Function Of Fragile X Mental Retardation Protein Research Articles

Loss of FMRP affects ovarian development and behaviour through multiple pathways in a zebrafish model of fragile X syndrome.

Fragile X syndrome (FXS) is an inherited neurodevelopmental disorder and the leading genetic cause of autism spectrum disorders. FXS is caused by loss of function mutations in Fragile X mental retardation protein (FMRP), an RNA binding protein that is known to regulate translation of its target mRNAs, predominantly in the brain and gonads. The molecular mechanisms connecting FMRP function to neurodevelopmental phenotypes are well understood. However, neither the full extent of reproductive phenotypes, nor the underlying molecular mechanisms have been as yet determined. Here, we developed new fmr1 knockout zebrafish lines and show that they mimic key aspects of FXS neuronal phenotypes across both larval and adult stages. Results from the fmr1 knockout females also showed that altered gene expression in the brain, via the neuroendocrine pathway contribute to distinct abnormal phenotypes during ovarian development and oocyte maturation. We identified at least three mechanisms underpinning these defects, including altered neuroendocrine signaling in sexually mature females resulting in accelerated ovarian development, altered expression of germ cell and meiosis promoting genes at various stages during oocyte maturation, and finally a strong mitochondrial impairment in late stage oocytes from knockout females. Our findings have implications beyond FXS in the study of reproductive function and female infertility. Dissection of the translation control pathways during ovarian development using models like the knockout lines reported here may reveal novel approaches and targets for fertility treatments.

Characterization of ribosome stalling and no-go mRNA decay stimulated by the fragile X protein, FMRP

Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal noncanonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNA•ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not poly(A)-binding protein, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of four putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes but only stimulates NGD of a small select set of transcripts, revealing a minor role of FMRP that would be misregulated in fragile X syndrome.

Characterization of Fragile X Mental Retardation Protein expression in human nociceptors and their axonal projections to the spinal dorsal horn.

Fragile X Mental Retardation Protein (FMRP) regulates activity-dependent RNA localization and local translation to modulate synaptic plasticity throughout the central nervous system. Mutations in the FMR1 gene that hinder or ablate FMRP function cause Fragile X Syndrome (FXS), a disorder associated with sensory processing dysfunction. FXS premutations are associated with increased FMRP expression and neurological impairments including sex dimorphic presentations of chronic pain. In mice, FMRP ablation causes dysregulated dorsal root ganglion (DRG) neuron excitability and synaptic vesicle exocytosis, spinal circuit activity, and decreased translation-dependent nociceptive sensitization. Activity-dependent, local translation is a key mechanism for enhancing primary nociceptor excitability that promotes pain in animals and humans. These works indicate that FMRP likely regulates nociception and pain at the level of the primary nociceptor or spinal cord. Therefore, we sought to better understand FMRP expression in the human DRG and spinal cord using immunostaining in organ donor tissues. We find that FMRP is highly expressed in DRG and spinal neuron subsets with substantia gelatinosa exhibiting the most abundant immunoreactivity in spinal synaptic fields. Here, it is expressed in nociceptor axons. FMRP puncta colocalized with Nav1.7 and TRPV1 receptor signals suggesting a pool of axoplasmic FMRP localizes to plasma membrane-associated loci in these branches. Interestingly, FMRP puncta exhibited notable colocalization with calcitonin gene-related peptide (CGRP) immunoreactivity selectively in female spinal cord. Our results support a regulatory role for FMRP in human nociceptor axons of the dorsal horn and implicate it in the sex dimorphic actions of CGRP signaling in nociceptive sensitization and chronic pain.

Role of Fragile X Mental Retardation protein in Amyloid Beta 1‐42 Induced Translational Suppression

AbstractBackgroundAlzheimer’s Disease (AD) is the most common cause of dementia where the accumulation of the peptide Amyloid‐Beta (Aβ) is the main theory linked to the pathogenesis of the disease. Fragile X Mental Retardation Protein (FMRP) is an RNA‐binding protein that functions to suppress global translation. Because Aβ is known to be a toxic peptide capable of inducing the Endoplasmic Reticulum (ER) stress response and because augmented translational suppression is one of the main molecular phenotypes in ER stress response, we ask whether and how FMRP is involved in Aß induced translational suppression.MethodPrimary mouse cortical neuronal cultures from wild type (WT) or Fmr1 KO pups were treated with Aβ (Aβ1–42) during 24 hours. For protein expression identification and quantification we used western blot and ImageJ software respectively. Protein translation was assessed by inhibition with puromycin. Neuronal networking assessment was made with multielectrode array system (MEA).ResultIn this study, we observed that treatments of synthetic Aβ peptides in culture primary mouse cortical neurons induces the expression of FMRP and FMRP‐dependent ER stress response and subsequent translational suppression. Also Aβ induces FMRP‐dependent neuronal hyposynchronicity . We further characterized that two translational regulators, eukaryotic translation initiation factor 2α (eIF2α) and eukaryotic translation elongation factor 2 (eEF2), are involved in Aβ induced translational suppression and are dysregulated in the absence of FMRP. Mechanistically we found that FMRP is responsible for regulating the levels of Protein Phosphatase 2 (PP2A) and Protein Phosphatase 1 (PP1) that differentially modulate the phosphorylation status and thereby activity of eIF2α and eEF2 following treatment of Aβ. Using a pharmacological approach to inhibit PP2A and PP1 with Okadaic Acid, we were able to rescue the impairment of eIF2α and eEF2 as well as the translational suppression and lack of neuronal hyposynchrony in the absence of FMRP.ConclusionThese results suggest a novel function of FMRP in regulating translational suppression and neuronal networking function following Aβ treatment by fine‐tuning two distinct signaling pathways involved in global translation.

