| ?Objective? Fragile X syndrome(FXS)is a genetic disease characterized by common neuropsychiatric symptoms such as intellectual retardation,overactivity,autism and concomitant epilepsy.The voltage-dependent potassium channel(Kv1.1)is encoded by the Kcna1 gene and belongs to the Shaker subfamily of voltage-gated potassium channels,which can regulate the excitability of central and peripheral nervous system neurons.Functional changes of Kv1.1 can cause excitatory disturbances of the nervous system,leading to diseases such as type 1 paroxysmal ataxia(EA1)and epilepsy.Our previous results found that cerebral cortex neurons from the fragile X syndrome model mice(Fmr1 knockout mice,Fmr1 KO mice)exhibited the increased electrical activity,including action potential facilitation,which was likely ascribed to decreased expression of voltage-dependent potassium channels(Kv1.1).Kv1.1 is considered to be an important regulator of the electrical excitability of the nervous system.Adeno-associated virus(AAV)-mediated gene transduction is an effective and safe way for manipulating protein expression.Therefore,we used the AAV to mediate exogenous expression of Kv1.1 in the prefrontal cortex of mice and explored whether the AAV-mediated expression of Kv1.1 in the prefrontal cortex could lead to the modulation of neuronal excitability and what role Kv1.1 plays in the pathophysiological process of fragile X syndrome.?Method? 1.Construct a plasmid expressing the mouse Kcna1 gene.The mouse Kcna1 gene was labeled with V5 at the C-terminus and inserted into p AAV,which was driven by the human synapsin 1 gene promoter labeled with the EGFP reporter gene.The empty p AAV without Kcnal gene and V5 tag was used as sham controls.They were named as p AAV-Kcna1-EGFP and p AAV-EGFP.2.The p AAV-Kcna1-EGFP was bilaterally injected in the prefrontal lobe of KO newborn pups in the treatment group(KO-Kcna1).Instead,the p AAV-EGFP was injected in KO littermates and WT pups,as the control groups(named as KO-sham and WT-sham)respectively.3.Fourteen days after AAV injection,electrophysiological recordings were performed on acute brain slice using whole-cell patch-clamp technique to measure spiking ability and potassium current.4.At their matured age(postnatal 28~40 days),we performed behavioral tests including Y-maze spontaneous alternation test,open field test,Morris water maze,and evaluation of pentylenetetrazole(PTZ)-induced seizure susceptibility.5.The prefrontal cortex of each group was isolated and subjected to Western blot analysis for examination of Kv1.1 protein expression and other related modulators for Kv1.1 modulation,including m TOR,Hu D and PERK.6.All data shown are presented as mean±SEM.Chi-square test was used to compare the incidence of seizure in the PTZ test.One-way ANOVA and post-hoc Bonferroni test was used to compare among the three groups.In all analyses,probability to detect the difference was set at the 5% level(P < 0.05).?Result? 1.Open field test and Y-maze spontaneous alternation test were used to analyze the space exploration behavior and anxiety behavior of experimental animals in a new environment.In open field test,we found that the KO-Kcna1 group stayed longer in the central area than the KO-sham group(P=0.030)and the WT-sham group(P=0.003),indicating the KO-Kcna1 group was more anxiolytic than the other two groups.Other parameters such as the total walking distance,the total walking distance in the central area,the number of crossing the central area,the rest time,and the running speed were not significantly altered among the groups.In the Y-maze spontaneous alternation test,no significant difference was found.2.Morris water maze was used to observe spatial memory and learning ability.During the training period,the escape distance was decreasing along with training in all groups.During the test session,the escape distance in the target quadrant was significantly longer than the other quadrants(P<0.05)for the WT-sham group,whereas the KO-Kcna1 and KO-sham groups lost the significant difference,indicating the Kv1.1 gene therapy did not have an effect on such behavioral impairment.3.In the evaluation of PTZ-induced seizure susceptibility,we found that the KO-sham group had higher incidence of generalized tonic-clonic seizure(GTCS)and seizure scores,when compared to the KO-Kcna1 and WT-sham groups(P <0.05).Although there was no significant difference in terms of seizure latency,the results suggest that the Kv1.1 gene therapy might restore the seizure susceptibilty of the KO mice.4.Using current clamp technique to evoke neuronal spiking,we found that spiking number and maximum spiking frequency(maximal spiking frequency,MSF)were increased in the KO-sham group compared with the WT-sham group(P<0.05).The Kv1.1 compensation via gene therapy in the KO-Kcna1 group exerted inhibiting effects on the facilitated spiking,evidenced by lower spiking number and MSF(P<0.05,v.s KO-sham).5.Peak amplitude of macroscopic potassium current in the KO-sham group was significantly lower than the WT-sham group(P<0.05),while the AAV infection in the KO-Kcna1 group led to a restore when compared to the two control groups.With the perfusion of Kv1.1-specific blocker DTX-?,the Kv1.1-specific current(IKv1.1)was isolated.The peak amplitude of IKv1.1 in the KO-sham group was significantly lower than that of WT-sham group,while the KO-Kcna1 group has an expected elevation in the IKv1.1,when compared to the KO-sham group(P<0.05).6.In the analysis of IKv1.1 voltage-dependent properties,the Vmid of activation in the KO-sham group was more depoloarized than that in the WT-sham group(P<0.05),and the activation conductance curve also showed a right shift in the KO-sham group.The exogenous expression compensation of Kv1.1 in the KO-Kcna1 group lowered the Vmid,but still had a higher Vmid than the WT-sham group(both P<0.05).There was no significant difference in the Vslope of voltage-dependent activation.In contrast,for the inactivation curve,the Vslope of inactivation in the KO-Kcna1 group was significantly higher than that of the other two groups(P<0.05),and no significant difference was found in the Vmid of IKv1.1 inactivation.7.Western bolt analysis confirmed that the protein expression levels of Kv1.1 in the KO-sham group were lower than the WT-sham group,and the AAV-mediated Kcna1 gene delivery restored the Kv1.1 protein level.Meanwile,we found that differences in the protein levels of either m TOR or p-m TOR among the three groups were in accord with that of Kv1.1.Protein expression levels of Hu D,PERK and p-PERK were not significantly different.We also found the phosphorylation level of m TOR in the KO-sham group was lower than the WT-sham group,and the Kv1.1 overexpression led to a rebound of m TOR phosphorylation level(P<0.05,KO-Kcna1 v.s KO-sham).On the contrary,phosphorylation percentage of PERK in the KO-sham group(85.3%±5.1%)was higher than the WT-sham(71.4%±2.6%),but lower than the KO-Kcna1(71.6%±3.0%,n=4,P<0.05).?Conclusion? 1.AAV-mediated gene therapy of Kv1.1 in the PFC of Fmr1 KO mice may alleviate anxiety behaviors and seizure-sensitive predisposition,but have no effects on spatial learning and memory.2.For neurons,exogeneous gene delivery of Kv1.1 in Fmr1 KO mice can rescue the decreased expression of Kv1.1,partly restore the corresponding current properties,and finally exhibit an inhibition of neuronal spiking activity.3.The m TOR signal pathway closely correlated with the expression level of Kv1.1,even in response to exogeneous gene delivery of Kv1.1.Both m TOR and PERK pathway might be involved in the regulation of Kv1.1 translation.4.Kv1.1 is an important modulator of neuronal excitability.Phenotypes of FXS might partly ascibe to the deficiency of Kv1.1.Targeting of Kv1.1 may offer a new therapeutic approach against FXS by fine-tuning neuronal excitability. |
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