking tissue plasminogen activator (tPA) by microinfusion of tPASTOP or TrkB activation by microinfusion of K252a 60 min prior to TLQP62 infusion almost completely abolished TLQP62-induced antidepressant-like effects, BDNF upregulation, and CREB/TrkB phosphorylation. In contrast, none of these effects were diminished by pretreatment with the non-specific 5-HT receptor antagonist metergoline, the selective 5-HT1A receptor antagonist NAN-190, the 5-HT synthase inhibitor parachlorophenylalanine, the selective α1-adrenoceptor antagonist prazosin, the β receptor antagonist propranolol, or the D2 receptor antagonist raclopride. Moreover, our study was also to investigate the antidepressant-like effects of TLQP62 (50, 250 and 500 nmol/kg; i.p.) on depression-related behaviors in comparison with fluoxetine (10 mg/kg; i.p.). While TLQP62 and fluoxetine showed similar antidepressant-like behavioral effects in the FST of mice. Our present results strongly suggest that activation of BDNF/TrkB/CREB signaling may be involved in the antidepressant-like effects of TLQP62. © 2014 Elsevier Inc. All rights reserved.

1. Introduction The monoamine hypothesis of depression postulates that insufficient serotonergic, noradrenergic, and/or dopaminergic transmission in the central nervous system (CNS) accounts for many or most symptoms of depression (Prins et al., 2011; Chopra et al., 2011). However, antidepressants regulating monoaminergic neurotransmission are beneficial for many patients, many others are drug refractory or experience intolerable side effects. Moreover, these drugs all show

⁎ Corresponding author at: Ningbo University School of Medicine, Zhejiang Provincial Key Laboratory of Pathophysiology, Ningbo University School of Medicine, Ningbo, Zhejiang 315211, PR China. Tel./fax: +86 57487609589. E-mail addresses: [email protected], [email protected] (C. Wang). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.pbb.2014.03.003 0091-3057/© 2014 Elsevier Inc. All rights reserved.

delayed onset of action and unpredictable efficacy in individual patients, prolonging the search for the most effective agent. Vgf (non-acronymic), originally identified as a nerve growth factor responsive gene (Salton, 1991), is expressed in neurons within the central and peripheral nervous system and in various endocrine cells. Neuropeptide VGF is abundant in the cortex, hypothalamus, hippocampus, cerebellum and olfactory system and present in other brain areas in different amounts (Salton et al., 2000). Recently, synthetic VGF C-terminal peptides TLQP62 have been found to increase the synaptic activity of cultured hippocampal cells (Alder et al., 2003) and to regulate depressive-like behavior in rodents (Hunsberger et al., 2007; Thakker-Varia et al., 2007), suggesting that TLQP62 modulates hippocampal signaling pathway. However, the mechanism(s) of antidepressant-like effects of TLQP62 in depression is unknown. Emerging evidence suggests that neuropeptide VGF is induced by brain-derived neurotrophic factor (BDNF) and involved in the

