Protein FMRP Research Articles

Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis.

SUMMARYTranscriptional 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.

The molecular view of mechanical stress of brain cells, local translation, and neurodegenerative diseases.

The assumption that chronic mechanical stress in brain cells stemming from intracranial hypertension, arterial hypertension, or mechanical injury is a risk factor for neurodegenerative diseases was put forward in the 1990s and has since been supported. However, the molecular mechanisms that underlie the way from cell exposure to mechanical stress to disturbances in synaptic plasticity followed by changes in behavior, cognition, and memory are still poorly understood. Here we review (1) the current knowledge of molecular mechanisms regulating local translation and the actin cytoskeleton state at an activated synapse, where they play a key role in the formation of various sorts of synaptic plasticity and long-term memory, and (2) possible pathways of mechanical stress intervention. The roles of the mTOR (mammalian target of rapamycin) signaling pathway; the RNA-binding FMRP protein; the CYFIP1 protein, interacting with FMRP; the family of small GTPases; and the WAVE regulatory complex in the regulation of translation initiation and actin cytoskeleton rearrangements in dendritic spines of the activated synapse are discussed. Evidence is provided that chronic mechanical stress may result in aberrant activation of mTOR signaling and the WAVE regulatory complex via the YAP/TAZ system, the key sensor of mechanical signals, and influence the associated pathways regulating the formation of F actin filaments and the dendritic spine structure. These consequences may be a risk factor for various neurological conditions, including autistic spectrum disorders and epileptic encephalopathy. In further consideration of the role of the local translation system in the development of neuropsychic and neurodegenerative diseases, an original hypothesis was put forward that one of the possible causes of synaptopathies is impaired proteome stability associated with mTOR hyperactivity and formation of complex dynamic modes of de novo protein synthesis in response to synapse-stimulating factors, including chronic mechanical stress.

The conserved alternative splicing factor caper regulates neuromuscular phenotypes during development and aging

RNA-binding proteins play an important role in the regulation of post-transcriptional gene expression throughout the nervous system. This is underscored by the prevalence of mutations in genes encoding RNA splicing factors and other RNA-binding proteins in a number of neurodegenerative and neurodevelopmental disorders. The highly conserved alternative splicing factor Caper is widely expressed throughout the developing embryo and functions in the development of various sensory neural subtypes in the Drosophila peripheral nervous system. Here we find that caper dysfunction leads to aberrant neuromuscular junction morphogenesis, as well as aberrant locomotor behavior during larval and adult stages. Despite its widespread expression, our results indicate that caper function is required to a greater extent within the nervous system, as opposed to muscle, for neuromuscular junction development and for the regulation of adult locomotor behavior. Moreover, we find that Caper interacts with the RNA-binding protein Fmrp to regulate adult locomotor behavior. Finally, we show that caper dysfunction leads to various phenotypes that have both a sex and age bias, both of which are commonly seen in neurodegenerative disorders in humans.

Loss of the fragile X syndrome protein FMRP results in misregulation of nonsense-mediated mRNA decay.

Loss of the fragile X protein FMRP is a leading cause of intellectual disability and autism1,2, but the underlying mechanism remains poorly understood. We report that FMRP deficiency results in hyperactivated nonsense-mediated mRNA decay (NMD)3,4 in human SH-SY5Y neuroblastoma cells and fragile X syndrome (FXS) fibroblast-derived induced pluripotent stem cells (iPSCs). We examined the underlying mechanism and found that the key NMD factor UPF1 binds directly to FMRP, promoting FMRP binding to NMD targets. Our data indicate that FMRP acts as an NMD repressor. In the absence of FMRP, NMD targets are relieved from FMRP-mediated translational repression so that their half-lives are decreased and, for those NMD targets encoding NMD factors, increased translation produces abnormally high factor levels despite their hyperactivated NMD. Transcriptome-wide alterations caused by NMD hyperactivation have a role in the FXS phenotype. Consistent with this, small-molecule-mediated inhibition of hyperactivated NMD, which typifies iPSCs derived from patients with FXS, restores a number of neurodifferentiation markers, including those not deriving from NMD targets. Our mechanistic studies reveal that many molecular abnormalities in FMRP-deficient cells are attributable-either directly or indirectly-to misregulated NMD.

Astroglial FMRP modulates synaptic signaling and behavior phenotypes in FXS mouse model.

