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.     

Olfactory bulb mitral cell dendritic pruning abnormalities in a mouse model of the Fragile-X mental retardation syndrome: further support for FMRP's involvement in dendritic development

The Fragile-X mental retardation syndrome is the leading form of inherited mental retardation. Dendritic analysis in a mouse model (FraX) found abnormal pruning in somatosensory cortex. To further characterize dendritic abnormalities and assess their occurrence in other brain regions, we examined mitral cells in FraX mice olfactory bulbs. FraX mice exhibited dendritic abnormalities consistent with somatosensory cortex, suggesting that deficient pruning is found in multiple brain regions.     

The Fragile X mental retardation protein

The clinical features of the Fragile X mental retardation syndrome are linked to the absence of the set of protein isoforms, derived from alternative splicing of the Fragile X mental retardation gene 1 (FMR1), and collectively termed FMRP. FMRP is an RNA binding protein that is part of a ribonucleoprotein particle associated to actively translating polyribosomes, and which can shuttle between nucleus and cytoplasm. Two highly homologous human proteins, FXR1P and FXR2P, share the same domain structure as FMRP, and probably similar functions. The properties of FMRP suggested that it is involved in nuclear export, cytoplasmic transport, and/or translational control of target mRNAs. In particular, it may play a role in regulation of protein synthesis at postsynaptic sites of dendrites, and in maturation of dendritic spines. Efforts are underway to identify the putative specific mRNA targets of FMRP, and study the effect of FMRP absence on the corresponding proteins. Other approaches have led to the identification of proteins that interact with FMRP. Some of them discriminate between FMRP and the homologous FXR1/2P proteins, and may thus be important for defining unique functions of FMRP that are deficient in Fragile X patients. The physiological functions of FMRP are notably approached through the study of a FMR1 knock-out mouse model. The recent identification in Drosophila melanogaster of genes encoding homologs of FMRP/FXRP and of their interacting proteins, open the way to use of Drosophila genetics to study FMRP function.     

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.     

Fragile X mental retardation protein in plasticity and disease

Fragile X syndrome is the most common cause of mental retardation known to be inherited. The syndrome results from the suppressed expression of a single protein, the fragile X mental retardation protein (FMRP). Understanding the function and regulation of FMRP can, therefore, offer insights into both the pathophysiology of fragile X syndrome and the molecular mechanisms of learning and memory. We provide an overview of current concepts of how FMRP functions in the nervous system, with special emphasis on recent evidence that FMRP has a role in metabotropic glutamate receptor-activated protein translation and synaptic plasticity.     

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.     

Alterations in the auditory startle response in Fmr1 targeted mutant mouse models of fragile X syndrome

Fragile X syndrome results from inadequate production of the fragile X mental retardation protein (FMRP). Mice with a mutation targeted to the Fmr1 gene lack FMRP and thus are a valuable animal model for studying the behavioral and neural phenotype of this human disorder. Mice of two genetic backgrounds containing the Fmr1 mutation, C57BL/6J (C57-KO) and an F1 hybrid (C57BL/6J mutant x FVB/NJ; F1-KO) did not differ from control mice in behavior in the elevated plus maze or the open field. Both the C57-KO and F1-KO mice exhibited greater startle responses than normal mice to low intensity (80 dB) white noise bursts and decreased responses to high intensity (120 dB) white noise bursts. These behavioral alterations appear to be specific to the Fmr1 mutation since they are present on both genetic backgrounds. Furthermore, the mice lacking FMRP resemble individuals with fragile X syndrome in their increased sensitivity to low intensity auditory stimuli. These findings should prove useful in determining how the absence of FMRP alters the brain and behavior, and in testing potential treatments for fragile X syndrome.     

Modest alterations in patterns of motor neuron dendrite morphology in the Fmr1 knockout mouse model for fragile X

Fragile X, an inheritable form of mental retardation, is caused by the inactivation of a gene on the X chromosome, FMR1 which codes for an RNA binding protein, fragile X mental retardation protein. Loss of this protein is associated with reduced complexities of neuronal dendrites and alterations in spine morphology in a number of cortical brain regions, and these deficits may underlie the cognitive impairment observed in fragile X patients. Among the many symptoms of fragile X are altered motor functions, although the neuronal basis for these remains unclear. In this study we investigated whether knockout of Fmr1 in the mouse model of fragile X altered dendrite morphology in developing spinal cord motor neurons. We find that Fmr1 knockout leads to modest alterations in the distribution of dendritic arbor across the span of the motor neuron dendritic tree in 2- and 4-week-old mice, compared to wild-type controls, consistent with slower rates of extension and abnormal pruning of intermediate dendritic segments. These studies suggest that some motor deficits in fragile X patients may be due to abnormal maturation of dendritic patterning within spinal motor neurons, and suggest that strategies aimed at preventing motor impairment in fragile X patients may be targeted at motor functions during early development.     

The mGluR theory of fragile X mental retardation

Many of the diverse functional consequences of activating group 1 metabotropic glutamate receptors require translation of pre-existing mRNA near synapses. One of these consequences is long-term depression (LTD) of transmission at hippocampal synapses. Loss of fragile X mental retardation protein (FMRP), the defect responsible for fragile X syndrome in humans, increases LTD in mouse hippocampus. This finding is consistent with the growing evidence that FMRP normally functions as a repressor of translation of specific mRNAs. Here we present a theory that can account for diverse neurological and psychiatric aspects of fragile X syndrome, based on the assumption that many of the protein-synthesis-dependent functions of metabotropic receptors are exaggerated in fragile X syndrome. The theory suggests new directions for basic research as well as novel therapeutic approaches for the treatment of humans with fragile X, the most frequent inherited cause of mental retardation and an identified cause of autism.     

