Fragile X mice: reduced long-term potentiation and N-Methyl-D-Aspartate receptor-mediated neurotransmission in dentate gyrus

Fragile X syndrome (FXS) is a monogenic mental retardation syndrome that frequently includes autism. The Fmr1-knockout (Fmr1-KO) mouse, like FXS-affected individuals, lacks the fragile X mental retardation protein (FMRP) and models autism as well as FXS. Limited human data and several mouse models have implicated the hippocampal dentate gyrus (DG) in autism. We therefore investigated whether the Fmr1-KO mouse exhibited functional changes in DG. We found diminished medial perforant path-granule cell long-term potentiation (LTP), complementing previous investigations of synaptic plasticity in Fmr1-KO demonstrating impaired LTP in CA1, neocortex, and amygdala and exaggerated long-term depression in CA1. We also found that peak amplitude of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) was smaller in Fmr1-KO than control. AMPA receptor-mediated EPSCs were comparable in the two strains, yielding a lower NMDA/AMPA ratio in Fmr1-KO mice and suggesting one mechanism by which absent FMRP might contribute to diminished LTP. The clinical hallmarks of autism include both excessive adherence to patterns and impaired detection of socially important patterns. The DG has a putative role in pattern separation (for time, space, and features) that has been attributed to granule cell number, firing rates, adult neurogenesis, and even perforant path LTP. DG also contributes to pattern completion in CA3 via its mossy fiber efferents, whose terminals include abundant FMRP in "fragile X granules." Together with the present data, these observations suggest that DG is a candidate region for further investigation in autism and that the Fmr1-KO model may be particularly apt.     

Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines

Fragile X syndrome (FXS) is a common form of mental retardation caused by the absence of functional fragile X mental retardation protein (FMRP). FXS is associated with elevated density and length of dendritic spines, as well as an immature-appearing distribution profile of spine morphologies in the neocortex. Mice that lack FMRP (Fmr1 knockout mice) exhibit a similar phenotype in the neocortex, suggesting that FMRP is important for dendritic spine maturation and pruning. Examination of Golgi-stained pyramidal cells in hippocampal subfield CA1 of adult Fmr1 knockout mice reveals longer spines than controls and a morphology profile that, while essentially opposite of that described in the Fmr1 knockout neocortex, appears similarly immature. This finding strongly suggests that FMRP is required for the processes of spine maturation and pruning in multiple brain regions and that the specific pathology depends on the cellular context.     

Evidence for a fragile X mental retardation protein-mediated translational switch in metabotropic glutamate receptor-triggered Arc translation and long-term depression

Group 1 metabotropic glutamate receptor (mGluR)-stimulated protein synthesis and long-term synaptic depression (mGluR-LTD) are altered in the mouse model of fragile X syndrome, Fmr1 knock-out (KO) mice. Fmr1 encodes fragile X mental retardation protein (FMRP), a dendritic RNA binding protein that functions, in part, as a translational suppressor. It is unknown whether and how FMRP acutely regulates LTD and/or the rapid synthesis of new proteins required for LTD, such as the activity-regulated cytoskeletal-associated protein (Arc). The protein phosphatase PP2A dephosphorylates FMRP, which contributes to translational activation of some target mRNAs. Here, we report that PP2A and dephosphorylation of FMRP at S500 are required for an mGluR-induced, rapid (5 min) increase in dendritic Arc protein and LTD in rat and mouse hippocampal neurons. In Fmr1 KO neurons, basal, dendritic Arc protein levels and mGluR-LTD are enhanced, but mGluR-triggered Arc synthesis is absent. Lentiviral-mediated expression of wild-type FMRP in Fmr1 KO neurons suppresses basal dendritic Arc levels and mGluR-LTD, and restores rapid mGluR-triggered Arc synthesis. A phosphomimic of FMRP (S500D) suppresses steady-state dendritic Arc levels but does not rescue mGluR-induced Arc synthesis. A dephosphomimic of FMRP (S500A) neither suppresses dendritic Arc nor supports mGluR-induced Arc synthesis. Accordingly, S500D-FMRP expression in Fmr1 KO neurons suppresses mGluR-LTD, whereas S500A-FMRP has no effect. These data support a model in which phosphorylated FMRP functions to suppress steady-state translation of Arc and LTD. Upon mGluR activation of PP2A, FMRP is rapidly dephosphorylated, which contributes to rapid new synthesis of Arc and mGluR-LTD.     

