PI3K/AKT/mTOR Signaling-Mediated Neuropeptide VGF in the Hippocampus of Mice Is Involved in the Rapid Onset Antidepressant-Like Effects of GLYX-13

Background: VGF (nonacryonimic) and phosphatidylinositol 3-kinase (PI3K)/AKT (also known as protein kinase B, PKB)/mammalian target of rapamycin (mTOR) signaling play pivotal roles in depression. However, whether phosphatidylinositol 3-kinase/AKT/mTOR signaling-mediated VGF participates in rapid-acting antidepressant-like actions of GLYX-13 is unclear. Methods: Herein, we evaluated the effects of acute treatment of GLYX-13 (0.5, 5, and 10mg/kg, i.p.) in the forced swim test. In addition, we assessed whether the acute treatment with GLYX-13 reverses the depressive-like behaviors induced by chronic unpredictable mild stress. Furthermore, we determined whether the Vgf knockdown in hippocampus of mice blocks the effects of GLYX-13. Moreover, we also demonstrated the effects of intra-hippocampus infusion of LY294002 (10 nmol/side), a specific phosphatidylinositol 3-kinase inhibitor prior to the treatment of GLYX-13 in the forced swim test. Lastly, whether alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mTOR activation involves in the antidepressant-like effects of GLYX-13 was examined. Results: Our results shown that GLYX-13 dose-dependently reversed the depressive-like behaviors in forced swim test. Additionally, GLYX-13 significantly reversed the downregulation of phosphorylation of AKT, mTOR, and eukaryotic elongation factor 2 as well as VGF induced by chronic unpredictable mild stress in hippocampus. Further, Vgf knockdown in hippocampus of mice significantly blocked the rapid-acting antidepressant-like effects and upregulation on phosphatidylinositol 3-kinase/AKT/mTOR/VGF signaling of GLYX-13. Moreover, intra-hippocampus infusion of LY294002 significantly abolished the antidepressant-like effects and upregulation on phosphatidylinositol 3-kinase/AKT/mTOR/VGF signaling of GLYX-13. Finally, antidepressant-like effects of GLYX-13 required AMPA receptor and mTOR activation, as evidenced by the ability of NBQX and rapamycin to block the effects of GLYX-13, respectively. Conclusions: Our results suggest that phosphatidylinositol 3-kinase/AKT/mTOR signaling-mediated VGF in hippocampus may be involved in the antidepressant-like effects of GLYX-13.


Introduction
Depression is a serious public health problem and one of the most common psychiatric disorders, with a lifetime prevalence of 17% in the United States (Kessler et al., 2005). Notably, to date, all drugs approved as antidepressant medications are either inhibitors of monoamine transporters or monoamine oxidase (Berton and Nestler, 2006) and take 3 to 8 weeks to exert their effects (Wong and Licinio, 2004). Patients that emerge as treatment resistant, defined as failing 2 or more trials of medication, are more severely ill with comorbid anxiety disorders and are at increased risk of suicide for an extended period of time (Schosser et al., 2012). Therefore, there is a pressing medical need to develop rapidly acting therapeutics that are capable of immediately relieving the depressive symptomology.
Recently it has been demonstrated that the N-methyl-Daspartic acid (NMDA) receptor (NMDAR) antagonist ketamine has rapid-acting antidepressant effects in patients that are treatment resistant (Mathew et al., 2012). Previous studies suggest that the rapid-acting antidepressant-like effects of ketamine are mediated by molecular alterations to the signaling pathway of phosphatidylinositol 3-kinase (PI3K)/AKT (also known as protein kinase B, PKB)/glycogen synthesis kinase 3 (GSK3)/mammalian target of rapamycin (mTOR)/brain derived neurotrophic factor (BDNF), which has been implicated in the adaptive response to stress, and are engaged in the development of mood-related disorders Zhou et al., 2013;Abelaira et al., 2014;Park et al., 2014;Zhou et al., 2014). Autry and colleagues (2011) showed that ketamine and other NMDAR antagonists produce fast-acting behavioral antidepressant-like effects in mouse models and that these effects depend on the rapid synthesis of BDNF, which may play an important role on the antidepressant modulator neuropeptide VGF (nonacronymic) production (Alder et al., 2003;Malberg and Monteggia, 2008). These findings indicate that the regulation of protein synthesis by spontaneous neurotransmission may serve as a viable therapeutic target for the discovery of fast-acting antidepressants. Unfortunately, ketamine causes unacceptable dissociative side effects and is a substance of abuse (Burgdorf et al., 2013). Recent studies demonstrate that GLYX-13, an NMDAR glycine site functional partial agonist, produces rapid onset antidepressant-like effects without the psychotomimetic side effects of NMDAR antagonists (Burgdorf et al., 2013;Moskal et al., 2014). However, it remains to be revealed whether PI3K/ AKT/mTOR also participates in the rapid-acting antidepressantlike effects of GLYX-13. Additionally, given that the VGF may act downstream of BDNF and exert rapid-acting antidepressant-like effects through TrkB receptor (Alder et al., 2003;Lin et al., 2014) and enhancing neurogenesis (Thakker-Varia et al., 2014) in hippocampus, many questions remain to be answered about the role of the VGF in antidepressant-like effects of GLYX-13.