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etiopathology of depression (Duman and Monteggia, 2006; Castrén et al., 2007; Calabrese et al., 2009; Kunugi et al., 2010; Schmidt and Duman, 2010). Indeed, leukocyte VGF mRNA levels were abnormally low in depressed patients and increased during clinically effective antidepressant drug administration (Cattaneo et al., 2010). In addition, chronic antidepressant treatment also upregulates the transcription factor cAMP response element binding protein (CREB) (Nibuya et al., 1996; Sairanen et al., 2005; Blendy, 2006; Gundersen et al., 2013) and CREB activation has been shown to induce antidepressant-like effects in animal models of depression (Conti and Blendy, 2004; Wallace et al., 2004). Furthermore, clinical studies have shown that reduced BDNF expression and reduced expression of the BDNF receptor tropomyosinrelated kinase B (TrkB) in depression and reversal by antidepressants (Thompson et al., 2011). These results suggest that BDNF mitigates depressive symptoms mainly by binding to TrkB, leading to autophosphorylation of TrkB tyrosine residues, and activation of downstream signaling molecules, including the extracellular signal-regulated kinases (ERKs) known to phosphorylate CREB (Chen et al., 2007; Numakawa et al., 2013). However, whether the BDNF/TrkB/CREB signaling is involved in the antidepressant-like effects of TLQP62 in mice still requires direct experimental demonstration. It has long been known that depletion of monoamines produces depressogenic effects in vulnerable individuals (Goodwin and Bunney, 1971). In addition, many antidepressants enhance synaptic 5-HT and upregulate the transcription of numerous genes and proteins, including neuropeptide VGF (Alfonso et al., 2005; Yamada and Higuchi, 2005; Holmes et al., 2006; Iwata et al., 2006; Sairanen et al., 2007). Furthermore, antidepressants that regulate serotonin and norepinephrine levels may act, at least partially, by direct or indirect activation of TrkB receptor signaling in the brain (Lee and Chao, 2001; Saarelainen et al., 2003). However, no direct evidence has yet been presented for a role of the monoamine system in the antidepressant effects of TLQP62. In this study, we evaluated the potential involvement of BDNF/TrkB/ CREB signaling and monoaminergic system in TLQP62-mediated antidepressant-like effects. 2. Materials and methods 2.1. Animals Experiments were conducted on young, healthy male imprinting control region (ICR) mice (22 g to 25 g) born and reared in the animal facility of Ningbo University Medical School, China. All animals were maintained at 22 ± 2 °C and 60% ± 5% relative humidity under a 12-h light/12-h dark cycle (lights on at 07:00 h) with ad libitum access to food and water when the stressors were not applied. All stressors were applied to animals outside of their housing area in a separate procedure room. All animal experiments were performed according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of Ningbo University Medical School. 2.2. Surgery for brain cannula implantation and drug treatment Intrahippocampal cannulation implantation was performed as described (Wang et al., 2012; Zhang et al., 2013). Briefly, animals were anesthetized with ketamine and xylazine (100 and 10 mg/kg i.p., respectively) and placed in a stereotaxic frame in the flat-skull position. Stainless steel guide cannulae were implanted bilaterally into the hippocampal CA1 at AP − 1.5 mm from bregma, ML ± 1.2 mm from the midline, and DV − 1.5 mm from dura (Zhang et al., 2013). The guide cannulae were anchored to the skull with dental cement and a stainless steel stylet was inserted to maintain patency for microinjections. The VGF-derived peptide TLQP62 (Biopeptide, San Diego, CA, USA) was dissolved in 0.01 M phosphate-buffered saline (PBS) before

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infusion, while tPASTOP (America Diagnostica Inc., Stamford, CT) and K252a (Sigma, MO, USA) were diluted in artificial cerebrospinal fluid (ACSF). At the time of drug delivery, infusion cannulas were tightly fitted into the guides. Microinfusions (0.5 μl/side) were carried out over 60 s with an infusion pump, and the cannulas were left in place for 60 additional seconds to avoid backflow. To verify the correct placement of the cannulae after intrahippocampal drug delivery, mice were sacrificed after behavioral tests and cryostat sections of hippocampus cut through the cortex determine the cannula track. Only animals with the correct cannula placement were used for further analysis. Four sets of experiments were conducted (As shown in Fig. 1). The first set experiment was performed to determine if acute TLQP62 treatment (0.25, 0.5 and 1 nmol/0.5 μl/side) modified behaviors in the open field test (OFT), forced swimming test (FST), and tail suspension test (TST) compared to PBS-infused control.The mice were employed for the OFT, FST, and TST successively with a 2 hour period between each test (Fig. 1a). The second set of experiment examined whether BDNF/ TrkB/CREB signaling participates in the antidepressant-like effects of repeated TLQP62 treatment. PBS or TLQP62 (1 nmol/0.5 μl/side) was microinjected daily for 21 days. tPASTOP (20 μM, 0.5 μl/side), K252a (72 μM, 0.5 μl/side), or their vehicles were administrated 60 min before TLQP62 or PBS each day. On day 20, 60 min after the last treatment (the infusion was done once only before the behavioral tests), then the OFT, FST, and TST were conducted successively with a 2 hour period between each test (Fig. 1b). The third set of experiment tested the involvement of the monoamine system in the antidepressant-like effects of TLQP62. The monoaminergic antagonists Metergoline (Met) (8 mg/kg, i.p.), Parachlorophenylalanine (Par) (150 mg / kg, i.p.), NAN-190 (NAN) (2 mg/kg, i.p.), Prazosin (Pra) (4 mg/kg, i.p.), Propranolol (Pro) (50 mg/kg, i.p.), and Raclopride (12 mg/kg, i.p.) (Rac) were administered i.p. daily 60 min prior to PBS or TLQP62 (1 nmol/0.5 μl/side) microinfusion. On day 20, 60 min after the last drug treatment (the infusion was done once only before the behavioral tests), then the mice were employed for the OFT, FST, and TST successively with a 2 hour period between each test (Fig. 1c). The fourth set of the experiment was performed in order to clarify the effects of TLQP62 on depressant-like behaviors induced by CUMS in comparision with the fluoxetine. The mice were randomly divided into six groups: Controlvehicle group, CUMS-vehicle group (0.9% Saline, i.p.), CUMS-TLQP62 group (50, 250 and 500 nmol/kg, dissolved in 0.9% Saline, i.p.) and CUMS-fluoxetine group (10 mg/kg, dissolved in 0.9% Saline, i.p.).TLQP62 and fluoxetine or their vehicle (0.9% Saline) was administered 60 min prior to CUMS for 21 days. Drugs were treated once a day from day 0 to day 20 as illustrated in Fig. 1d. From day 21 to 22, CUMS was absent and 60 min after the last drug treatment of each day, the open-field test (OFT) (day 21), forced-swimming test (FST) (day 22) were successively performed and then the animals were sacrificed for biochemical assays. 2.3. Chronic unpredictable mild stress (CUMS) procedure This animal model of stress consists of chronic exposure to variable unpredictable stressors, none of which is sufficient alone to induce long-lasting effects. Briefly, CUMS consisted of exposure to a variety of unpredictable stressors (randomly), including (1) 24-h food deprivation, (2) 24-h water deprivation, (3) 1-h exposure to an empty bottle, (4) 7-h cage tilt (45 °C), (5) overnight illumination, (6) 24-h soiled cage (200 ml water in 100 g sawdust bedding), (7) 6-min forced swimming at 12 °C, (8) 2-h physical restraint, and (9) 24-h exposure to a foreign object (e.g., a piece of plastic). All stressors were applied individually and continuously, day and night. The control animals were housed in a separate room and had no contact with the stressed groups. To prevent habituation and to ensure the unpredictability of the stressors, all stressors were randomly scheduled over a 1-week period and repeated throughout the 3 weeks experiments (As shown in Table 1 for detailed protocol).

