Effect And Mechanism Of Potassium Channel Tetramerization Domain Containing 12 (Kctd12) In Depressive-like Behaviors Of Mice

Objective:Potassium channel tetramerization domain containing 12(Kctd12),an important auxiliary subunit of the GABA_B receptor,has been associated with a variety of psychiatric diseases.The dentate gyrus(DG)region plays a unique and key role in the pathogenesis of depression and the effect of antidepressant.A large number of studies have shown that neurogenesis and the activity of granule cell(GC)in DG play a key role in the regulation of depressive-like behaviors,and are also important targets of antidepressants.Moreover,microglia modulate neurogenesis and neuronal activity through multiple functions,and play an important role in the pathogenesis of depression.Our previous study has confirmed that Kctd12 in DG is involved in the regulation of depressive-like behaviors induced by chronic social defeat stress in mice,but the specific role of Kctd12 in depression and the undelying mechanism are still unclear.Here,we investigated the role and the mechanism of Kctd12 in the regulation of depressive-like behaviors.Methods:Chronic social defeat stress(CSDS),subthreshold social defeat stress,and Lipopolysaccharide(LPS)inflammatory mouse models combined with social interaction test(SIT),tail suspension test(TST),forced swim test(FST),and sucrose preference test(SPT)were used to evaluate depressive-like behaviors in mice;Quantitative real-time PCR(Q-PCR),Western blotting(WB)and immunofluorescence techniques were used to detect the expression of target gene and protein;Adeno-associated virus(AAV)was stereotactically injected into DG to interfere with the expression of the target gene;The 5-bromo-2-deoxyuridine(Brd U)were used to mark and evaluate the changes of neurogenesis;c-Fos labeling and whole-cell patch clamp technique were used to detect the activity of GC;Immunofluorescence technique was used to detect the morphology of microglia and the expression of inflammatory marker protein in the DG of mice;BV2 cells were cultured and combined with Q-PCR,WB and immunofluorescence experiments to detect the expression of Kctd12 and inflammation-related factors.Results:(1)CSDS induced depressive-like behaviors in mice,which was manifested as the reduced social interaction ratio of mice(P<0.001),the reduced time in the interaction zone in SIT(P<0.001),the decreased sucrose preference ratio(P<0.01)and the increased immobility time in TST(P<0.01).The expression of Kctd12 protein in the DG of CSDS mice was significantly increased compared with normal control mice(P<0.001).(2)Overexpression of Kctd12 in the DG of normal mice(Kctd12_OE mice)induced desperate behavior,which was manifested by the increased immobility time in TST(P<0.05)and FST(P<0.01).After subthreshold social defeat stress,the social interaction ratio of Kctd12_OE mice decreased significantly compared with the control mice(P<0.05),but the anxiety-like behavior or spontaneous activity was unaltered.In contrast,silence of Kctd12 in the DG of CSDS mice improved depressive-like behaviors,manifested as the increased social interaction ratio(P<0.001)in SIT,and the decreased immobility time in TST(P<0.01).(3)The treatment of fluoxetine in CSDS mice reduced the immobility time in TST(P<0.01),increased the social interaction ratio in SIT(P<0.05)and decreased the expression of Kctd12 in DG(P<0.05).(4)Compared with normal control mice,the expression of GB2(P<0.05)and Kir 3.2(P<0.001)in the DG of CSDS mice increased significantly,and the overexpression of GB2 C-terminal domain significantly elevated the protein level of Kctd12(P<0.05),but the overexpression of Kctd12 did not alter the expression of GB2.(5)Immunofluorescence assay showed that Kctd12 protein was expressed on neurons and microglia,but not on astrocyte.(6)Neurogenesis in the DG of CSDS mice was inhibited,presenting as the reduced cell number of Brd U⁺cells(P<0.05)and Brd U⁺/DCX⁺cells(P<0.01).Overexpression of Kctd12 also reduced the cell number of Brd U⁺cells(P<0.01)and Brd U⁺/DCX⁺cells(P<0.01)in DG,which was similar to that of CSDS mice.Silence of Kctd12expression in CSDS mice improved the neurogenesis(P<0.05).(7)Compared with control mice,the excitability of granule cell in the DG of CSDS mice was significantly reduced,which was manifested as decreased expression of c-Fos in GC(P<0.001),decreased resting membrane potential(P<0.01),increased rheobase(P<0.05)and decreased action potentials(APs)frequency responding to depolarizing current injections in GC(P<0.05).Overexpression of Kctd12 in the DG of normal mice can mimic the excitability changes in GC of stressed mice.Silencing the expression of Kctd12 in the DG of stressed mice reversed the decrease in the excitability of GC,reflected by the significantly increased expression of c-Fos(P<0.001).(8)Microglial density(P<0.01)and M1-type marker CD86(P<0.001)increased in the DG of CSDS mice.Overexpression of Kctd12 also increased microglial density(P<0.001),promoted the activation of microglia(P<0.001)and increased the expression of CD86(P<0.01).Silence of Kctd12 reduced the expression level of CD86(P<0.05)and increased the expression of M2-type marker CD206(P<0.001)in the DG of CSDS mice.(9)Intraperitoneal injection of LPS induced depressive-like behaviors in mice,elevated the m RNA level of Kctd12 in DG(P<0.05),and fluoxetine inhibited the increase of Kctd12(P<0.01).(10)In BV2 cells,LPS increased the expression of Kctd12(P<0.001),while IL-4inhibited the expression of Kctd12(P<0.01).Overexpression of Kctd12 promoted the expression of LPS-induced pro-inflammatory factors IL-1?and IL-6(P<0.01),inhibited the expression of IL-4-induced anti-inflammatory factors CD206(P<0.001)and Arg1(P<0.05).Silencing the expression of Kctd12 promoted the expression of anti-inflammatory factors CD206 and Arg1(P<0.001).Fluoxetine(P<0.05)inhibited the expression of Kctd12 induced by LPS.Conclusion:Both CSDS and LPS induced depressive-like behaviors in mice,and increased the expression of Kctd12 in the DG of mice.Overexpression of Kctd12 in DG induced depressive-like behaviors in normal mice,while reducing the expression of Kctd12 in the DG of CSDS mice improved depressive-like behaviors.The increased expression of Kctd12 mediated the enhancement of GABA_B2-Kir 3.2 signaling,and promoted depressive-like behaviors by impairing neurogenesis,inhibiting the excitability of GC and promoting the expression of pro-inflammatory factors in microglia.The classic antidepressant fluoxetine could play an antidepressant effect by inhibiting the expression of Kctd12,suggesting that Kctd12 may be a key target for the treatment of depression.Our study might provide a new perspective for the treatment of depression.