The Effect And Mechanism Of Increased Mitochondrial Fission In Depressive-like Behavior Induced By Chronic Social Defeat Stress

Part I The effect of chronic social defeat stress on mitochondrial function and morphology in the medial prefrontal cortex of mice Objective: Mitochondria are characterized by their dynamics,and their morphological and functional changes are affected by dynamic changes in mitochondrial fission and fusion.Mitochondrial morphological abnormalities were observed in animal models as well as in patients with many neurodegenerative diseases,and followed by mitochondrial dysfunction.Autopsy results of patients with major depression disorder(MDD)indicate that mitochondrial dysfunction mainly exists in the prefrontal cortex.Thus abnormal mitochondrial dynamics may be involved in depression.In this part the effect of chronic social defeat stress(CSDS)model will be used to evaluate its effect on the function and morphology of mitochondria in medial prefrontal cortex(mPFC)of mice.Methods: First we construct a chronic social defeat stress animal model.After the mice were exposed to CSDS,social interaction test(SIT)and sucrose preference test(SPT)were utilized to evaluate the depressive-like behavior of mice;Bioluminescence assay was used to detect the level of intracellular ATP;JC-1fluorescent probe was used to measure mitochondrial membrane potential;DHE fluorescent probe was used to detect reactive oxygen species(ROS);Western blotting was used to detect the expression of mitochondrial respiratory chain complex I;After mitochondria in the medial prefrontal cortex(mPFC)were labeled by adeno-associated virus(AAV-Mito-7-mcherry),confocal microscopy and transmission electron microscopy were used to observe mitochondrial morphological changes.Results:(1)CSDS induced the depression-like behavior in mice.Compared with thenormal control group(Control: 90.81 ± 1.41%),sucrose preference ratio of susceptible mice decreased to 56.70 ± 3.24%(P < 0.001),and there was no significant change in resilient mice(Resilient: 91.19 ± 3.65 %).Social interaction ratio decreased from 1.60 ± 0.12 to 0.62 ± 0.07(P < 0.001),with no significant change in resilient mice(Resilient: 1.40 ± 0.09).Time spent in the target area also decreased significantly(Control: 71.30 ± 3.52 sec,Susceptible: 36.36 ± 4.41 sec,P < 0.001 vs Control,Resilient: 75.02 ± 6.35 sec).(2)CSDS induced mitochondrial dysfunction.Compared with the control group(1.00 ± 0.11 nmol/μg),the ATP level of susceptible mice decreased to 0.48 ± 0.04 nmol/μg(P < 0.001),and membrane potential decreased from 1.00 ± 0.09 to 0.70 ± 0.07(P < 0.05),the expression of respiratory chain complex I was reduced(Control: 1.00 ± 0.04,CSDS: 0.46 ± 0.08,P < 0.01)and production of ROS was increased(Control: 1.00 ± 0.11,CSDS: 2.91 ± 0.42,P < 0.01).(3)Compared with the long tubular mitochondria and extensive networking in control group,the mitochondria of susceptible mice was fragmentated and short(Control:1.04 ± 0.08,CSDS:0.54 ± 0.05,P < 0.01).Conclusion: Depressive-like behavior was sucessfully induced after exposure of mice to CSDS.CSDS susceptible mice had increased mitochondrial fission and impaired mitochondrial function in mPFC,indicating that increased mitochondrial fission is involved in depressive-like behavior in depressive mice.Part Ⅱ The effect and mechanism of increased mitochondrial fission on depressive-like behavior of mice Objective: Dynamin-related protein(Drp1),which is encoded by Dnm1 l,is considered to be one of the most important molecules involved in mitochondrial fission.Phosphorylation of Drp1 at Ser 616 enhances the activity of Drp1 and promotes the recruitment of Drp1 lead to mitochondrial fission.Recent studies have shown that mitochondrial fragmentation and elevated level of Drp1 phosphorylation are observed in neuropsychiatric diseases such as Alzheimer’s disease(AD)and drug addiction,suggesting that Drp1 mediates the higher level of mitochondrial fission and mitochondrial dysfunction may be responsible for the pathology of these diseases.In part one,increased mitochondrial fission and mitochondrial dysfunction had been detected in CSDS susceptible mice,with the underlying