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Fig. 1. Drug treatment and test schedules. (a) Investigation of the antidepressant-like effects of acute TLQP62 administration. TLQP62 was administrated seven days after cannula implantation surgery. The mice were employed for the OFT, FST, and TST successively with a 2 h period between each test (Fig. 1a). (b) Antidepressant-like effects of repeated TLQP62 administration. PBS or TLQP62 (1 nmol/0.5 μl/side) was microinjected daily for 21 days. tPASTOP (20 μM, 0.5 μl/side), K252a (72 μM, 0.5 μl/side), or their vehicles were administrated 60 min before TLQP62 or PBS each day. On day 20, 60 min after the last treatment (the infusion was done once only before the behavioral tests), then the OFT, FST, and TST were conducted successively with a 2 h period between each test.(c) The monoaminergic antagonists were administered i.p. daily 60 min prior to PBS or TLQP62 (1 nmol/0.5 μl/side) microinfusion. On day 20, 60 min after the last drug treatment (the infusion was done once only before the behavioral tests), then the mice were employed for the OFT, FST, and TST successively with a 2 h period between each test. (d) TLQP62 and fluoxetine or their vehicle (0.9% Saline) was administered 60 min prior to CUMS for 21 days. Drugs were treated once a day from day 0 to day 20 as illustrated in Fig. 1d. From day 21 to 22, CUMS was absent and 60 min after the last drug treatment of each day, the the open-field test (OFT) (day 21), forced-swimming test (FST) (day 22) were successively performed and then the animals were sacrificed for biochemical assays.

2.4. Behavioral paradigms 2.4.1. Open field test The OFT was conducted (Zhang et al., 2013, with minor modifications) first to ensure that any changes in activity during the FST or TST were not due to non-specific changes in motor activity. Briefly, mice were placed individually in a white Plexiglas box (50 × 50 × 39 cm) with a bottom divided into four identical squares. Line crossings (four paws placed into a new square) and rearings (with both front paws raised from the floor) were recorded over 5 min in a dimly lit room. After each test, the apparatus was cleaned with 5% ethanol to remove scent clues.

2.4.2. Forced swimming test The FST was conducted in a sound-attenuated room eliminated by white light (40 lux) as described (Sarkisyan et al., 2010). Briefly, mice were placed individually in a clear plastic cylinder (height: 25 cm; diameter: 10 cm) containing 10 cm of fresh water at 23 ± 2 °C for 6 min, and the duration of immobility was scored during the last 4 min. The total time during which the mouse made only small movements necessary to keep the head above water was considered the duration of immobility. To eliminate the influence of potential alarm substances, fresh water was introduced prior to each test. All test sessions were recorded with a video camera. Trained observers blind to the drug treatments scored the video recordings.

