Increased EphA4-ephexin1 signaling in the medial prefrontal cortex plays a role in depression-like phenotype

Accumulating evidence suggests a role of the ephrin receptor EphA4 and the downstream protein ephexin1 in synaptic plasticity, which is implicated in depression. We examined whether EphA4–ephexin1 signaling plays a role in the pathophysiology of depression, and the antidepressant-like effect of EphA4 inhibitor rhynchophylline. We found increased ratios of p-EphA4/EphA4 and p-ephexin1/ephexin1 in the prefrontal cortex (PFC) and hippocampus but not in the nucleus accumbens (NAc), of susceptible mice after social defeat stress. Furthermore, the p-EphA4/EphA4 ratio was higher in the parietal cortex of depressed patients compared with controls. Systemic administration of rhynchophylline, produced a rapid antidepressant-like effect in a social defeat stress model by inhibiting EphA4–ephexin1 signaling and activating brain-derived neurotrophic factor-TrkB signaling in the PFC and hippocampus. Pretreatment with rhynchophylline before each social defeat stress could prevent the onset of the depression-like phenotype after repeated social defeat stress. Overexpression of EphA4 in the medial PFC owing to infection with an EphA4 adeno-associated virus caused the depression-like phenotype 3 weeks later and rhynchophylline had a rapid antidepressant-like effect in these mice. These findings suggest that increased EphA4–ephexin1 signaling in the PFC plays a role in the pathophysiology of depression.

various types of synapses [19][20][21] . It is well known that changes in the dendritic length and spine density in the prefrontal cortex (PFC) and hippocampus contribute to the neurobiology of depression and that the action of antidepressants is mediated by blocking or reversing these changes [22][23][24] . Therefore, it is important to determine whether EphA4-ephexin1 signaling plays a role in the pathophysiology of depression.
In the present study, we examined the role of EphA4-ephexin1 signaling in the pathophysiology of depression. First, we tested whether the expression of EphA4 and ephexin1 as well as their phosphorylated forms was altered in the brain regions of mice with a depression-like phenotype and depressed patients. Second, we examined the effects of a novel EphA4 inhibitor, rhynchophylline [25][26][27] , and demonstrated its antidepressant-like effect in a social defeat stress model of depression. Third, we examined whether pretreatment with rhynchophylline before each stress could prevent the onset of the depression-like phenotype after repeated social defeat stress. Finally, we determined whether overexpression of EphA4-venus by adeno-associated virus (AAV) infection in the medial PFC caused the depression-like phenotype. Subsequently, we examined the antidepressant-like effect of rhynchophylline in mice after EphA4 AAV infection.
Furthermore, the p-EphA4/EphA4 and p-epexin1/ephexin1 ratios were significantly higher in the PFC, CA3, and DG from learned helplessness (LH: susceptible) rats compared with the control rats, but not in the non-LH (resilience) rats, (Fig. 1d-f). By contrast, LH stress did not change the ratio in the CA1 and NAc of rats ( Fig. 1e and f). One-way ANOVA revealed statistical results for EphA4 (PFC: F 1,16 = 23.882, P < 0.001; NAc: F 1, 16  Using postmortem brain samples from the Neuropathology Consortium of the Stanley Medical Research Institute 28,29 , we found that the p-EphA4/EphA4 ratio was significantly higher in the parietal cortex (Brodmann area 7: BA7) of subjects with depression and bipolar disorder, but not schizophrenia, than in that of controls (one-way ANOVA, F 1,59 = 5.302, P = 0.003) (Fig. 1g). In contrast, there were no differences in the cerebellum among the four groups (one-way ANOVA, F 1,59 = 1.534, P = 0.216) (Fig. 1h). These findings suggest that the increased p-EphA4/EphA4 ratio in the parietal cortex may be implicated in the pathogenesis of mood disorders such as depression and bipolar disorder.
