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Fragile X Messenger Ribonucleoprotein (FMRP) is necessary for experience-dependent, developmental synapse elimination and the loss of this process may underlie the excess dendritic spines and hyperconnectivity of cortical neurons in Fragile X Syndrome, a common inherited form of intellectual disability and autism. Little is known of the signaling pathways that regulate synapse elimination and if or how FMRP is regulated during this process. We have characterized a model of synapse elimination in CA1 neurons of organotypic hippocampal slice cultures that is induced by expression of the active transcription factor Myocyte Enhancer Factor 2 (MEF2) and relies on postsynaptic FMRP. MEF2-induced synapse elimination is deficient in Fmr1 KO CA1 neurons, and is rescued by acute (24 h), postsynaptic and cell autonomous reexpression of FMRP in CA1 neurons. FMRP is an RNA binding protein that suppresses mRNA translation. Derepression is induced by posttranslational mechanisms downstream of metabotropic glutamate receptor signaling. Dephosphorylation of FMRP at S499 triggers ubiquitination and degradation of FMRP which then relieves translation suppression and promotes synthesis of proteins encoded by target mRNAs. Whether this mechanism functions in synapse elimination is not known. Here we demonstrate that phosphorylation and dephosphorylation of FMRP at S499 are both necessary for synapse elimination as well as interaction of FMRP with its E3 ligase for FMRP, APC/Cdh1. Using a bimolecular ubiquitin-mediated fluorescence complementation (UbFC) assay, we demonstrate that MEF2 promotes ubiquitination of FMRP in CA1 neurons that relies on activity and interaction with APC/Cdh1. Our results suggest a model where MEF2 regulates posttranslational modifications of FMRP via APC/Cdh1 to regulate translation of proteins necessary for synapse elimination., (Copyright © 2023. Published by Elsevier Inc.)

The ubiquitination/proteasome system is important for the spatiotemporal control of protein synthesis and degradation at synapses, while dysregulation may underlie autism spectrum disorders (ASDs). However, methods allowing direct visualization of the subcellular localization and temporal dynamics of protein ubiquitination are lacking. Here we report the development of Single-Molecule Ubiquitin Mediated Fluorescence Complementation (SM-UbFC) as a method to visualize and quantify the dynamics of protein ubiquitination in dendrites of live neurons in culture. Using SM-UbFC, we demonstrate that the rate of PSD-95 ubiquitination is elevated in dendrites of FMR1 KO neurons compared with wild-type controls. We further demonstrate the rapid ubiquitination of the fragile X messenger ribonucleoprotein, FMRP, and the AMPA receptor subunit, GluA1, which are known to be key events in the regulation of synaptic protein synthesis and plasticity. SM-UbFC will be useful for future studies on the regulation of synaptic protein homeostasis., Competing Interests: Declaration of interests G.J.B. serves on advisory board of Cell Reports., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hyperexcitability is a shared neurophysiological phenotype across various genetic neurodevelopmental disorders, including Fragile X syndrome (FXS). Several patient symptoms are associated with hyperexcitability, but a puzzling feature is that their onset is often delayed until their second and third year of life. It remains unclear how and why hyperexcitability emerges in neurodevelopmental disorders. FXS is caused by the loss of FMRP, an RNA-binding protein which has many critical roles including protein synthesis-dependent and independent regulation of ion channels and receptors, as well as global regulation of protein synthesis. Here, we discussed recent literature uncovering novel mechanisms that may drive the progressive onset of hyperexcitability in the FXS brain. We discussed in detail how recent publications have highlighted defects in homeostatic plasticity, providing new insight on the FXS brain and suggest pharmacotherapeutic strategies in FXS and other neurodevelopmental disorders.

The RNA binding protein FMRP regulates the synthesis of synaptic and nuclear proteins within different compartments of a neuron., Competing Interests: YY, GB No competing interests declared, (© 2022, Yoon and Bassell.)