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3. Results

Table 1 Schedule of stressors used in the 21 days of CUMS procedure.

3.1. TLQP62 mitigates depressant-like behavior in mice

stressor

Duration

Day

Food deprivation Exposure to a foreign object Water deprivation

24-h 24-h 24-h

Monday

Forced swimming at 12 °C Soiled cage Overnight illumination

6-min 24-h Overnight

Tuesday

Food deprivation Cage tilt (45 °C) Physical restraint

24-h 7-h 2-h

Wednesday

Exposure to an empty bottle Cage tilt (45 °C) Overnight illumination

1-h 7-h Overnight

Thursday

Soiled cage Forced swimming at 12 °C Physical restraint

24-h 6-min 2-h

Friday

3.2. The antidepressant-like effects of TLQP62 were reversed by blockade of tPA or TrkB receptors

Exposure to a foreign object Forced swimming at 12 °C Cage tilt (45 °C)

24-h 6-min 7-h

Saturday

Pretreatment with the tPA antagonist tPASTOP or the TrkB antagonist K252a but not vehicle 60 min prior to TLQP62 significantly reduced the antidepressant-like effect of TLQP62 compared to TLQP62 alone in

Soiled cage Exposure to an empty bottle Overnight illumination

24-h 1-h Overnight

Sunday

Acute intrahippocampal infusion of TLQP62 reduced immobility in the FST [F(3, 36) = 39.86; p b 0.0001; one-way ANOVA, Fig. 2a] at each dose tested (0.25 nmol/side, p N 0.05; 0.5 nmol/side, p b 0.01; and 1 nmol/side, p b 0.01) compared to PBS-treated control. Similarly, TLQP62 reduced immobility in the TST [F(3, 36) = 10.60, p b 0.0001; Fig. 2b] at each dose (0.25 nmol/side, p N 0.05; 0.5 nmol/side, p b 0.01; and 1 nmol/side, p b 0.01; Fig. 2b). In contrast, TLQP62 treatment did not alter locomotor activity as measured by line crossing [F (3, 36) = 0.253, p = 0.859, Fig. 2c] and rearing [F (3, 36) = 0.282, p = 0.838, Fig. 2c].

2.4.3. Tail suspension test Experiments were performed under acoustic and visual isolation. Mice were suspended 50 cm above the floor with an adhesive tape placed at approximately 1 cm from the tip of their tail and at least 15 cm away from the nearest objects. Animals were allowed to hang for 6 min and the duration of immobility was recorded during the last 4 min. Mice were considered immobile only when they hung completely motionless. The immobility time was recorded by an observer blind to the treatment.

2.5. Immunoblotting Brain tissues were sonicated in RIPA lysis buffer (Upstate, Temecula, CA) containing protease and phosphatase inhibitors (Pierce Biotechnology, Rockford, IL). Lysates were centrifuged at 16,000 ×g for 30 min and total supernatant protein (80 μg gel lane) separated by SDS-PAGE and transferred to PVDF membranes (0.22 μm; Millipore, CA). Membranes were then incubated with rabbit anti-BDNF (1:500;), rabbit anti-pCREB (Ser133, 1:1000), rabbit anti-CREB (1:1000), rabbit anti-pTrkB (1:1500), rabbit anti-TrkB (1:1000; all from Millipore), or anti-β-actin (1:1000; Chemicon, Temecula, CA) at 4 °C overnight. The membranes were then incubated with Alexa Fluor 700-conjugated goat anti-rabbit antibody (1:10000; Invitrogen, Eugene, OR) for 60 min. Target bands were detected and quantified using a fluorescence scanner (Odyssey Infrared Imaging System, LI-COR Biotechnology, Lincoln, NE). For band stripping, membranes were incubated with stripping buffer (Chemicon) for 15 min. All the lysate samples were analyzed at least in triplicate.

2.6. Statistical analysis All measurements were performed by an independent investigator blind to the experimental conditions. Data are presented as mean ± standard error of the mean (SEM). Group means were compared by one-way analysis of variance (ANOVA) followed by Newman–Keuls Multiple Comparison Tests using GraphPad Prism software (Version 5.0, Prism software for PC, GraphPad). The criterion for significance was p b 0.05.