Antidepressant effects of rhynchophylline are in part mediated by changes in the EphA4-ephexin1-Cdk5 signaling, brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase (TrkB) signaling and postsynaptic density protein (PSD)-95. We examined the The ratios of p-EphA4/EphA4 and p-ephexin1/ ephexin1 in the mouse brain regions from social defeat stress (susceptible) mice and control (unsusceptible) mice. Data represent the mean ± S.E.M. (n = 5 or 6). *P < 0.05 compared with the control group. N.S.: not significant (Student's t-test). (d) Schedule of learned helplessness (LH) and brain sample collection. (e and f) The ratios of p-EphA4/EphA4 and p-Ephexin1/Ephexin1 in the rat brain regions from control, LH, and non-LH rats (n = 5-6). Data represent the mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.01 compared with the control group. N.S.: not significant. (g and h): The ratios of p-EphA4/EphA4 in the postmortem brain samples (parietal cortex and cerebellum) from control, depression, schizophrenia, and bipolar disorder subjects. Data represent the mean ± S.E.M. (n = 15). *P < 0.05 compared with control group, N.S.: not significant. molecular mechanism underlying the antidepressant-like effect of rhynchophylline ( Fig. 4a-g). Social defeat stress significantly attenuated the increased p-EphA4/EphA4 and p-ephexin1/ephexin1 ratios in the PFC, CA3, and DG, but not in the CA1 and NAc, of susceptible mice. In the susceptible mice, rhynchophylline (25 mg/kg) significantly attenuated the increased p-EphA4/EphA4 and p-ephexin1/ephexin1 ratios in the PFC, CA3, and DG, but not in the CA1 and NAc (two-way ANOVA, p-EphA4/EphA4 ratio: PFC: stress: F 1,23 = 7.655, P = 0.012, treatment: F 1,23 = 6.514, P = 0.019, interaction:  Furthermore, social defeat stress significantly increased the levels of p-Fyn/Fyn ratio in the PFC, CA3, and DG, but not in the CA1 and NAc, of susceptible mice. Rhynchophylline significantly attenuated the increased p-Fyn/ Fyn ratio in the PFC, CA3, and DG, but not in the CA1 and NAc, of susceptible mice ( Figure S1).
Signaling by BDNF and its receptor TrkB plays a key role in the depression-like phenotype [30][31][32][33][34][35][36] , so we examined the role of BDNF-TrkB signaling in the selected brain regions after rhynchophylline treatment. In susceptible mice, social defeat stress significantly decreased the levels of BDNF and p-TrkB/TrkB ratio in the PFC, CA3, and DG, but not in the NAc and CA1. A single administration of rhynchophylline (25 mg/kg) significantly attenuated the decreased levels of BDNF and the p-TrkB/TrkB ratio in the PFC, CA3, and DG, but not in the NAc and CA1 (two-way ANOVA, BDNF: PFC: stress: F 1,21 = 7.606, P = 0.013, treatment: F 1,21 = 15.908, P = 0.001, interaction: F 1,21 = 19.563, P < 0.001, NAc: stress:   (Fig. 4g). These results suggest that rhynchophylline shows an antidepressant-like effect by activating BDNF-TrkB signaling and synaptogenesis in the PFC, CA3, and DG in mice with the depression-like phenotype.
Moreover, the administration of EphA4 AAV significantly decreased the expression of BDNF, the p-TrkB/ TrkB ratio, and the PSD-95 level in the PFC (Fig. 6j-l). The administration of rhynchophylline (25 mg/kg) also significantly attenuated the decreased levels of BDNF and PSD-95, and the p-TrkB/TrkB ratio in the PFC  (Fig. 6j-l). These results suggest that the administration of EphA4 AAV into the mPFC activated EphA4-ephexin1 signaling and the subsequent inhibition of BDNF-TrkB signaling in the PFC, thereby resulting in the depression-like phenotype. Rhynchophylline showed a rapid antidepressant-like effect by blocking EphA4-ephexin1 signaling in the PFC.

