Mechanisms Of GABRG2(I107T) Mutation-induced Epileptic Encephalopathy

Objective To explore the effect of GABRG2（I107T）mutation on the expression and transportation of GABA_Areceptors as well as the structure and function of synapses by in vivo and in vitro models,which will expand the molecular mechanisms underlying GABRG2 mutations related epileptic encephalopathy.Methods1.The mutant plasmid GABRG2^320T>Cwas constructed by Gateway technique and was co-injected with transposase m RNA into the embryo of AB zebrafish.The transgenic zebrafish line,Tg(h GABRG2^I107T),was screened with EGFP labeling the embryonic heart.The control transgenic zebrafish line,Tg(h GABRG2^WT),was also established by injection with wild-type（WT）plasmid.2.Electroencephalogram（EEG）recordings were performed to quantify the epileptic seizures in WT and I107T transgenic zebrafish.3.The behavioral detection system was applied to analyze the changes in swimming behavior of WT and I107T transgenic zebrafish under normal light and light flash stimulation,and the circadian rhythm changes were also compared.4.The expression of epileptic marker gene c-fos in WT and I107T transgenic zebrafish was detected by whole embryo in situ hybridization（WISH）and real time quantitative PCR（q PCR）.5.The temporal and spatial expressions of GABA_Areceptor subunits in WT and I107T transgenic zebrafish in vivo were detected by WISH,q PCR,Western blot,whole embryo immunofluorescence staining and transcriptome sequencing.6.The synaptic ultrastructure in the telencephalon of WT and I107T transgenic zebrafish was evaluated by transmission electron microscopy（TEM）.7.The changes of excitatory and inhibitory neuronal markers in WT and I107T transgenic zebrafish were detected by q PCR,and the expression of synaptic associated proteins was detected by whole embryo immunofluorescence staining and Western blot.8.The changes of gene expression pattern in the brain of I107T transgenic zebrafish were analyzed by transcriptome sequencing.9.Co-expression ofα1,β2,WT or I107T mutantγ2 subunits in HEK293T cells with PEI transfection was used to establish a cell model in vitro.10.The expression and transportation of GABA_Areceptor subunits in transfected cells were detected by immunofluorescence staining,q PCR and Western blot.11.The change of metabolism in the cell model was analyzed by measuring the content of mitochondrial ATP.Results1.The results of sequence alignment showed that GABRG2 was highly conserved among different species.The transgenic zebrafish lines,Tg(h GABRG2^I107T)and Tg(h GABRG2^WT),were obtained by Gateway method and embryonic screening.2.At 1 month post fertilization,the EEG of I107T transgenic zebrafish showed different degrees of discharge,accompanied by spontaneous recurrent behavioral seizures.In infancy,the basic swimming behavior was more active,with tonic-clonic seizures,in I107T transgenic zebrafish.The I107T transgenic zebrafish was highly sensitive to light stimulation and frequent light flashes.Although the circadian rhythm did not change significantly,the swimming activity of I107T transgenic zebrafish increased,especially during the daylight,which is similar to human epilepsy phenotype.3.In Tg(h GABRG2^I107T)zebrafish line,the expression of epilepsy marker gene c-fos in the brain increased significantly at 5 days post fertilization（dpf）.4.Compared with WT zebrafish,the expression of GABRA1 gene in Tg(h GABRG2^I107T)zebrafish decreased slightly at 3 dpf,and returned to normal level at 5 dpf.The total and membraneα1 subunit expressions remained unchanged.The totalγ2 subunit expression increased while the expression in cell membrane decreased at 5 dpf.Immunofluorescence staining showed the aggregation ofγ2 in the cytoplasm in telencephalon.5.In Tg(h GABRG2^I107T)zebrafish line,the number of synaptic vesicles increased in the telencephalon,but the total number of synapses did not change significantly,and there was abnormal aggregation of vesicles in the pre-synaptic terminals.6.In Tg(h GABRG2^I107T)zebrafish line,the inhibitory synaptic marker GAD65/67 did not change significantly at 3 dpf,and was down-regulated at 5 dpf.On the other hand,the expression of excitatory synaptic marker PSD95 decreased at 3 dpf and increased at 5 dpf.The expression of vesicle associated protein SV2A decreased at 5 dpf.7.Transcriptome sequencing data showed that there were 4734differentially expressed genes in the brain of Tg(h GABRG2^I107T)zebrafish at 3 dpf and 3549 differential genes at 5 dpf,most of which were enriched in endoplasmic reticulum protein processing,metabolic pathway and TGF-βsignal pathway.8.The transfection model of HEK293T cells was established by co-transfectingα1,β2 and WT or mutantγ2 subunits at the ratio of1:1:1 with PEI reagent.9.Immunofluorescence staining showed thatα1,β2 andγ2subunits were co-localized in the cells.Compared with the WT control group,the expression of totalα1 and surfaceα1 in mutantγ2（I107T）transfected cells did not change.Although the total expression ofβ2subunit did not change significantly,its expression at cell surface decreased.The total expression ofγ2 subunit increased while the surfaceγ2 decreased.10.Compared with the WT control group,the mitochondrial ATP level in mutantγ2（I107T）transfected cells decreased significantly.Conclusions1.The transgenic zebrafish epileptic model expressing human mutant GABRG2（I107T）was successfully established.In addition,the cell transfection model co-expressingα1,β2,WT or I107T mutantγ2subunits were constructed.2.Theγ2（I107T）mutation increased the aggregation of mutant proteins in cytoplasm,resulting in the destruction of receptor assembly and transportation,thus affecting its expression on the cell surface.3.In the telencephalon of Tg(h GABRG2^I107T)zebrafish larvae,abnormal aggregation of synaptic vesicles was shown and the expression of synapse related protein changed,leading to the imbalance of excitatory/inhibitory neurotransmission.4.The transcriptome of Tg(h GABRG2^I107T)zebrafish changed and the disruption of metabolic pathway might be involved in the occurrence of epileptic encephalopathy.