Fig. 2. The effects of TLQP62 on the depressant-like behaviors and locomotor activity following a single treatment in mice. (a) FST, (b) TST, and (c) OFT. Each bar represents the mean ± SEM (n = 10). **p b 0.01 vs. PBS-treated group (one-way ANOVA followed by Newman–Keuls Multiple Comparison Test).

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the FST [F (3, 36) = 9.951, tPASTOP, p b 0.01; K252a, p b 0.01; Fig. 3a] and TST [F (3, 36) = 7.041, tPASTOP, p b 0.01; K252a, p b 0.01; Fig. 3b] but had no effect on line crossing [F (3, 36) = 0.213, p = 0.887, Fig. 3c] and rearing [F (3, 36) = 1.616, p = 0.203, Fig. 3c] in the OFT. Thus, blockade of tPA and BDNF-TrkB signaling interfered with the antidepressant-like effects of TLQP62 but had no effect on general locomotor activity. 3.3. tPA or TrkB receptor blockade inhibited TLQP62-induced up-regulation of BDNF/TrkB/CREB signaling Mice treated for 21 consecutive days with TLQP62 exhibited significant increases in BDNF expression as well as phosphorylation CREB (pCREB) and phosphorylation TrkB (pTrkB), suggesting elevated hippocampal BDNF/TrkB/CREB signaling compared to PBS-treated group (Fig. 4). Pretreatment with tPASTOP or K252a but not their vehicles 60 min prior to TLQP62 injection significantly reversed these changes in BDNF [F (3, 36) = 10.54, p = 0.0037; Fig. 4b], pCREB [F (3, 36) = 5.897, p = 0.0200; Fig. 4c], and pTrkB [F (3, 36) = 26.150, p = 0.0002; Fig. 4e]. Compared to Vehicle plus PBS group, TLQP62 significantly increased BDNF (p b 0.01), pCREB (p b 0.05) and pTrkB (p b 0.01), whereas tPASTOP and K252a significantly blocked

the TLQP62-induced up-regulation of BDNF [tPASTOP, p b 0.01; K252a, p b 0.01; Fig. 4b], pCREB [tPASTOP, p b 0.05; K252a, p b 0.05; Fig. 4c], and pTrkB [tPASTOP, p b 0.05; K252a, p b 0.05; Fig. 4e] respectively. However, neither CREB [F (3, 36) = 0.1691, p = 0.9143; Fig. 4d] nor TrkB [F (3, 36) = 0.1453, p = 0.9298; Fig. 4f] expression was altered by all treatments. 3.4. Modulators of monoaminergic signaling had no effects on TLQP62induced antidepressant-like activity Consistent with our previous experiments, TLQP62 had a significant effect on immobility in the FST [F (7, 72) = 2.183, p = 0.0456; Fig. 5a] and TST [F (7, 72) = 2.507, p = 0.0230; Fig. 5b] compared to PBStreated group. The Newman–Keuls Multiple Comparison Test revealed significantly decreased immobility compared to the Vehicle plus PBS group in the FST (p b 0.05) and TST (p b 0.05). Neither pretreatment with metergoline, parachlorophenylalanine, NAN-190, prazosin, propranolol, nor raclopride blocked the antidepressant-like actions of TLQP62, indicating that TLQP62 produces antidepressant-like actions independently of monoamine system in mice. To determine whether all these treatments affected general health or motor activity, the locomotor activity was examined in mice treated with TLQP62 alone or in combination with these pharmacological inhibitors in the OFT. One-way ANOVA revealed no changes in locomotor activity as evidenced by line crossing [F (7, 72) = 0.3120, p = 0.9463; Fig. 5c] and rearing [F (7, 72) = 0.1417, p = 0.9945; Fig. 5d] after 21 days of drug administration. 3.5. Modulators of monoaminergic signaling had no effects on TLQP62induced changes in BDNF/TrkB/CREB signaling Mice were pretreated with metergoline, parachlorophenylalanine, NAN-190, prazosin, propranolol, raclopride, or vehicles 60 min prior to injection of TLQP62 or PBS. In contrast to tPASTOP and K252a, the increases in BDNF [F (7, 16) = 5.816, p = 0.0017; p b 0.01; Fig. 6b], pCREB [F (7, 16) = 5.115, p = 0.0033; p b 0.01; Fig. 6c], and pTrkB [F (7, 16) = 0.4.494, p = 0.0061; p b 0.01; Fig. 6e] induced by TLQP62 were not altered by metergoline (BDNF, p N 0.05; pCREB, p N 0.05; and pTrkB, p N 0.05), parachlorophenylalanine (BDNF, p N 0.05; pCREB, p N 0.05; and pTrkB, p N 0.05), NAN-190 (BDNF, p N 0.05; pCREB, p N 0.05; and pTrkB, p N 0.05), prazosin (BDNF, p N 0.05; pCREB, p N 0.05; and pTrkB, p N 0.05), propranolol (BDNF, p N 0.05; pCREB, p N 0.05; and pTrkB, p N 0.05), or raclopride (BDNF, p N 0.05; pCREB, p N 0.05; and pTrkB, p N 0.05). 3.6. Effect of TLQP62 in comparison with fluoxetine on depressant-like behaviors induced by CUMS