Animals
Experiments were conducted on young, healthy, male ICR mice (22-25 g) born and reared in the animal facility of Ningbo University Medical School and Zhengzhou University, China. All animals were maintained at 22 ± 2°C and 60% ± 5% relative humidity under a 12-h-light/-dark cycle (lights on at 7:00 am) 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 and Zhengzhou University.

Drugs and Treatment
The drugs used included:GLYX-13 (Tocris Bioscience) and desipramine (Des) (Sigma, St. Louis, MO). Previous studies demonstrate that a low dose of dimethyl sulfoxide (DMSO) in saline vehicle did not make the changes in pharmacological effects and toxic effects of compounds (Wang et al., 2012;Li et al., 2011); both GLYX-13 and Des were dissolved in 0.9% saline containing 1% DMSO in our current work. These solutions, freshly prepared before administration, were given by i.p. in a volume of 10 mL/kg body weight. LY294002, NBQX, and rapamycin were purchased from Tocris Bioscience (Tocris Bioscience), dissolved in artificial cerebrospinal fluid (ACSF), and administrated by intra-hippocampal (i.h.) infusion. The i.h. injection was performed by employing a "free hand" method under light ether anesthesia according to the procedure described previously Lin et al., 2014). Briefly, animals were anaesthetized 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 at AP −1.5 mm from bregma, ML ± 1.2 mm from the midline, and DV −2.0 mm from dura. The guide cannulae were anchored to the skull with dental cement, and a stainless steel stylet was inserted to maintain patency for microinjection. The mice were allowed to recover for 7 days and were handled every other day to reduce the stress associated with handling at the time of testing. The drugs were then microinjected into the bilateral hippocampus of the mice. The injection cannula was left in place for another 60 seconds before being slowly withdrawn to avoid back flow.

Chronic Unpredictable Mild Stress (CUMS)
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), as shown in Table 1. The control animals were housed in a separate room and had no contact with the stressed groups. To prevent habituation and ensure the unpredictability of the stressors, all stressors were randomly scheduled during a 1-week period and repeated throughout the 3-week experiments.

Construction of Vgf-shRNA-Lentivirus
Small interfering RNAs targeting the mouse Vgf gene were designed by the Shanghai GeneChem, Co. Ltd, China. The optimal sequence of small interfering RNAs against mice VGF (5'-CCAATTCCAGGCTCGAATG-3') was then cloned into the plasmid pGCL-GFP, which encodes an human immunodeficiency virus (HIV)-derived lentiviral vector containing a multiple cloning site for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter-GFP fluorescent protein (marker gene) cassette flanked by loxP sites. A negative control lentiviral vector containing NS-shRNA was constructed by a similar process (NS-lentivirus, 5'-TTCTCCGAACGTGTCACGT-3'). These modified plasmids were further cotransfected into HEK 293T cells with lentiviral packaging plasmids to generate an Vgf-shRNA-expressing lentivirus (Vgf-shRNA-lentivirus) or a control shRNA-expressing lentivirus.
HEK293 cells were plated in 6-well plates (6 × 10 5 cells/well) and cultured for 24 hours before the transduction of lentiviral vectors. After 2 days of infection, the medium was replaced and cells were further incubated until 48 hours as required.