Fragile X syndrome (FXS) is caused by the loss of fragile X mental retardation protein (FMRP), an RNA-binding protein that can regulate the translation of specific mRNAs. In this study, we developed an FXS human forebrain organoid model and observed that the loss of FMRP led to dysregulated neurogenesis, neuronal maturation and neuronal excitability. Bulk and single-cell gene expression analyses of FXS forebrain organoids revealed that the loss of FMRP altered gene expression in a cell-type-specific manner. The developmental deficits in FXS forebrain organoids could be rescued by inhibiting the phosphoinositide 3-kinase pathway but not the metabotropic glutamate pathway disrupted in the FXS mouse model. We identified a large number of human-specific mRNAs bound by FMRP. One of these human-specific FMRP targets, CHD2, contributed to the altered gene expression in FXS organoids. Collectively, our study revealed molecular, cellular and electrophysiological abnormalities associated with the loss of FMRP during human brain development., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Transcriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to the loss of the RNA-binding protein FMRP. In addition to regulating mRNA translation and protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether loss of FMRP-mediated translational control is related to impaired cell fate specification in the developing human brain remains unknown. Here, we use human patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells and organoids to model neurogenesis in FXS. We developed a high-throughput, in vitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development. Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Repeat-associated non-AUG-initiated translation of expanded CGG repeats (CGG RAN) from the FMR1 5'-leader produces toxic proteins that contribute to neurodegeneration in fragile X-associated tremor/ataxia syndrome. Here we describe how unexpanded CGG repeats and their translation play conserved roles in regulating fragile X protein (FMRP) synthesis. In neurons, CGG RAN acts as an inhibitory upstream open reading frame to suppress basal FMRP production. Activation of mGluR5 receptors enhances FMRP synthesis. This enhancement requires both the CGG repeat and CGG RAN initiation sites. Using non-cleaving antisense oligonucleotides (ASOs), we selectively blocked CGG RAN. This ASO blockade enhanced endogenous FMRP expression in human neurons. In human and rodent neurons, CGG RAN-blocking ASOs suppressed repeat toxicity and prolonged survival. These findings delineate a native function for CGG repeats and RAN translation in regulating basal and activity-dependent FMRP synthesis, and they demonstrate the therapeutic potential of modulating CGG RAN translation in fragile X-associated disorders.

Cortical hyperexcitability is a hallmark of fragile X syndrome (FXS). In the Fmr1 knockout (KO) mouse model of FXS, cortical hyperexcitability is linked to sensory hypersensitivity and seizure susceptibility. It remains unclear why homeostatic mechanisms fail to prevent such activity. Homeostatic intrinsic plasticity (HIP) adjusts membrane excitability through regulation of ion channels to maintain activity levels following activity perturbation. Despite the critical role of HIP in the maturation of excitability, it has not been examined in FXS. Here, we demonstrate that HIP does not operate normally in a disease model, FXS. HIP was either lost or exaggerated in two distinct neuronal populations from Fmr1 KO cortical cultures. In addition, we have identified a mechanism for homeostatic intrinsic plasticity. Compromising HIP function during development could leave cortical neurons in the FXS nervous system vulnerable to hyperexcitability., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Defects in the phosphoinositide 3-kinase (PI3K) pathway are shared characteristics in several brain disorders, including the inherited intellectual disability and autism spectrum disorder, fragile X syndrome (FXS). PI3K signaling therefore could serve as a therapeutic target for FXS and other brain disorders. However, broad inhibition of such a central signal transduction pathway involved in essential cellular functions may produce deleterious side effects. Pharmacological strategies that selectively correct the overactive components of the PI3K pathway while leaving other parts of the pathway intact may overcome these challenges. Here, we provide the first evidence that disease mechanism-based PI3K isoform-specific inhibition may be a viable treatment option for FXS. FXS is caused by loss of the fragile X mental retardation protein (FMRP), which translationally represses specific messenger RNAs, including the PI3K catalytic isoform p110β. FMRP deficiency increases p110β protein levels and activity in FXS mouse models and in cells from subjects with FXS. Here, we show that a novel, brain-permeable p110β-specific inhibitor, GSK2702926A, ameliorates FXS-associated phenotypes on molecular, cellular, behavioral, and cognitive levels in two different FMRP-deficient mouse models. Rescued phenotypes included increased PI3K downstream signaling, protein synthesis rates, and dendritic spine density, as well as impaired social interaction and higher-order cognition. Several p110β-selective inhibitors, for example, a molecule from the same chemotype as GSK2702926A, are currently being evaluated in clinical trials to treat cancer. Our results suggest that repurposing p110β inhibitors to treat cognitive and behavioral defects may be a promising disease-modifying strategy for FXS and other brain disorders.