Lithium reverses increased rates of cerebral protein synthesis in a mouse model of fragile X syndrome

Individuals with fragile X syndrome (FXS), an inherited form of cognitive disability, have a wide range of symptoms including hyperactivity, autistic behavior, seizures and learning deficits. FXS is caused by silencing of FMR1 and the consequent absence of fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that associates with polyribosomes and negatively regulates translation. In a previous study of a mouse model of FXS (Fmr1 knockout (KO)) we demonstrated that in vivo rates of cerebral protein synthesis (rCPS) were elevated in selective brain regions suggesting that the absence of FMRP in FXS may result in dysregulation of cerebral protein synthesis. Lithium, a drug used clinically to treat bipolar disorder, has been used to improve mood dysregulation in individuals with FXS. We reported previously that in the Fmr1 KO mouse chronic dietary lithium treatment reversed or ameliorated both behavioral and morphological abnormalities. Herein we report that chronic dietary lithium treatment reversed the increased rCPS in Fmr1 KO mice with little effect on wild type mice. We also report our results of analyses of key signaling molecules involved in regulation of mRNA translation. Our analyses indicate that neither effects on the PI3K/Akt nor the MAPK/ERK 1/2 pathway fully account for the effects of lithium treatment on rCPS. Collectively our findings and those from other laboratories on the efficacy of lithium treatment in animal models support further studies in patients with FXS.     

A mouse model of the fragile X premutation: effects on behavior, dendrite morphology, and regional rates of cerebral protein synthesis

Carriers of FMR1 premutation alleles have 55-200 CGG repeats in the 5' untranslated region of the gene. These individuals are at risk for fragile X associated primary ovarian insufficiency (females) and, in late life, fragile X associated tremor and ataxia syndrome (males, and to a lesser extent, females). Premutation carrier status can also be associated with autism spectrum disorder, attention deficit hyperactivity disorder, and some cognitive deficits. In premutation carriers, FMR1 mRNA levels are often higher than those with normal sized alleles. In contrast, in subjects with full mutation alleles, (>200 repeats) the FMR1 gene is silenced and FMR1 mRNA and its product, FMRP, are absent. We have studied a male knock-in (KI) mouse model of the fragile X premutation (120-140 repeats) during young adulthood. In comparison to wild type, KI mice were hyperactive, exhibited less anxiety in both the open field and the elevated zero maze, were impaired on the passive avoidance test, and showed some subtle deficits on a test of social interaction. Motor learning as assessed by the rotarod test was normal. Dendritic arbors were less complex and spine densities and lengths increased in medial prefrontal cortex, basal lateral amygdala, and hippocampus compared with wild type. Regional rates of cerebral protein synthesis measured in vivo in KI mice were increased. KI mice also had elevated levels of Fmr1 mRNA and decreased levels of FMRP. Our results highlight similarities in phenotype between KI and Fmr1 knockout mice and suggest that the decreased concentration of FMRP contributes to the phenotype in young adult KI mice.     

Rgs4 mRNA expression is decreased in the brain of Fmr1 knockout mouse

In the Fmr1 knockout mice, a model for fragile X mental retardation syndrome, the levels of regulator of G-protein signaling (Rgs) 4 but not Rgs2 mRNA were considerably reduced (65% from control) in the cerebral cortex and hippocampal CA1 region. The expression of Rgs4 was normal in animals lacking a related protein, FXR2P, indicating that the decrease in Rgs4 expression was specific for the absence of FMRP, and suggests a role for FMRP in G-protein signaling.     

Neuropeptide Release Is Impaired in a Mouse Model of Fragile X Mental Retardation Syndrome

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Social behavior deficits in the Fmr1 mutant mouse

Mice exhibiting deficits in social behavior may provide valuable models for autistic-like behavioral problems. We tested social interactions in male mice from three inbred strains: C57BL/6J (B6), BALB/cJ (C) and DBA/2J (D2). All three strains showed gradual habituation of the number of social interactions with an ovariectomized female over four subsequent 2min sessions, returning to initial levels when presented with another stimulus mouse. Next, we studied males with a knockout mutation in the Fmr1 gene on a B6 background. KO animals showed strongly reduced levels of social interaction, which were about similar as those of habituated controls. This social behavior deficit suggests that Fmr1 KO mice could possibly be used as models for autistic behaviors.     