Experimental Treatments
Five sets of experiments were conducted. The first set was performed to assess the fast-acting antidepressant-like effects of acute GLYX-13 administration ( Figure 1a). The second set of experiments was to investigate whether 21-day CUMS alters VGF and PI3K/AKT/mTOR signaling in the hippocampus of mice and to determine whether acute treatment of GLYX-13 (0.5, 5, and 10 mg/kg, i.p.) reverses the depressive-like behaviors induced by CUMS in mice ( Figure 2a). The third set was to demonstrate whether knock down of VGF in hippocampus by Vgf-shRNA-lentivirus blocks the antidepressant-like action of GLYX-13 (10 mg/kg, i.p.) (Figure 4a). Lentiviral vectors containing NS-shRNA or Vgf-shRNA-lentivirus (5 × 10 7 TU/μL, 1 μL/side) were infused at a rate of 0.2 μL/min with an infusion pump, and the cannulas were left in place for 60 additional seconds to avoid back flow. The fourth set of experiments was to investigate whether i.h. infusion of LY294002 (10 nmol/side), a specific PI3K inhibitor, 30 minutes prior to the administration of GLYX-13 (10 mg/kg, i.p.) significantly blocks the antidepressant-like behaviors in the open field test (OFT) (Figure 6a). The fifth set of experiments was to investigate whether i.h. infusions of NBQX (2 μg/side), a AMPA receptor inhibitor (NBQX), or mTOR inhibitor (rapamycin) 30 minutes prior to the administration of GLYX-13 (10 mg/kg, i.p.) blocks the antidepressant-like behaviors of GLYX-13 (Figure 8a, e).

OFT
The OFT was conducted first to ensure that any changes in activity during the forced swim test (FST) were not due to nonspecific changes in motor activity. Briefly, mice were placed individually in a white Plexiglas box (50 × 50 × 39 cm) with a bottom divided into 4 identical squares. Line crossings (4 paws placed into a new square) and rearings (with both front paws raised from the floor) were recorded for 5 minutes in a dimly lit room. After each test, the apparatus was cleaned with 5% ethanol to remove scent clues.

FST
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 minutes, and the duration of immobility was scored during the last 4 minutes. The total time during which the mouse made only small movements necessary to keep the head above water was considered the duration of immobility.

Data Analysis
Data are expressed as the means ± SEM. Data were analyzed by 1-way analysis of variance (ANOVA) or 2-way ANOVA followed by Newman-Keuls posthoc test using GraphPad Prism software (Version 5.0, Prism software for PC, GraphPad). The criterion for significance was P < .05.

GLYX-13 Administration Exerts Rapid-Acting Antidepressant-Like Effects in Mice
Using the FST in mice, we first assessed the antidepressant-like effects of acute GLYX-13 administration. The mice received an i.p. injection of GLYX-13 (0.5, 5, and 10 mg/kg) or Des (10 mg/kg) 30 minutes before the OFT and then immediately followed by the

Vgf Knockdown in Hippocampus Blocks the Rapid-Acting Antidepressant-Like Effects of GLYX-13 in Mice
NS-shRNA or Vgf -shRNA were microinfused into bilateral hippocampus of mice following a 7-day acclimatization. GLYX-13 (10 mg/kg, i.p.) or its vehicle was administrated beginning from 7 days after the viral infusion (day 1), and then 30 or 60 minutes later, the OFT or FST was conducted, respectively  upregulation effects of GLYX-13 on the expression of pAKT [P < .01; Figure 5b], pmTOR [P < .05; Figure 5d], and VGF [P < .01; Figure 5g] were completely prevented by treatment of animals with the Vgf-shRNA in hippocampus. In addition, the downregulation effects of GLYX-13 on the expression of peEF2 (P < .01) (Figure 5f) also was completely blocked by treatment of animals with the Vgf-shRNA in hippocampus. By contrast, none of the treatments affected the AKT (Figure 5c) and mTOR (Figure 5e) levels in the hippocampus.

Inhibition of PI3K Activity Blocks the Regulation Effects of GLYX-13 on pAKT, pmTOR, peEF2, and VGF in the Hippocampus of Mice
As shown in Figure 7

