Background: It is widely accepted that cognitive processes, such as learning and memory, are affected in depression, but the molecular mechanisms underlying the interactions of these 2 disorders are not clearly understood. Recently, glycogen synthase kinase-3 beta (GSK-3β)/β-catenin signaling was shown to play an important role in the regulation of learning and memory. Methods: The present study used a rat model of depression, chronic unpredictable stress, to determine whether hippocampal GSK-3β/β-catenin signaling was involved in learning and memory alterations. Results: Our results demonstrated that chronic unpredictable stress had a dramatic influence on spatial cognitive performance in the Morris water maze task and reduced the phosphorylation of Ser9 of GSK-3β as well as the total and nuclear levels of β-catenin in the hippocampus. Inhibition of GSK3β by SB216763 significantly ameliorated the cognitive deficits induced by chronic unpredictable stress, while overexpression of GSK3β by AAV-mediated gene transfer significantly decreased cognitive performance in adult rats. In addition, chronic unpredictable stress exposure increased the expression of the canonical Wnt antagonist Dkk-1. Furthermore, chronic administration of corticosterone significantly increased Dkk-1 expression, decreased the phosphorylation of Ser9 of GSK-3β, and resulted in the impairment of hippocampal learning and memory. Conclusions: Our results indicate that impairment of learning and memory in response to chronic unpredictable stress may be attributed to the dysfunction of GSK-3β/β-catenin signaling mediated by increased glucocorticoid signaling via Dkk-1. Keywords: chronic unpredictable mild stress, depression, glycogen synthase kinase-3 beta, learning and memory, β-catenin Received: January 10, 2018; Revised: March 3, 2018; Accepted: April 16, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 1 provided the original work is properly cited. For commercial re-use, please contact email@example.com Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 2 | International Journal of Neuropsychopharmacology, 2018 Significance Statement Growing evidence indicates the concurrence and interrelationship of depression and cognitive impairments. However, the detailed molecular mechanisms underlying the interactions of these 2 disorders have not been fully understood. Recently, glyco- gen synthase kinase-3 beta (GSK-3β)/β-catenin signaling was shown to play an important role in the regulation of learning and memory. The present study provides the first evidence that impairment of learning and memory in response to chronic unpre- dictable stress (CUS) may be attributed to the dysfunction of GSK-3β/β-catenin signaling mediated by increased glucocorticoid signaling via Dkk-1. Understanding the mechanisms that underlie hippocampal damage in response to stress/glucocorticoids may shed new light on the pathophysiology of mood disorders and stress-related cognitive dysfunctions and may lead to the identification of new therapeutic targets. Introduction Depression, with 10% to 20% lifetime prevalence, is one of the dentate gyrus regions (Hooper et al., 2007). However, it remains most common psychiatric illness that involves the disturbance unclear whether GSK-3β/β-catenin signaling is involved in the of mood (Wong and Licinio, 2001). It is not only life threaten- deficits of learning and memory related to depression. ing but also has a negative impact on cognitive processes, espe- In the present study, we aimed to investigate whether the cially learning and memory (Dolan, 2002T ; rivedi and Greer, 2014; GSK-3β/β-catenin signaling would be related to the learning and Dillon, 2015; McFarland and Vasterling, 2017P ; an et al., 2017). memory changes in a rat chronic unpredictable stress (CUS) Growing evidence has shown that patients suffering from major model, one of the most valid and relevant rodent models of depression often experience memory deficits even after the depression (Willner, 2005; Duric et al., 2010; Banasr et al., 2010). remission of mood symptoms (Airaksinen et al., 2004; Weiland- In addition, we explored the mechanisms involved in regulation Fiedler et al., 2004; Reppermund et al., 2009). Furthermore, of the GSK-3β/β-catenin signaling pathway induced by CUS. rodents that experience repeated stress demonstrate deficits in tasks assessing learning or memory (Song et al., 2006). However, Materials and Methods the detailed molecular mechanisms underlying the interactions of these 2 disorders are not clearly understood. Animals Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase. Four different phosphorylated regions have been Experiments were performed on adult male Sprague-Dawley described in GSK-3, and phosphorylation of regulatory serine rats (Experimental Animal Center, Shanghai Medical College of residues (Ser21 in GSK-3α and Ser9 in GSK-3β) correlates with Fudan University) weighing 200 g. Animals were housed 4/cage the inhibition of its kinase activity (Hughes et al., 1993 W ; ang with food and water available ad libitum. All rats were kept on et al., 1994). The GSK-3β/β-catenin pathway has been studied a 12-h-light/-dark cycle (lights on at 7 am) in the same colony extensively in the context of the canonical Wnt pathway, which room, with temperature (21°C± 2°C) and humidity (55% ± 5%) is an important regulator of mammalian neural development remaining constant. All experiments were conducted in accord- (Logan and Nusse, 2004; Ciani and Salinas, 2005). In the Wnt/β- ance with the National Institutes of Health Guide for the Care catenin pathway, Wnt