Effect And Mechanism Of Caspase-1 In Chronic Stress-induced Depression-like Behaviors Of Mice

Part ? The role of caspase-1 in chronic stress-induced depression-like behaviors in miceObjective:Caspase-1,which belongs to a member of the cysteine-aspartic acid protease family,exists intracellularly as an inactive proenzyme until it is proteolytically processed by inflammasomes.It has been implicated to play an important role in the neurological diseases through inflammatory response.Studies have also shown that exposure to lipopolysaccharide(LPS)can produce depression-like behaviors via caspase-1 activation.However,it still remains unknown whether caspase-1 plays an important role in chronic stress-induced depression-like behaviors.Several recent studies report cognitive impairment in patients with major depressive disorder(MDD),and caspase-1 regulates learning and memory via IL-1? signaling pathway.Thus,we use various depression models of mice to investigate the potential role of caspase-1 in depression.Methods:Chronic social defeat stress(CSDS)paradigm was utilized to establish mouse models of depression;Enzyme-linked immunosorbent assay(ELISA)was used to determine the serum IL-1? and corticosterone levels in mice subjected to chronic social defeat stress(CSDS);Western blotting and quantitative real-time PCR(qRT-PCR)were performed to determine the levels of caspase-1 in peripheral blood mononuclear cells(PBMC)and brain of mice.The level of caspase-1 protein was analyzed in mice hippocampus induced by chronic unpredictable mild stress(CUMS);Western blotting and qRT-PCR were used to analyze the level of caspase-1 mRNA and pro-Caspase-1 protein in the hippocampus of WT and Caspase-1-/-mice,respectively;Nissl's staining and immunofluorescence were employed to examine gross brain function and anatomy in WT and Caspase-1-/-mice;Open field(OF)and elevated plus maze(EPM)test were used to determine the impact of caspase-1 knockout on locomotive activity and basal anxiety levels of mice;Caspase-1-/-mice were used to determine the resilience to chronic stress-induced depression-like behaviors;Adeno-associated virus(AAV)-mediated overexpression techniques,subthreshold social defeat stress,forced swim test(FST)and tail suspension test(TST)were used to investigate the effect of caspase-1 overexpression in the hippocampus on depression-and anxiety-like behavior of mice;Intracerebroventricular(i.c.v.)microinjection techniques were utilized to investigate the effect of exogenous caspase-1 specific inhibitor AC-YVAD-CMK on immobility time in the TST and FST,and CSDS-induced depression-like behaviors in mice.Results:(1)Susceptible mice displayed a significant decrease in depression-like behaviors and memory impairment when compared with nondefeated control mice.Caspase-1 mRNA was significantly increased to 3.31 ± 0.73(n = 8,P<0.01 vs Control)and 2.40 ± 0.38(n = 6,P<0.01 vs Control)in the PBMC and hippocampus of susceptible mice,respectively.There were no significant differences in the striatum and amygdala.In addition,the level of caspase-1 mRNA in the hippocampus was negatively correlated with social avoidance behavior(Pearson's,r =-0.481,P<0.01).Furthermore,the hippocampal caspase-1 expression in mice exposure to CSDS was significantly increased to 1.69 ± 0.08(n = 10,P<0.01 vs Control).Interestingly,the level of caspase-1 protein was also significantly elevated in the mice hippocampus induced by CUMS.(2)Real-time PCR(RT-PCR)confirmed that knockout of caspase-1 was a success,and there was no detectable level of caspase-1 in the hippocampus of Caspase-1-/-mice 5.08 ± 0.33%(n = 5-6,P<0.001 vs Control).Deletion of caspase-1 did not change calcium/calmodulin-dependent protein kinase type ?(CaMKII)signaling 83.5±3.8%(n = 5-6).There was no gross morphology abnormality of hippocampus in Caspase-1-/-mice in Nissl's staining and immunofluorescence.Caspase-1-/-mice showed anxiolytic behavior in the OF and EPM test,and no differences were observed between the groups in the locomotor activity,total solution intake,vocalizing and jumping.