Research in the past decades has unfolded the multifaceted role of Fragile X mental retardation protein (FMRP) and how its absence contributes to the pathophysiology of Fragile X syndrome (FXS). Excess signaling through group 1 metabotropic glutamate receptors is commonly observed in mouse models of FXS, which in part is attributed to dysregulated translation and downstream signaling. Considering the wide spectrum of cellular and physiologic functions that loss of FMRP can affect in general, it may be advantageous to pursue disease mechanism based treatments that directly target translational components or signaling factors that regulate protein synthesis. Various FMRP targets upstream and downstream of the translational machinery are therefore being investigated to further our understanding of the molecular mechanism of RNA and protein synthesis dysregulation in FXS as well as test their potential role as therapeutic interventions to alleviate FXS associated symptoms. In this review, we will broadly discuss recent advancements made towards understanding the role of FMRP in translation regulation, new pre-clinical animal models with FMRP targets located at different levels of the translational and signal transduction pathways for therapeutic intervention as well as future use of stem cells to model FXS associated phenotypes., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

RNA-binding proteins (RBPs) acting at various steps in the post-transcriptional regulation of gene expression play crucial roles in neuronal development and synaptic plasticity. Genetic mutations affecting several RBPs and associated factors lead to diverse neurological symptoms, as characterized by neurodevelopmental and neuropsychiatric disorders, neuromuscular and neurodegenerative diseases, and can often be multisystemic diseases. We will highlight the physiological roles of a few specific proteins in molecular mechanisms of cytoplasmic mRNA regulation, and how these processes are dysregulated in genetic disease. Recent advances in computational biology and genomewide analysis, integrated with diverse experimental approaches and model systems, have provided new insights into conserved mechanisms and the shared pathobiology of mRNA dysregulation in disease. Progress has been made to understand the pathobiology of disease mechanisms for myotonic dystrophy, spinal muscular atrophy, and fragile X syndrome, with broader implications for other RBP-associated genetic neurological diseases. This gained knowledge of underlying basic mechanisms has paved the way to the development of therapeutic strategies targeting disease mechanisms., (Copyright © 2016 the authors 0270-6474/16/3611418-09$15.00/0.)

Fragile X syndrome (FXS), the most-frequently inherited form of intellectual disability and the most-prevalent single-gene cause of autism, results from a lack of fragile X mental retardation protein (FMRP), an RNA-binding protein that acts, in most cases, to repress translation. Multiple pharmacological and genetic manipulations that target receptors, scaffolding proteins, kinases and translational control proteins can rescue neuronal morphology, synaptic function and behavioural phenotypes in FXS model mice, presumably by reducing excessive neuronal translation to normal levels. Such rescue strategies might also be explored in the future to identify the mRNAs that are critical for FXS pathophysiology.

Fragile X syndrome (FXS), an inherited intellectual disability often associated with autism, is caused by the loss of expression of the fragile X mental retardation protein. Tremendous progress in basic, preclinical, and translational clinical research has elucidated a variety of molecular-, cellular-, and system-level defects in FXS. This has led to the development of several promising therapeutic strategies, some of which have been tested in larger-scale controlled clinical trials. Here, we will summarize recent advances in understanding molecular functions of fragile X mental retardation protein beyond the well-known role as an mRNA-binding protein, and will describe current developments and emerging limitations in the use of the FXS mouse model as a preclinical tool to identify therapeutic targets. We will review the results of recent clinical trials conducted in FXS that were based on some of the preclinical findings, and discuss how the observed outcomes and obstacles will inform future therapy development in FXS and other autism spectrum disorders.

Fragile X syndrome (FXS) is caused by the loss of the fragile X mental retardation protein (FMRP), an RNA binding protein that regulates translation of numerous target mRNAs, some of which are dendritically localized. Our previous biochemical studies using synaptoneurosomes demonstrate a role for FMRP and miR-125a in regulating the translation of PSD-95 mRNA. However, the local translation of PSD-95 mRNA within dendrites and spines, as well as the roles of FMRP or miR-125a, have not been directly studied. Herein, local synthesis of a Venus-PSD-95 fusion protein was directly visualized in dendrites and spines using single-molecule imaging of a diffusion-restricted Venus-PSD-95 reporter under control of the PSD-95 3'UTR. The basal translation rates of Venus-PSD-95 mRNA was increased in cultured hippocampal neurons from Fmr1 KO mice compared with WT neurons, which correlated with a transient elevation of endogenous PSD-95 within dendrites. Following mGluR stimulation with (S)-3,5-dihydroxyphenylglycine, the rate of Venus-PSD-95 mRNA translation increased rapidly in dendrites of WT hippocampal neurons, but not in those of Fmr1 KO neurons or when the binding site of miR125a, previously shown to bind PSD-95 3'UTR, was mutated. This study provides direct support for the hypothesis that local translation within dendrites and spines is dysregulated in FXS. Impairments in the regulated local synthesis of PSD-95, a critical regulator of synaptic structure and function, may affect the spatiotemporal control of PSD-95 levels and affect dendritic spine development and synaptic plasticity in FXS., (Copyright © 2015 the authors 0270-6474/15/357116-15$15.00/0.)