ligands bind to the Frizzled receptors and Use of Laboratory Animals and were approved by Jiang Su and the co-receptors LRP5/6, which leads to the phosphoryl- Animal Care and Use Committee. ation of Disheveled (Dvl) (Wharton, 2003). The activation of Dvl leads to the inhibition of GSK3β, which allows β-catenin to be CUS Procedure and Drug Treatment stabilized, to accumulate in the cytoplasm and to translocate to the nucleus where it activates the transcription of T-cell fac- Animals were exposed to a variable sequence of mild and unpre- tor/lymphoid enhancer factor (TCF/LEF) target genes, including dictable stressors for 35 days as previously described (Xi et al., the cell-cycle regulatory genes cyclin D1 and c-myc (Logan et al., 2011). The CUS procedure contained 9 different stressors ran- 2004) and genes important for synaptic plasticity and memory domly arranged day and night across 35 consecutive days: 20 h of (Arrázola et al., 2009). Wnt signaling is not only modulated by food and water deprivation, 18 h of water deprivation, 17 h of 45° the presence or absence of Wnt ligands but also by antagonists cage tilt, overnight illumination, 21 h of wet cage, 5 minutes of such as the secreted Dickkopf (Dkk) glycoproteins, which bind to swimming in water at 4°C, 30 minutes on a 160-Hz rocking bed, LRP5/LRP6, thereby preventing its interaction with Wnt ligands 1-minute tail pinch, and 2-h immobilization. The behavioral tests (Niehrs, 2006). were operated and scored by trained and experienced observ- It is now widely accepted that the GSK-3β/β-catenin pathway ers who were blind to the condition of the animals. Three weeks plays an important role in the regulation of learning and mem- after the beginning of the CUS, rats received SB216763 (2 mg/kg, ory (Maguschak and Ressler, 2008, 2011; King et al., 2013; Liu i.p.) or saline treatment every other day for another 2 weeks. The et al., 2017). In vivo activation of Wnt signaling increases exci- dose of SB216763 and alternate day treatment schedule used tatory synaptic transmission and improves episodic memory in in this experiment was selected based on previous study (Mao adult wild-type mice (Vargas et al., 2014). Fortress et al. report et al., 2009). SB216763 has been previously reported to cross the that learning rapidly activates GSK-3β/β-catenin signaling in the blood-brain barrier after i.p. injection (Selenica et al., 2007). dorsal hippocampus and suggest that canonical Wnt signaling is necessary for hippocampal memory consolidation (Fortress Sucrose Preference Test et al., 2013). Moreover, inhibition of GSK-3β facilitates the induc- The sucrose preference test was performed as previously tion of long-term potentiation, which is the best characterized described (Xi et al., 2011). The animals were allowed to consume molecular and cellular component of the plasticity thought to water and 1% sucrose solution for 1 h after 20-h food and water underlie learning and memory, in the hippocampal CA1 and Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 Hui et al. | 3 deprivation. The positions of the 2 bottles (right/left) were varied GSK-3α (1:1000, Cell Signaling); phospho-Ser21-GSK-3α (1:1000, randomly across animals and were reversed after 30 minutes. Abcam); GSK-3β (1:1000, Cell Signaling); phospho-Ser9-GSK- The sucrose preference was calculated according to the follow- 3β (1:1000, Cell Signaling); β-catenin (1:2000, BD Bioscience); ing ratio: sucrose preference (%)= [sucrose intake (g)/sucrose α-tubulin (1:2000, Invitrogen); Wnt1 (1:1000, Abcam); Wnt3a intake (g) + water intake (g)]× 100%. (1:1000, Abcam); Wnt7a (1:1000, Abcam); Dkk-1(1:500, Santa Cruz Biotechnology). After washing, the membranes were incubated with a secondary antibody solution (goat anti-mouse, or goat Forced Swim Test anti-rabbit IgG-HRP, 1:5000, Santa Cruz) at room temperature for 2 hours followed by detection using the enhanced chemilumin- The forced swim test was conducted as previously described (Xi escence method. et al., 2011). Briefly, rats were forced to swim individually in a cylindrical glass container (40 cm height, 30 cm diameter), which Construction and Preparation of Recombinant AAV contained tap water (25°C ± 1°C) of 28 cm depth. Rats underwent a preswimming session of 15 minutes followed 24 hours later by The rat GSK-3β cDNA was amplified from a rat hippocampal a swimming test of 5 minutes. All test sessions were recorded by cDNA library and subcloned into an AAV2/8 backbone, which a video camera from the opposite side of the cylinder. The time was generated from a pAAV-MCS-EGFP vector by digesting with spent with minimal activity to keep respiration, passively float- EcoRI/NheI. The control plasmid expressed only EGFP. The virus ing in the water, was measured as immobility. was generated with a triple-transfection, helper-free method as previously described (Zolotukhin et al., 1999). Briefly, human embryonic kidney-293 cells were cultured in DMEM with 10% FBS Morris Water Maze Test and plated at a density of 8× 10 cells in T-75 flasks. The following The Morris water maze test was performed as previously day, the cells were transfected with pAAV-GSK-3β, pHelper, and described (Xi et al., 2011). The water maze was a 160-cm diam- pAAV-RC plasmids (Gene Chem) with a standard calcium phos- eter black circular pool filled with opaque water (30 cm depth) phate method. After 48 hours, cells were harvested and lysed at 25°C ±1°C. An escape platform (11 cm diameter) was placed (3 freeze/thaw cycles in dry ice-ethanol and 37°C baths). Then in the middle of one of the quadrants (1 cm below the water benzonase