Study On The Genetic Analysis And Molecular Mechanism Of Genetic Epilepsy With Febrile Seizures Plus (GEFS+) Induced By GABRG2 Mutation

Part?.Construction of GABRG2 knockout cell line and discussion of the effect of temperature on related proteinsObjective To construct a GABRG2 knockout(KO)mouse hippocampal neuron cell line(HT22)by CRISPR/Cas9 system and analyze the epigenetic characteristics of cell genes by RNA-seq technique,and to explore the effects of other protein expressions under different temperature conditions.Methods(1)Search for the DNA sequence and protein information of GABRG2 gene in National Center for Biotechnology Information(NCBI)website,according to the sequence characteristics of GABRG2 gene,the second exon region of third transcript was selected for targeted deletion.(2)The single guide RNA(g RNA)was designed in the first 1/3 region of the exon corresponding to the protein conservative region.(3)The g RNA sequence was screened by CRISPR design software and website(http://www.e-crisp.org/E-CRISP/),a series of sg RNA sequences were generated,and the first three sg RNAs with high score were selected to reduce homologous sequences and off-target effects.(4)The selected g RNA sequence was synthesized and inserted into PX459 plasmid vector,and selecting the positive clones,and identified by restriction enzyme digestion.(5)The constructed PX459-GABRG2-1/2/3 plasmid was transfected into HT22 cells,and the genomic DNA was extracted and sequenced to verify the plasmid activity.(6)The protein and m RNA expression of GABRG2 were detected by Western blot and RT-q PCR techniques,respectively,to verify the knockout effect of GABRG2 gene.The group with the highest knockout efficiency as the knockout group,and the wild type(WT)was used as the control group.(7)The total RNA from the control group and the experimental group was extracted,and the transcriptome sequencing was performed.We screened the differentially expressed genes(DEGs),and performed pathways and protein-protein interaction analysis.(8)The expression of GABA_Areceptors and MMP family related proteins was detected under different temperature conditions.Results(1)DNA sequencing and Enzyme digestion resluts showed that the GABRG2g RNA was successfully inserted into PX459 plasmid,and the plasmid was constructed successfully.(2)Sanger sequencing results after TA cloning showed that the plasmids PX459-GABRG2-01,PX459-GABRG2-2 and PX459-GABRG2-03 occurred base deletions,point mutations and point mutations,respectively,and the constructed plasmid had good CRISPR/Cas9 activity.(3)The protein and m RNA expression levels of GABRG2 in the GABRG2 KO group were significantly lower than those in the control,and the knockout efficiency in the PX459-GABRG2-03 group was the highest,reaching more than 90%.(4)RNA-seq showed a total of 2157 differential expression genes,of which up-regulated and down-regulated genes were 1173 and 984,respectively.(5)Kyoto Encyclopedia of Genes and Genomes(KEEG)enrichment results indicated that the pathways mainly included PI3K-Akt pathway,MAPK pathway,p53 pathway and Rap1 pathway;Gene Ontology(GO)enrichment analysis showed that the differential expression genes were mainly concentrated in cell biological process,metabolism,development,immunity,apoptosis,cell membrane,extracellular,synaptic and receptor activity,transporter activity,enzyme activity regulation and other processes;protein-protein interaction(PPI)results showed that GABRG2 is closely related to other subunits of the GABA_A receptor,and was in the core position.(6)Under different temperature conditions,the expression of GABRA1/GABRB1/GABRB3 and CACNA1A decreased with the increase of temperature in GABRG2 knockout cells,while the expression of GABRA5 gradually increased with the increase of temperature.The expression of MMP family related factors such MMP3 and MMP9 increased.Conclusion(1)The GABRG2 gene knockout in vitro cell model was successfully constructed by CRISPR/Cas9 technology,which provided clues for studying the pathogenic mechanism of GABRG2 in genetic epilepsy with febrile seizures plus(GEFS+).