Discussion
Interestingly, our present study provided the first evidence that the rapid-acting antidepressant-like effect of GLYX-13 in the FST involves PI3K activation, increased mTOR signaling, and VGF activation. In addition, our results further demonstrated that the GLYX-13-induced antidepressant-like effects required AMPA/kainate receptor and mTOR activation, as evidenced by the ability of NBQX and rapamycin to abolish the antidepressant-like effect of GLYX-13, respectively. Moreover, the fast-acting antidepressant effect elicited by GLYX-13 is associated with VGF in hippocampus of mice. Increasing evidence has shown that the well-documented delay in antidepressant efficacy (Adell et al., 2005) may be attributable to neural adaptive mechanisms to reverse the damage of stress in the hippocampus, including changes in neurotrophic factors (Castren, 2005;Dranovsky and Hen, 2006;Warner-Schmidt and Duman, 2006). The neurotrophic hypothesis of depression is based on the stress-induced downregulation and rapid-acting antidepressant-induced upregulation of BDNF and neuropeptide VGF (nonacronymic) expression in the brain (Adlard and Cotman, 2004;Thakker-Varia et al., 2010;Nasca et al., 2013;Lin et al., 2014). VGF is upregulated by both BDNF and 5-HT treatment, and VGF protein in the hippocampus is reduced in animals subjected to behavioral models of depression (Thakker-Varia et al., 2007). This hypothesis is supported by previous research indicating that the administration VGFderived peptide TLQP-62 into the brain produced sustained and (a) Representative immunoblots of pAKT, AKT, pmTOR, mTOR, peEF2, and VGF detected by Western blotting with tissues from the hippocampus; the rest of the panels are quantifications of the immunoblotting bands of pAKT (b), AKT (c), pmTOR (d), mTOR (e), peEF2 (f), and VGF (g). The data are expressed as mean ± SEM (n = 3 per group). *P < .05, **P < .01, compared with ACSF + Vehicle group; ##P < .01, compared with ACSF + GLYX-13 group. rapidly antidepressant-like effects (Lin et al., 2014). Additionally, growing studies suggest that the rapid-acting antidepressantlike effects of ketamine are mediated by molecular alterations to the mTOR, a serine/threonine kinase and key component of the insulin-signaling pathway and then reduces the expression of eEF2, which prevents protein translation of BDNF (Li et al., 2010;Autry et al., 2011;Monteggia et al., 2013). Furthermore, BDNF regulates neuronal mTOR function via AKT and PI3K, creating a positive feedback loop of BDNF production following the activation of mTOR by ketamine (Hay and Sonenberg, 2004;Hoeffer and Klann, 2010), suggesting that the production of BDNF occurs rapidly and may underlie the fast behavioral response to ketamine. However, to date, whether neuropeptide VGF also plays an important role in the rapid onset antidepressant-like effects of antidepressants remains unknown. In addition, given that the GLYX-13 is a recently developed tetrapeptide (Thr-Pro-Pro-Thr) that acts as a glycine-site modulator at the NMDAR (Moskal et al, 2005) with therapeutic potential as a cognitive enhancer (Moskal et al., 2005) and antidepressant (Moskal et al., 2014), whether the PI3K/AKT/mTOR/VGF signaling also involve in the rapid-acting antidepressant-like effects of GLYX-13 is not clear. In support of this hypothesis, we confirmed that GLYX-13, a novel NMDAR glycine-site functional partial agonist, produces an antidepressantlike effect in the FST of mice. Our present results are consistent with previous studies that revealed that GLYX-13 produced rapid-acting antidepressant-like effects in the rat Porsolt test and novelty-induced hypophagia test (Burgdorf et al., 2013). The previous studies demonstrated that the following reasons may explain the rapid-acting antidepressant-like effects of GLYX-13 in the current work. First, GLYX-13 has been shown to readily cross the blood-brain barrier, showing a brain uptake index of 80% (Moskal et al, 2005). Second, it enhances the magnitude of long-term potentiation of synaptic transmission while reducing long-term depression (Zhang et al., 2008). Third, GLYX-13 has been shown to have a critical role in the induction of metaplasticity (Yang et al, 2011). Our current data also showed that CUMS for 3 weeks caused a reduction of the pAKT, pmTOR, and VGF expression and a increase of peEF2 in hippocampus of mice and that these stress-induced changes were prevented by acute GLYX-13 treatment. To the best of our knowledge, this study is the first to provide evidence for the increased PI3K/AKT/mTOR/ VGF signaling following acute treatment with GLYX-13. Thus, the corrective action of GLYX-13 on brain PI3K/AKT/mTOR/ peEF2 signaling-mediated VGF levels may be in line with its rapid-acting antidepressant activity in mice.