was added to the mixture (50 U/mL, final concentra- surface). The behavior of the animal was monitored by a video tion), and the lysate was incubated for 30 minutes at 37°C, cen- camera mounted in the ceiling above the center of the pool. In trifuged for 20 minutes at 3700 g, and filtered through a 0.45-μm the acquisition trials, the rats were trained 120 seconds per trial sterile syringe filter. Subsequently, viruses were purified using and 4 trials per day starting at 4 different positions with 30-min- a modified caesium chloride centrifugation. The virus was then ute intervals for consecutive 4 days. Each trial began with the rat titered using an AAV ELISA kit (Progen) and stored at -80°C. in the pool facing the sidewalls. If the rat failed to escape within 120 seconds, it was guided to the platform by the experimenter. Stereotaxic Surgery and Infusions When the rat escaped onto the platform, the rat was allowed to stay on the platform for 30 seconds before being returned to Rats were anesthetized with Nembutal (i.p. 55 mg/kg) and its home cage. The hidden platform was removed on day 5, and mounted in a rat stereotaxic apparatus. Bilateral viral injections memory retrieval was examined by a probe trial that lasted for were performed with coordinates -4.3 mm (anterior/poster - 180 seconds. The escape latency in the acquisition trials, the ior), -2.0 mm (lateral), and -4.2 mm (dorsal/ ventral) relative to number of crossings over the platform location, and the time the bregma (Duric et al., 2010). Each hippocampal hemisphere spent in the target quadrant during the probe test were recorded was infused with a total of 2 μL of purified virus over a 15-min- by a computerized video tracking system. ute period followed by 5 minutes of rest. Behavioral tests were performed 4 weeks after virus infusion. After behavioral test- ing, animals were perfused with phosphate-buffered saline fol- Western Blotting lowed by 400 mL of 4% paraformaldehyde (dissolved in 0.1 M After the behavioral test, hippocampal tissues were imme- PBS, pH 7.4). Brains were immediately removed from the skull, diately frozen on dry ice after dissection and stored at -80°C. postfixed overnight, followed by an incubation overnight in PBS Western blotting was performed according to a standard proto- containing 30% sucrose at 4°C. Brains were cut into 25-μm sec- col. Nuclear and cytoplasmic proteins were extracted using the tions using a microtome to allow for staining with GFP (1:1000, NE-PER Nuclear Protein Extraction Kit (Thermo). In brief, 100-mg Abcam) and β-catenin (1:1000, BD Bioscience). samples were resuspended in 1000 μL cytoplasmic extraction reagent I and homogenized with a probe sonicator. The mixture Corticosterone Administration was incubated in an ice bath for 10 minutes. Then, 55 μL cyto- plasmic extraction reagent II was added to the mixture, violently Corticosterone (CORT, Sigma) was administered at a dose of 40 mg/kg (Gregus et al., 2005) suspended in 0.9% (w/v) physio- vortexed for 5 seconds, and incubated in an ice bath for 1 min- ute. The solution was then centrifuged at 16 000 g for 10 min- logical saline with 2% (v/v) polyoxyethylene glycol sorbitan monooleate (Sigma-Aldrich). Control animals were injected with utes at 4°C. After removal of the supernatant, 500 μL of nuclear protein extraction reagent was added to the nuclear precipitate vehicle (physiological saline). All injections were delivered s.c. once per day between 9:00 am and 11:30 am for 21 consecutive and vortexed on the highest setting for 15 seconds every 10 minutes for a total of 40 minutes. The mixture was centrifuged days. The acute CORT treatment group received a vehicle injec- tion once per day for 20 days, followed by a single CORT injection at 16 000 g for 15 minutes at 4°C, and protein concentrations in the supernatant were detected by the Bradford method. Equal on day 21. The chronic CORT treatment group received a CORT injection once per day for 21 consecutive days, and the control quantities of protein were loaded onto a 10% polyacrylamide gel containing 0.2% SDS for separation. The separated proteins group received a vehicle injection along the same time course. The sucrose preference test, forced swim test and Morris water were transferred onto a PVDF membrane (Millipore) and incu- bated overnight at 4°C with the following primary antibodies: maze test were carried out 24 hours after the last injection. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 4 | International Journal of Neuropsychopharmacology, 2018 acquisition trials of Morris water maze test were carried out Measurement of Plasma CORT on 4 consecutive days, repeated-measures ANOVA was initially Rats were killed by rapid decapitation the next morning (8:00 performed. In all other cases, 1-way or 2-way ANOVA was used. am to approximately 10:00 am) after the Morris water maze Posthoc analyses were performed by the Bonferroni’s test for test. Blood samples were collected into EDTA in microcentri- selected or multiple comparisons when P < .05. fuge tubes on ice, centrifuged at 800 g for 14 minutes at 4°C, and the plasma was collected and centrifuged further at 800 g for 7 minutes at 4°C. Plasma was stored at -80°C until ana- Results lysis. Plasma CORT was analyzed by radioimmunoassay using the ImmuChem Corticosterone Double Antibody RIA kit (cata- Impairment of Spatial Cognitive Performance log no. 07-120102, MP Biomedicals). The assay sensitivity was Induced by CUS 0.8 μg/dL and the intra- and inter-assay CVs were 6.8% and 7.6%, Before CUS, there were no significant differences among the respectively. groups