Discussion
In the present study, we demonstrated a key role for EphA4-ephexin1 signaling in the pathophysiology of depression and our main findings are as follows. First, we found that EphA4-ephexin1 signaling was activated in the PFC, CA3, and DG of mice with the depression-like phenotype. Furthermore, the p-EphA4/EphA4 ratio was higher in the parietal cortex from depressed patients compared with the controls. Second, a novel EphA4 inhibitor, rhynchophylline, had a rapid antidepressant-like effect and prophylactic effects in a social defeat stress model. Finally, a bilateral injection of EphA4 AAV into the mPFC produced the depression-like phenotype in mice. Interestingly, rhynchophylline had a rapid antidepressant-like effect by inhibiting increased EphA4-ephexin1 signaling in the mPFC. In addition, rhynchophylline showed a rapid antidepressant-like effect in susceptible mice at 10 weeks old and 12 weeks old, suggesting an antidepressant-like effect of rhynchophylline in late adolescent and adult mice. These findings suggest that the activation of EphA4-ephexin1 signaling in the PFC plays a key role in the pathophysiology of depression, thereby indicating that EphA4-ephexin1 signaling could be a novel therapeutic target for depression.
EphA4 is expressed in the PFC and hippocampus of the adult brain, where it acts as a negative regulator of NMDA receptor neurotransmission 14 . EphA4 activation by ephrins triggers forward signaling, which leads to the retraction of dendritic spines via the activation of ephexin1 12,[19][20][21] . EphA4 also causes the removal of synaptic and surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors during homeostatic plasticity 18 . It is well known that alterations in the dendritic spine morphology in the PFC and hippocampus play a role in the neurobiology of depression [22][23][24] . In this study, rhynchophylline produced a rapid antidepressant-like effect by normalizing increased EphA4-ephexin1 signaling in the PFC and hippocampus of susceptible mice and by normalizing the decreased spine density in the PFC and hippocampus of susceptible mice. Furthermore, we found an increased EphA4 signaling in the parietal cortex from depressed patients. In contrast, Li et al. 39 reported decreased expression of EphA4 in the hippocampus from rats with depression-like phenotype in the chronic mild stress model although the p-EphA4/EphA4 ratio was not measured. We also found increased p-EphA4/ EphA4 and p-ephexin1/ephexin1 ratios in the PFC and hippocampus of LH rats compared with the control rats, but not in the non-LH (resilient) rats. EphA4-ephexin1 signaling was activated in the susceptible LH rats, so it is possible that EphA4-ephexin1 signaling in the PFC and hippocampus may partly contribute to stress resilience. Given the role of EphA4-ephexin1 signaling in the dendritic spine morphology 25,40 , these findings suggest that EphA4-ephexin1 signaling might play a key role in the pathophysiology of depression, and the antidepressant-like effect of rhynchophylline might be at least in part due to an impairment of ephrin reverse signaling.
Accumulating evidence suggests that Cdk5 also plays an important role in the pathophysiology of depression. It has been reported that stress can increase the Cdk5 levels in the PFC and hippocampus of rodents 41,42 . In contrast, loss of Cdk5 function in the ventral tegmental area (VTA) induced anxiety-and depression-like behaviors 43 . A recent study demonstrated that the overexpression of Cdk5 by Cdk5-ZFP1-p65 in the NAc of mice could make mice more resilient to social defeat stress 44 . These findings suggest that abnormal Cdk5 levels in the PFC and hippocampus, as well as the VTA-NAc pathway might have causative roles in depression. In this study, we found that the p-Cdk5 (Tyr 15)/Cdk5 ratio increased in the PFC and hippocampus of mice with the depression-like phenotype, but not in the NAc. Interestingly, rhynchophylline significantly attenuated the increased p-Cdk5 (Tyr 15)/Cdk5 ratio in the PFC and hippocampus, thereby suggesting that Cdk5 might play a role in the antidepressant-like effect of rhynchophylline in the PFC and hippocampus.