(3)When compared to WT-CSDS mice,Caspase-1-/-mice were resistant to CSDS-induced depressant-like behavior.Moreover,Caspase-1-/-knockout prevented the cognitive impairment caused by CSDS.(4)In a chronic restraint stress(CRS)model of depression,caspase-1 gene ablation also prevented chronic stress-induced depressive behaviors as shown by increased sucrose consumption and decreased immobility time in FST(WT:77.1±10.7 sec;WT-CSDS:138.9 ± 13.4 sec;Caspase-1-/-:64.4± 7.3 sec;Caspase-1-/--CSDS:68.2 ± 9.8 sec)(n = 10-11,P<0.01 vs WT-CSDS)and TST(WT:69.0 ± 6.2 sec;WT-CSDS:118.7±10.6 sec;Caspase-1-/-:60.9±8.6 sec;Caspase-1-/--CSDS:58.3±7.6 sec)(n = 10-11,P<0.01 vs WT-CSDS).(5)AAV-mediated caspase-1 overexpression(AAV-caspase-1)in mice hippocampus not only directly induced depression-like behaviors in TST and FST,but also increased the susceptibility to subthreshold social defeat stress.Hippocampal caspase-1 overexpression led to an increased in immobility time of mice from 85.2 ± 8.6 sec to 122.6 ± 15.4 sec and 49.6 ± 7.0 sec to 87.2 ± 13.2 sec in FST and TST,respectively(n = 10-11,P<0.01 vs AAV-GFP).In the social interaction test,AAV-caspase-1 showed a decreased time in interaction zone from 54.0 ±3.7 sec to 33.5 ± 6.4 sec in subthreshold social defeat stress(n = 10-11,P<0.01 vs AAV-GFP).(6)A single i.c.v.infusion of AC-YVAD-CMK significantly decreased immobility time in a dose-dependent manner in TST as well as FST(TST:Control:116.5 ± 7.3 sec;DMSO:119.3 ±10.4 sec;5?g/kg AC-YVAD-CMK:89.0 ± 10.0 sec;10 ?g/kg AC-YVAD-CMK:85.3 ± 3.7 sec;FST:Control:106.6 ±8.4 sec;DMSO:105.6 ± 5.5 sec;5 ?g/kg AC-YVAD-CMK:75.2 ± 5.0 sec;10?g/kg AC-YVAD-CMK:60.8±7.7 sec)(n = 9-10,P<0.05 vs DMSO).After 10 days repeated intracerebroventricular infusion of AC-YVAD-CMK 30 min prior to daily social defeat,mice exhibited normal social interaction(Target:DMSO:47.0±3.4 sec;10 ?g/kg AC-YVAD-CMK:49.1 ± 4.0 sec;CSDS-DMSO:18.8 ± 2.5 sec;CSDS-10 ?g/kg AC-YVAD-CMK:46.2±5.6 sec)(n = 9-11,P<0.01 vs CSDS-DMSO)and sucrose preference(DMSO:82.4 ± 4.5%;10 ?g/kg AC-YVAD-CMK:81.8 ± 3.9%;CSDS-DMSO:56.6 ± 5.9%;CSDS-10 ?g/kg AC-YVAD-CMK:88.0 ± 3.5%)(n = 9-11,P<0.01 vs CSDS-DMSO).Conclusion:Caspase-1 in mice hippocampus plays a critical role in the development of depression.Upregulation of caspase-1 expression in the hippocampus increases depression-like behaviors and cognitive impairment,whereas genetic deletion and pharmacological inhibition of caspase-1 produce antidepressant-like behavior.Part ? The mechanism of caspase-1 in chronic stress-induced depressive behaviors of miceObjective:Caspase-1(interleukin-1(3-converting enzyme)is required to cleave the immature form of IL-1? and IL-18 into the active protein.Proinflammatory cytokine IL-1? is thought to be the main effector of inflammation in the brain,and has been shown to regulate cognitive function and synaptic plasticity.Although MDD exhibits multiple molecular phenotypes,accumulating evidence suggests that neuroinflammation and glutamatergic neurotransmission abnormalities are sustaining and exacerbating reasons for depression.However,little is known about whether activation of caspase-1 causes depression through inhibiting glutamatergic neurotransmission.Therefore,we tested the mechanism of caspase-1 in CSDS-induced synaptic plasticity in the hippocampus.Methods:CSDS was utilized to establish mouse models of depression;In vitro field potential recordings and patch-clamp recordings methods were employed to record the field excitatory postsynaptic potentials(fEPSPs)and miniature excitatory postsynaptic currents(mEPSCs)in the Schaffer collateral-CA1 afferents and CA1 pyramidal neurons,respectively;Surfacereceptor cross-linking with BS3 assay and western blotting were performed to determine the levels of surface and total AMPA and NMDA receptors in WT and Caspase-1-/-mice subjected to CSDS.The expression levels of PSD95 and DAPI was investigated by immunofluorescence in WT and Caspase-1-/-mice subjected to CSDS;qRT-PCR experiments were performed to quantitative analyze NLRP1,NLPR3,AIM2,ASC and IL-1? mRNA in hippocampus of mice exposure to CSDS;Serum IL-1? was determined by ELISA in WT and Caspase-1-/-mice exposure