exposed to the sucrose preference test (> P .05) and the forced swimming test (P > .05). After CUS for 5 weeks, stressed Statistical Analysis rats showed a significant decrease in sucrose preference (P < .05; All data are expressed as the mean ± SEM. Paired Student’s t test Figure 1A) and a significant increase in immobility time (P < .01; was used to compare 2 experimental groups. Considering the Figure 1B). Figure 1. Effects of chronic unpredictable stress (CUS) on behavioral tests. (A) Results of sucrose preference in sucrose preference test. (B) Immobility time in forced swimming test. (C) In the acquisition trials of the Morris water maze test, CUS rats showed longer escape latency during training days 2 to 4. (D–E) In the probe trial, CUS impaired memory retrieval as indicated by fewer crossing times over the platform position and less time spent in the target quadrant. (F–G) There was no significant difference of swim distance and swim speed among groups. Data are presented as mean± SEM (n = 6/group). *P < .05, **P < .01 vs control group. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 Hui et al. | 5 Figure 1C showed the average escape latency onto a hid- Because phosphorylation on Ser9 inactivates GSK-3β, a den platform in the acquisition trials of the Morris water reduction in the inactivation of GSK-3β decreases cytosolic lev- maze test. The curves were similar between groups, with els of β-catenin and its translocation from the cytoplasm to the increasingly shorter latency on consecutive days. There was nucleus. Therefore, the effects of CUS exposure on the total cel- a significant effect of day [F(3, 40) = 81.971, P < .001] and CUS lular level of β-catenin and the nuclear level of β-catenin were [F(1, 40) = 61.964, P < .001] on latency to find the platform. On measured. Figure 2C showed that both total cellular levels and further day-by-day analysis, the CUS group latencies were sig- nuclear levels of β-catenin were significantly decreased com- nificantly longer than the control group on day 2 (P < .01), day pared with the control group (P < .05 for total β-catenin, P < .01 for 3 (P < .01), and day 4 (P < .05), while no significant difference in nuclear β-catenin; Figure 2D). swimming velocity was observed between the 2 experimental groups (data not shown). In the probe trial, the CUS group dis- CUS-Induced Cognitive Impairment Is Reversed by played fewer crossings (P < .01; Figure 1D) and less time swim- GSK-3β Inhibition ming in the target quadrant (P < .05; Figure 1E) compared with the control group. During the period of memory retrieval, the To study the role of GSK-3β /β-catenin signaling in the cogni- swim distance and swim speed were similar among groups tive function of rats exposed to CUS, we used SB216763, a spe- (both P> .05; Figure 1F–G). cific chemical inhibitor of GSK3β . ANOVA analysis revealed significant effects of CUS [F(1, 20) = 18.714, P < .001] and SB216763 [F(1, 20) = 7.831, P = .01] on the sucrose preference. Posthoc ana- Effects of CUS on the GSK-3β/β-Catenin Signaling lysis showed that the sucrose preference of CUS+saline animals Pathway was significantly decreased compared with control+saline rats CUS exposure had no significant effect on the total protein levels (P < .01), and this was reversed by chronic SB216763 administration of either GSK-3α or GSK-3β (both P> .05; Figure 2A–B) in the hippo- (P < .05) (Figure 3A). In the forced swimming test, similar effects campus. We further examined the phosphorylation state of GSK-3 of CUS [F(1, 20) = 13.031, P = .002] and SB216763 [F(1, 20) = 7.219, and found that phosphorylation only on the Ser9 residue of GSK- P = .014] were demonstrated on the immobility time, with longer 3β was significantly decreased after CUS exposure compared with immobility in CUS+saline (P < .01, compared with control+saline the control group (P < .05; Figure 2A–B), while phosphorylation on group) and a reversal of this effect in CUS+SB216763 (P < .05, com- Ser21 of GSK-3α was not significantly changed (P> .05). pared with CUS+saline group) (Figure 3B). Figure 2. Effects of chronic unpredictable stress (CUS) on GSK-3β/β-catenin expression. (A) Representative western blotting of total GSK-3α, phospho-Ser21-GSK-3α, total GSK-3β, phospho-Ser9-GSK-3β, and α-tubulin proteins. (B) Quantification of western-blotting signals of GSK3 and α-tubulin proteins. (C) Representative western blotting of total β-catenin, nuclear β-catenin, and α-tubulin proteins. (D) Quantification of western blotting signals of β-catenin and α-tubulin proteins. Data were ratios compared with α-tubulin protein. Values represent means ± SEM (n = 6/group). * P < .05, ** P < .01 vs control group. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 6 | International Journal of Neuropsychopharmacology, 2018 Figure 3. Influence of GSK-3β inhibition on behavior tests in chronic unpredictable stress (CUS) rats. (A–B) Effects of CUS and SB216763 treatment on sucrose preference and immobility time in forced swimming test. (C) SB216763 treatment restored the CUS-induced longer latencies in the acquisition trials of Morris water maze test. (D–E) In the probe trial, SB216763 treatment restored the CUS-induced fewer crossing times over the platform position and less time spent in the target quadrant. (F) Western-blotting analysis showing the effects of SB216763 treatment on hippocampal β-catenin expression. (G) Quantification of western-blotting signals of β-catenin and α-tubulin proteins. Data are presented as mean ± SEM (n = 6/group). *P < .05, **P < .01 vs control+saline group, #P < .05 vs CUS+saline group, ##P < .01 vs CUS+saline group. We next assessed learning and memory performance in P = .052 for CUS; F(1, 20) = 3.826, P = .065 for SB216763]. Posthoc the Morris water maze test. In the acquisition trials, there tests demonstrated that SB216763 administration significantly was a significant effect of day [F(3, 80) = 202.115, P < .001], CUS reversed the decreased crossings (P < .01; Figure 3D) and time [F(1, 80) = 40.840, P < .001] and SB216763 [F(1, 80) = 23.846, P < .001] swimming in the target quadrant (P < .05; Figure 3E) induced by on latency to find the platform. The CUS+SB216763 group CUS. There were no differences in the swim distance and swim showed significantly shorter latencies than the CUS+saline speed among the groups (data not shown). Western-blotting group on day 3 (P < .01), and day 4 (P < .01, Figure 3C) in day-by- analysis of whole-hippocampal homogenates showed that day analysis. In the probe trial, ANOVA revealed main effects for chronic SB216763 administration significantly increased total CUS and SB216763 treatment on crossing times [F(1, 20) = 12.468, cellular β-catenin (P < .05) but had no significant effect on the lev- P = .002 for CUS; F(1, 20) = 13.852, P = .001 for SB216763], but no els of nuclear β-catenin compared with the control+saline group significant effect on the time in target quadrant [F(1, 20) = 4.258, (Figure 3F–G). Furthermore, SB216763 significantly prevented the Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 Hui et al. | 7 CUS-induced decrease of both total cellular levels and nuclear analysis showed that acute CORT treatment had no significant levels of β-catenin compared with the CUS+saline group (P < .05 effect on the latencies in acquisition trials compared with the for total β-catenin, P < .01 for nuclear β-catenin; Figure 3F–G). control group (all >P .05; Figure 6C). However, chronic CORT Further ANOVA analysis showed significant effects for CUS and treatment significantly elevated the latencies in acquisition tri- SB216763 treatment on both total cellular levels [F(1,= 20) 16.723, als on day 2 (P < .01), day 3 (P < .01), and day 4 (P < .01; Figure 6C) P = .001 for CUS; F(1, 20) = 18.863, P < .001 for SB216763] and compared with the control group. In the probe trial, acute CORT nuclear levels of β-catenin [F(1, 20) = 11.670, P = .003 for CUS; F(1, treatment had no significant effect on the crossing times or the 20) = 16.987, P = .001 for SB216763]. time in the target quadrant (both >P .05; Figure 6D–E), while the chronic CORT treatment group displayed decreased crossings (P < .01; Figure 6D) and less time swimming in the target quad- Cognitive Impairment of Viral GSK-3β Expression rant (P < .05; Figure 6E) compared with the control group. During To directly determine the influence of increased expression of the memory retrieval phase, the swim distance and swim speed GSK-3β on memory and depression behaviors in rats, an AAV were similar among groups (data not shown). vector was designed to express GSK-3β as well as the marker We further examined the levels of plasma CORT of all 4 GFP to allow for detection of infected neurons. AAV-control or groups of rats. Compared with the control group, acute CORT AAV-GSK-3β was bilaterally injected into the dorsal hippocam- treatment had no significant effect on plasma corticosterone pus of adult rats, although we also observed GFP cells outside (P > .05; Figure 6F), while the chronic CORT treatment group hippocampus, probably as a result of the virus traveling up the and CUS group displayed higher levels of CORT (both P < .01; cannula track (Figure 4A). To verify the expression and function Figure 6F). In addition, Western-blotting analysis showed that of AAV-GSK-3β, we performed immunofluorescence to detect chronic, but not acute, CORT treatment significantly increased both GFP, expressed by virus-infected neurons, and β-catenin, a hippocampal levels of Dkk-1 (P < .05) and decreased phosphor - downstream target of GSK-3β. We found that AAV-GSK-3β injec- ylation of Ser9 on GSK-3β (P < .05; Figure 6G–H), whereas both tion significantly decreased levels of β-catenin colocalization treatments had no significant effect on the levels of total GSK-3β with GFP compared with that in AAV-control rats (Figure 4B). (both P> .05). We confirmed this in a separate cohort by Western-blotting analysis of rat hippocampus. AAV-GSK-3β infusion significantly Discussion increased GSK-3β levels (P < .01) and decreased total cellu- lar level of β-catenin compared with AAV-control rats (P < .01), The results of the present study demonstrated that exposure to demonstrating that GSK-3β overexpression caused functional CUS had a dramatic influence on spatial cognitive performance activation of GSK-3β/β-catenin pathway in the hippocampus in the Morris water maze task and decreased the phosphoryl- (Figure 4C). ation of Ser9 of GSK-3β as well as the total and nuclear levels of Four weeks after virus infusion, AAV-GSK-3β rats showed β-catenin in the hippocampus. Inhibition of GSK3β by SB216763 a significant decrease in sucrose preference (P < .05; Figure 4D) significantly ameliorated the cognitive deficits induced by CUS, and a significant increase in immobility time (P < .05; Figure 4E). while overexpression of GSK3β by AAV-mediated gene trans- Animals infused with AAV-GSK-3β displayed longer latencies fer significantly decreased cognitive performance in adult on day 2 (P < .05), day 3 (P < .01), and day 4 (P < .05, Figure 4F) in rats. Moreover, CUS exposure increased the expression of the the acquisition trials of the Morris water maze test relative to canonical Wnt