Association Of Up-regulated Ras/Raf/ERK1/2Signaling With Autism

BACKGROUND&OBJECTIVEAutism is seriously neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors,hardly diagnosis and incurable. ASD disproportionately affect male children,about4males per1female.The reported prevalence of autism spectrum disorders (ASD) in continental Europe, the UK and the US has increased5-10fold over the past20years.Although medication prescriptions are common, there is little evidence they do any good for ASD. Behavioral treatment is the most effective approach to improving social integration and language in youths with ASD. The costs of supporting children with ASDs were estimated to be pound2.7billion each year. The lifetime per capita incremental societal cost of autism is$3.2million in USA. Individuals with autism are large financial burden affecting not only those families but also potentially society in general,so ASD become a public health problem.The etiology of ASD is still not precisely defined, growing scientific evidence shows it is complex and encompasses the roles of genes, the environment Abnormalities of chromosomes are thought to account for10%to20%of autism cases.The most promising genes are exclusively involved in the physiological and pathophysiological processes of neurogenesis, neuronal migration, synaptogenesis, such as NLGNS (NLGN1, NLGN3, and NLGN4X), MECP2、FMR1、TSC1/2、 SHANK3、CNTNAP2. It is suggest that susceptibility to ASD has moderate genetic heritability and a substantial shared with environmental component. The individual compounds that contributed most to these associations included mercury, cadmium, nickel, trichloroethylene, and vinyl chloride,especially during prenatal and perinatal period. The G X E interactions either modulate the degree of dysfunction of the core clinical features of ASD or have an impact on neurobiological circuits that are at greater risk for dysfunction, because genetic vulnerability pushes the system closer to disorder threshold.New studies have identified individuals with microdeletions encompassing the MAPK1gene that encodes ERK2in distal chromosome22q11who exhibit cognitive deficits and features of the DiGeorge syndrome (DGS) spectrum.Studies reported that a deletion of a locus on chromosome16, including the MAPK3gene that encodes ERK1, is associated with autism. A number of recent studies have demonstrated a death-promoting role for Ras/Raf/Erkl/2in both in vitro and in vivo models of neuronal death. A possible association between abnormal neural cell death and autism. Our laboratory showed that Ras/Raf/Erk1/2proteins are increased in the frontal cortex of autistic brains. These findings suggest a possible association between Ras/Raf/Erkl/2and autism.The inbred mouse strain BTBR T+tf/J (BTBR) incorporates multiple behavioral phenotypes relevant to all three diagnostic symptoms of autism.:low social interactions, reduced vocalizations in social settings, and high levels of repetitive self-grooming, is currently used as a model for understanding mechanisms that may be responsible for the pathogenesis of autism.U0126, a mitogen-activated protein kinase kinase (Mek)1/2inhibitor, inhibited migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP.Now it is widely used in vivo and in vitro.Taken together, we hypothesize that Ras/Raf/Erkl/2signaling could be abnormally regulated in the brains of BTBR mice that have been used as a promising animal model for autism research. In this study, we examined the entire Ras/Raf/Erkl/2signaling pathway in the brain tissues of different age BTBR mice and age-matched B6control mice, detected when Ras/Raf/Erkl/2proteins begin to increase and normal.And then used Mekl/2inhibitor to reduced the expression of phospho-Mekl/2and phosphor-Erkl/2, detected the behavioral change of mice brains.METHODS1. Detected expression of Ras/Raf/Erkl/2cascade in the brain tissues of BTBR mice during different age period by Western Blot.Seperated brain from BTBR mice during newborn,3weeks,6weeks old,and age-matched C57BL/6mice as control, used Western Blot to detect the expression of P-A-Raf, P-B-Raf. P-C-Raf、P-Erk1/2、P-Mek1/2after protein homogenate.2.Treat BTBR mice by Mekl/2inhibitorFrom3days old,injected BTBR mice and age-mateched B6mice with Mekl/2inhibitor,U0126,intraperitoneally,once a day,continue for3weeks. Western Blot to detect the expression of P-Erk1/2、P-Mekl/2after protein homogenate.3.Behavioral test after treatment with Mekl/2inhibitor.In order from least stressful to most stressful,all the behavior tests running in order: Tests should have at least2-3days in between each one.4.1mmuflurescence detected the expression of VGLUT1/VGAT,Map2and PSD in nerve system after U0126injection.5.DiI labeling detected the number of mature dendritic spines in cortex after U0126injection.STATISTICAL ANALYSISExpresssion of protein was analysised by independent sample T test.Different time stayed in three chambers was analysised by Repeated Measures of ANOVA,the time of grooming and sniff was analysised by ANOVA. Open arm entry in EPM was analysised by ANOVA.Times stayed in light and dark boxes was analysised by ANOVA.Rearing time was analysised by ANOVA.Number of unburied marble was analysised by ANOVA. Expressionsof VGLUT1/VGAT,Map2and PSD were analysis by ANOVA.Number of mature spines was analysis by ANOVA.LSD was used to analysis difference between two groups when there was significant difference between three groups.All statistical evaluations were performed using the SPSS13.0statistical software package.P<0.05was significant difference.RESULT1. Abnormal expressions of Ras/Raf/Erkl/2pathway proteins in BTBR mcie.Expressions of Ras/Raf/Erk1/2pathway proteins upregulate in newborn BTBR mice(P-B-Raf:F=7.192, P<0.001; P-C-Raf:F=2.661, P=0.024; P-Mek1/2F=8.912, P<0.001,P-Erk1/2F=3.733, P=0.004;. There are no difference between BTBR and B6mice during3weeks old (P-B-Raf:F=1.663, P=0.127; P-C-Raf:F=1.019, P=0.332; P-Mek1/2:F=2.188, P=0.053; P-Erkl/2:F=1.341, P=0.210) till adult (P-B-Raf:F=2.127, P=0.059; P-C-Raf:F=0.394, P=0.702; P-Mek1/2:F=0.961, P=0.359; P-Erk1/2F=0.916, P=0.381)2.Behavior change of BTBR mice after treatment with Mekl/2inhibitor.B6and BTBR used U0126treated showed more time to stay with stranger rather than novel object(F=3.941,P=0.010; F=2.146,P=0.043).And there is significant difference between three groups with immobile time in open field test,post-hoc analysis showed difference between BTBR U0126AND BTBR DMSO groups.But other behavior tests showed no different between groups.3.Mekl/2inhibitor regulated expression of VGLUT1,VGAT,Map2and PSD in neutral systemMekl/2inhibitor down-regulated ration of VGLUT1and VGAT in CA1of hippocampus.Mekl/2inhibitor up-regulated expression of Map2in CA1and CA3of hippocampus.Mekl/2inhibitor up-regulated expression of PSD in CA1of hippocampus and cortex.4.Mekl/2inhibitor up-regulated formation of mature dendritic spines in cortex.CONCLUSION1.Ras/Raf/Erkl/2pathway upregulated in BTBR mice from newborn till three weeks old.2.Mek1/2inhibitor could downregulate P-Mek1/2and P-Erk1/2,improved social ability and activity of BTBR mice,but no effect on anxiety,learning and memory,repetitive behaviors.3.Mekl/2inhibitor down-regulated ration of VGLUT1and VGAT in CA1of hippocampus.Mekl/2inhibitor up-regulated expression of Map2in CA1and CA3of hippocampus.Mekl/2inhibitor up-regulated expression of PSD in CA1of hippocampus and cortex.4.Mekl/2inhibitor up-regulated formation of mature dendritic spines in cortex.