Expression Of MicroRNA-134 And Limk1 In Brain Tissue Of FMR1 Knockout Mice

IntroductionFragile X syndrome is the most common form of single-gene inherited mental retardation. The clinical phenotype is characterized by mental retardation, cognitive deficits, seizure and behavioral dysfunction, autism, enlarged testes (macro orchidism) in post pubescent males and mildly abnormal facial features (a prominent jaw, high forehead and large ears). FXS is the result of massive CGG trinucleotide expansion in the 5'untranslated region of the fragile X mental retardation 1 gene (FMR1). The expansion mutation leads to abnormal hypermethylation which results in the transcriptional silencing of FMR1 and the loss of FMR1 product-- fragile X mental retardation protein (FMRP). A significant neuropathologic lesion associated with FXS is abnormally thin and tortilis dendritic immature spine. It has been identified that the dendritic spine is the main structure of postsynaptic membrane. The paramorphia of dendritic spine might involve in the impairment of synaptic plasticity which is possible pathogenesis of fragile X syndrome. Previous studies have indicated FMRP is a selective RNA-binding protein associated with ribosomes and down-regulate the expression of proteins related to development. It was reported that FMRP might contribute to the mature of miRNAs。The recent studies demonstrate that FMRP is one of component of RNA-induced silencing complex (RISC) that function silence gene. However, the molecular mechanism that the absence of FMRP causes a series of abnormal phenotypes is still unclear. It is identified that MiR-134 can affect dendritic spine development through down-regulating Limk1 mRNA expression. Taken above data together, it is speculated that absence of FMRP causes the dysfunction of miR-134, which might contribute to the paramorphia in FXS. The present study determines the expression of miR-134 and Limk1 in brain tissue of FMR1 knockout mice during the different development period, in order to explore the role of miR-134 and Limk1 in abnormal development of dendritic spine in FXS.Materials and MethodsFVB strain male mice, including FMR1 knockout (KO) and their wild type (WT) counterparts were used and were genotyped by PCR before experiments. They were housed under standard laboratory conditions with 19～21℃and with free access to mouse chow and water. Experimented animals were divided into 6 groups according to their genotype and age(n=11 for each group): the newborn KO and WT(KO0d and WT0d),the KO and WT mice of postnatal 2 week(sKO2w and WT2w) and the KO and WT mice of postnatal 6 weeks(KO6w and WT6w). Six mice were used to examine the expression of Limk1 by immunohistochemistry method; five mice were used to examine the transcriptional level of miR-134 by qRT-PCR.RESULT:1. The distribution of Limk1 expression in brain during the different development periodLimk1 immunoreactive fibers were abundantly found in brain regions in newborn mice, especially in cortex, hippocampus, thalamus opticus and cerebellum, in where few immunoreactive cells are detected. the strong immunoreactive cells become intensive in incertitude zone, ventral poster lateral nucleus of thalamus opticus, Purkinje's cells of cerebellum and auditory nucleus in the postnatal 2 and 6 weeks mice, but only sporadic weakly immunoreactive signal was observed in cerebral cortex and hippocampus, The distribution of Limk1 in each brain region has no significant difference between KO mice and WT mice. 2. The level of Limk1 expression in the brain of KO and WT miceThe mean optical density and positive cell count of Limk1 in hippocampus,brain cortex ,thalamus opticus and cerebellum of KO0d, KO2W and KO6Wgroup are significantly higher than the age-matched WT group (P<0.05).3. The level of miR-134 in the brain of WT and KO miceThe transcriptional level of miR-134 in the brain of KO mice have no significant difference when compared to the age-matched WT mice(P >0.05).4. The level of miR-134 in the brain during the development periodThe transcriptional level of miR-134 was significantly decreased in both 6 week KO and WT mice when compared with the newborn and 2 week same genotype mice (P<0.05),but no significant difference between 2 week and the newborn mice in same genotype.ConclusionThe expressions of Limk1 that is developmentally regulated showed obviously time series in the different brain regions of KO and WT mice. The transcriptional level of miR-134 in brain tissues maintains high level during the developmental stage of the nervous system(newborn ~2 weeks)and decreases in adult(6 weeks),which demonstrates that it may play a important role in regulating the development of nervous system.The increased Limk1 level in KO mice demonstrates that FMRP down-regulate the expressions of Limk1. The transcriptional level of miR-134 is unchanged in KO mice, which suggests that the increase of Limk1 level might be a result of dysfunction of miR-134 caused by the absence of FMRP. The increased Limk1 level might be involved in the abnormal development of dentritic spine in FXS.