The Effects And Mechanisms Of Wnt2 And Wnt3 In Chronic Restraint Stress-induced Depression-like Behaviors

Background:Wnts are secreted glycoproteins that act as ligands to activate different signaling pathways including canonical Wnt/β-catenin pathway, non-canonical planar cell polarity (PCP) pathway and Wnt/Ca2+ pathway. Previous studies have reported that Wnt signaling pathways play essential roles in embryogenesis and nervous systerm development, including synapse formation, dendritic morphogenesis and hippocampal formation. Recently, Wnt/β-catenin signaling is proved to play a role in adult neurogenesis, which is required for the treatment of depression, suggesting that Wnt signaling pathway may participate in depression behaviors via neurogenesis. Indeed, Wnt signaling components, including GSK-3β, have been reported to participate in depression behaviors and antidepressant effect. However, there are fewer reports about Wnt and depression.Chronic stress is a risk factor for developing depression disorder. Growing evidence has showed that Wnt signaling components are altered under stress condition. Chronic unpredictable stress decreased the expression of Frizzled 6 (Fzd6), a Wnt receptor, in the hippocampus. The expression of disheveled-2 (DVL2), a key molecule of Wnt signaling cascade, was downregulated in the nucleus accumbens (NAc) of mice subjected to chronic social defeat stress.Recently, it has reported that acute and chronic stress could increase the expression of Dickkopf-1 (Dkk-1), an inhibitor of Wnt/β-catenin signaling, in the hippocampus. Previou studies focused on the components of Wnt/β-catenin signaling. However, it remains puzzled whether Wnts, acting as ligands to activate Wnt/p-catenin signaling, was involved in stress-related depression behavior.In the present study, using the chronic restraint stress (CRS) paradigm and molecular and cellular biology techniques, we aimed to investigate whether selective Wnts participated in stress-induced depression-like behaviors and its underlying mechnism.Objective:1. To investigate whether Wnts participated in CRS-induced depression-like behaviors.2. To investigate the mechanism of Wnts in CRS-induced depression-like behaviors.Methods:1. Chronic restraint stressMice were assigned randomly into chronic restraint stress (CRS) group and no CRS (No-CRS) group. The CRS group was restrained during the morning (8:00 A.M.-10:00 A.M.) for 2 h daily for 14 days in well ventilated polypropylene restrainers.2. Stereotaxic surgery and Lentivirus MicroinjectionMice were intraperitoneally anesthetized and placed in a stereotaxic apparatus. Mice received ventral hippocampus (VH) micro-infusions,1μl lentivirus/side and the injection coordinate relative to bregma was as follows: anteroposterior (AP),-2.8 mm; lateral (L),±2.5 mm; dorsoventral (V),-2.3 mm.3. Behavioral tests3.1 Open field testSpontaneous exploratory activity was assessed in the open field test. Mice were placed in the center of the field and the total distance traveled in 10 min was recorded as a measure of locomotor activity.3.2 Sucrose preference testOn the last day of CRS, mice were deprived of water (beginning at 08:00 p.m.). Mice were accustomed to sucrose for 3 d with bottles containing sucrose solution (1%). Sucrose preference was assessed for 2 d. Sucrose preference was presented as:sucrose preference (%)=sucrose intake/total fluid intake x100.3.3 Forced swim testThe swim test was performed in a glass cylinder (25 cm height,10 cm diameter, containing18 cm of water at 22℃). Mice were forced to swim for 6 min.3.4 Tail suspension testThe tail of mice was wrapped with tape around 2 mm of tail protruding. Mice were suspended by tails for 6 min. Mice that climbed their tails were removed from the experimental analysis.4 BrdU administration and ImmunohistochemistryFor evaluation of cell proliferation, mice were intraperitoneally injected with 50 mg/kg 5-Bromo-2-deoxyUridine (BrdU, Sigma) in 0.9% NaCI and perfused 2 h later. For survival analysis, mice were given four injections of BrdU every 24 h and killed 4 weeks after the last injection. Mice were anesthetized with 5% chloral hydrateand and perfused with 4% paraformaldehyde (PFA) and postfixed overnight. Brain sections were acquired at 30 μm for immunohistochemistry analysis.For BrdU staining, brain sections were incubated with primary antibodies: sheep anti-BrdU and mouse anti-NeuN. Images were acquired using a confocal fluorescence microscopy with a Carl Zeiss LSM-780 