Stress induced changes in cortical and hypothalamic c-fos expression are altered in fragile X mutant mice

Fragile X (FraX) syndrome is characterized by mental retardation and a behavioral phenotype that includes stress-related behaviors. Recently, FraX children were shown to have elevated glucocorticoid hormones under basal conditions and an exaggerated hormonal response to stress. In the present study, fragile X mental retardation 1-knockout (Fmr1-KO) and wild-type (WT) mice were subjected to immobilization stress for 30 min or 2 h, killed with paired controls, and the hippocampus, neocortex, and hypothalamic paraventricular nucleus (PVN) assessed by in situ hybridization for effects on c-fos mRNA. The main effect of stress in hippocampus was a reduction in mRNA levels within CA3-CA1 pyramidal cells in both genotypes. Stress significantly reduced CA1 c-fos mRNA in Fmr1-KOs at 30 min (-41%) and 2 h (-57%), whereas in WTs levels were significantly reduced only at 2 h (-57%). In neocortex, 30 min stress significantly increased c-fos mRNA in Fmr1-KOs only (+53%); however, by 2 h levels were reduced in both genotypes versus respective controls. In the paraventricular nucleus, c-fos mRNA levels were significantly, and equally, increased in both genotypes at 30 min. However, at 2 h, mRNA levels were still elevated in the Fmr1-KOs, whereas they had returned to control values in the WTs. Finally, immobilization stress significantly increased serum corticosterone levels in both genotypes at 30 min and 2 h, with Fmr1-KOs exhibiting greater levels than WTs; levels were statistically different at 2 h. These data indicate a greater response to stress in FraX mutants than in WTs, and further support the hypothesis of a dysregulated hypothalamic-pituitary-adrenal (HPA) axis in FraX syndrome.     

Rescue of behavioral phenotype and neuronal protrusion morphology in Fmr1 KO mice

Lack of fragile X mental retardation protein (FMRP) causes Fragile X Syndrome, the most common form of inherited mental retardation. FMRP is an RNA-binding protein and is a component of messenger ribonucleoprotein complexes, associated with brain polyribosomes, including dendritic polysomes. FMRP is therefore thought to be involved in translational control of specific mRNAs at synaptic sites. In mice lacking FMRP, protein synthesis-dependent synaptic plasticity is altered and structural malformations of dendritic protrusions occur. One hypothesized cause of the disease mechanism is based on exaggerated group I mGluR receptor activation. In this study, we examined the effect of the mGluR5 antagonist MPEP on Fragile X related behavior in Fmr1 KO mice. Our results demonstrate a clear defect in prepulse inhibition of startle in Fmr1 KO mice, that could be rescued by MPEP. Moreover, we show for the first time a structural rescue of Fragile X related protrusion morphology with two independent mGluR5 antagonists.     

AFQ056, a new mGluR5 antagonist for treatment of fragile X syndrome

Fragile X syndrome, the most common form of inherited intellectual disability, is caused by a lack of FMRP, which is the product of the Fmr1 gene. FMRP is an RNA-binding protein and a component of RNA-granules found in the dendrites of neurons. At the synapse, FMRP is involved in regulation of translation of specific target mRNAs upon stimulation of mGluR5 receptors. In this study, we test the effects of a new mGluR5 antagonist (AFQ056) on the prepulse inhibition of startle response in mice. We show that Fmr1 KO mice have a deficit in inhibition of the startle response after a prepulse and that AFQ056 can rescue this phenotype. We also studied the effect of AFQ056 on cultured Fmr1 KO hippocampal neurons; untreated neurons showed elongated spines and treatment resulted in shortened spines. These results suggest that AFQ056 might be a potent mGluR5 antagonist to rescue various aspects of the fragile X phenotype.     

The fragile X-cerebellum connection

Fragile X syndrome (FXS) is an inherited form of mental retardation that results from the loss of function of the fragile X mental retardation protein (FMRP). A recent report demonstrated alterations in the structure and plasticity of synapses on cerebellar Purkinje cells in Fmr1 knockout mice, which are a model of FXS. These synaptic alterations are associated with deficits in the cerebellar learning both in the mice and humans with FXS. This work forges an important link between the FMR1 gene, altered synaptic plasticity in the cerebellum and mental retardation.     