A noncanonical RNA-binding domain of the fragile X protein, FMRP, elicits translational repression independent of mRNA G-quadruplexes

Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome, the leading form of inherited intellectual disability and the most common monogenic cause of autism spectrum disorders. FMRP is an RNA-binding protein that controls neuronal mRNA localization and translation. FMRP is thought to inhibit translation elongation after being recruited to target transcripts via binding RNA G-quadruplexes (G4s) within the coding sequence. Here, we directly test this model and report that FMRP inhibits translation independent of mRNA G4s. Furthermore, we found that the RGG box motif together with its natural C-terminal domain forms a noncanonical RNA-binding domain (ncRBD) that is essential for translational repression. The ncRBD elicits broad RNA-binding ability and binds to multiple reporter mRNAs and all four homopolymeric RNAs. Serial deletion analysis of the ncRBD identified that the regions required for mRNA binding and translational repression overlap but are not identical. Consistent with FMRP stalling elongating ribosomes and causing the accumulation of slowed 80S ribosomes, transcripts bound by FMRP via the ncRBD cosediment with heavier polysomes and were present in puromycin-resistant ribosome complexes. Together, this work identifies a ncRBD and translational repression domain that shifts our understanding of how FMRP inhibits translation independent of mRNA G4s.

Cellular distribution of the Fragile X mental retardation protein in the inner ear: a developmental and comparative study in the mouse, rat, gerbil, and chicken.

The Fragile X mental retardation protein (FMRP) is an mRNA binding protein that is essential for neural circuit assembly and synaptic plasticity. Loss of functional FMRP leads to Fragile X syndrome (FXS), a neurodevelopmental disorder characterized by sensory dysfunction including abnormal auditory processing. While the central mechanisms of FMRP regulation have been studied in the brain, whether FMRP is expressed in the auditory periphery and how it develops and functions remains unknown. In this study, we characterized the spatiotemporal distribution pattern of FMRP immunoreactivity in the inner ear of mice, rats, gerbils, and chickens. Across species, FMRP was expressed in hair cells and supporting cells, with a particularly high level in immature hair cells during the prehearing period. Interestingly, the distribution of cytoplasmic FMRP displayed an age-dependent translocation in hair cells, and this feature was conserved across species. In the auditory ganglion (AG), FMRP immunoreactivity was detected in neuronal cell bodies as well as their peripheral and central processes. Distinct from hair cells, FMRP intensity in AG neurons was high both during development and after maturation. Additionally, FMRP was evident in mature glial cells surrounding AG neurons. Together, these observations demonstrate distinct developmental trajectories across cell types in the auditory periphery. Given the importance of peripheral inputs to the maturation of auditory circuits, these findings implicate involvement of FMRP in inner ear development as well as a potential contribution of periphery FMRP to the generation of auditory dysfunction in FXS.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation.

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

The Drosophila fragile X mental retardation protein modulates the neuronal cytoskeleton to limit dendritic arborization

Dendritic arbor development is a complex, highly regulated process. Post-transcriptional regulation mediated by RNA-binding proteins plays an important role in neuronal dendrite morphogenesis by delivering on-site, on-demand protein synthesis. Here, we show how the Drosophila fragile X mental retardation protein (FMRP), a conserved RNA-binding protein, limits dendrite branching to ensure proper neuronal function during larval sensory neuron development. FMRP knockdown causes increased dendritic terminal branch growth and a resulting overelaboration defect due, in part, to altered microtubule stability and dynamics. FMRP also controls dendrite outgrowth by regulating the Drosophila profilin homolog chickadee (chic). FMRP colocalizes with chic mRNA in dendritic granules and regulates its dendritic localization and protein expression. Whereas RNA-binding domains KH1 and KH2 are both crucial for FMRP-mediated dendritic regulation, KH2 specifically is required for FMRP granule formation and chic mRNA association, suggesting a link between dendritic FMRP granules and FMRP function in dendrite elaboration. Our studies implicate FMRP-mediated modulation of both the neuronal microtubule and actin cytoskeletons in multidendritic neuronal architecture, and provide molecular insight into FMRP granule formation and its relevance to FMRP function in dendritic patterning.