The PI3K enhancer PIKE links PI3K catalytic subunits to group 1 metabotropic glutamate receptors (mGlu1/5) and activates PI3K signaling. The roles of PIKE in synaptic plasticity and the etiology of mental disorders are unknown. Here, we show that increased PIKE expression is a key mediator of impaired mGlu1/5-dependent neuronal plasticity in mouse and fly models of the inherited intellectual disability fragile X syndrome (FXS). Normalizing elevated PIKE protein levels in FXS mice reversed deficits in molecular and cellular plasticity and improved behavior. Notably, PIKE reduction rescued PI3K-dependent and -independent neuronal defects in FXS. We further show that PI3K signaling is increased in a fly model of FXS and that genetic reduction of the Drosophila ortholog of PIKE, CenG1A rescued excessive PI3K signaling, mushroom body defects, and impaired short-term memory in these flies. Our results demonstrate a crucial role of increased PIKE expression in exaggerated mGlu1/5 signaling causing neuronal defects in FXS., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Distinct isoforms of the PI3K catalytic subunit have specialized functions in the brain, but their role in cognition is unknown. Here, we show that the catalytic subunit p110β plays an important role in prefrontal cortex (PFC)-dependent cognitive defects in mouse models of Fragile X syndrome (FXS), an inherited intellectual disability. FXS is caused by loss of function of the fragile X mental retardation protein (FMRP), which binds and translationally represses mRNAs. PFC-selective knockdown of p110β, an FMRP target that is translationally upregulated in FXS, reverses deficits in higher cognition in Fmr1 knockout mice. Genetic full-body reduction of p110β in Fmr1 knockout mice normalizes excessive PI3K activity, restores stimulus-induced protein synthesis, and corrects increased dendritic spine density and behavior. Notably, adult-onset PFC-selective Fmr1 knockdown mice show impaired cognition, which is rescued by simultaneous p110β knockdown. Our results suggest that FMRP-mediated control of p110β is crucial for neuronal protein synthesis and cognition., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1(-/y); Cpeb1(-/-) double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1(-/y) mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.

The fragile X mental retardation protein (FMRP) plays a key role for neurotransmitter-mediated signaling upstream of neuronal protein synthesis. Functional loss of FMRP causes the inherited intellectual disability fragile X syndrome (FXS), and leads to increased and stimulus-insensitive neuronal protein synthesis in FXS animal models. Previous studies suggested that excess protein synthesis mediated by dysregulated signal transduction contributes to the majority of neurological defects in FXS, and might be a promising target for therapeutic strategies in patients. However, possible impairments in receptor-dependent protein synthesis have not been evaluated in patient cells so far. Using quantitative fluorescent metabolic labeling, we demonstrate that protein synthesis is exaggerated and cannot be further increased by cytokine stimulation in human fragile X lymphoblastoid cells. Our previous work suggested that loss of FMRP-mediated regulation of protein expression and enzymatic function of the PI3K catalytic subunit p110β contributes to dysregulated protein synthesis in a mouse model of FXS. Here, we demonstrate that these molecular mechanisms are recapitulated in FXS patient cells. Furthermore, we show that treatment with a p110β-selective antagonist rescues excess protein synthesis in synaptoneurosomes from an FXS mouse model and in patient cells. Our work suggests that dys-regulated protein synthesis and PI3K activity in patient cells might be suitable biomarkers to quantify the efficacy of drugs to ameliorate molecular mechanisms underlying FXS, and could be used for drug screens to refine treatment strategies for individual patients. Moreover, we provide rationale to pursue p110β-targeting treatments as potential therapy in FXS, and possibly other autism spectrum disorders.