(2)GABRG2gene mutation can reduce the expression of most of GABA_A receptor subunits,which may lead to the impairment of GABAergic signaling pathway,resulting in the decrease of inhibitory neurotransmitter release in vivo.(3)By regulating PI3K Akt and MAPK signaling pathways,it can affect neuronal apoptosis,development,receptor activity,transporter activity,enzyme activity and related gene expression.These changes may play an important role in the occurrence of GEFS+.Part ?.The effect of GABRG2 knockout on the release and function of presynaptic membrane vesicles in HT22 cellsObjective To evaluate the function of presynaptic membrane neurotransmitter release after GABRG2 gene knockout and the transmission activity of GABAergic inhibitory synapses.Methods(1)Synaptic vesicle release staining with FM4-64 dye was used to observe the endocytosis and exocytosis of the cells,and to detect the function of cytotransmitter release after GABRG2 knockout.(2)The whole cell patch clamp technique was used to detect the m IPSC changes of GABRG2 knockout cells.Results(1)Compared with the wild group,the number of FM4-64 positive clusters in GABRG2 knockout group was significantly reduced(WT: 4.2 ± 0.21 clusters/10 ?m;KO: 1.6 ± 0.18 clusters/10 ?m;t-test;P < 0.0001).In GABRG2 KO group,the fluorescence intensity of FM4-64 was also significantly decreased(P < 0.0001).(2)The frequency of m IPSC in KO group was significantly lower than that in WT(0.79 ± 0.04 Hz vs.1.87 ± 0.16 Hz;P < 0.001),and the amplitude of m IPSC was also lower than that in the control group(10.18 ± 0.87 p A vs.24.00 ± 1.10 p A;P <0.001).In addition,there was a significant difference in the peak current between WT group and Ko group(498 ± 35.5 p A vs.53.4 ± 7.0;P < 0.001).Conclusion The dysfunction of ?2 subunit affects the release of neurotransmitters from cell membrane and reduces the evoked frequency and amplitude of the action potential of m IPSCs,which may be an important factor in the occurrence of GEFS+.Part ?.Establishment of GABRG2 knockout mouse model and explore the effect of temperature on epileptic behaviorObjective To construct the hippocampus and neocortex-specific GABRG2 knockout mouse model,and to explore the effect of different temperatures on the epileptic behavior of GABRG2 knockout mice.Methods GABRG2fl/wt mice were first constructed by the CRISPR/Cas9-mediated genome engineering techniques,and the GABRG2fl/fl homozygous mice were obtained by the crossing GABRG2fl/wt heterozygous mice with GABRG2fl/wt mice.Then the GABRG2fl/wtCre+ mice were generated by crossing GABRG2fl/fl mice with the Emx1-IRES-Cre homozygous mice and as objective group,WT in littermates as control mice.The genomic DNA was extracted from the toes or tail of the offspring mice.Agarose gel electrophoresis was used to identify the genotypes of mice.(2)RT-q PCR,Western blot and immunohistochemical were used to detect the m RNA and protein expression of GABRG2 in hippocampus and neocortex.(3)Nissl staining was used to analyze the loss of neurons in cortex and hippocampus.(4)The temperature heating instrument was used to heat the mice,and the electroencephalogram(EEG)was used to record the seizure.(5)PTZ was used to induce seizures,and the severity of heat-and PTZ-induced epileptic seizures was compared.Results(1)The GABRG2fl/wtCre+ mice was selected as experimental subjects,and the expression of m RNA and protein of GABRG2 in the hippocampus and neocortex of GABRG2fl/wtCre+ mice were significantly lower than those in the control group.(2)Tissue staining showed that the expression of GABRG2 in the hippocampus and neocortex of GABRG2fl/wtCre+ mice was significantly decreased,mainly in CA3 region and IV—V layer of neocortex.(3)The GABRG2fl/wtCre+ mice had more seizures during the heating process compared with WT group.The electroencephalogram(EEG)showed that the number of spikes in the KO group was significantly higher than that in the control group.The GABRG2fl/wtCre+ heterozygous mice were more sensitive to heat induced seizures,which could induce generalized forced clonic seizures,while the WT mice were not.