Fig. 3. Influence of repeated TLQP62 treatment with or without tPASTOP or K252a pretreatment on behavior in the OFT, FST, and TST. (a) FST, (b) TST, and (c) OFT. Each bar represents the mean ± SEM (n = 10). **p b 0.01 vs. Vehicle + PBS-treated group, ##p b 0.01 vs. Vehicle + TLQP62-treated group (one-way ANOVA followed by Newman–Keuls Multiple Comparison Test).

Using the CUMS model established, the effects of TLQP62 and fluoxetine on the stress-induced behavioral deficits were examined. For behavioral tests, ANOVA indicated there were significant differences among all treatments in their effect on open field behaviors: As shown in Fig 7, line crossing [F (5,54) = 3.349, p = 0.0.0105, Fig. 7a] and rearing F (5,54) = 6.651, p b 0.0001, Fig. 7b]. Post hoc tests revealed vehicle-treated CUMS mous exhibited a significant decrease in line crossing and rearing compared to vehicle-treated control mouse, but TLQP62 treatment had no significant effect on any open field measure. These results suggested that repeated treatment with the TLQP62 or fluoxetine at the used does not affect the general health nor decrease overall motor activity in mice. As shown in Fig. 8, antidepressant-like effects of TLQP62 as well as fluoxetine were assessed in mice with the FST. The one-way ANOVA revealed significant differences for treatments in FST [F (5,54) = 6.941, p b 0.0001, Fig. 8]. Post hoc analyses indicated that exposure to stressors in the vehicle treated group significantly increased the immobility time in FST (p b 0.01) as compared to non stressed mice with vehicle treated group. However, the TLQP62 dose-

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Fig. 4. Effects of repeated TLQP62 treatment with or without tPASTOP or K252a pretreatment on BDNF, pCREB, CREB, pTrkB, and TrkB protein expressions in the mouse hippocampus. Protein expressions were determined by Western blot and densitometric analysis. The upper panel (a) shows representative immunoblots and the lower panels densitometry of BDNF (b), pCREB (c), CREB (d), pTrkB (e) and TrkB (f) expressed as fold changes in optical density values normalized to β-actin. Each bar represents the mean ± SEM (n = 3 mice per group). *p b 0.05, **p b 0.01 vs. Vehicle + PBS; #p b 0.05, ##p b 0.01 vs. Vehicle + TLQP62.

dependently significantly reversed these effects in FST (p b 0.01 for 250 nmol/kg and p b 0.01 for 500 nmol/kg), with similar antidepressant-like effect of fluoxetine. 4. Discussion In this study, we showed that acute treatment of TLQP62 rapidly produces antidepressant-like effects as evidence by reduced immobility in the FST and TST. In addition, repeated TLQP62 administration upregulated the BDNF/TrkB/CREB signaling pathway. Further, both antidepressant-like effects and enhanced BDNF/TrkB/CREB signaling were blocked by tPASTOP or K252a respectively, whereas pharmacological antagonists of monoaminergic transmission had no significant effect. In parallel, our present study also demonstrated that repeated TLQP62 (i.p.) treatment significantly reversed depressive-like behaviors induced by CUMS, showing similar effects with fluoxetine on depression. These results further confirmed that across TLQP62 may cross the blood–brain barrier and play an important role in antidepressant actions. Neurotrophins like BDNF and neurotrophin receptors such as TrkB appears both necessary and sufficient for the behavioral effects produced by antidepressant drugs (Castrén and Rantamäki, 2008; Schmidt and Duman, 2010). We speculated that the Vgf gene, a target of BDNF-induced transcriptional activation, may be a critical downstream mediator of rapid antidepressant action (Cattaneo et al., 2010;