microscope fitted with standard filter sets and a standard (1 Airy disk) pinhole (Microstructural platform of Shandong University). BrdU and+ BrdU+ NeuN+ cells were counted using MetaMorph software package (Molecular Devices) with an unbiased stereological protocol.5 Quantitative Real-time PCRTotal RNA was extracted and purified total RNA (500 ng) was then reversely transcribed to cDNA. Quantitative Real-time PCR was performed in a Cycler using SYBR Green. Each sample was analysed in duplicate and the relative levels of mRNA were normalized for each well of the β-actin mRNA levels using the 2-△△CT method.6 Protein extraction and Western blotSamples of mice brain were isolated and homogenized. The concentration of the protein was detected using the BCA protein assay. Samples were loaded to detect immunoreactivity by western blot. Densitometry analysis on the bands was caculated by Quantity one.Results:1. Selective reduction of Wnt2 and Wnt3 in the VH after chronic restraint stressTo investigate the role of Wnts in chronic restriant stress, we initially examined various Wnts mRNA and protein levels after CRS using Real-time PCR and western blot. Real-time PCR results showed that among the 6 different Wnts (Wnt2, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt7a), only Wnt2 and Wnt3 mRNA levels were significantly reduced in the VH. There were no significant changes for all of the 6 different Wnts in the DH after CRS. Western blots analysis showed that the protein levels of Wnt2 and Wnt3 have been significantly decreased after CRS in the VH but not in the DH. We did not detect significant decrease in Wnt3a protein level both in the VH and DH. The selective reduction of Wnt2 and Wnt3 expression in the VH following CRS suggested that Wnt2 and Wnt3 may play roles in stress-induced depression-like behavior.2. Knockdown of Wnt2 or Wnt3 could mimic CRS-induced depression-like behaviorsAs the above results showed that CRS could decrease the expression of Wnt2 and Wnt3, we next investigated whether endogenous Wnt2 and Wnt3 are functionally related to depression-like behaviors using lentivirus-mediated RNA interference. Mice in Lenti-siWnt2 or Lenti-siWnt3 group showed decreased sucrose intake consumption and increased immobility time in FST and TST compared with mice in Lenti-siSCR.3. Overexpression of Wnt2 and Wnt3 could buffer CRS-induced depression-like behaviorsWe next tested whether overexpressing Wnt2 or Wnt3 in the VH is sufficient for alleviating CRS-induced depression-like behaviors. We found that sucrose consumption was significantly increased and the immobility time was siginificantly decreased in FST and TST in CRS-Lenti-Wnt2 or CRS-Lenti-Wnt3 group compared with CRS-Lenti-GFP group. In addition, we found that the antidepressant effect of Lenti-Wnt2+Wnt3 group was much more pronounced than Lenti-Wnt2 or Lenti-Wnt3 group.4. Wnt/β-catenin signaling and neurogenesis participated in CRS-induced depression-like behaviors.We found that CRS could lead to Wnt/(3-catenin signaling and neurogenesisb deficits. Knockdown of Wnt2 or Wnt3 could mimic CRS-induced decrease of Wnt/p-catenin signaling and neurogenesis deficits. In addition, overexpression of Wnt2 or Wnt3 could rescue CRS-induced decrease of Wnt/β-catenin signaling and neurogenesis deficits. These results suggested that Wnt/β-catenin signaling and neurogenesis participated in CRS-induced depression-like behaviors.5. Wnt2 and Wnt3 could mediate the effect of antidepressantsIn the present study, we try to investigate whether Wnt2 or Wnt3 is necessary for the effects of antidepressant treatment under stress condition. We found that two weeks intraperitoneal injection of fluoxetine can significantly reverse the CRS-induced decrease in Wnt2 and Wnt3 levels compared with CRS+SAL group. We further examined the role of Wnt2 or Wnt3 in antidepressant effect using lentivirus-mediated knockdown of Wnt2 or Wnt3. We found that knockdown of Wnt2 or Wnt3 abolished the antidepressant effect of fluoxetine.Conclusion:1. Wnt2 and Wnt3 in the VH participated in depression behaviors.2. Wnt2 and Wnt3 mediated Wnt/β-catenin signaling and neurogenesis participated in CRS-induced depression-like behaviors.3. We determined that Wnt2 and Wnt3 in the VH could mediate the antidepressant effect of fluoxetine.Significance:In our study, we provided direct evidence of Wnt participating in CRS-induced depression-like behaviors. Importantly, Wnt2 or Wnt3 could be potential new targets for the intervention of depression.