It is well known that BDNF-TrkB signaling plays a key role in depression [30][31][32][33][34][35][36] . Previous studies of postmortem brains showed that the BDNF levels were decreased in the PFC and hippocampus of psychiatric disorder patients who had committed suicide compared with non-psychiatric controls 45,46 , which suggests that the decreased BDNF levels in the PFC and hippocampus may have causative roles in the pathophysiology of depression. We found decreased levels of BDNF in the PFC and hippocampus of rodents with the depression-like phenotype 37,38,[47][48][49] . Similar to the NMDA receptor antagonist ketamine 47, 50 , rhynchophylline produced a rapid antidepressant-like effect by normalizing decreased BDNF-TrkB signaling in the PFC and hippocampus of mice with depression-like phenotype. However, unlike ketamine, rhynchophylline did not have a sustained antidepressant-like effect.
The previous studies demonstrated that increased BDNF-TrkB signaling in the VTA-NAc pathway plays key a role in the depression-like phenotype 30,31,37,38,45,47 . A study using postmortem brain samples showed increased levels of BDNF in the NAc of patients with depression relative to controls 51 . We reported that lipopolysaccharide-induced inflammation and social defeat stress increased BDNF levels in the NAc, resulting in depression-like phenotype in mice. Interestingly, ANA-12, a TrkB antagonist, showed an antidepressant-like effect by blocking TrkB in the NAc 30,37,47,52,53 . Therefore, increased BDNF-TrkB signaling in NAc may play a causative role in the pathophysiology of depression. However, rhynchophylline did not alter BDNF, p-TrkB/TrkB ratio and PSD-95 in the NAc. Taken together, it is likely that rhynchophylline could show antidepressant-like effect by activating BDNF-TrkB signaling in the PFC and hippocampus, but not in NAc, of susceptible mice. However, the precise mechanisms underlying the relationship between EphA4 signaling and BDNF-TrkB signaling are currently unknown.
EphA4 signaling was the first extracellular cue to be identified as phosphorylating ephexin1, and Cdk5 promote its activity in central nervous system synapses 19,20,54 . EphA4 stimulation by an ephrin-A ligand enhances the Cdk5 activity via the phosphorylation of Cdk5 at Tyr15 19,20 . Importantly, ephexin1, a Rho GEF, is phosphorylated by Cdk5 in vivo. Ephexin1 has been reported to transduce signals from activated EphA4 to RhoA, thereby resulting in growth cone collapse during axon guidance 19,20 . The loss of ephexin1 in cultured hippocampal neurons or in vivo perturbs the ability of ephrin-A to induce EphA4-dependent spine retraction. Therefore, the phosphorylation of ephexin1 by Cdk5 is required for EphA4-dependent spine retraction. BDNF-TrkB signaling is also implicated in synapse and spine development. Thus, BDNF triggers serine phosphorylation of TrkB in the juxtamembrane region at S478 by Cdk5 54, 55 . Overexpression of a TrkB construct that lacks the Cdk5 phosphorylation site also inhibits neurite outgrowth by cultured hippocampal neurons in response to BDNF, which suggests that serine phosphorylation might be crucial for TrkB signaling pathway 55 . In addition, the inhibition of Cdk5 or absence of the Cdk5 activator p35 completely abolishes BDNF-induced spine formation and enlargement 55 . These results indicate that Cdk5 may be a downstream protein in the BDNF-TrkB signaling pathway. Therefore, it is likely that Cdk5 plays an important role in both the EphA4-ephexin1 and BDNF-TrkB signaling pathways. Based on the present findings, we propose a framework for the pathological changes in the PFC and hippocampus after experiencing social defeat stress and the possible therapeutic mechanism of EphA4 inhibition (Fig. 7). In the current study, we found that stress activates EphA4-ephexin1 signaling and inhibits BDNF-TrkB signaling in the PFC and hippocampus, which reduced the abundance of the synaptic protein PSD-95. Rhynchophylline could normalize increased EphA4-ephexin1 signaling and decreased BDNF-TrkB signaling in the PFC and hippocampus of susceptible mice, which resulted in its rapid antidepressant-like effect. However, the precise mechanisms that underlie the relationship between EphA4-ephexin1 signaling and BDNF-TrkB signaling are currently unknown.
