Disruption Of AT-hook 1 Domain In MeCP2 Protein Caused Behavioral Abnormality In Mice

BackgroundMutations in the X-linked gene,methyl-CpG binding protein 2(MECP2)cause autism spectrum disorders in which Rett syndrome(RTT,OMIM#312750)count for the most cases.Classical RTT is characterized by a normal development period of 6-18 months,followed by a regression period with gradual loss of acquired motor and language skills,intellectual disability and handstereotypies.MeCP2 contains three highly conserved AT-hook-like domains.Our previous study identified mutation in AT-hook 1 domian of MeCP2 in a RTT patient.There is no report on functional study of AT-hook 1 domain.To investigate the role of AT-hook 1 domain in MeCP2 protein,we therefore developed a line of mice carrying deletion of eight conserved amino acids located in AT-hook 1 domain.Animal behavioral study was performed to observe RTT like phonotype.Molecluar levels of several proteins assessed to elucidate the role of AT-hook 1 domain in MeCP2 proteinPurpose1.To generate MeCP2 mutant mice carrying deletion of AT-hook 1 domain.2.To confirm whether Mecp2?AT-hook1/y mice have behavioral abnormalitythrough behavioral test.3.To confirm the levels of histone 3 dimethylation in cerebrum or cerebellum of Mecp2?AT-hook1/y mice due to deletion of AT-hook 1 domain in MeCP24.To confirm the RNA expression levels of candicate genes expressed in inhibitory neuron in cerebrum or cerebellum of Mecp2?AT-hook1/y mice.Method1.Generation of MeCP2 mutant mice carrying deletion of AT-hook 1 domain using by CRISPR/Cas9 technology(Mecp2?AT-hook1/ymice).Genomic sequencing was used to verify the mutation.2.Behavioral Assays Two groups animals were used,including Mecp2?AT-hook1/y mutant male mice(MUT)and wild-type(WT)male littermates.2.1 Accelerating rotarod was used to test balance and motor coordination function when mice was at 24 weeks old and 34 weeks old.2.2 Open field and High plus maze were used to test locomotor activity and anxiety function when mice were at 24 weeks old and 34 weeks old.2.3 Active avoidance test was used to acess learning and memory function when mice was at 24 weeks old and 40 weeks old.2.4 Nest-building assay and direct social interaction were used to test social ability of mice at 48 weeks old.2.5 Tail flick and hot plate test were used to measure pain sensitivity when mice were at 24 weeks old.3.Western blot analysis was used to test levels of histone 3 dimethylation in cerebrum or cerebellum of Mecp2?AT-hook1/y mice at 24 weeks old.4.QPCR was used to test RNA levels of candicate genes expressed in inhibitory neuron in cerebrum or cerebellum of Mecp2?AT-hook1/y mice at 24 weeks old5.Immunohistochemistry and Nissl staning were used to test histopathologic change in brain of muatant mice.6.Statistical analysisAll date were analysied by Statistical software SPSS 20.0 analysisand GraphPad Prism 6.05.Comparison between two groups was conducted by Student't test.All the experimental data were expressed by the mean ± standard deviation(X ± SEM).P<0.05 was considered significant.Data of shuttle box active avoidance test were analyzed by repeated-measures ANOVA followed by least significant difference(LSD)test.Results1.Genomic sequencing of the F0 mice confirmed the mutant mice lacking the expected 24bp sequence in exon 4,indicating successful generation of a Mecp2 mutant mouse model(Mecp2?AT-hook1/y mice)carrying deletion of AT-hook 1 domain with CRISPR/Cas9 technology.2.General appearance and histopathology:Lifespan,body weight,brain size and brain morphology in Mecp2?AT-hook1/y male mice were not changed.Gross body tremor or hindlimb clasping was absent inMecp2?AT-hook1/y mice.Nissl staining was performed to check the morphology of mice brain.There was no remarkable difference in neurons of cortex,hippocampus and cerebellum between two groups.Furthermore,staining of two commonly used inflammation markers,GFAP for astrocytes and Iba-1 for microgliocytes in different brain sections were performed No evidence of inflammation was observed.3.Behaviral test:In rotarod test,we found that the latency of falling off the rod of Mecp2?AT-hook1/y ymice(24 weeks old and 34 weeks old)were shorter than WT