Fragile X syndrome (FXS) is one of the most common inherited intellectual disability (ID) disorders, in which the loss of FMRP protein induces a range of cellular signaling changes primarily through excess protein synthesis. Although neuron-centered molecular and cellular events underlying FXS have been characterized, how different CNS cell types are involved in typical FXS synaptic signaling changes and behavioral phenotypes is largely unknown. Recent evidence suggests that selective loss of astroglial FMRP is able to dysregulate glutamate uptake, increase spine density, and impair motor-skill learning. Here we investigated the effect of astroglial FMRP on synaptic signaling and FXS-related behavioral and learning phenotypes in astroglial Fmr1 cKO and cON mice in which FMRP expression is selectively diminished or restored in astroglia. We found that selective loss of astroglial FMRP contributes to cortical hyperexcitability by enhancing NMDAR-mediated evoked but not spontaneous miniEPSCs and elongating cortical UP state duration. Selective loss of astroglial FMRP is also sufficient to increase locomotor hyperactivity, significantly diminish social novelty preference, and induce memory acquisition and extinction deficits in astroglial Fmr1 cKO mice. Importantly, re-expression of astroglial FMRP is able to significantly rescue the hyperactivity (evoked NMDAR response, UP state duration, and open field test) and social novelty preference in astroglial Fmr1 cON mice. These results demonstrate a profound role of astroglial FMRP in the evoked synaptic signaling, spontaneously occurring cortical UP states, and FXS-related behavioral and learning phenotypes and provide important new insights in the cell type consideration for the FMRP reactivation strategy.

FMR1 loss in a human stem cell model reveals early changes to intrinsic membrane excitability

Fragile X mental retardation 1 (FMR1) encodes the RNA binding protein FMRP. Loss of FMRP drives Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and a leading monogenic cause of autism. While cortical hyperexcitability is a hallmark of FXS, the reported phenotypes and underlying mechanisms, including alterations in synaptic transmission and ion channel properties, are heterogeneous and at times contradictory. Here, we report the generation of new isogenic FMR1y/+ and FMR1y/- human pluripotent stem cell (hPSC) lines using CRISPR-Cas9 to facilitate the study of how complete FMRP loss, independent of genetic background, drives molecular and cellular alterations relevant for FXS. After differentiating these stem cell tools into excitatory neurons, we systematically assessed the impact of FMRP loss on intrinsic membrane and synaptic properties over time. Using whole-cell patch clamp analyses, we found that FMR1y/- neurons overall showed an increased intrinsic membrane excitability compared to age-matched FMR1y/+ controls, with no discernable alternations in synaptic transmission. Surprisingly, longitudinal analyses of cell intrinsic defects revealed that a majority of significant changes emerged early following in vitro differentiation and some were not stable over time. Collectively, this study provides a new isogenic hPSC model which can be further leveraged by the scientific community to investigate basic mechanisms of FMR1 gene function relevant for FXS. Moreover, our results suggest that precocious changes in the intrinsic membrane properties during early developmental could be a critical cellular pathology ultimately contributing to cortical hyperexcitability in FXS.

Replication Stress Induces Global Chromosome Breakage in the Fragile X Genome

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of a functional FMRP protein. FMRP is known as a translation repressor whose nuclear function is not understood. We investigated the global impact on genome stability due to FMRP loss. Using Break-seq, we map spontaneous and replication stress-induced DNA double-strand breaks (DSBs) in an FXS patient-derived cell line. We report that the genomes of FXS cells are inherently unstable and accumulate twice as many DSBs as those from an unaffected control. We demonstrate that replication stress-induced DSBs in FXS cells colocalize with R-loop forming sequences. Exogenously expressed FMRP in FXS fibroblasts ameliorates DSB formation. FMRP, not the I304N mutant, abates R-loop-induced DSBs during programmed replication-transcription conflict. These results suggest that FMRP is a genome maintenance protein that prevents R-loop accumulation. Our study provides insights into the etiological basis for FXS.

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

Splicing of exon 9a in FMR1 transcripts results in a truncated FMRP with altered subcellular distribution

FMRP is an RNA-binding protein, loss of which causes fragile X syndrome (FXS). FMRP has several isoforms resulted from alternative splicing (AS) of fragile X mental retardation 1 (FMR1) gene, but their biological functions are still poorly understood. In the analysis of alternatively spliced FMR1 transcripts in the blood cells from a patient with FXS-like phenotypes (normal CGG repeats and no mutation in coding sequence of FMR1), we identified three novel FMR1 transcripts that include a previously unidentified microexon (46 bp), terming the exon 9a. This microexon exists widely in unaffected individuals, inclusion of which introduces an in-frame termination codon. To address whether these exon 9a-containing transcripts could produce protein by evading nonsense-mediated decay (NMD), Western blot was used to analysis blood cell lysate from unaffected individuals and a 34 kDa protein that consistent in size with the molecular weight of the predicted truncated protein produced from mRNA with this microexon was found. Meanwhile, treatment of peripheral blood mononuclear cells with an inhibitor of NMD (Cycloheximide) did not result in significant increase in exon 9a-containing transcripts. Using confocal immunofluorescence, we found the truncated protein displayed both nuclear and cytoplasmic localization in HEK293T and HeLa cells due to lacking C-terminal domains including KH2, NES, and RGG, while the full-length FMRP protein mainly localized in the cytoplasm. Therefore, we hypothesize that the inclusion of this microexon to generate exon 9a-containing transcripts may regulate the normal functionality of FMRP, and the dysregulation of normal FMRP due to increased exon 9a-containing alternatively spliced transcripts in that patient may be associated with the manifestation of FXS phenotype.