Fragile X syndrome (FXS) is an inherited neurodevelopmental disease caused by loss of function of the fragile X mental retardation protein (FMRP). In the absence of FMRP, signaling through group 1 metabotropic glutamate receptors is elevated and insensitive to stimulation, which may underlie many of the neurological and neuropsychiatric features of FXS. Treatment of FXS animal models with negative allosteric modulators of these receptors and preliminary clinical trials in human patients support the hypothesis that metabotropic glutamate receptor signaling is a valuable therapeutic target in FXS. However, recent research has also shown that FMRP may regulate diverse aspects of neuronal signaling downstream of several cell surface receptors, suggesting a possible new route to more direct disease-targeted therapies. Here, we summarize promising recent advances in basic research identifying and testing novel therapeutic strategies in FXS models, and evaluate their potential therapeutic benefits. We provide an overview of recent and ongoing clinical trials motivated by some of these findings, and discuss the challenges for both basic science and clinical applications in the continued development of effective disease mechanism-targeted therapies for FXS.

Fragile X syndrome (FXS) is an inherited neurologic disease caused by loss of fragile X mental retardation protein (FMRP), which is hypothesized to mediate negative regulation of mRNA translation at synapses. A prominent feature of FXS animal models is exaggerated signaling through group 1 metabotropic glutamate receptors (gp1 mGluRs), and therapeutic strategies to treat FXS are targeted mainly at gp1 mGluRs. Recent studies, however, indicate that a variety of receptor-mediated signal transduction pathways are dysregulated in FXS, suggesting that FMRP acts on a common downstream signaling molecule. Here, we show that deficiency of FMRP results in excess activity of phosphoinositide 3-kinase (PI3K), a downstream signaling molecule of many cell surface receptors. In Fmr1 knock-out neurons, excess synaptic PI3K activity can be reduced by perturbation of gp1 mGluR-mediated signaling. Remarkably, increased PI3K activity was also observed in FMRP-deficient non-neuronal cells in the absence of gp1 mGluRs. Here, we show that FMRP regulates the synthesis and synaptic localization of p110beta, the catalytic subunit of PI3K. In wild type, gp1 mGluR activation induces p110beta translation, p110beta protein expression, and PI3K activity. In contrast, both p110beta protein synthesis and PI3K activity are elevated and insensitive to gp1 mGluR stimulation in Fmr1 knock-out. This suggests that dysregulated PI3K signaling may underlie the synaptic impairments in FXS. In support of this hypothesis, we show that PI3K antagonists rescue three FXS-associated phenotypes: dysregulated synaptic protein synthesis, excess AMPA receptor internalization, and increased spine density. Targeting excessive PI3K activity might thus be a potent therapeutic strategy for FXS.

Fragile X syndrome is the most common inherited form of cognitive deficiency in humans and perhaps the best-understood single cause of autism. A trinucleotide repeat expansion, inactivating the X-linked FMR1 gene, leads to the absence of the fragile X mental retardation protein. FMRP is a selective RNA-binding protein that regulates the local translation of a subset of mRNAs at synapses in response to activation of Gp1 metabotropic glutamate receptors (mGluRs) and possibly other receptors. In the absence of FMRP, excess and dysregulated mRNA translation leads to altered synaptic function and loss of protein synthesis-dependent plasticity. Recent evidence indicates the role of FMRP in regulated mRNA transport in dendrites. New studies also suggest a possible local function of FMRP in axons that may be important for guidance, synaptic development, and formation of neural circuits. The understanding of FMRP function at synapses has led to rationale therapeutic approaches.

The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. We report a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs were deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity.

Fragile X syndrome (FXS), a common inherited form of mental retardation, is caused by the functional absence of the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates the translation of specific mRNAs at synapses. Altered synaptic plasticity has been described in a mouse FXS model. However, the mechanism by which the loss of FMRP alters synaptic function, and subsequently causes the mental impairment, is unknown. Here, in cultured hippocampal neurons, we used siRNAs against Fmr1 to demonstrate that a reduction of FMRP in dendrites leads to an increase in internalization of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit, GluR1, in dendrites. This abnormal AMPAR trafficking was caused by spontaneous action potential-driven network activity without synaptic stimulation by an exogenous agonist and was rescued by 2-methyl-6-phenylethynyl-pyridine (MPEP), an mGluR5-specific inverse agonist. Because AMPAR internalization depends on local protein synthesis after mGluR5 stimulation, FMRP, a negative regulator of translation, may be viewed as a counterbalancing signal, wherein the absence of FMRP leads to an apparent excess of mGluR5 signaling in dendrites. Because AMPAR trafficking is a driving process for synaptic plasticity underlying learning and memory, our data suggest that hypersensitive AMPAR internalization in response to excess mGluR signaling may represent a principal cellular defect in FXS, which may be corrected by using mGluR antagonists.

Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein that is hypothesized to regulate local mRNA translation in dendrites downstream of gp1 metabotropic glutamate receptors (mGluRs). However, specific FMRP-associated mRNAs that localize to dendrites in vivo and show altered mGluR-dependent translation at synapses of Fmr1 knock-out mice are unknown so far. Using fluorescence in situ hybridization, we discovered that GluR1/2 and postsynaptic density-95 (PSD-95) mRNAs are localized to dendrites of cortical and hippocampal neurons in vivo. Quantitative analyses of their dendritic mRNA levels in cultured neurons and synaptoneurosomes did not detect differences between wild-type and Fmr1 knock-out (KO) mice. In contrast, PSD-95, GluR1/2, and calcium/calmodulin-dependent kinase IIalpha (CaMKIIalpha) mRNA levels in actively translating polyribosomes were dysregulated in synaptoneurosomes from Fmr1 knock-out mice in response to mGluR activation. [35S]methionine incorporation into newly synthesized proteins similarly revealed impaired stimulus-induced protein synthesis of CaMKIIalpha and PSD-95 in synaptoneurosomes from Fmr1 KO mice. Quantitative analysis of mRNA levels in FMRP-specific immunoprecipitations from synaptoneurosomes demonstrated the association of FMRP with CaMKIIalpha, PSD-95, and GluR1/2 mRNAs. These findings suggest a novel mechanism whereby FMRP regulates the local synthesis AMPA receptor (AMPAR) subunits, PSD-95, and CaMKIIalpha downstream of mGluR-activation. Dysregulation of local translation of AMPAR and associated factors at synapses may impair control of the molecular composition of the postsynaptic density and consequently alter synaptic transmission, causing impairments of neuronal plasticity observed in Fmr1 knock-out mice and fragile X syndrome.

Genetic deficiency of the mRNA binding protein FMRP results in the most common inherited form of mental retardation, Fragile X syndrome. We investigated the localization and function of FMRP during development of hippocampal neurons in culture. FMRP was distributed within granules that extended into developing axons and growth cones, detectable at distances over 300 microm from the cell body. In mature cultures, FMRP granules were present in both axons and dendrites, with pockets of higher concentrations appearing intermittently, along distal axon segments and near synapses. MAP1b mRNA, a known FMRP target, was also localized to axon growth cones. Morphometric analysis of growth cones from the FMR1 KO revealed both excess filopodia and reduced motility. At later stages during synapse formation, FMR1 KO neurons exhibited excessive filopodia and long spines along dendrites, yet there was a marked decrease in the density of spine-like protrusions juxtaposed to presynaptic terminals. In contrast, there was no difference in the density of shaft synapses between FMR1 KO and WT. Brief depolarization of WT neurons resulted in increased numbers of filopodia and spine synapses, whereas no additional morphologic changes were observable in dendrites of FMR1 KO neurons that already had increased density of filopodia-spines. These findings suggest that alterations in the regulation of axonal growth and innervation in FMR1 KO neurons may contribute to the dendritic and spine pathology in Fragile X syndrome. This work has broader implications for understanding the role of mRNA binding proteins in developmental and protein-synthesis-dependent plasticity.

Fragile X syndrome is caused by the absence of the mRNA-binding protein Fragile X mental retardation protein (FMRP), which may play a role in activity-regulated localization and translation of mRNA in dendrites and at synapses. We investigated whether neuronal activity and glutamatergic signals regulate trafficking of FMRP and its encoding Fmr1 mRNA into dendrites or at synapses. Using high-resolution fluorescence and digital imaging microscopy in cultured hippocampal neurons, FMRP and Fmr1 mRNA were localized in granules throughout dendrites and within spines. KCl depolarization rapidly increased FMRP and Fmr1 mRNA levels in dendrites. Metabotropic glutamate receptor (mGluR) activation, in particular mGluR5 activation, was necessary for localization of FMRP into dendrites. Blockade of either PKC or internal calcium prevented mGluR-dependent localization of both FMRP and Fmr1 mRNA in dendrites. The activity-dependent localization of FMRP was not dependent on protein synthesis. Fluorescence recovery after photobleaching analysis of live neurons transfected with enhanced green fluorescent protein-FMRP revealed increased granule trafficking in response to KCl depolarization. In contrast to its dendritic localization, mGluR activation diminished FMRP, but not Fmr1 mRNA, localization at synapses. These results demonstrate regulation of FMRP and Fmr1 mRNA trafficking in dendrites and synapses in response to specific glutamatergic signals.

