Regulation And Mechanism Of Twist1 In Chronic Stress-induced Depressive-like Behaviors Of Mice

PartⅠRegulation of Twist1 on depressive-like behaviors induced by chronic stressObjective: Depression is known as a severe mental disorder,in severe case,even leads to suicides,which brings heavy burden to families and society.A lot of clinical researches have proven that cancer patients have a greater morbidity rate of depression disease.This comorbidity is defined as cancer induced depression(CID).However,its mechanism is poorly understood.Twist1 is a basic helix-loop-helix(b HLH)transcription factor that promotes numerous cancer cells metastasis through epithelial mesenchymal transition(EMT),such as breast cancer,pancreatic cancer and prostate cancer.At present,the function of Twist1 in the brain and its regulation on CID are not clear.Here,our study investigated Twist1 expression partern in the medial prefrontal cortex(m PFC)and its role in chronic stress-induced depressive-like behaviors in mice.Methods: Application of chronic social defeat stress(CSDS),chronic restraint stress(CRS)and subcutaneous inoculation of 4T1 model.Depressive-like behaviors were evaluated by social interaction test,sucrose preference test(SPT),forced swimming test(FST)and tail suspension test(TST).Western blotting was used to detect the protein levels.Quantitative polymerase chain reaction(q PCR)was used to detect the m RNA expression.Lentivirus and adeno-associated virus infection by stereotaxic injection were used to knock down and overexpress Twist1.Results:(1)The susceptible mice in CSDS model introduced typical depressive-like behaviors,such as social avoidance and anhedonia,reflected by a significant decrease in the social interaction time(Control: 55.41 ± 3.93 sec;Susceptible:20.14 ± 3.13 sec;Resilient: 54.5 ± 4.02 sec,p < 0.01 vs Control)and sucrose preference(Control: 84.44 ± 1.71%;Susceptible: 62.08 ± 2.37%;Resilient: 84.63 ± 1.24%,p < 0.01 vs Control).(2)Twist1 had highest expression in the hippocumpus and lowest expression in the striatum withcolocalization with neuronal marker Neu N.(3)Twist1 protein levels in the m PFC of susceptible mice increased by 7.93 ± 1.02 and m RNA expression increased by 0.47 ± 0.16.(4)Twist1 protein levels in the hippocumpus of susceptible mice increased by 0.60 ± 0.17 compared with control mice.(5)Twist1 protein in the peripheral blood mononuclear cell(PBMC)of susceptible mice increased by 0.40 ± 0.15,but not in resilient mice.(6)CRS displayed typical depressive-like behaviors,such as anhedonia and despair behavior,reflected by a significant decrease in sucrose preference(Control: 83.53 ± 3.14%;CRS: 51.68 ± 8.20%;p < 0.01 vs Control)and increased immobility in the TST(Control: 79.00 ± 10.94 sec;CRS: 131.40 ± 7.40 sec,p < 0.01 vs Control).Twist1 protein levels in the m PFC of CRS model also increased by 0.48 ± 0.14 compared with control mice(1.00 ± 0.06).(7)Repeat injection of fluoxetine improved depressive-like behaviors including increased social interaction time(Vehicle: 20.73 ± 2.81sec;Fluoxetine: 86.66 ± 13.50 sec,p < 0.01 vs Vehicle)and decreased immobility in the TST(Vehicle: 197.00 ± 10.55 sec;Fluoxetine;58.83 ± 19.73 sec;p < 0.01 vs Vehicle)and decreased Twist1 protein levels in the m PFC by 1.76 ± 0.53.