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The role of gephyrin palmitoylation in anxiety behavior of ratsObjective:Gephyrin is originally identified as a key anchoring protein of synaptic GABA_A receptors and a core scaffolding protein for virtually all inhibitory synapses.Since gephyrin interacts with key proteins involved in various neuropsychiatric disorders,dysfunction of gephyrin may underlie many diseases,such as epilepsy,autsim and schizophrenia.Palmitoylation is an important posttanslational modification that regulates proteins transport,location and function.Recently,it has been found that the interaction between GABA_A receptor and gephyrin is controlled by palmitoylation.Considering the GABA_A receptor plays a key role in anxiety,we specualte that palmitoylation of gephyrin may be involved in the regulation of anxiety behavior and investigate its role.Methods:We screened out the low and high anxious rats using elevated plus maze(EPM)test.We detected the palmitoylation of proteins in the basolateral amygdala(BLA)using the method of acyl-biotinyl exchange(ABE).We tested the effect of NtBuHA on anxiey-like behavior through EPM and open field test(OFT)and investigated its mechanism using electrophysiological recordings and ABE methods.We used a GABA_AR?3 peptide that disrupted the interaction between gephyrin and GABA_A receptor and detected its effect on mIPSCs and anxiety-like behavior via EPM and OFT.Results:(1)According to the time spent on the open arm in the EPM test,rats were classified as three groups:the LA(low-anxiety)group,the IA(intermediate-anxiety)group and the HA(high-anxiety)group.The LA group was classified as the time spent on the open arm at the top40%,and the HA group was classified as the time at the bottom 10%.(2)ABE method showed the palmitoylation of global proteins was significantly decreased in the HA group.The expression and palmitoylation of GABA_A receptor was not alterred,but the palmitoylation of gephyrin was reduced in the HA group(LA group:100.0±4.6%,HA group:68.9±4.6%,n=5,P<0.01).(3)Chronic corticosterone exposured rats displayed anxiety-like behaviors and an obvious reduction in palmitoylated proteins in BLA,especially gephyrin(Veh group:100.0±4.0%,Cort group:63.2±9.1%,n=6,P<0.01).(4)NtBuHA(1.0 mM)decreased the palmitoylation of global proteins in the BLA and increased the anxiety behaviors of rats.Whole-cell patch-clamp recording showed that NtBuHA significantly lowered the amplitude of mIPSCs(Veh group:12.9±0.5 pA,NtBuHA group:10.4±0.6 pA,n=16 cells from 8 rats,P<0.01).NtBuHA also reduced the palmitoylation of gephyrin(Veh group:100.0±8.9%,NtBuHA group:66.2±5.9%,n=7,P<0.01).(5)GABA_AR?3 peptide did not change the expression of GABA_A receptor subunits,but weaken the interactions between GABA_A receptor and gephyrin,and significantly lowered the amplitude of mIPSCs(Veh group:13.05±0.8 pA,NtBuHA group:10.2±0.4 pA,n=12-13 cells from 10 rats,P<0.01).Conclusion:In the HA rats,the palmitoylation of protein decreased significantly.NtBuHA weakened the palmitic modification of gephyrin in the BLA and significantly increased the anxiety behaviors.Meanwhile,a GABA_AR?3 peptide that disrupts the interaction between GABA_A receptor subunits and gephyrin can mimic the effect of NtBuHA on anxiety behavior.Taken together,these results that palmition of gephyrin may serve as a novel target for regulating anxiety-like behavior.Part ? The role and mechanisms of gephyrin palmitoylation underlying the anxiolytic effect of diazepamObjective: Studies have show that the binding of benzodiazepines(BDZs)to GABAA receptor induced receptor conformational changes,promoted the binding of GABA to GABAA receptor and increased the open frequency of Cl channel,thereby enhancing the inhibitory effect ofGABA on nervous system.Recent research also showed that the level of gephyrin palmitoylation increased with inhibitory synapase activity.Therefore,in combination with the above results about the role of gephyrin palmitoylation in the anxiety,we further explored the role and mechanism of gephyrin palmitoylation in the diazepam(DZP)-mediated anxiolytic effect.Methods: We used quantitative real-time polymerase chain reaction(q RT-PCR)to detect the m RNA of aspartate-histidine-histidine-cysteine(DHHC)in the BLA of rats.Western blotting was used to detect the expression of DHHC12 and gephyrin in various brain regions of rats.The palmitoylation of proteins was detected via ABE method.We genetically knockdowned DHHC12 in the BLA via lentic virus(LV)-expressing sh RNA.We overexpressed DHHC12 via adeno-associated virus(AAV)vector.Results:(1)q RT-PCR data showed the m RNA of DHHC12 was significantly decreased in the BLA of HA rats(LA group: 100.0 ± 4.4%,HA group: 82.5 ± 3.4%,n = 8,P < 0.01).(2)Western blotting showed a high level of gephyrin in the m PFCand BLA,but a high level of DHHC12 in the BLA.(3)Injection of DZP(i.p.)increased the palmitoylation level of global protein in BLA,including gephyrin and DHHC12(gephyrin: Veh group: 100.0 ± 5.0%,DZP group: 154.4 ± 6.8%,n = 6,P < 0.001;DHHC12: Veh group: 100.0 ± 13.4%,DZP group: 157.7 ± 14.9%,n = 8,P < 0.01).(4)LV-DHHC12 significantly decreased the expression of DHHC12 in the BLA(LV-e GFP-sh RNA group: 100.0 ± 6.6%,LV-DHHC12-sh RNA group: 55.0 ± 10.1%,n = 4,P < 0.05).We found that silencing DHHC12 not only increased anxiety-like behavior,but also abolished the anxiolytic effect of DZP.(5)AAV-DHHC12 significantly overexpressed DHHC12 in the BLA(AAV-e GFP group: 100.0 ± 9.1%,AAV-DHHC12 group: 310.1 ± 46.3%,n = 8,P < 0.001).The results from EPM and OFT showed that overexpressed DHHC12 in BLA had an anxiolytic effect,which can be abolished by FMZ.Conclusion: Palmitoylation of gephyrin plays an important role in the anxiolytic effect of DZP.The possible mechanism may be that DZP increased DHHC12 activity by self-palmitoylation via a GABAA receptor activity-dependent manner and triggered gephyrin palmitoylation,which may contribute to its anxiolytic effects. |
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