The Fragile X Syndrome‐Related Protein, FMRP, Modulates Innate Immune Gene Expression in Macrophages

Fragile X mental retardation protein (FMRP) is an RNA‐binding protein with a wide range of functions, including mRNA translation, mRNA transport, and mitochondrial organization. Loss of FMRP due to silencing of the FRM1 gene is associated with intellectual disability and has been implicated in viral pathogenesis and cancer risk. However, the contribution of FMRP to innate immunity in response to bacterial infection remains understudied. Given the function of FMRP in post‐transcriptional regulation, we hypothesized that loss of FMRP would impair repression of inflammatory cytokines in response to infection with the bacterial pathogen Listeria monocytogenes. We used RNA‐seq as an unbiased approach to identify global gene expression changes in FMRP‐deficient murine macrophages, both at rest and during infection with Listeria monocytogenes. We identified 22 differentially expressed genes that were upregulated in resting FMRP KO macrophages, including aconitate decarboxylase 1 (ACOD1, IRG1), erythroid differentiation regulator 1 (Erdr1), and Cxcl10, as well as 6 downregulated genes, including microRNA 6236 and mitochondrially encoded ATP synthase 8 (MT‐ATP8). Unbiased canonical pathway analysis of the upregulated genes in resting FMRP KO macrophages revealed significant enrichment of genes related to phagosome maturation and immunity. Within the downregulated genes, unbiased canonical pathway analysis revealed pathways related to EIF2 signaling and BEX2 Signaling. Analysis of L. monocytogenes infected FMRP KO macrophages revealed 31 upregulated genes, including the E3 ubiquitin ligase gene midline 1 (Mid1, Trim18) and the complement genes C1QA and C1QB, as well as 8 downregulated genes including plasminogen activator urokinase (PLAU). Canonical pathway analysis of the downregulated genes included DNA‐damage signaling and role of pattern recognition receptors in recognition of bacteria and viruses. Surprisingly, when comparing differentially enriched genes in infected macrophages to uninfected macrophages, a greater number of innate immune transcripts were upregulated in resting macrophages lacking FMRP. Together, these data suggest that FMRP may also have a role in innate immunity.

FMRP promotes transcription-coupled homologous recombination via facilitating TET1-mediated m5C RNA modification demethylation

RNA modifications regulate a variety of cellular processes including DNA repair.The RNA methyltransferase TRDMT1 generates methyl-5-cytosine (m5C) on messen-ger RNA (mRNA) at DNA double-strand breaks (DSBs) in transcribed regions, pro-moting transcription-coupled homologous recombination (HR). Here, we identifiedthat Fragile X mental retardation protein (FMRP) promotes transcription-coupled HRvia its interaction with both the m5C writer TRDMT1 and the m5C eraser ten-eleventranslocation protein 1 (TET1). TRDMT1, FMRP, and TET1 function in a temporalorder at the transcriptionally active sites of DSBs. FMRP displays a higher affinity forDNA:RNA hybrids containing m5C-modified RNA than for hybrids without modifica-tion and facilitates demethylation of m5C by TET1 in vitro. Loss of either the chroma-tin- or RNA-binding domain of FMRP compromises demethylation of damage-inducedm5C in cells. Importantly, FMRP is required for R-loop resolving in cells. Due to unre-solved R-loop and m5C preventing completion of DSB repair, FMRP depletion or lowexpression leads to delayed repair of DSBs at transcriptionally active sites and sensitizescancer cells to radiation in a BRCA-independent manner. Together, ourfindings presentan m5C reader, FMRP, which acts as a coordinator between the m5C writer and eraserto promote mRNA-dependent repair and cell survival in cancer.

Fragile X Syndrome: role of the FMR1 gene in neurodevelopmental disorders

Fragile X syndrome (FXS) is a multisystem disorder characterized by a large clinical spectrum including neuropsychological and behavioral disturbances such as intellectual deficiency (ID), global developmental delay, seizures and autism spectrum disorders (ASD). This syndrome is the result of a full mutation of the FMR1 gene. Here, we report the case of two patients with FXS.
 Patient 1 is a 9-years-old child with ID, psychomotor developmental delay and facial dysmorphia. The family survey found a history of school failure in maternal uncle and cousin. FMR1 gene analysis identified pre-mutation and full mutation.
 Patient 2, aged 10, presents ID, epilepsy, absence of language, psychomotor retardation, behavioral disorder and facial dysmorphia. The brothers and three maternal cousins have ID. FMR1 gene analysis revealed full mutation.
 The FMR1 gene, located on Xq27.3, is involved in the development and maintenance of neural circuits essential for cognitive functions. FMR1 encodes the fragile X mental retardation protein (FMRP). This protein has a local role in synaptic plasticity, dendrite and axon development, and underlying learning and memory. The trinucleotide repeat expansions inactivate the FMR1 gene leading to the absence of FMRP and RNA/proteins dysregulation that causes alterations in synaptic connectivity clearly observed in FXS patient brains and a decrease in the number of dendrites and spines. FXS research emerged as a model of translational neuroscience. Therefore, deeper knowledge of FMRP functions would lead to rational therapeutic approaches. Furthermore, understanding the role of FMRP at synapses offers hope for the treatment of other ASD affecting learning and behavior.
 Keywords: X Fragile Syndrome, FMR1, FMRP, Neurodevelopmental disorders.