(4)Compared with wild-type littermates,the WT group had a longer incubation period of seizure by PTZ induced than GABRG2fl/wtCre+ mice,while GABRG2fl/wtCre+ mice had longer seizure duration,higher mortality and severity of PTZ induced.Conclusion(1)The hippocampus-and neocortex-specific GABRG2 knockout mice model were successfully constructed.(2)GABRG2fl/wtCre+ mouse model reproduce many characteristics of human febrile seizures,which is a good model for studying genetic epilepsy with febrile seizures plus.(3)GABRG2fl/wtCre+ mice have significant temperature susceptibility.Part ?.Epigenetic characteristics of GABRG2 knockout miceObjective To explore the epigenetic characteristics of the genetic epilepsy with febrile seizure plus caused by GABRG2 gene knockout.Methods(1)Using RNA-seq technology and based on Illumina sequencing platform,the total RNA was extracted from hippocampus and neocortex tissue for the c DNA library construction and bioinformatics analysis.(2)Using the ATAC-seq technology and based on Illumina sequencing platform,the nucleus of the cell were extracted from hippocampus and neocortex,and T5 transposase was added to purify them,then the library sequencing and bioinformatics analysis were constructed.Results(1)The RNA-seq sequencing analysis suggested that the number of DEGs in the hippocampus of GABRG2fl/wtCre+ mice and wild-type mice was 667 at 37?,which the number of up-regulated genes and down-regulated genes were 496 and 171,respectively.In neocortex,the number of DEGs was 552,which the number of up-regulated genes and down regulated genes was 170 and 383,respectively.Under the heat-induced seizures(40?),the number of DEGs in hippocampus was 681,which the number of up-regulated genes and down regulated genes were 440 and 241 respectively.In neocortex,the number of differentially expressed genes was 743,which the number of up-regulated genes and down regulated genes was 382 and 361 respectively.GO enrichment results showed that it mainly included neuropeptide signal pathway,neurotransmitter metabolism process,protein extracellular matrix,extracellular matrix,receptor activity regulation and G-protein coupled receptor binding.KEEG results indicated that it mainly included NF-?B pathway,IL-17 pathway,Toll-like receptor pathway,MAPK pathway,c AMP pathway and PI3K-Akt pathway.(2)ATAC-Seq results showed that the number of clean bases in each group was more than 17.5G.The number of difference peaks in hippocampus and neocortex between GABRG2fl/wtCre+ mice and WT mice at normal temperature is 10,6187 and 11,8620,respectively.Under the condition of heat-induced seizures(40?),the number of differential peaks in hippocampus and neocortex was 116,754 and 121,874,respectively.Gene annotation and signal pathway analysis were performed based on the number of differential peaks.At normal body temperature,there were 16,418 DEGs in hippocampus,which the number of up-regulated and down-regulated genes were 1,767 and 14,651,respectively.There were 12,716 DEGs in neocortex,which the number of up-regulated and down-regulated genes was 852 and 1,1864,respectively.Under heat-induced seizures,the total number of differentially expressed genes in the hippocampus was 6,530,which the number of up-regulated and down-regulated genes was 4,410 and 2,120,respectively.In neocortex,the number of DEGs was 5,249,which the number of up-regulated and down regulated genes was 2,884 and 2,365 respectively.In addition,we also analyzed the differentially expressed motifs in differentially expressed peaks,and selected the most significant 25 motifs as experimental subjects.Then these differentially motifs were enriched with known transcription factors,and 14 transcription factors were found to be the most closely related.Conclusion(1)The results of RNA-seq and ATAC-seq are consistent,and the common pathways include m TOR pathway,PI3K-Akt pathway,MAPK pathway,and Toll-like receptor pathway.(2)Differentially motif enrichment analysis showed that 14 important transcription factors were involved.