Hunsberger et al., 2007; Thakker-Varia et al., 2007). In addition, recent studies have demonstrated that the Vgf gene and specific VGF Cterminal peptide TLQP62 play a role in the regulation of depressive behavior (Hunsberger et al., 2007; Thakker-Varia et al., 2007), like BDNF, but the mechanism(s) of antidepressant-like action of the TLQP62 are incompletely understood. In the present study, the acute TLQP62 infusion rapidly produces antidepressant-like effects. However, TLQP62 had no effect on the number of line crossing and rearing in the OFT, suggesting that reduced immobility in the FST and TST was not attributable to nonspecific locomotor activation. A close functional interaction between neuropeptide VGF and BDNF was also suggested by VGF colocalization with TrkB (Salton et al., 2000), suggesting that VGF-derived peptides could potentially mediate a subset of the behavioral and electrophysiological alterations that are ascribed to BDNF signaling. In support of this hypothesis, the inhibition of tPA or TrkB by tPASTOP or K252a, respectively, have been reported to block the synaptic activity produced by TLQP62 (Bozdagi et al., 2008), thereby reducing activity-dependent BDNF secretion and TrkB activation, and interfering with the antidepressant actions of TLQP62 reported by our current work. In addition, the downstream intracellular signaling cascades activated by TrkB may converge on nuclear transcription factor CREB (Xing et al., 1998; Marsden, 2013), a known mediator of antidepressant actions. CREB is likely the most important regulator of BDNF gene expression as evidence by the CRE elements in the promoter region of BDNF (Finkbeiner et al., 1997), while BDNF may induce CREB

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Fig. 5. Effects of 5-HT receptor, 5-HT synthase, α1 receptor, β receptor, and D2 receptor antagonists on TLQP62-induced behavioral changes in the FST, FST, and OFT. (a) FST, (b) TST, and (c–d) OFT. Each bar represents the mean ± SEM (n = 10). *p b 0.05 vs. Vehicle + PBS-treated group (one-way ANOVA followed by Newman–Keuls Multiple Comparison Test). Met: Metergoline, Par: Parachlorophenylalanine, NAN: NAN-190, Pra: Prazosin , Pro: Propranolol, and Rac: Raclopride.

phosphorylation either directly or through activation of other effector such as NMDA receptors (Rakhit et al., 2005). More and more studies also demonstrated that antidepressants promote an increase in the Fig. 6. Effects of 5-HT receptor, 5-HT synthase, α1 receptor, β receptor, and D2 receptor antagonists on TLQP62-induced changes in BDNF, pCREB, CREB, pTrkB, and TrkB protein expressions in the hippocampus.Protein levels were determined by Western blot analysis. The upper panel (a) shows representative immunoblots and the lower panels densitometry for BDNF (b), pCREB (c), CREB (d), pTrkB (e), and TrkB (f) expressed as fold change in optical density values normalized to β-actin. Each bar represents the mean ± SEM (n = 3 mice per group). **p b 0.01 vs. Vehicle + PBS. Met: Metergoline, Par: Parachlorophenylalanine, NAN: ana-190, Pra: Prazosin , Pro: Propranolol, and Rac: Raclopride.

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Fig. 7. Effect of TLQP62 and fluoxetine on CUMS-induced locomotor activity changes in mice. (a) Line crossing, (b) Rearing. Each bar represents the mean ± SEM (n = 10). *p b 0.05,**p b 0.01 vs. No-CUMS treated plus Vehicle group, #p b 0.05, ##p b 0.01 vs. CUMS treated plus Vehicle group.

level of serotonin and/or noradrenaline in the synaptic cleft, resulting in the phospho-activation of CREB, which then induces BDNF gene transcription (Nibuya et al., 1996; Czéh and Simon, 2005). Consistent with these hypotheses above, tPASTOP and K252a blocked the TLQP62induced antidepressant-like behaviors and reversed the up-regulation of pCREB in our study. One limitation of the current study is the specificity of K252a for TrkB signaling. K252a has been reported to inhibit other kinases, including mixed-lineage kinase 3 (MLK3) (Roux et al., 2002). Therefore, the involvement of other tyrosine kinases in the antidepressant-like effects of TLQP62 cannot be excluded. Future studies should be aimed at using the selective TrkB inhibitor ANA-12 to understand of TLQP62 in the modulation of depressive-like behavior and particularly in relation to TrkB/CREB signaling. Previous studies demonstrated that depression is strongly associated with impairments in 5-HT, NA, and DA neurotransmission (Frazer, 2000; Millan, 2004; Nutt, 2006), and these monoaminergic systems are thought to be critical targets for the treatment of depression (Frazer, 2000; Millan, 2004; Nutt, 2006). However, pharmacological antagonists and modulators of the monoaminergic system had no