antagonist Dkk-1. Furthermore, chronic adminis- AAV-control-infused animals. In the probe trial, the AAV-GSK- tration of CORT, the key mediator of stress-induced depressive- 3β group displayed decreased crossings (P < .01; Figure 4G) and like behavioral changes and synaptic dysfunction, significantly less time swimming in the target quadrant (P < .05; Figure 4H) increased Dkk-1 expression, decreased the phosphorylation of compared with the AAV-control group, with no changes in swim Ser9 of GSK-3β, and resulted in impairment of hippocampal distance or swim speed (data not shown). learning and memory. These results suggest that elevated CORT levels could play a role in the regulation of the GSK-3β/β-catenin signaling that underlies learning and memory deficits in CUS. Effects of CUS on Wnt Ligands and Antagonists It is now widely accepted that cognitive dysfunctions includ- Because GSK-3β and β-catenin are the key downstream regula- ing attention, executive function, and memory persist in patients tors in canonical Wnt signaling, we next investigated the effects suffering from major depression (Morimoto and Alexopoulos, of CUS exposure on protein levels of Wnt ligands and antago- 2013; Trivedi et al., 2014; Dillon, 2015; Gałecki et al., 2015). An nists. Western blotting (Figure 5A) indicated that there were effect size analysis of cognitive functioning in 726 patients with no significant changes in the levels of Wnt1, Wnt3a or Wnt7a, major depressive disorder, conducted using meta-analytic prin- which are classified as canonical Wnt ligands, compared to the ciples, found that the type of memory task most affected by control group (all >P .05; Figure 5B). However, there was a signifi- depression was recollection (Zakzanis et al., 1998). Patients suf- cant increase (P < .05; Figure 5B) in the expression of Wnt signal- fering from chronic major depression display volume reductions ing antagonist Dkk-1. of the hippocampus (Campbell et al., 2004; Koolschijn et al., 2009), a region important for memory formation. The present experiments showed a deficit of spatial memory in rats exposed Chronic Treatment of Corticosterone Induces Dkk-1 to CUS, supporting the hypothesis that depressed subjects show Expression and Cognitive Impairment differential impairment on memory tasks that are dependent To further examine the effects of CUS exposure on the regula- on the hippocampus. Our results are consistent with a previous tion of Dkk-1, we hypothesized that stress-induced increases in report showing a deficit of spatial memory in the water maze CORT might be the key mediator. Figure 6A and B showed that task following chronic stress or learned helplessness in mice chronic, but not acute, CORT treatment rats showed a signifi- (Song et al., 2006) and the findings of spatial memory deficits in cant decrease in sucrose preference (P < .01) and a significant other animal models of depression (Sun and Alkon, 2004; Wright increase in immobility time (P < .01). Morris water maze test et al., 2006; Bondi et al., 2008). Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 8 | International Journal of Neuropsychopharmacology, 2018 Figure 4. Influence of GSK-3β overexpression on behavior tests in rats. (A) Rats received bilateral intrahippocampal infusions of AAV-control or AAV-GSK-3β-GFP. Representative images of GFP protein expression in dorsal hippocampus. Blue DAPI staining showed the nuclei. Scale bars = 100 μm. (B) Representative colocalization images from dorsal hippocampal neurons positive for GFP and β-catenin. Scale bars = 25 μm. (C) Western-blotting analysis showing the effects of AAV-GSK-3β injec- tion on the expression of GSK-3β and β-catenin in hippocampus. (D–E) Effects of AAV-GSK-3β infusion on sucrose preference and immobility time in forced swimming test. (F) AAV-GSK-3β infusion rats showed longer escape latency during training days 2 to 4 in the acquisition trials of Morris water maze test. (G–H) In the probe trial, AAV-GSK-3β infusion rats showed fewer crossing times over the platform position and less time spent in the target quadrant. Data are presented as mean ± SEM (n = 6/ group). *P < .05, **P < .01 vs AAV-control group. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 Hui et al. | 9 Figure 5. Effects of chronic unpredictable stress (CUS) on Wnt ligand and antagonist expression. (A) Representative western blotting of Wnt1, Wnt3a, or Wnt7a, Dkk-1, and α-tubulin proteins. (B) Quantification of western-blotting signals of Wnt ligands, antagonists, and α-tubulin proteins. Data were ratios compared with α-tubulin protein. Values represent means ± SEM (n = 6/group). *P < .05 vs control group. The GSK-3β/β-catenin pathway has been shown to be regu- signaling pathway has been shown to be involved in the regu- lated by chronic stress. Prenatal chronic mild stress significantly lation of hippocampal long-term potentiation (Chen et al., 2006; increased the expression of hippocampal GSK-3β (Li et al., 2014). Hooper et al., 2007; Franklin et al., 2014), which is an activity- Decreased levels of phosphorylated GSK-3β and β-catenin in dependent enhancement of synaptic strength and is considered the hippocampus have been demonstrated in rats subjected to one of the physiological mechanisms that underlies learning forced swim stress for 14 consecutive days (Liu et al., 2012). In and memory in the hippocampus (Citri and Malenka, 2008). addition, chronic restraint stress significantly decreased phos- Importantly, β-catenin, present at pre- and postsynaptic ter - phorylation levels of Ser9 of GSK-3β in the prefrontal cortex minals, associates with