to CSDS;Intracerebroventricular(i.c.v.)microinjection techniques were utilized to investigate the effects of exogenous IL-1(3 receptor antagonist(IL-1ra)on depression-like behaviors and synaptic plasticity;Microinfusion techniques were performed to study the effect of exogenous IL-1? on depression-and anxiety-like behavior in WT and Caspase-1-/-mice.Results:(1)The electrophysiological results showed that WT mice displayed decreased induction and maintenance of LTP in Schaffer collateral-CA1(WT:142.7±4.6%;WT-CSDS:116.6 ± 3.7%;Caspase-1-/-:168.1± 5.7%;Caspase-1-/-CSDS:167.9± 6.2%)(n = 8-11 slices from 4-7 mice,P<0.001 vs WT-CSDS)and mEPSC amplitude(WT:14.0 ± 0.9 pA;WT-CSDS:9.0±0.4 pA;Caspase-1-/-:15.5±1.1pA;Caspase-1-/--CSDS:14.7±0.8 pA)(n=10-12 cells from 5-6 mice per group,P<0.01 vs WT-CSDS)in CA1 pyramidal neurons during CSDS,while there was no effects of CSDS in the Caspase-1-/-mice.However,there were no significant differences in LTD and mEPSC frequency between groups.(2)Following exposure to CSDS,Caspase-1-/-mice revealed no difference in the surface expression of GluAl(WT:100.0 ± 10.5%;WT-CSDS:60.2 ± 3.4%;Caspase-1-/-:126.4±8.5%;Caspase-1-/--CSDS:120.1± 5.9%)(n = 7,P<0.01 vs WT-CSDS)and GluA2(WT:100.0±8.7%;WT-CSDS:63.9±3.7%;Caspase-1-/-:113.5 ± 7.7%;Caspase-1-/--CSDS:112.6±9.6%)(n = 7,P<0.01 vs WT-CSDS)in the hippocampus while there was significant decease in WT control mice,and no changes in total levels of GluAl and GluA2 and total and surface expression of NMDAR between groups.In addition,caspase-1 gene deletion completely blocked the reduction of PSD95 caused by CSDS relative to the WT control group.(3)In ELSIA experiment,CSDS increased the level of IL-1? in the serum in WT mice,but not in Caspase-1-/-mice(WT:66.4±19.3 pg/ml;WT-CSDS:283.6 ± 24.8 pg/ml;Caspase-1-/-:76.0± 10.9 pg/ml;Caspase-1-/--CSDS:86.7±9.9 pg/ml)(n = 10-12,P<0.001 vs WT-CSDS).Furthermore,knockout of caspase-1 also prevented the elevation of IL-1? mRNA in the hippocampus caused by CSDS(WT:1.00±0.26;WT-CSDS:18.48±4.62;Caspase-1-/-:1.44± 0.41;Caspase-1-/--CSDS:2.84±0.90)(n = 5-6,P<0.01 vs WT-CSDS).However,when compared with WT mice,CSDS exposure increased the expression of NLRP3 mRNA in Caspase-1-/-mice,not ASC mRNA.Meanwhile,there were no changes in NLRP1 and AIM2 mRNA between groups.(4)Western blotting showed that knockout of caspase-1 prevented the elevated level of IL-1? protein in the hippocampus caused by CSDS(WT:100.0±11.2%;WT-CSDS:155.1± 6.5%;Caspase-1-/-95.3±3.9%;Caspase-1-/--CSDS:97.7± 8.5%)(n = 6-7,P<0.01 vs WT-CSDS).Moreover,knockout of caspase-1 also prevented the decreased level of BDNF protein in the hippocampus induced by CSDS.(5)Repeated i.c.v.infusion of IL-1ra prevented CSDS-induced social avoidance and sucrose preference reduction in a dose-dependent manner.It was found that IL-1ra(90 p,g/kg,i.c.v.)reduced the expression of phosphorylated p38 MAPK and increased the level of BDNF and PSD95 in CSDS-treated mice.Furthermore,IL-1ra blocked the decrease in surface expression of AMPAR subunits(GluA1:Vehicle:100.0 ±10.2%;CSDS-vehicle:57.1±3.6%;90 ?g/kg IL-1ra:102.0±8.8%;CSDS-90 ?g/kg IL-1ra:99.8±9.8%;GluA2:Vehicle:100.0±4.7%;CSDS-vehicle:59.8±6.4%;90 ?g/kg IL-1ra:96.8±10.8%;CSDS-90 ?g/kg IL-1ra:93.3± 7.6%)(n = 5,P<0.05 vs WT-CSDS)and mEPSC amplitude(Vehicle:20.7±1.3 pA;CSDS-vehicle:14.0±1.0 pA;90 ?g/kg IL-1ra:20.7±1.0 pA;CSDS-90 ?g/kg IL-1ra:19.2±0.8 pA)(n = 10-12 cells from 4-6 mice,P<0.01 vs CSDS-vehicle)caused by CSDS,whereas the total proteins and mEPSC frequency remained unchanged.(6)Following IL-1? exposure for 10 days(5 ?g/kg,i.c.v.),both WT and Caspase-1-/-mice exhibited depression-and anxiety-like behavior.Conclusion:Our findings demonstrate that an increase in caspase-1/IL-1? axis facilitates AMPAR internalization in the hippocampus which dysregulates glutamatergic synaptic transmission,eventually resulting in depression-like behaviors.Meanwhile,chronic intracerebroventricular infusion of IL-1p induces depression-and anxiety-like behavior of Caspase-1-/-mice,and highlights caspase-1-IL-1? as a novel therapeutic potential target for the treatment of mood disorders.