molecular mechanism to be elucidated.In part two,we will explore whether Drp1 also plays a pivotal role in increased mitochondrial fission in mediating CSDS-induced depressive-like behavior in mice.Methods: After exposure to chronic social defeat stress and subthreshold social defeat stress,SIT and SPT were used to evaluate the depressive-like behavior in mice;Western blotting was used to detect the expression of Drp1 protein and its phosphorylation in different brain regions of susceptible mice.The recruitment of Drp1 to mitochondria was observed by using virus labeling mitochondria and immunofluorescence to label Drp1;After pharmacological inhibition of Drp1,silencing and overexpression of Drp1 by lentivirus in mPFC,depressive-like behavior was detected.Immunofluorescence was used to observe the recruitment of Drp1 to mitochondria and the production of ROS in mPFC after Mdivi-1;Western blotting was used to detect the effect of the expression of membrane of GluA1 by silencing Drp1 in mPFC.Results:(1)Western blotting experiments showed that compared with the control group(Control: 1.00 ± 0.04),the expression of phosphorylated Drp1 in mPFC of susceptible mice increased(Susceptible: 1.33 ± 0.08,P < 0.01),with no change in resilient mice.There was no significant change in the expression of Drp1 total protein.(2)There was no significant change in phosphorylated Drp1 and total protein level in nucleus accumbens(NAc)and hippocampus of susceptible mice.(3)Compared with the control group(1.00 ± 0.04),the colocalization of Drp1 with mitochondria in mPFC increased in susceptible mice(CSDS: 1.48 ± 0.15,P < 0.05).It means the number of Drp1 recruited from the cytoplasm to the mitochondrial outer membrane was increased.(4)Mdivi-1,the Drp1 selective inhibitor,improved the depressive-like behavior in mice after administration to mPFC.Compared with the CSDS + Vehicle group(0.38 ± 0.10),the social contact ratio of the CSDS + Mdivi-1 group increased to1.38 ± 0.16(P < 0.01),and time spent in the target area increased from 21.33 ± 7.42 sec to 77.85 ± 12.19 sec(P < 0.001).The sucrose preference ratio increased from35.00 ± 8.17 % to 87.23 ± 4.26 %(P < 0.001).The recruitment of Drp1 to mitochondria was decreased.(5)Western blotting experiments showed that Drp1 silencing virus(LV-Dnm1l-RNAi)effectively down-regulated the expression of Drp1(LV-GFP: 1.00 ± 0.10,LV-Dnm1l-RNAi: 0.42 ± 0.02,P < 0.01).(6)Silencing virus inhibiting the expression of Drp1 in mPFC improved depressive-like behavior in mice.Compared with CSDS+LV-GFP group(0.55 ± 0.10),the social interaction ratio of CSDS + LV-Dnm1l-RNAi group increased to 1.35 ± 0.17.(P < 0.001),time spent in the target area increased from 31.69 ± 5.99 sec to 54.26 ± 5.50 sec(P < 0.05).(7)LV-Dnm1 overexpressing Drp1 in mPFC increased susceptibility to stress in mice.Both social interaction ratio(Stress + LV-GFP: 1.36 ± 0.21,Stress + LV-Dnm1l: 0.83± 0.13,P < 0.05),and time spent in the target area(Stress + LV-GFP: 60.33 ± 4.36 Sec,Stress + LV-Dnm1l: 39.54 ± 7.85 sec,P < 0.05)decreased significantly.(8)Drp1 is expressed more on neurons.Mdivi-1 reduced production of ROS in mPFC of susceptible mice(CSDS + Vehicle: 1.00 ± 0.04,CSDS + Mdivi-1: 0.52 ± 0.11,P <0.05).(9)Compared with the control group(1.00 ± 0.06),the expression of membrane of GluA1 in mPFC of susceptible mice was decreased(0.49 ± 0.04,P < 0.05),which is alleviated by silencing of Drp1(CSDS + LV-Dnm1l-RNAi: 1.03 ± 0.25,P < 0.05 vs CSDS + LV-GFP),with no change in GluA1 total protein.Conclusion: CSDS activated Drp1 in mPFC,promoting the recruitment of this protein from the cytoplasm to the mitochondrial outer membrane,and increasing miochondrial fission.Overexpression of Drp1 in mPFC increased the susceptibility to stress in mice.Pharmacological inhibition of Drp1 in mPFC improved depressive-like behavior while reducing ROS production.Knocking down Drp1 in mPFC blocked the effect of CSDS induced reduction of membrane GluA1.This suggests that CSDS induced activation of Drp1 in mPFC mediates increased mitochondrial fission and impaired mitochondrial function,possibly via further deliberating the excitatory synaptic transmission in depressive mice.