the cytoplasmic domain of cadherin (Huang et al., 2015). Furthermore, GSK-3β/β-catenin signaling and directly links to the actin cytoskeleton through α-catenin has been implicated in both the pathophysiology and treatment (Gumbiner, 1996). Alterations of cadherin-catenin complexes are of depression (Crofton et al., 2017Xu ; et al., 2017). For example, thought to influence synaptic size and strength (Murase et al., increases in GSK-3β activity have been found in the prefrontal 2002), suggesting direct participation in synaptic remodelling. cortex of postmortem depressed suicide victims (Karege et al., Furthermore, GSK-3β/β-catenin has an important role in the 2012). The GSK-3β gene may play a role in determining the regulation of synaptic neurotransmission in hippocampal neu- regional gray matter volume differences of the right hippocam- rons (Ahmad-Annuar et al., 2006; Cerpa et al., 2008). In addition, pus and bilateral superior temporal gyri in patients with recur - as a key component of the Wnt signaling pathway, β-catenin rent major depressive disorder (Inkster et al., 2009). Okamoto may activate TCF/LEF target genes that are important for neuro- et al. reported that GSK-3β/β-catenin signaling in the hippocam- genesis, synaptic plasticity, and neuronal death and survival pus is regulated by different classes of antidepressant therapies, (Clevers, 2006; Hui et al., 2015). Further work is required to deter - including SSRIs, SNRIs, dual 5-HT/NE reuptake inhibitors, and mine the mechanisms associated with learning and memory chronic electroconvulsive shock (Okamoto et al., 2010). Our find- impairments in response to stress. ings that CUS exposure decreased the phosphorylation of Ser9 Growing evidence indicates the concurrence and interrela- of GSK-3β as well as the total and nuclear levels of β-catenin in tionship of depression and cognitive impairments (Kuzis et al., the hippocampus are consistent with previous studies. 1997; Payne et al., 1998; Zubenko et al., 2003). However, the On the other hand, abnormal Wnt/GSK-3β/β-catenin signal- detailed molecular mechanisms underlying the interactions of ing has been implicated in the pathophysiology of learning and these 2 disorders have not been fully understood. It has been memory deficits. Pharmacological stabilization of β-catenin suggested that decreased brain derived neurotrophic factor and with LiCl resulted in enhanced learning, whereas genetic dele- cAMP-response element-binding protein levels in hippocam- tion of Ctnnb1 (encoding β-catenin) in the amygdala resulted in pus could be involved (Song et al., 2006). In the present study, deficient learning (Maguschak et al., 2008). Furthermore, activa- our results showed that CUS exposure impaired spatial cogni- tion of the canonical Wnt signaling pathway in hippocampus tive performance and decreased the phosphorylation of Ser9 of not only improves episodic memory and enhances long-term GSK-3β and β-catenin levels in hippocampus, while inhibition of potentiation in adult wild-type mice but also rescues memory GSK-3β significantly ameliorated the cognitive deficits induced loss and improves synaptic dysfunction in APP/PS1-transgenic by CUS, indicating an important function of GSK-3β/β-catenin mice (Vargas et al., 2014), a model of Alzheimer’s disease, which signaling in the interactions between these 2 disorders. is characterized by a progressive deterioration of cognitive We further investigated the levels of Wnt ligands and antag- function (Toledo and Inestrosa, 2010). In agreement with pre- onists to explore the mechanism of the phosphorylation of GSK- vious studies, the present study demonstrated that inhibition 3β caused by CUS. The results showed no significant differences of GSK3β by SB216763 improved the cognitive deficits in the in the levels of Wnt1, Wnt3a, or Wnt7a but did show significantly Morris water maze task induced by CUS, while overexpression increased expression of Dkk-1. Although previous studies sup- of GSK3β in the hippocampus decreased cognitive performance port a critical role for Wnt ligands and antagonists in learning in adult rats. and memory (Tabatadze et al., 2012; Fortress et al., 2013), our The possible mechanisms of GSK-3β/β-catenin signaling study suggested that only Dkk1 might be involved in the regu- in regulating learning and memory are as follows. First, this lation of learning and memory impairments induced by CUS, Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 10 | International Journal of Neuropsychopharmacology, 2018 Figure 6. Effects of corticosterone (CORT) treatment on behavior tests and Dkk-1 expression in rats. (A–B) Effects of CORT treatment on sucrose preference and immo- bility time in forced swimming test. (C) Effects of CORT treatment on escape latency during training days 2 to 4 in the acquisition trials of Morris water maze test. (D–E) In the probe trial, rats treated with chronic CORT showed fewer crossing times over the platform position and less time spent in the target quadrant. (F) Effects of CORT treatment and CUS on plasma corticosterone levels. (G) Western-blotting analysis showing the effects of CORT treatments on hippocampal total GSK-3β, phospho-Ser9- GSK-3β, and Dkk-1 expression. (H) Quantification of western-blotting signals of total GSK-3β, phospho-Ser9-GSK-3β, Dkk-1, and α-tubulin proteins. Data are presented as mean ± SEM (n = 6/group). *P < .05, **P < .01 vs control group. reflecting differences between Wnt ligands and antagonists in treatment enhanced the expression of Dkk-1 in the hippocam- response to chronic stress. pus of mice, while stress-induced