Transgenic Zebrafish Line Expressing Mutant Human GABRG2 For Modeling Of Genetic Epilepsy And Studying The Meshnisms Involved

Objective To establish a transgenic zebrafish line-expressing mutant human GABRG2(F343L),explore the mechanisms involved and screen effective antiepileptic drugs.Methods1.The expressing plasmid of hGABRG2wt and hGABRG2F343L were constructed into pDestTol2CG2 vector by Gateway cloning technique.2.The constructed hGABRG2wt or hGABRG2F343L plasmid were injected into wild type zebrafish(AB)embryos with Tol2 transposase mRNA respectively.The zebrafish with green fluorescent expressing in the heart were selected at 24～48 h after fertilization.3.F1 generations of Tg(hGABRG2wt)and Tg(hGABRG2F343L)zebrafish line was generated by crossing adult FO F343L transgenic zebrafish with wild type zebrafish.Genotyping was performed by PCR and DNA sequencing.4.A DanioVision setup with an infrared camera was used to record the locomotor activity of the transgenic zebrafish at 120 hpf in normal light condition,upon light flash or PTZ induction.5.Expression of c-fos in transgenic zebrafish was detected by real time quantitative PCR ans whole mout in situ hybridization.6.Expressions of GABAA receptor subunit were detected by real time quantitative PCR,Western blot and immunofluoresent staining.7.Postsynaptic membrane proteins including gephyrin and GAD65/67 were detected by Western blot and immunofluoresent staining.8.Transcriptomic assay was performed to analysis differential expressing genes in the brain of transgenic zebrafish.GO and KEGG pathway enrichment were analysed.9.Different anti epileptic drugs were tested in transgenic zebrafish by locototor activity tracking.Results1.Transgenic zebrafish line expressing human mutant GABRG2(F343L)were generated and exhibited spontaneous seizure-like activity.2.Increased locomotor activities,as well as sensitive response to light flash and PTZ induction were discovered in Tg(hGABRG2F343L)zebrafish line.3.Seizure-related c-fos gene transcripts were up-regulated in Tg(hGABRG2F343L)zebrafish line.4.The expression of GABRG2 mRNA and protein decreased in Tg(hGABRG2F343L)zebrafish line at both 72 hpf and 120 hpf.mRNA of GABRAl decreased at 72 hpf and recoverd to nomal level at 120 hpf,while protein level did not change significantly.mRNA of GABRB2 also decreased at 72 hpf while increased at 120 hpf.Protein expression of GABRB2 did not change at 72 hpf and increased at 120 hpf.5.The expressions of gephyrin and GAD65/67 were down-regulated in Tg(hGABRG2F343L)zebrafish line.6.953 and 524 differentiated expressed genes were detected at 72 hpf and 120 hpf in the brains of Tg(hGABRG2F343L)zebrafish line compared to Tg(hGABRG2wt)line.7.There was no significant effect of traditional anti epileptic drugs in Tg(hGABRG2F343L)zebrafish line with 30 min incubation,while HD AC inhibitor SAHA reduced locomotor activities,showing a dose dependent manner.Conclusions1.A transgenic zebrafish line expressing human mutant GABRG2(F343L)was successfully established,which displayed epileptic phenotype and was more sensitive to seizure stimulation.2.The down-regulation of GABRG2 in Tg(hGABRG2F343L)zebrafish line might be responsible for the decrease of gephyrin and GAD65/67,thus changing the functions of ion channel and synapse.3.GABRG2 mutation altered the developmental brain transcriptome in zebrafish.4.SAHA showed anti epileptic effect in Tg(hGABRG2F343L)zebrafish line.