(8)Injection of LV-Twist1 in m PFC of susceptible mice expressed sh RNA targeting Twist1 rescued the depressive-like behaviors,including increased social interaction time(CSDS+LV-GFP: 20.92 ± 3.14 sec;CSDS+LV-Twist1: 47.97 ± 4.92 sec,p < 0.01 vs CSDS+LV-GFP)and sucrose preference(CSDS+LV-GFP: 70.47 ± 1.62%;CSDS+LV-Twist1: 87.51 ± 2.46%,p < 0.05 vs CSDS+LV-GFP)and decreased immobility in the FST(CSDS+LV-GFP: 145.00 ± 11.70 sec;CSDS+LV-Twist1: 84.25 ± 7.86 sec,p < 0.01 vs CSDS+LV-GFP)and TST(CSDS+LV-GFP: 129.30 ± 8.96 sec;CSDS+LV-Twist1: 70.77 ± 12.05 sec,p < 0.01 vs CSDS+LV-GFP).(9)Injection of AD-Twist1 in m PFC of normal C57BL/6J mice to overexpress Twist1 protein reduced social interaction time(AD-GFP: 43.52 ± 3.10 sec;AD-Twist1: 19.24 ± 2.09 sec,p < 0.01 vs AD-GFP)and sucrose preference(AD-GFP: 73.67 ± 4.40%;AD-Twist1: 51.89 ± 5.51%,p < 0.01 vs AD-GFP),indicating its pro-susceptibility effect.(10)Female mice subcutaneously inoculated with 4T1 mammary carcinoma cells exhibited depressive-like behaviors,including decreased sucrose preference(Control: 79.75 ± 3.28%: Tumor: 47.21 ± 5.06%,p < 0.01 vs Control)and increased immobile time in TST(Control: 110.4 ± 5.89 sec;Tumor:142.3 ± 13.24 sec,p < 0.01 vs Control)and FST(Control: 72.14 ± 9.05 sec;Tumor: 115 ± 11.87 sec,p < 0.05 vs Control).The Twist1 protein levels in m PFC of 4T1 group also increased by 0.45 ± 0.20.Conclusion: Chronic stress and subcutaneously inoculated with 4T1 induces an increase in Twist1 expression in m PFC of mice.Overexpression of Twist1 in m PFC increases the susceptibility of mice to stress.Conversely,knockdown of Twist1 in m PFC produces antidepressant action.Part Ⅱ Regulation of Twist1 on miR-214-PPAR-δ pathway in m PFC of the susceptible mice in CSDS modelObjective: Twist1 regulates neural cell differentiation by promoting miR-214 expression in vitro.Peroxisome proliferator-activated receptor-δ(PPAR-δ),as a target molecular of miR-214,improves depressive-like behaviors via neurogenesis.Our study investigated miR-214-PPAR-δ pathway in m PFC in chronic stress model and asked whether Twist1 modulated depressive-like behaviors through miR-214-PPAR-δ pathway.Methods: CSDS model,social interaction test,SPT,FST and TST were used to detect depressive-like behaviors.Western blotting and q PCR were used to detect protein and RNA level,respectively.Lentivirus and recombinant adenovirus infection were used to regulate gene expression.Lentivirus-mediated gene transfer with crispr/cas9 system was used to knock down PPAR-δ expression.N2 a and HT22 cell culture in vitro.Results:(1)Dnm3os expressed in the m PFC and located to nuclear.Transfection of N2a cells with si RNA targeting Dnm3 os decreased Dnm3 os and miR-214 expression by 0.35 ± 0.08 and 0.58 ± 0.08,respectively.(2)Social defeat increasesd the binding of Twist1 to the promoter of Dnm3 os by 1.59 ± 0.38,leading to the increase in the expression of miR-214 and Dnm3 os in m PFC of CSDS treated mice by 1.36 ± 0.18 and 1.00 ± 0.18,respectively,but decreased the levels of PPAR-δ protein by 0.46 ± 0.77.