Altered mitochondrial function in cells carrying a premutation or unmethylated full mutation of the FMR1 gene

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5' UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.

FMRP has a cell-type-specific role in CA1 pyramidal neurons to regulate autism-related transcripts and circadian memory.

Loss of the RNA binding protein FMRP causes Fragile X Syndrome (FXS), the most common cause of inherited intellectual disability, yet it is unknown how FMRP function varies across brain regions and cell types and how this contributes to disease pathophysiology. Here we use conditional tagging of FMRP and CLIP (FMRP cTag CLIP) to examine FMRP mRNA targets in hippocampal CA1 pyramidal neurons, a critical cell type for learning and memory relevant to FXS phenotypes. Integrating these data with analysis of ribosome-bound transcripts in these neurons revealed CA1-enriched binding of autism-relevant mRNAs, and CA1-specific regulation of transcripts encoding circadian proteins. This contrasted with different targets in cerebellar granule neurons, and was consistent with circadian defects in hippocampus-dependent memory in Fmr1 knockout mice. These findings demonstrate differential FMRP-dependent regulation of mRNAs across neuronal cell types that may contribute to phenotypes such as memory defects and sleep disturbance associated with FXS.

Fragile X syndrome: clinical presentation, pathology and treatment.

Fragile X syndrome is the monogenetic condition that produces more cases of autism and intellectual disability. The repetition of CGG triplets (> 200) and their methylation entail the silencing of the FMR1 gene. The FMRP protein (product of the FMR1 gene) interacts with ribosomes by controlling the translation of specific messengers, and its loss causes alterations in synaptic connectivity. Screening for fragile X syndrome is performed by polymerase chain reaction. Current recommendation of the American Academy of Pediatrics is to test individuals with intellectual disability, global developmental retardation or with a family history of presence of the mutation or premutation. Hispanic countries such as Colombia, Chile and Spain report high prevalence of fragile X syndrome and have created fragile X national associations or corporations that seek to bring patients closer to available diagnostic and treatment networks.

Calsyntenin-1 Negatively Regulates ICAM5 Accumulation in Postsynaptic Membrane and Influences Dendritic Spine Maturation in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder that causes intellectual disability, as well as the leading monogenic cause of autism spectrum disorders (ASD), in which neurons show aberrant dendritic spine structure. The reduction/absence of the functional FMRP protein, coded by the X-linked Fmr1 gene in humans, is responsible for the syndrome. Targets of FMRP, CLSTN1, and ICAM5, play critical roles in the maturation of dendritic spines, synapse formation and synaptic plasticity. However, the implication of CLSTN1 and ICAM5 in dendritic spine abnormalities and the underlying neuropathologic processes in FXS remain uninvestigated. In this study, we demonstrated that CLSTN1 co-localizes and co-transports with ICAM5 in cultured cortical neurons. Also we showed that shRNA-mediated downregulation of CLSTN1 in cultured WT neurons increases ICAM5 on the surface of synaptic membrane, subsequently affecting the maturation of dendritic spines. Whereas, normalization of CLSTN1 level in Fmr1 KO neurons reduces ICAM5 abundance and rescues impaired dendritic spine phenotypes. Most importantly, CLSTN1 protein is reduced in the postnatal medial prefrontal cortex of Fmr1 KO mice, which is correlated with increased ICAM5 levels on the surface of synapses and excessive filopodia-like spines. In conclusion, this study demonstrates that CLSTN1 plays a critical role in dendritic spine formation and maturation in FXS by regulating ICAM5 redistribution.