Since the identification of the FMR1 gene basic research has been focused on the molecular characterization of the FMR1 gene product, the fragile X mental retardation protein (FMRP). Recent developments in fragile X research have provided new insights and knowledge about the physiological function of FMRP in the cell and the nerve cell in particular. Currently, compelling evidence suggests a role for FMRP in the transport/translation of dendritically localized mRNAs. In addition, the identification of some of the target mRNAs of FMRP have led to an increased interest in the neurobiology of the syndrome. This review highlights the role of FMRP in dendritic mRNA transport/translation in relation to synaptic plasticity, a molecular mechanism implicated in learning and memory., (Copyright 2004 Wiley-Liss, Inc.)

The article reports on a study, according to which function for the fragile X mental retardation protein in RNA editing necessary for synaptic development. It states ability of a neuron to modulate protein composition affecting synaptic development and function lies in a fragile balance. It reports the loss of fragile X mental retardation protein (FMRP), is the cause of fragile X syndrome, which is the most common inherited form of intellectual disability.

Background: Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP). Methods: Electrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced. Results: Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca2+-activated K+ currents and the persistent Na+ current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na+ channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines. Conclusions: Pharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered. [ABSTRACT FROM AUTHOR]

An essential aspect of goal-directed decision-making is selecting actions based on anticipated consequences, a process that involves the orbitofrontal cortex (OFC) and potentially, the plasticity of dendritic spines in this region. T o investigate this possibility, we trained male and female mice to nose poke for food reinforcers, or we delivered the same number of food reinforcers non-contingently to separate mice. We then decreased the likelihood of reinforcement for trained mice, requiring them to modify action-outcome expectations. In a separate experiment, we blocked action-outcome updating via chemogenetic inactivation of the OFC. In both cases, successfully selecting actions based on their likely consequences was associated with fewer immature, thin-shaped dendritic spines and a greater proportion of mature, mushroom-shaped spines in the ventrolateral OFC. This pattern was distinct from spine loss associated with aging, and we identified no effects on hippocampal CA1 neurons. Given that the OFC is involved in prospective calculations of likely outcomes, even when they are not observable, constraining spinogenesis while preserving mature spines may be important for solidifying durable expectations. To investigate causal relationships, we inhibited the RNA-binding protein fragile X mental retardation protein (encoded by Fmrl), which constrains dendritic spine turnover. Ventrolateral OFC-selective Fmrl knockdown recapitulated the behavioral effects of inducible OFC inactivation (and lesions; also shown here), impairing action-outcome conditioning, and caused dendritic spine excess. Our findings suggest that a proper balance of dendritic spine plasticity within the OFC is necessary for one's ability to select actions based on anticipated consequences. [ABSTRACT FROM AUTHOR]

Fragile X syndrome, the most common inherited form of intellectual disability, is caused by the CGG trinucleotide expansion in the 5’-untranslated region of the Fmr1 gene on the X chromosome, which silences the expression of the fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA, which encodes for the postsynaptic density protein 95, and together with microRNA-125a to mediate the reversible inhibition of the PSD-95 mRNA translation in neurons. The miR-125a binding site within the PSD-95 mRNA 3’-untranslated region (UTR) is embedded in a G-rich region bound by FMRP, which we have previously demonstrated folds into two parallel G-quadruplex structures. The FMRP regulation of PSD-95 mRNA translation is complex, being mediated by its phosphorylation. While the requirement for FMRP in the regulation of PSD-95 mRNA translation is clearly established, the exact mechanism by which this is achieved is not known. In this study, we have shown that both unphosphorylated FMRP and its phosphomimic FMRP S500D bind to the PSD-95 mRNA G-quadruplexes with high affinity, whereas only FMRP S500D binds to miR-125a. These results point towards a mechanism by which, depending on its phosphorylation status, FMRP acts as a switch that potentially controls the stability of the complex formed by the miR-125a-guided RNA induced silencing complex (RISC) and PSD-95 mRNA. [ABSTRACT FROM AUTHOR]