(3)Injection of LV-Twist1 into m PFC of susceptible mice decreased miR-214 expression(CSDS+LV-GFP: 2.31 ± 0.28;CSDS+LV-Twist1: 1.19 ± 0.28,p < 0.01 vs CSDS+LV-GFP)and Dnm3os(CSDS+LV-GFP: 1.62 ± 0.13;CSDS+LV-Twist1: 0.84 ± 0.11,p < 0.01 vs CSDS+LV-GFP)expression and increased the level of PPAR-δ protein(CSDS+LV-GFP: 0.54 ± 0.05;CSDS+LV-Twist1: 0.77 ± 0.03,p < 0.01 vs CSDS+LV-GFP),but had no impact on β-catenin.(4)Overexpression of Twist1 in HT22 cells increased the expression of miR-214(AD-GFP: 1.01 ± 0.04;AD-Twist1: 2.19 ± 0.17,p < 0.01 vs AD-GFP)and Dnm3os(AD-GFP: 1.05 ± 0.13;AD-Twist1: 1.68 ± 0.13,p < 0.01 vs AD-GFP)and decreased the levels of PPAR-δ(AD-GFP: 1.00 ± 0.09;AD-Twist1: 0.51 ± 0.05,p < 0.01 vs AD-GFP)and β-catenin protein(AD-GFP: 1.00 ± 0.11;AD-Twist1: 0.57 ± 0.03,p < 0.01 vs AD-GFP).(5)Chronic intracerebroventricular injection of selective PPAR-δ antagonist GSK3787 for 7 days after CSDS decreased social interaction time(Vehicle: 48.44 ± 4.75 sec;GSK3787: 21.04 ± 4.52 sec,p < 0.01 vs Vehicle)and sucrose preference(Vehicle: 86.28 ± 1.78%;GSK3787: 64.81 ± 5.77%,p < 0.01 vs Vehicle),increased immobile time in FST(Vehicle: 78.5 ± 9.48 sec;GSK3787: 109.42 ± 8.69 sec,p < 0.05 vs Vehicle)and TST(Vehicle: 91.9 ± 10.04 sec;GSK3787: 149.5 ± 16.61 sec,p < 0.01 vs Vehicle),suggesting the inhibition of antidepressant action of LV-Twist1.(6)Overexpression of miR-214 through injection of LV-miR-214 in m PFC of normal mice decreased sucrose preference(LV-GFP: 83.39 ± 1.85%;LV-miR214: 62.51 ± 5.10%,p < 0.01 vs LV-GFP)and increased immobile time in FST(LV-GFP: 110.5 ± 6.46 sec;LV-miR214: 146.5 ± 8.16 sec,p < 0.01 vs LV-GFP)and TST(LV-GFP: 119.0 ± 11.18 sec;LV-miR214: 158.6 ± 5.49 sec,p < 0.01 vs LV-GFP),indicating depressive-like behaviors.(7)Downregulation of PPAR-δ through injection of LV-sg RNA in m PFC also decreased sucrose preference(LV-GFP: 83.39 ± 1.85%;LV-sg RNA: 67.54 ± 8.29%,p < 0.05 vs LV-GFP)and increased immobile time in FST(LV-GFP: 110.5 ± 6.46 sec;LV-miR214: 146.5 ± 8.16 sec,p < 0.01 vs LV-GFP)and TST(LV-GFP: 119.0 ± 11.18 sec;LV-miR214: 158.6 ± 5.49 sec,p < 0.01 vs LV-GFP).(8)Chronic intracerebroventricular injection of selective PPAR-δ agonist GW0742 for 7 days rescued the depressive-like behaviors,including increased social interaction time(Vehicle: 24.48 ± 4.34 sec;GW0742: 72.89 ± 5.42 sec,p < 0.01 vs Vehicle)and sucrose preference(Vehicle: 57.00 ± 5.49%;GW0742: 78.89 ± 2.64%,p < 0.01 vs Vehicle).(9)Injection of LV-PPAR-δ in m PFC of susceptible mice to overexpress PPAR-δ also had antidepressant effect,including increased social interaction time(CSDS+LV-GFP: 26.09 ± 5.14 sec;CSDS+LV-PPAR-δ: 75.53 ± 6.74 sec,p < 0.01 vs CSDS+LV-GFP)and sucrose preference(CSDS+LV-GFP: 62.66 ± 7.24%;CSDS+LV-PPAR-δ: 79.41 ± 2.16%,p < 0.05 vs CSDS+LV-GFP).Conclusion: The miR-214-PPAR-δ pathway is upregulated in the m PFC of CSDS model.Upregulation of miR-214 and downregulation of PPAR-δ in the m PFC induces depressive-like behaviors of mice.Twist1 modulates depression-related behavioral abnormalities through enhancing miR-214-PPAR-δ pathway.Part Ⅲ Regulation of Twist1 on dendrite morphogenesis of neurons in m PFC of the susceptible mice in CSDS modelObjective: Brain is a high energy consumption