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.     

Intense and specialized dendritic localization of the fragile X mental retardation protein in binaural brainstem neurons: A comparative study in the alligator,chicken, gerbil,and human

Neuronal dendrites are structurally and functionally dynamic in response to changes in afferent activity. The fragile X mental retardation protein (FMRP) is an mRNA binding protein that regulates activity‐dependent protein synthesis and morphological dynamics of dendrites. Loss and abnormal expression of FMRP occur in fragile X syndrome (FXS) and some forms of autism spectrum disorders. To provide further understanding of how FMRP signaling regulates dendritic dynamics, we examined dendritic expression and localization of FMRP in the reptilian and avian nucleus laminaris (NL) and its mammalian analogue, the medial superior olive (MSO), in rodents and humans. NL/MSO neurons are specialized for temporal processing of low‐frequency sounds for binaural hearing, which is impaired in FXS. Protein BLAST analyses first demonstrate that the FMRP amino acid sequences in the alligator and chicken are highly similar to human FMRP with identical mRNA‐binding and phosphorylation sites, suggesting that FMRP functions similarly across vertebrates. Immunocytochemistry further reveals that NL/MSO neurons have very high levels of dendritic FMRP in low‐frequency hearing vertebrates including alligator, chicken, gerbil, and human. Remarkably, dendritic FMRP in NL/MSO neurons often accumulates at branch points and enlarged distal tips, loci known to be critical for branch‐specific dendritic arbor dynamics. These observations support an important role for FMRP in regulating dendritic properties of binaural neurons that are essential for low‐frequency sound localization and auditory scene segregation, and support the relevance of studying this regulation in nonhuman vertebrates that use low frequencies in order to further understand human auditory processing disorders. J. Comp. Neurol. 522:2107–2128, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

A neuropsychological investigation of male premutation carriers of fragile X syndrome

It is currently thought that fragile X syndrome (FraX; the most common inherited form of learning disability) results from having more than 200 cytosine–guanine–guanine (CGG) trinucleotide repeats, with consequent methylation of the fragile X mental retardation (FMR1) gene and loss of FMR1 protein (FMRP). It was also considered that premutation carriers (with 55–200 CGG repeats) are unaffected, although a tremor/ataxia syndrome has recently been described in older adult male carriers. We reported that premutation expansion of CGG trinucleotide repeats affects brain anatomy, which, together with other studies, indicates that the molecular model for FraX needs modification. However, there are few studies on the cognitive ability of adult male premutation carriers. Thus, we selected 20 male premutation carriers on the basis of their genetic phenotype, and compared them to 20 male controls matched on age, IQ and handedness. We investigated intellectual functioning, executive function, memory, attention, visual and spatial perception, and language and pragmatics. The premutation carriers had significant impairments on tests of executive function (Verbal Fluency, Trail Making Test and Tower of London) and memory (Names sub-test of the Doors and People, Verbal Paired Associates Immediate Recall and Visual Paired Associates Delayed Recall sub-tests of the WMS-R, and Category Fluency Test for natural kinds). We therefore suggest that CGG trinucleotide repeats in the premutation range affect specific neuronal circuits that are concordant with specific neuropsychological deficits; and that these deficits reflect an emerging neuropsychological phenotype of premutation FraX.     

Autism, Alzheimer disease, and fragile X: APP, FMRP, and mGluR5 are molecular links

The present review highlights an association between autism, Alzheimer disease (AD), and fragile X syndrome (FXS). We propose a conceptual framework involving the amyloid-β peptide (Aβ), Aβ precursor protein (APP), and fragile X mental retardation protein (FMRP) based on experimental evidence. The anabolic (growth-promoting) effect of the secreted α form of the amyloid-β precursor protein (sAPPα) may contribute to the state of brain overgrowth implicated in autism and FXS. Our previous report demonstrated that higher plasma sAPPα levels associate with more severe symptoms of autism, including aggression. This molecular effect could contribute to intellectual disability due to repression of cell-cell adhesion, promotion of dense, long, thin dendritic spines, and the potential for disorganized brain structure as a result of disrupted neurogenesis and migration. At the molecular level, APP and FMRP are linked via the metabotropic glutamate receptor 5 (mGluR5). Specifically, mGluR5 activation releases FMRP repression of APP mRNA translation and stimulates sAPP secretion. The relatively lower sAPPα level in AD may contribute to AD symptoms that significantly contrast with those of FXS and autism. Low sAPPα and production of insoluble Aβ would favor a degenerative process, with the brain atrophy seen in AD. Treatment with mGluR antagonists may help repress APP mRNA translation and reduce secretion of sAPP in FXS and perhaps autism.     

Reduced cortical synaptic plasticity and GluR1 expression associated with fragile X mental retardation protein deficiency

Lack of expression of the fragile X mental retardation protein (FMRP), due to silencing of the FMR1 gene, causes the Fragile X syndrome. Although FMRP was characterized previously to be an RNA binding protein, little is known about its function or the mechanisms underlying the Fragile X syndrome. Here we report that the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit, GluR1, was decreased in the cortical synapses, but not in the hippocampus or cerebellum, of FMR1 gene knockout mice. Reduced long-term potentiation (LTP) was also found in the cortex but not in the hippocampus. Another RNA binding protein, FXR; the N-methyl-D-aspartate receptor subunit, NR2; and other learning-related proteins including c-fos, synapsin, myelin proteolipid protein, and cAMP response element binding protein were not different between FMR1 gene knockout and wild-type mice. These findings suggest that the depressed cortical GluR1 expression and LTP associated with FMRP deficiency could contribute to the Fragile X phenotype.