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significant effects on TLQP62-induced antidepressant activity in our present study. Firstly, it has been shown that long-term antidepressant treatment increase the number of postsynaptic 5-HT1A receptors in the dorsal hippocampus and the forebrain (Welner et al., 1989; Chaput et al., 1991; Haddjeri et al., 1998), and that selective activation of postsynaptic 5-HT1A receptors facilitates the response to antidepressants in clinical studies (Blier et al., 1997). In the current work, both metergoline and NAN-190 did not reverse the antidepressantlike effects of TLQP62, suggesting that TLQP62 activates an antidepressant signaling pathway independently of 5-HT1A receptor stimulation. However, we have not yet investigated the involvement of other 5-HT subtypes on the effects of TLQP62 in our study. Secondly, parachlorophenylalanine, an inhibitor of tryptophan hydroxylase, depleted endogenous 5-HT stores in mice without affecting NA and DA levels (Redrobe et al., 1998). Additionally, parachlorophenylalanine inhibitors blocked the antidepressant effects of selective serotonin reuptake inhibitors or serotonin-specific reuptake inhibitor (SSRIs) in the FST and the TST (Page et al., 1999; O'Leary et al., 2007). However, we found no effect of parachlorophenylalanine on the antidepressantlike actions of TLQP62, indicating that presynaptic 5-HT release is not necessary for the antidepressant-like effects of TLQP62. Thirdly, adrenoceptors have been shown to underlie some of the antidepressant-like responses of drugs in rodent models of depression (Kitada et al., 1983; Danysz et al., 1986; Masuda et al., 2001), but both the α1-adrenoceptor antagonist prazosin and the β-adrenoceptor antagonist propranolol did not significantly change the antidepressantlike effects of TLQP62. Finally, deficits in dopaminergic signaling produce several symptoms of depression, including anhedonia and decreased motivation (Jackson and Westlind-Danielsson, 1994; Chaudhury et al., 2013). However, the D2 antagonist raclopride did not significantly inhibit the antidepressant-like effects of TLQP62. Therefore, the antidepressant-like actions of TLQP62 in mice appear to be dependent on the downstream of monoaminergic neurotransmission. However, one drawback of our experiments (First, second and third) design is that three different behavioral tests (OFT, FST, TST) within a short session (2 hours). Therefore, we cannot exclude that the potential interactions among the tests in the current work. However, given that OFT is the relative lower stressful test for mice, so it had no significantly affected on the next test of FST. Further studies are needed to clarify this crucial issue. In conclusion, our results strongly suggest that activation of BDNF/ TrkB/CREB signaling may be involved in the antidepressant-like effects of TLQP62. Future experiments need to address critical outstanding issues, such as whether perturbing BDNF/TrkB/CREB signaling blocks hippocampal neurogenesis, whether administration of TLQP62 to the midbrain also exerts antidepressant effects through monoaminergic and (or) the BDNF/TrkB/CREB pathway and whether monoaminergicand TLQP62-dependent pathways act synergistically to reduce depressive-like behaviors. Furthermore, given that the recently studies have reported that another VGF-derived peptide TLQP21 may act via the C1q and/or the C3a receptors (Chen et al., 2013; Hannedouche et al., 2013), additional studies are required to determine the TLQP62 receptors in the future. Acknowledgements

Fig. 8. Effect of TLQP62 and fluoxetine on CUMS-induced depressive-like behavior in FST of mice. Each bar represents the mean ± SEM (n = 10). **p b 0.01 vs. No-CUMS treated plus Vehicle group, ##p b 0.01 vs. CUMS treated plus Vehicle group.

This work was supported by grants from the National Natural Science Foundation of China (No. 81201050 to Dr. Chuang Wang, No. 30901802 to Dr. Xin Zhao, and No. 81000599 to Dr. Xiaofei Wei), Natural Science Foundation of Zhejiang province (No. LQ12H09001 to Dr. Chuang Wang and No. LY12H09002 to Dr. Xin Zhao), Natural Science Foundation of Ningbo (No. 2012A610249 to Dr. Chuang Wang and 2012A610252 to Dr. Junfang Zhang), Innovative Research Team of Ningbo (2009B21002) and Natural Science Foundation of Ningbo University (No. XKL11D2111 to Dr. Chuang Wang), as well as the Student Research and Innovation Program (SRIP) of Ningbo University

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