mice,which indicated that mutant mice were easier to fall from the rod than WT mice.In open field,mutant mice travelled less distance in comparison to wild type mice at 24 weeks-old and 34 weeks-old.Although a trend was observed that mutant mice(24 weeks old)had spent less time in center area than WT mice,the difference was not significant.However,mutant mice spent significant less time in center area at 34 weeks compared to WT mice,which indicate appearanc of anxiety in mutant mice.To further confirm the anxitey results,elevated plus maze test was carried out.Mutant mice not only preferred to stay in the close arm,but also spent less time and travelled less distance in the open arms,a sign of anxiety.Taken together,the findings demonstrate that mutant mice impaired locomotor activity and increased anxiety-like behavior.In active avoidance test,the mutant male mice at 24 weeks displayed similar or significantly increased numbers of active avoidance responses compared to the age-matched WT miceat day 1 to 3 or day 4 respectively.In contrary,the older mutant mice(40weeks-old)exhibited less tendency of avoidance responses compared to the age-matched WT mice,with a pronounced reduction at the 4th day test.The results indicate that disruption of AT-hook 1 domain in MeCP2 in mice caused enhanced cognitive functionat early ages but cognitive deficit when mice grow older.In social ability test,however,the AT-hook 1 mutant mice showed normal social interaction behavior.In Tail flick test,no significant difference in tail flick test between two genotypes was observed.However,the latency to response to heat pain in mutant mice significantly increased compared to WT mice in hot plate assay,these results reveal that the pain reflex is normal in spinal cord,but impaired in brain.4.Western blot:Expression of MeCP2 significantly increased in the cerebrum but decreased in the cerebellum in Mecp2?AT-hook1/y male mice at age 24 weeks.In addition,levels of MeCP2 in mutant mice at age 48-weeks were elevated consistently in the cortex but were not significantly changed in the hippocampus,striatum and cerebellum,there was even a tendency of lower levels of MeCP2 in the cerebellum.The level of three markers of H3 dimethylation were measured,H3 lysine 4(H3K4me2),H3K9me2 or H3K27me2,both in cerebellum and cerebrum.Mutant mice displayed with elevated level of H3K27me2,with no difference in H3K4me2 or H3K9me2 in cerebrum.In cerebellum,levels of all three H3 dimethylation markers were significantly decreased in mutant mice compared to WT mice.5.QPCR:In the cerebrum,mutant mice showed significantly down-regulation on the expression of genes including cabp7,kcng4,nxph4,rassf8 and up-regulation on genes including gabra3,scg2,robo2.In cerebellum,the RNA level of kcng4 was higher and RNA levels of cabp7 and nxph4 were lower in mutant mice compared to WT,and no difference was found on the expression of other genesbetween the two groups.Conclusions1.In this study,we established a mutant mouse model(Mecp2?AT-hook1/y)carrying deletion of eight conserved amino acids in AT-hook 1 motif by CRISPR/Cas9 technology.Acording to behavorioral assay,we found that the Mecp2?AT-hook1/y mutant male mice exhibited pronounced motor incoordination,cognitive deficit,anxiety-like behavior and reduction in pain sensitivity.These findings provide evidence that conserved domain of AT-hook 1 is critical region for the function of MeCP2 protein,suggesting that mutation in AT-hook 1 motif of MeCP2 might lead to several behavioral changes related to RTT or autism spectrum disorders.2.Through western blot analysis,we found Mecp2?AT-hook1/y mutant male mice changed levels of H3 dimethylation(H3K4me2 H3K9me2 H3K27me2)both in cerebrum and cerebellum.In addition,we found that altered gene expression pattern related to inhibitory neurons in cerebrum and cerebellum of Mecp2?AT-hook1/y mutant male mice by QPCR.Our studies provide evident clue that disruption of AT-hook 1 domain in MeCP2 may affect levels of H3 dimethylation,sequently change some genes RNA expression in vivo.These findings partly suggest the critical regions in or nearby AT-hook 1 motif for the function of MeCP2 through underling molecular mechanism.