Fragile X syndrome is a common cause of intellectual disability and autism spectrum disorder. The gene underlying the disorder, fragile X mental retardation 1 (FMR1), is silenced in most cases by a CGG-repeat expansion mutation in the 5' untranslated region (UTR). Recently, we identified a variant located in the 3'UTR of FMR1 enriched among developmentally delayed males with normal repeat lengths. A patient-derived cell line revealed reduced levels of endogenous fragile X mental retardation protein (FMRP), and a reporter containing a patient 3'UTR caused a decrease in expression. A control reporter expressed in cultured mouse cortical neurons showed an expected increase following synaptic stimulation that was absent when expressing the patient reporter, suggesting an impaired response to neuronal activity. Mobility-shift assays using a control RNA detected an RNA-protein interaction that is lost with the patient RNA, and HuR was subsequently identified as an associated protein. Cross-linking immunoprecipitation experiments identified the locus as an in vivo target of HuR, supporting our in vitro findings. These data suggest that the disrupted interaction of HuR impairs activity-dependent translation of FMRP, which may hinder synaptic plasticity in a clinically significant fashion. [ABSTRACT FROM AUTHOR]

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP), which represses and reversibly regulates the translation of a subset of mRNAs in dendrites. Protein synthesis can be rapidly stimulated by mGluR-induced and protein phosphatase 2a (PP2A)-mediated dephosphorylation of FMRP, which is coupled to the dissociation ofFMRPand targetmRNAsfrom miRNA-induced silencing complexes. Here, we report the rapid ubiquitination and ubiquitin proteasome system (UPS)-mediated degradation ofFMRPin dendrites upon DHPG (3,5-dihydroxyphenylglycine) stimulation in cultured rat neurons. Using inhibitors to PP2A and FMRP phosphomutants, degradation of FMRP was observed to depend on its prior dephosphorylation. Translational induction of an FMRP target, postsynaptic density-95mRNA,required both PP2A and UPS. Thus, control ofFMRPlevels at the synapse by dephosphorylation-induced and UPS-mediated degradation provides a mode to regulate protein synthesis. [ABSTRACT FROM AUTHOR]

A prominent characteristic of the inherited intellectual impairment disease fragile X syndrome (FXS) is neuronal hyperexcitability, resulting in a variety of symptoms, such as hyperactivity, increased sensitivity to sensory stimuli, and a high incidence of epileptic seizures. These symptoms account for a significant part of the disease pattern, but the underlying molecular mechanisms of neuronal hyperexcitability in FXS remain poorly understood. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), which regulates synaptic protein synthesis and is a key player to limit signaling pathways downstream of metabotropic glutamate receptors 1/5 (mGlu1/5). Recent findings suggest that FMRP might also directly regulate voltage-gated potassium channels. Here, we show that total and plasma membrane protein levels of Kv4.2, the major potassium channel regulating hippocampal neuronal excitability, are reduced in the brain of an FXS mouse model. Antagonizing mGlu5 activity with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) partially rescues reduced surface Kv4.2 levels in Fmr1 knock-out (KO) mice, suggesting that excess mGlu1/5 signal activity contributes to Kv4.2 dysregulation. As an additional mechanism, we show that FMRP is a positive regulator of Kv4.2 mRNA translation and protein expression and associates with Kv4.2 mRNA in vivo and in vitro. Our results suggest that absence of FMRP-mediated positive control of Kv4.2 mRNA translation, protein expression, and plasma membrane levels might contribute to excess neuronal excitability in Fmr1 KO mice, and thus imply a potential mechanism underlying FXS-associated epilepsy. [ABSTRACT FROM AUTHOR]

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retarda- tion protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthe- sis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho- FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for protein- synthesis-dependent synaptic plasticity. [ABSTRACT FROM AUTHOR]

Fragile X syndrome is a common form of inherited mental retardation and is caused by loss of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that influences the translation of target messages. Here, we identify protein phosphatase 2A (PP2A) as an FMRP phosphatase and report rapid FMRP dephosphorylation after immediate group I metabotropic glutamate receptor (mGluR) stimulation (<1 min) in neurons caused by enhanced PP2A enzymatic activity. In contrast, extended mGluR activation (1-5 min) resulted in mammalian target of rapamycin (mTOR)-mediated PP2A suppression and FMRP rephosphorylation. These activitydependent changes in FMRP phosphorylation were also observed in dendrites and showed a temporal correlation with the translational profile of select FMRP target transcripts. Collectively, these data reveal an immediate-early signaling pathway linking group I mGluR activity to rapid FMRP phosphorylation dynamics mediated by mTOR and PP2A. [ABSTRACT FROM AUTHOR]