hippocampal damage does Because the elevated activity of the hypothalamic-pitui- not occur in mice that lack a Dkk-1 gene transcriptional enhan- tary-adrenal axis has key implications in the pathogenesis of cer (Doubleridge) (Matrisciano et al., 2011). Importantly, the several stress-related psychiatric illnesses (de et al., 2005), we Dkk-1 gene promoter contains at least 3 glucocorticoid-respon- hypothesize that the mechanisms underlying the increased sive elements, and the induction of Dkk-1 by dexamethasone Dkk-1 expression induced by CUS may involve glucocortic- mainly resulted from the activation of transcription through oid signaling. A recent study has shown that dexametha- glucocorticoid-responsive elements in the Dkk-1 gene pro- sone, a glucocorticoid hormone receptor agonist, induces an moter in human osteoblasts (Ohnaka et al., 2004). Our results upregulation of Dkk-1 in human neural stem/progenitor cells of increased Dkk-1 expression after chronic administration of (Moors et al., 2012). In addition, mild restraint stress and CORT CORT are consistent with these previous findings. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy040/4982724 by guest on 13 July 2018 Hui et al. | 11 The action of glucocorticoids on target tissues is mediated by Banasr M, Chowdhury GM, Terwilliger R, Newton SS, Duman interactions with the glucocorticoid receptor (GR) or mineralo- RS, Behar KL, Sanacora G (2010) Glial pathology in an ani- corticoid receptor (MR). Although the mechanism of CORT in mal model of depression: reversal of stress-induced cellular, increasing the expression of Dkk-1 is not clear, studies implicate metabolic and behavioral deficits by the glutamate-modulat- GR-dependent regulation. The upregulation of Dkk-1 in primary ing drug riluzole. Mol Psychiatry 15:501–511. cultured hippocampal neurons induced by CORT was attenuated Bondi CO, Rodriguez G, Gould GG, Frazer A, Morilak DA (2008) Chronic by the GR blocker mifepristone but not by spironolactone, which unpredictable stress induces a cognitive deficit and anxiety-like blocks MR (Matrisciano et al., 2011). In addition, our hypothesis behavior in rats that is prevented by chronic antidepressant is also consistent with the evidence that excessive activation of drug treatment. Neuropsychopharmacology 33:320–331. GRs produces neurotoxic effects in the hippocampus, while acti- Campbell S, Marriott M, Nahmias C, MacQueen GM (2004) Lower vation of MRs is neuroprotective (Crochemore et al., 2005). hippocampal volume in patients suffering from depression: We acknowledge that the current studies on learning and a meta-analysis. Am J Psychiatry 161:598–607. memory deficits of CUS rats do not necessarily extrapolate to Cerpa W, Godoy JA, Alfaro I, Farías GG, Metcalfe MJ, Fuentealba R, cognitive declines in depressed patients, which are involved in Bonansco C, Inestrosa NC (2008) Wnt-7a modulates the syn- diminished ability to think and concentrate or indecisiveness, aptic vesicle cycle and synaptic transmission in hippocampal with devastating effects on executive functions, short- and long- neurons. J Biol Chem 283:5918–5927. term learning, and memory. In addition, effects of CUS on Wnt/ Chen J, Park CS, Tang SJ (2006) Activity-dependent synaptic wnt GSK-3β/β-catenin signaling pathway were studied in the whole release regulates hippocampal long term potentiation. J Biol hippocampus; further studies will be needed to determine Chem 281:11910–11916. which subregions of hippocampus are specific to these effects. Ciani L, Salinas PC (2005) Wnts in the vertebrate nervous system: In summary, our results suggest that learning and memory from patterning to neuronal connectivity. Nat Rev Neurosci deficits resulting from long-term stress exposure are associ- 6:351–362. ated with the GSK-3β/β-catenin signaling pathway that links Citri A, Malenka RC (2008) Synaptic plasticity: multiple forms, the upregulation of Dkk-1 induced by chronic CORT treatment. functions, and mechanisms. Neuropsychopharmacology Understanding the mechanisms that underlie hippocampal 33:18–41. damage in response to stress/glucocorticoids may shed new Clevers H (2006) Wnt/beta-catenin signaling in development and light on the pathophysiology of mood disorders and stress- disease. Cell 127:469–480. related cognitive dysfunctions and may lead to the identifica- Crochemore C, Lu J, Wu Y, Liposits Z, Sousa N, Holsboer F, tion of new therapeutic targets. Almeida OF (2005) Direct targeting of hippocampal neurons for apoptosis by glucocorticoids is reversible by mineralocor - ticoid receptor activation. Mol Psychiatry 10:790–798. Funding Crofton EJ, Nenov MN, Zhang Y, Scala F, Page SA, McCue DL, Li D, This research was supported by National Natural Science Hommel JD, Laezza F, Green TA (2017) Glycogen synthase kin- ase 3 beta alters anxiety-, depression-, and addiction-related Foundation of China (81201051, Guangjun Xi; 81401619, Jiaojie Hui), Natural Science Foundation of Jiangsu Province behaviors and neuronal activity in the nucleus accumbens shell. Neuropharmacology 117:49–60. (BK2012097, Guangjun Xi), and Medical Young Talents Program of Jiangsu Province (QNRC2016191, Guangjun Xi; QNRC2016178, de Kloet ER, Joëls M, Holsboer F (2005) Stress and the brain: from adaptation to disease. Nat Rev Neurosci 6:463–475. Jiaojie Hui). Dillon DG (2015) The neuroscience of positive memory deficits in depression. Front Psychol 6:1295. 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International Journal of Neuropsychopharmacology – Oxford University Press
Published: Apr 23, 2018
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