Drosophila Netrin-B controls mushroom body axon extension and regulates courtship-associated learning and memory of a Drosophila fragile X syndrome model

Mushroom body (MB) is a prominent structure essential for olfactory learning and memory in the Drosophila brain. The development of the MB involves the appropriate guidance of axon lobes and sister axon branches. Appropriate guidance that accurately shapes MB development requires the integration of various guidance cues provided by a series of cell types, which guide axons to reach their final positions within the MB neuropils. Netrins are axonal guidance molecules that are conserved regulators of embryonic nerve cord patterning. However, whether they contribute to MB morphogenesis has not yet been evaluated. Here, we find that Netrin-B (NetB) is highly expressed in the MB lobes, regulating lobe length through genetic interactions with the receptors Frazzled and Uncoordinated-5 from 24 h after pupal formation onwards. We observe that overexpression of NetB causes severe β lobe fusion in the MB, which is similar to the MB defects seen in the Drosophila model of fragile X syndrome (FXS). Our results further show that fragile-X mental retardation protein FMRP inhibits the translational activity of human ortholog Netrin-1 (NTN1). Knock-down of NetB significantly rescues the MB defects and ameliorates deficits in the learning and memory in FXS model Drosophila. These results indicate a critical role for NetB in MB lobe extension and identify NetB as a novel target of FMRP which contributes to learning and memory.

Retinal alterations in a pre-clinical model of an autism spectrum disorder

BackgroundAutism spectrum disorders (ASD) affect around 1.5% of people worldwide. Symptoms start around age 2, when children fail to maintain eye contact and to develop speech and other forms of communication. Disturbances in glutamatergic and GABAergic signaling that lead to synaptic changes and alter the balance between excitation and inhibition in the developing brain are consistently found in ASD. One of the hallmarks of these disorders is hypersensitivity to sensory stimuli; however, little is known about its underlying causes. Since the retina is the part of the CNS that converts light into a neuronal signal, we set out to study how it is affected in adolescent mice prenatally exposed to valproic acid (VPA), a useful tool to study ASD endophenotypes.MethodsPregnant female mice received VPA (600 mg/kg, ip) or saline at gestational day 11. Their male adolescent pups (P29–35) were behaviorally tested for anxiety and social interaction. Proteins known to be related with ASD were quantified and visualized in their retinas by immunoassays, and retinal function was assessed by full-field scotopic electroretinograms (ERGs).ResultsEarly adolescent mice prenatally exposed to VPA displayed impaired social interest and increased anxiety-like behaviors consistent with an ASD phenotype. The expression of GABA, GAD, synapsin-1, and FMRP proteins were reduced in their retinas, while mGluR5 was increased. The a-wave amplitudes of VPA-exposed were smaller than those of CTR animals, whereas the b-wave and oscillatory potentials were normal.ConclusionsThis study establishes that adolescent male mice of the VPA-induced ASD model have alterations in retinal function and protein expression compatible with those found in several brain areas of other autism models. These results support the view that synaptic disturbances with excitatory/inhibitory imbalance early in life are associated with ASD and point to the retina as a window to understand their subjacent mechanisms.

Drosophila melanogaster as a Model to Study the Multiple Phenotypes, Related to Genome Stability of the Fragile-X Syndrome.

Fragile-X syndrome is one of the most common forms of inherited mental retardation and autistic behaviors. The reduction/absence of the functional FMRP protein, coded by the X-linked Fmr1 gene in humans, is responsible for the syndrome. Patients exhibit a variety of symptoms predominantly linked to the function of FMRP protein in the nervous system like autistic behavior and mild-to-severe intellectual disability. Fragile-X (FraX) individuals also display cellular and morphological traits including branched dendritic spines, large ears, and macroorchidism. The dFmr1 gene is the Drosophila ortholog of the human Fmr1 gene. dFmr1 mutant flies exhibit synaptic abnormalities, behavioral defects as well as an altered germline development, resembling the phenotypes observed in FraX patients. Therefore, Drosophila melanogaster is considered a good model to study the physiopathological mechanisms underlying the Fragile-X syndrome. In this review, we explore how the multifaceted roles of the FMRP protein have been addressed in the Drosophila model and how the gained knowledge may open novel perspectives for understanding the molecular defects causing the disease and for identifying novel therapeutical targets.

Fragile X associated tremor/ataxia syndrome: its clinical presentation, pathology, and treatment

The fragile X associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disease associated with the repetition of CGG triplets (55-200 CGG repetitions) in the FMR1 gene. The premutation of the FMR1 gene, contrasting with the full mutation (more than 200 CGG repetitions), presents an increased production of messenger and a similar or slightly decreased production of FMRP protein. FXTAS affects 40% of men and 16% of women carriers of the premutation. It presents with a wide constellation of neurological signs such as intention tremor, cerebellar ataxia, parkinsonism, executive function deficits, peripheral neuropathy and cognitive decline leading to dementia among others. In this review, we present what is currently known about the molecular mechanism, the radiological findings and the pathology, as well as the complexity of the diagnosis and management of FXTAS.

Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells.

Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5′ untranslated region (UTR) of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the Clustered Regularly Interspaced Palindromic Repeats/deficient CRISPR associated protein 9 (CRISPR/dCas9) system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCas9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 messenger RNA (mRNA) expression despite promoter and repeat methylation. However, these changes at the transcript level were not coupled with a significant elevation in FMRP protein expression in FXS cells. These studies demonstrate that directing a transcriptional activator to CGG repeats is sufficient to selectively reactivate FMR1 mRNA expression in Fragile X patient stem cells.

Loss of fragile X protein FMRP impairs homeostatic synaptic downscaling through tumor suppressor p53 and ubiquitin E3 ligase Nedd4-2

Synaptic scaling allows neurons to homeostatically readjust synaptic strength upon chronic neural activity perturbations. Although altered synaptic scaling has been implicated to underlie imbalanced brain excitability in neurological disorders such as autism spectrum disorders and epilepsy, the molecular dysregulation and restoration of synaptic scaling in those diseases have not been demonstrated. Here, we showed that the homeostatic synaptic downscaling is absent in the hippocampal neurons of Fmr1 KO mice, the mouse model of the most common inherited autism, fragile X syndrome (FXS). We found that the impaired homeostatic synaptic downscaling in Fmr1 KO neurons is caused by loss-of-function dephosphorylation of an epilepsy-associated ubiquitin E3 ligase, neural precursor cell expressed developmentally down-regulated gene 4-2, Nedd4-2. Such dephosphorylation of Nedd4-2 is surprisingly caused by abnormally stable tumor suppressor p53 and subsequently destabilized kinase Akt. Dephosphorylated Nedd4-2 fails to elicit 14-3-3-dependent ubiquitination and down-regulation of the GluA1 subunit of AMPA receptor, and therefore impairs synaptic downscaling. Most importantly, using a pharmacological inhibitor of p53, Nedd4-2 phosphorylation, GluA1 ubiquitination and synaptic downscaling are all restored in Fmr1 KO neurons. Together, our results discover a novel cellular mechanism underlying synaptic downscaling, and demonstrate the dysregulation and successful restoration of this mechanism in the FXS mouse model.

S66. Therapeutic effects of an AMPA receptor antagonist on fragile X syndrome and cerebellar coordination disorders/involuntary movements

Introduction To examine the effectiveness of an AMPA receptor antagonist (perampanel: PER) for treating cerebellar coordination disorders/involuntary movements with the genetic loss of FMRP protein, which regulates AMPA receptor expression in Purkinje cells. Fragile X syndrome causes mental retardation and cerebellar motor disorders through CGG repeat expansion in the 5′-untranslated region of the FMR1 gene (chX 27.3). The FMRP protein encoded by the FMR1 gene is responsible for translational repression of the signaling of a metabotropic glutamate receptor, mGluR. In FMRP-protein knockout animals, abnormal phenotypes are caused by mGluR-dependent LTD, resulting in morphological abnormalities of the hippocampal neuronal dendrites, which were improved by mGluR antagonist administration ( Bear et al., 2004 ). In Japan, PER is covered by insurance as a combination therapy for epilepsy. Methods The present study was approved by the ethics committee of our hospital, with consent obtained from patients and their families. Two patients with fragile X syndrome (16-year-old male with FMR1-full mutation and 68-year-old female with FMRI-premutation) were included. One case of Usher syndrome and two cases of LCCA were included as disease controls. The administration of PER was started at 2 mg/day, and maintained at 4 mg/day in week 2 and later to examine effectiveness for 12 weeks. Motor symptoms and involuntary movements were evaluated using an international standard score, ICARS, and MDS-UPDRS part III-3, respectively 15–18. AMPA-R protein (130 m/kDa) expressed on the peripheral blood lymphocytes before drug administration was examined by Western blotting (MyBioSource, AMPA receptor (GluR4) rabbit polyclonal antibody). Results Motor and gait functions were markedly improved in the cases of fragile X syndrome (ICARS-motor; −9, gait; −3). Involuntary movement scores were improved in all cases (−12). AMPA-R protein on lymphocytes was detected in the cases of fragile X syndrome, but was not in the disease controls. Conclusion PER is effective for treating cerebellar involuntary movements in fragile X syndrome, and may have LTP/disinhibiting effects through AMPA-R blocking in Purkinje cells. Changes in lymphocyte AMPA-R expression level suggested that AMPA-R expression pattern might be changed in fragile X syndrome.