Patients with depression become chronically ill, with several relapses (early return of symptoms within the expected duration of a current episode, possibly 3-12 months) or later recurrences (new episodes) following initial short-term improvement or remission 56,57 . The recurrence rates are over 85% within a decade of an index depressive episode, with an average of approximately 50% or more within six months of apparent clinical remission 57 . Therefore, the prevention of relapse and recurrence is very important for the management of depression. In this study, we demonstrated the prophylactic effects of rhynchophylline in social defeat stress models of depression, which suggests that rhynchophylline could prevent the onset of the depression-like phenotype caused by social defeat stress. Considering the inclusion of rhynchophylline in traditional Chinese/Japanese medicine 26,27,58 , it is likely that rhynchophylline could be used as a natural prophylactic compound to prevent or minimize the relapses caused by stress during the remission state in depressed patients.
A previous study showed that rhynchophylline acts as non-competitive antagonist of NMDA receptor 59 . Subsequently, it is reported that rhynchophylline exerts its protective action by inhibiting NMDA, muscarinic M 1 , and serotonin 5-HT 2 receptor-mediated neurotoxicity during ischemia 60 . These findings suggest that rhynchophylline is not a selective EphA4 inhibitor. Therefore, further detailed experiments using a selective EphA4 inhibitor will be needed.
In conclusion, these findings suggest that increased EphA4-ephexin1 signaling in the PFC and hippocampus play key roles in the depression phenotype after repeated social defeat stress in mice, and that rhynchophylline has a rapid antidepressant-like effect. Therefore, EphA4-ephexin1 signaling could be a novel therapeutic target for depression.

Materials and Methods
Animals and animal care. Male C57BL/6 mice (n = 182), aged 8 or 10 weeks (body weight 20-25 g; Japan SLC Inc., Hamamatsu, Japan) or 5 weeks (body weight 15-20 g; Japan SLC Inc., Hamamatsu, Japan), and male adult CD1 mice (n = 60) aged 13-15 weeks (body weight >40 g; Japan SLC, Inc., Hamamatsu, Japan) were used in the experiments. Animals were housed in polycarbonate cage (22.5 × 33.8 × 14.0 cm) under controlled temperatures with a 12 h: 12 h light: dark cycle (lights on between 07:00-19:00 h), where food (CE-2; Japan CLEA, Ltd., Tokyo, Japan) and water were available ad libitum. The protocol was approved by Chiba University Institutional Animal Care and Use Committee, and the Animal Experiment Committee of the University of Tokyo. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. Animals were deeply anaesthetized with isoflurane before being killed by cervical dislocation. All efforts were made to minimize animal suffering.
Drug administration. On the day of injection, fresh solutions were prepared by dissolving compounds in sterile endotoxin-free isotonic saline. The dose of rhynchophylline (25 mg/kg; Catalog number: R0105, Tokyo Chemical Industry, Tokyo, Japan) was based on a previous report 25 and the preliminary experiment. Rhynchophylline was dissolved in saline using ultrasound. Control (unsusceptible) mice and susceptible mice were randomly divided into two groups, respectively.