Quantitative proteomics of forebrain subcellular fractions in fragile X mental retardation 1 knockout mice following acute treatment with 2‐Methyl‐6‐(phenylethynyl)pyridine: Relevance to developmental study of schizophrenia

The fragile X mental retardation 1 knockout (Fmr1 KO) mouse replicates behavioral deficits associated with autism, fragile X syndrome, and schizophrenia. Less is known whether protein expression changes are consistent with findings in subjects with schizophrenia. In the current study, we used liquid chromatography tandem mass spectrometry (LC‐MS/MS) proteomics to determine the protein expression of four subcellular fractions in the forebrains of Fmr1 KO mice vs. C57BL/6 J mice and the effect of a negative allosteric modulator of mGluR5—2‐Methyl‐6‐(phenylethynyl)pyridine (MPEP)—on protein expression. Strain‐ and treatment‐specific differential expression of proteins was observed, many of which have previously been observed in the brains of subjects with schizophrenia. Western blotting verified the direction and magnitude of change for several proteins in different subcellular fractions as follows: neurofilament light protein (NEFL) and 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterase (CNP) in the total homogenate; heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC) and heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) in the nuclear fraction; excitatory amino acid transporter 2 (EAAT2) and ras‐related protein rab 3a (RAB3A) in the synaptic fraction; and ras‐related protein rab 35 (RAB35) and neuromodulin (GAP43) in the rough endoplasmic reticulum fraction. Individuals with FXS do not display symptoms of schizophrenia. However, the biomarkers that have been identified suggest that the Fmr1 KO model could potentially be useful in the study of schizophrenia.     

Fierce: a new mouse deletion of Nr2e1; violent behaviour and ocular abnormalities are background-dependent

A new spontaneous mouse mutation named fierce (frc) is deleted for the nuclear receptor Nr2e1 gene (also known as Tlx, mouse homolog of Drosophila tailless). The fierce mutation is genetically and phenotypically similar to Nr2e1 targeted mutations previously studied on segregating genetic backgrounds. However, we have characterized the fierce brain, eye, and behavioural phenotypes on three defined genetic backgrounds (C57BL/6J, 129P3/JEms, and B6129F1). The data revealed many novel and background-dependent phenotypic characteristics. Whereas abnormalities in brain development, hypoplasia of cerebrum and olfactory lobes, were consistent on all three backgrounds, our novel finding of enlarged ventricles in 100% and overt hydrocephalus in up to 30% of fierce mice were unique to the C57BL/6J background. Developmental eye abnormalities were also background-dependent with B6129F1-frc mice having less severe thinning of optic layers and less affected electroretinogram responses. Impaired regression of hyaloid vessels was observed in all backgrounds. Furthermore, retinal vessels were deficient in size and number in 129P3/JEms-frc and B6129F1-frc mice but almost entirely absent in C57BL/6J-frc mice. We present the first standardized behavioural tests conducted on Nr2e1 mutant mice and show that C57BL/6J-frc and B6129F1-frc mice have deficits in sensorimotor assays and are hyperaggressive in both sexes and backgrounds. However, C57BL/6J-frc mice were significantly more aggressive than B6129F1-frc mice. Overall, this extensive characterization of the fierce mutation is essential to its application for the study of behavioural, and brain and eye developmental disorders. In addition, the background-dependent differences revealed will enable the identification of important genetic modifiers.     

微管相关蛋白1B在Fmr1基因敲除小鼠脑组织内的表达变化(英文)

目的探讨脆性X智能低下蛋白(fragile X mental retardation protein,FMRP)对微管相关蛋白1B(microtubuleassociated protein 1B,MAP1B)是否具有调控作用。方法应用免疫组化、免疫印记和原位杂交的方法,对1周龄和6周龄的Fmr1基因敲除型(KO)和同龄野生型(WT)小鼠脑组织MAP1B及MAP1B mRNA进行分析。结果免疫组化的结果显示 :6周龄KO小鼠各个脑区MAP1B的平均光密度值(MOD)值均显著低于同龄WT小鼠(P < 0.05),1周龄KO小鼠仅在小脑和海马显著降低(P < 0.01) ;各脑区MAP1B的MOD值在6周龄小鼠均比同基因型的1周龄小鼠显著降低(P < 0.05)。免疫印记和原位杂交结果分别显示MAP1B及MAP1B mRNA在KO小鼠的海马组织均显著降低(P <0.05)。结论MAP1B和MAP1B mRNA在Fmr1基因敲除小鼠脑组织的表达均显著减少,提示FMRP 可能正性调节MAP1B的表达。