Further, VGF shRNA-mediated Vgf knockdown in hippocampus has been used to investigate mechanisms underlying the acute behavioral actions of GLYX-13. Downregulation of VGF in hippocampus produced depressive-like behaviors, as indicated by increased immobility in the FST. Furthermore, we compared the antidepressant-like effect of GLYX-13 with that of combined GLYX-13 and Vgf shRNA. If VGF was important, inhibition of Figure 8. Administration of the AMPA receptor inhibitor NBQX or mTOR inhibitor rapamycin attenuates the rapid-acting antidepressant-like effectst of GLYX-13 in mice. Experimental procedure for the assessment of the role of AMPA receptor (a) or mTOR (e) in the antidepressant-like effects of GLYX-13 (10 mg/kg, i.p.). Cannula implantations were microinfused into bilateral hippocampus of mice following 7-day acclimatization. Mice were treated with NBQX (2 μg/side) or rapamycin (2 nmol/ side) and 30 minutes later followed by i.p.). Then the open field test (OFT) was performed 30 minutes later and the FST was conducted 30 minutes after the OFT. (b, f) Immobility time of mice was measured. Pretreatment with NBQX (b) or rapamycin (f) reversed the reduction of immobility time produced by GLYX-13. All the treatments had no effects on locomotor activity, reflected by the line crossings (c, g) and rearings (d, h) in mice. The data are expressed as mean ± SEM (n = 9 per group). **P < .01, compared with ACSF + Vehicle group; ##P < .01, compared with ACSF + GLYX-13 group. the VGF in hippocampus should block the rapid-acting antidepressant-like effects of GLYX-13. Indeed, the downregulation of VGF in hippocampus significantly increased the immobility in the FST compared with the NS shRNA-treated group and also blocked the antidepressant-like effects of GLYX-13 when combined with GLYX-13, supporting the important role of VGF in the mediation of rapid-acting antidepressant activity of GLYX-13. Furthermore, our present study also demonstrated that Vgf knockdown in hippocampus decreased the expressions of pAKT, pmTOR, and VGF and increased the levels of peEF2. The following reasons may explain the relationship between PI3K/AKT/mTOR and VGF in our present work. The VGF may act downstream of BDNF and exert rapid-acting antidepressant-like effects through TrkB receptor (Alder et al., 2003;Lin et al., 2014) in hippocampus. In addition, BDNF-induced TrkB activation stimulates the PI3K/ AKT signaling pathways, which play a principal role in promoting neuronal survival and synaptic plasticity and antidepressant-like actions (Numakawa et al., 2010(Numakawa et al., , 2013, suggesting that VGF may feedback regulate the PI3K/AKT/mTOR signaling in hippocampus of mice. Our results not only are consistent with rapid-acting antidepressant-like effects of GLYX-13 (Burgdorf et al., 2013;Moskal et al., 2014) and VGF (Lin et al., 2014) but also extended the findings for the first time to the role of VGF in the rapid-acting antidepressant-like effects of GLYX-13 and in the regulation of PI3K/AKT/mTOR signaling.
As the downstream regulatory signaling pathway of GLYX-13 in our current work, PI3Ks are a large family of intracellular signal transducers. The role of the PI3K pathway has been implicated in the regulation of cell growth, survival, proliferation, and movement (Astle et al., 2011;Nedachi et al., 2011). Numerous studies have also implicated PI3K in depression and anxiety (Leibrock et al., 2013;Moretti et al., 2014). Another interesting result of our present study showed that inhibition of AKT, following blockade of PI3K by LY294002, prevented GLYX-13-produced antidepressant-like effects and upregulation on PI3K/ AKT/mTOR/VGF signaling. These results not only indicate that blocking PI3K is most likely involved in the fast-acting effects of GLYX-13 but also confirms that rapid increases in VGF produced by activation of PI3K/AKT/mTOR signaling are useful markers of novel rapid-acting antidepressants. One limitation of the current study is that several reports have shown that LY294002 is not exclusively selective for the PI3Ks and could in fact act on other lipid kinases and additional apparently unrelated proteins (Gharbi et al., 2007). Therefore, the involvement of other tyrosine kinases in the antidepressant-like effects of GLYX-13 cannot be excluded. Future studies should be aimed at using the selective PI3K inhibitor to understand of PI3K/AKT signaling in the modulation of depressive-like behavior and particularly in relation to the rapid-acting antidepressant-like effects of GLYX-13. Additionally, it has recently been reported that the antidepressant-like effects of GLYX-13 were prevented by preadministration of NBQX (Burgdorf et al., 2013). The requirement for AMPA receptor is consistent with our current finding that NBQX also blocked the rapid-acting antidepressant-like effects of GLYX-13. Further, our novel finding that mTOR is involved in the VGF upregulation contributes to the antidepressant-like effects of GLYX-13. Our results showed that pretreatment with rapamycin completely abolished the effects of GLYX-13.

Conclusion
To our knowledge, our present study is the first to report that GLYX-13 activate the PI3K/AKT/mTOR signaling pathway, leading to VGF activation in hippocampus of mice. Taken together, alterations in the expression of the proteins investigated in the present study may contribute to the molecular basis of depressive-like behaviors and changes in brain of mice and could provide novel insights into the development of new therapeutic approaches having greater efficacy against major depression.