Nedd4-2 Mediated Kir4.1 Channel Protein Ubiquitination Modification On The Pathogenesis Of Epilepsy

Objective: Neural precursor cell expressed developmentally down-regulated gene4-like(Nedd4-2)encodes a ubiquitin E3 ligase.Recent studies have found that the Nedd4-2 gene has mutations in some patients with epilepsy,and pointed out that Nedd4-2 mediates the ubiquitination of AMPA receptor subunit Glu A1,which affects neuronal activity and susceptibility to seizures.Recent studies have shown that the occurrence of epilepsy is closely related to ion channels,neurotransmitters,and synaptic connections.Many of the substrates modified by ubiquitination are ion channel proteins that affect the excitability of the central nervous system.Ubiquitination modification can further affect the excitability of neurons by regulating the expression level of these channel proteins,which is closely related to the occurrence of epilepsy.However,whether there are other Nedd4-2 substrates that are not known to be associated with epilepsy remains to be further studied.In this study,using CRISPR/Cas9 technology,We constructed a novel Nedd4-2+/-mouse model with half level of both Nedd4-2 long and short isoforms in the brain.In this study,pentylenetetrazole(PTZ)induced epilepsy in the experimental group Nedd4-2+/-(KO)mice and the control group Nedd4-2+/+(WT)mice,using Label-free proteomics detection and in vitro and in vivo experiments to compare KO and WT mouse hippocampal tissue protein expression differences,in order to find new Nedd4-2 target protein,reveal the new mechanism of Nedd4-2 involved in epilepsy.Methods:1.Nedd4-2 knockout mice were constructed by Shanghai Southern Model Biotechnology Co.,Ltd.2.Mice were given intraperitoneal injection of PTZ 35mg/kg every day,and the seizure score was graded by Racine.3.Label-free-LC-MS/MS was used to detect differentially expressed proteins in the hippocampus of KO mice and WT mice with PTZ-induced epilepsy,and perform Cluster,GO and KEGG Biological information analysis,screen out the differential protein related to epilepsy.4.The mouse tail DNA was extracted and the mouse genotype was identified by PCR.5.Detection of Nedd4-2 and Kir4.1 expression by Rt-qPCR6.Detection of Nedd4-2 expression in the hippocampus,cortex and medulla of mice by Western Blot.7.Nissl staining detects the changes in the number of neurons in mouse brain tissue.8.Immunohistochemical technique was used to detect the expression of Kir4.1 in mouse brain tissue.9.Detection of binding Nedd4-2 and Kir4.1 and Kir4.1 binding and Ub in brain tissue by Co-IP.10.Cell transfection.The expression of Nedd4-2 and Kir4.1 was detected by Western Blot.Co-IP verifies the change of Kir4.1 ubiquitination degree after transfection.11.The Kir4.1 expression vector and the mutant vector were transfected into the cells respectively,and the binding of Nedd4-2 to Kir4.1 was verified by Co-IP.12.The Kir4.1 expression vector and Kir4.1 mutant T312 A were respectively transfected into the cells,and treated with Cycloheximide(100ug/ml).Western Blot was used to detect Kir4.1 expression.13.The cells were transfected with the 14-3-3 expression vector and treated with R18.The expression changes of Kir4.1 were detected by Western Blot,and the binding degree of Nedd4-2 and Kir4.1 was verified by Co-IP.14.Kir4.1 expression vector and Nedd4-2 expression vector or empty vector were co-transfected into HEK293 cells,Kir4.1 expression vector and Kir4.1 mutant T312 A were transfected into HEK293 cells,respectively,and Ba2+-sensitive inward potassium ion current was recorded.Result:1.The results of Rt-qPCR and Western Blot showed that the expression levels of Nedd4-2 in the hippocampus,cortex and medulla of the experimental group and the control group decreased by about half.2.After the mice were injected with PTZ,according to the Racine scoring standard,the results showed that the epileptic seizure score of KO mice was consistently higher than that of the WT group.3.After the mice were injected with PTZ,according to the Racine scoring standard,the results showed that the epileptic seizure score of KO mice was consistently higher than that of the WT group.4.Detection of Nedd4-2 expression in the hippocampus,cortex and medulla of mice by Western Blot.The results showed that the expression of Nedd4-2 was significantly reduced compared with the control group WT mice(P <0.01),Kir4.1expression was significantly increased(P <0.01).5.Immunohistochemistry results showed that the expression of Kir4.1 was increased in KO mice compared with WT mice.6.The results of Co-IP showed that compared with the WT group,the level of Kir4.1 ubiquitination in the hippocampus of KO mice was significantly down-regulated(P<0.01).7.Co-IP experiments confirmed that Nedd4-2 interacts with Kir4.1 in the hippocampus of KO and WT mice.In addition,the binding of Kir4.1 to Nedd4-2 was significantly decreased in KO group compared with WT group(P < 0.01).8.The cells were transfected with Nedd4-2 interference vector,and the expression of Kir4.1 protein was increased compared with the control group.Co-IP results showed that the ubiquitination level of Kir4.1 was significantly reduced.9.The Kir4.1 expression vector and the three mutant vectors were co-transfected with Nedd4-2 into c6 cells.The results of Co-IP experiments showed that the interaction between Kir4.1 expression vector of T312 A mutation and Nedd4-2 was significantly weakened.10.The Kir4.1 expression vector of T312 A mutation and wild type Kir4.1expression vector were co-transfected with the Nedd4-2 expression vector to c6 cells,and treated with Cycloheximide.Compared with the WT group,the mutant Kir4.1 still maintained a higher level(P< 0.0001).11.The results of Co-IP showed that 14-3-3 interacted with Kir4.1.c6 cells were transfected with the 14-3-3 expression vector.Compared with the empty group,the expression level of Kir4.1 was reduced,and the interaction between Kir4.1 and Nedd4-2 was enhanced.On the contrary,given the 14-3-3 inhibitor R18 treatment,compared with the control group,the expression level of Kir4.1 increased,and the interaction between Kir4.1 and Nedd4-2 was enhanced.12.In HEK293 cells,the Ba2+-sensitive inwardly rectifying K+ current was significantly decreased in cells with Nedd4-2 expression than vacant control;Compared with the transfection of wild-type Kir4.1 expression vector,the Ba2+-sensitive K+current of T312 A mutant Kir4.1 vector was 3.5% of the wild-type level.Conclusions:1.In the hippocampal tissues of KO and WT mice with PTZ-induced epilepsy,a total of 3379 proteins were identified,and 55 differential proteins were screened.Compared with the WT group,there were 21 high expressions and 13 low expressions in the KO group.2.Nedd4-2 haploinsufficiency caused increased seizure susceptibility.3.Nedd4-2 is the ubiquitin E3 ligase of Kir4.1.Nedd4-2 interacts with the motif of threonine 312-proline of Kir4.1 and mediates its ubiquitination modification and degradation.4.In KO mouse brain tissue,Kir4.1 expression was significantly up-regulated,and Kir4.1 ubiquitination modification level was down-regulated.5.Adaptor protein 14-3-3 facilitated Nedd4-2-mediated Kir4.1 ubiquitination.