organ.Neurons need abundant ATP to maintain their normal morphology and physiological function.Deficiency of ATP leads to cell atrophy even apoptosis.Previous researches have suggested neuron atrophy in the PFC of depression patients with abnormal mitochondrial function,and mitochondrial reinforcing agents have antidepressant action.In addition,PPAR-δ promotes mitochondrial biogenesis and function,increasing ATP production.The study investigated whether upregulation of Twist1 induced depressive-like behaviors through targeting PPAR-δ signaling by impairing mitochondrial function and dendritice morphogenesis.Methods: Social interaction test,SPT,FST and TST were used to detect depressive-like behaviors.Lentivirus and recombinant adenovirus infection were used to regulate gene expression.Lentivirus-mediated gene transfer with CRISPR-Cas9 system was used to knock down PPAR-δ expression.Confocal microscopy was used to observe fluorescent images.Mitochondrial membrane potential(MMP)was measured by application of JC-1.Bioluminescence was used to detect ATP levels.q PCR was used to test mitochondrial DNA(mt DNA)copies.Results:(1)mt DNA copies,MMP and ATP expression in the m PFC of susceptible mice were decreased(mt DNA: Control: 1.04 ± 1.11;Susceptible: 0.69 ± 0.04;MMP: Control: 100.0 ± 4.50%;Susceptible: 67.61 ± 5.77%;ATP: Control: 99.94 ± 5.60%;Susceptible: 68.12 ± 7.04%,p < 0.05 or 0.01 vs Control),indicating the impairment of mitochondrial function.(2)Tranfecting primary cortical neurons with AAV-Twist1 impaired MMP(AAV-Flag: 1.00 ± 0.11;AAV-Twist1: 0.48 ± 0.07,p < 0.01 vs AAV-Flag)and decreased ATP levels(AAV-Flag: 108.9 ± 10.19%;AAV-Twist1: 67.64 ± 7.87%,p < 0.01 vs AAV-Flag)but returned to normal levels after treatment with selective PPAR-δ agonist GW0742(Untreated: 0.48 ± 0.07;GW0742: 1.00 ± 0.13,p < 0.01 vs Untreated).(3)CSDS reduced total dendritice length(Control+LV-GFP: 795.5 ± 127.7 μm;CSDS+LV-GFP: 324.8 ± 24.48 μm,p < 0.01 vs Control+LV-GFP)and branches number(Control+LV-GFP: 4.78 ± 0.68;CSDS+LV-GFP: 1.92 ± 0.21,p < 0.01 vs Control+LV-GFP)and dendritic branching complexity of m PFC neurons and recovered to normal levels after LV-Twist1 and LV-PPAR-δ treatment.(4)Transfecting primary cortical neurons with AAV-Twist1 to overexpress Twist1 protein reduced total dendrite length(AAV-GFP: 718.5 ± 44.58 μm;AAV-Twist1: 461.1 ± 45.60 μm,p < 0.01 vs AAV-GFP)and branching number(AAV-GFP: 7.50 ± 0.77;AAV-Twist1: 3.28 ± 0.43,p < 0.01 vs AAV-GFP)and dendritic branching complexity.(5)transfecting cortical primary neurons with LV-sg RNA to knock down PPAR-δ expression also reduced total dendrite length(LV-GFP: 721.4 ± 59.07 μm;LV-sg RNA: 431.0 ± 51.22 μm,p < 0.01 vs LV-GFP)and branching number(LV-GFP: 7.88 ± 0.67;LV-sg RNA: 3.06 ± 0.46,p < 0.01 vs LV-GFP)as well as dendritic branching complexity.Conclusion: The mitochondrial function is impaired in the m PFC of susceptible mice.Upregulation of Twist1 and downregulation of PPAR-δ lead to dendrite morphogenesis abnormalities.These results suggest that Twist1 induces depressive-like behaviors through downregulation of PPAR-δ,impairment of mitochondrial function,attenuation of ATP levels and dendrite morphogenesis.