Social defeat procedure. The social defeat procedure was performed as reported previously 47,50,[61][62][63] . Each day, the C57BL/6 mice (8 weeks or 10 weeks old) were exposed to a different CD1 aggressor mouse for 10 min for a total of 10 days. When the social defeat session ended, the resident CD1 mouse and the intruder mouse were housed in one half of the cage separated by a perforated Plexiglas divider to allow visual, olfactory, and auditory contact for the remainder of the 24-h period. At 24 h after the last session, all mice were housed individually. On day 11, a social avoidance test was performed to identify subgroups of mice that were susceptible and not susceptible to social defeat stress. A social interaction test was performed on the day after the last social defeat session. In this test, an open field arena (42 × 42 cm) was divided into an interaction zone and two opposing corner zones. A plastic mesh target box (10 × 4.5 cm) was placed in the interaction zone. A test mouse was allowed to roam around the open field arena for 2.5 min with no social target (CD1 mouse) in the mesh box (which is denoted as "no target" in the figures showing the results of the social-interaction experiments). Next, a novel CD1 mouse was placed in a metal/plastic mesh target box in the interaction zone (which is denoted as "target" in the figures showing the results of the social-interaction experiments) and the test mouse was placed back in the open arena for another 2.5 min. Using a stopwatch, the amount of time spent in the interaction zone (defined as the 8 cm-wide area surrounding the wire mesh cage) was measured with or without the social target during 2.5 min 63 . The interaction ratio was calculated as 100 × (time spent in an interaction zone with an aggressor)/(time spent in an interaction zone without an aggressor). An interaction ratio of 100 was set as the cutoff, where mice with scores <100 were defined as "susceptible" to social defeat stress and those with scores ≥100 were defined as "unsusceptible. " Approximately 70% of mice were susceptible in this study. The resilience mice were removed from the experiments.
Stress paradigm: learned helplessness (LH) model. The LH paradigm and behavioral tests were performed using the Gemini Avoidance System (San Diego Instruments, San Diego, CA, USA) 37,48,49 . The apparatus was divided into two compartments by a retractable door. On days 1 and 2, male Sprague-Dawley rats (200-300 g; 7 weeks old, Charles River Japan Co., Tokyo, Japan) were subjected to 30 inescapable electric foot shocks (0.65 mA, 30-s duration, at random intervals averaging 18-42 s). On day 3, a two-way conditioned avoidance test was performed as a post-shock test to determine whether the rats exhibited the predicted escape deficits. This screening process comprised 30 trials where the electric foot shocks (0.65 mA, 6-s duration, at random intervals averaging 30 s) were preceded by a 3-s conditioned stimulus tone, which remained active until the shock was terminated. The numbers of escape failures and the latency to escape were recorded in each trial. Rats with more than 25 escape failures in the 30 trials were regarded as having satisfied the criterion for the depression-like phenotype (susceptible). Approximately 65% of the rats satisfied this criterion. Rats with fewer than 25 failures that did not satisfy the criterion were defined as non-LH rats (resilience). On day 8, the animals were decapitated. The left brain hemispheres were used for conducting the western blot, as reported previously 37, 48, 49 . Behavioral tests. The behavioral tests were performed by two observers who were blinded to the group assignment of animals. These tests were performed as follows. Locomotion test: The mice were placed in experimental cages (length × width × height: 560 × 560 × 330 mm), where the locomotor activity of mice was counted using a SCANET MV-40 system (Melquest Co. Ltd, Toyama, Japan) and the cumulative exercise was recorded for 60 min. Cages were cleaned between test sessions. Tail suspension test (TST): The mice were taken from their home cage and a small piece of adhesive tape was placed approximately 2 cm from the tip of their tail. A single hole was punched in the tape and mice were hung individually on a hook. The immobility time of each mouse was recorded for 10 min. Mice were considered immobile only when they hung passively and completely motionless. Forced swimming test (FST): The mice were placed individually in a cylinder (diameter: 23 cm; height: 31 cm) containing 15 cm of water, which was maintained at 23 ± 1 °C. Animals were tested in an automated forced-swimming apparatus using a SCANET MV-40 system (MELQUEST Co. Ltd, Toyama, Japan). The immobility time was calculated from the activity time as (total) -(active) time, using the analysis software provided with the apparatus. The cumulative immobility time was scored for 6 min during the test. Sucrose preference test (SPT): Mice were habituated to a 1% sucrose solution for 24 h before the test day. Mice were deprived of water and food for 4 h from 13:00 pm to 17:00 pm, before a preference test for 1 h with water and 1% sucrose, which were