FMRP Sustains Presynaptic Function via Control of Activity-Dependent Bulk Endocytosis.

Synaptic vesicle (SV) recycling is essential for the maintenance of neurotransmission, with a number of neurodevelopmental disorders linked to defects in this process. Fragile X syndrome (FXS) results from a loss of fragile X mental retardation protein (FMRP) encoded by the FMR1 gene. Hyperexcitability of neuronal circuits is a key feature of FXS, therefore we investigated whether SV recycling was affected by the absence of FMRP during increased neuronal activity. We revealed that primary neuronal cultures from male Fmr1 knock-out (KO) rats display a specific defect in activity-dependent bulk endocytosis (ADBE). ADBE is dominant during intense neuronal activity, and this defect resulted in an inability of Fmr1 KO neurons to sustain SV recycling during trains of high-frequency stimulation. Using a molecular replacement strategy, we also revealed that a human FMRP mutant that cannot bind BK channels failed to correct ADBE dysfunction in KO neurons, however this dysfunction was corrected by BK channel agonists. Therefore, FMRP performs a key role in sustaining neurotransmitter release via selective control of ADBE, suggesting intervention via this endocytosis mode may correct the hyperexcitability observed in FXS.SIGNIFICANCE STATEMENT Loss of fragile X mental retardation protein (FMRP) results in fragile X syndrome (FXS), however whether its loss has a direct role in neurotransmitter release remains a matter of debate. We demonstrate that neurons lacking FMRP display a specific defect in a mechanism that sustains neurotransmitter release during intense neuronal firing, called activity-dependent bulk endocytosis (ADBE). This discovery provides key insights into mechanisms of brain communication that occur because of loss of FMRP function. Importantly it also reveals ADBE as a potential therapeutic target to correct the circuit hyperexcitability observed in FXS.

Reciprocal regulation of spontaneous synaptic vesicle fusion by Fragile X mental retardation protein and group I metabotropic glutamate receptors.

Fragile X mental retardation protein (FMRP) is a neuronal protein mediating multiple functions, with its absence resulting in one of the most common monogenic causes of autism, Fragile X syndrome (FXS). Analyses of FXS pathophysiology have identified a range of aberrations in synaptic signaling pathways and plasticity associated with group I metabotropic glutamate (mGlu) receptors. These studies, however, have mostly focused on the post-synaptic functions of FMRP and mGlu receptor activation, and relatively little is known about their presynaptic effects. Neurotransmitter release is mediated via multiple forms of synaptic vesicle (SV) fusion, each of which contributes to specific neuronal functions. The impacts of mGlu receptor activation and loss of FMRP on these SV fusion events remain unexplored. Here we combined electrophysiological and fluorescence imaging analyses on primary hippocampal cultures prepared from an Fmr1 knockout (KO) rat model. Compared to wild-type (WT) hippocampal neurons, KO neurons displayed an increase in the frequency of spontaneous excitatory post-synaptic currents (sEPSCs), as well as spontaneous SV fusion events. Pharmacological activation of mGlu receptors in WT neurons caused a similar increase in spontaneous SV fusion and sEPSC frequency. Notably, this increase in SV fusion was not observed when spontaneous activity was blocked using the sodium channel antagonist tetrodotoxin. Importantly, the effect of mGlu receptor activation on spontaneous SV fusion was occluded in Fmr1 KO neurons. Together, our results reveal that FMRP represses spontaneous presynaptic SV fusion, whereas mGlu receptor activation increases this event. This reciprocal control appears to be mediated via their regulation of intrinsic neuronal excitability.

FMRP Interacts with RARα in Synaptic Retinoic Acid Signaling and Homeostatic Synaptic Plasticity.

The fragile X syndrome (FXS) is an X-chromosome-linked neurodevelopmental disorder with severe intellectual disability caused by inactivation of the fragile X mental retardation 1 (FMR1) gene and subsequent loss of the fragile X mental retardation protein (FMRP). Among the various types of abnormal synaptic function and synaptic plasticity phenotypes reported in FXS animal models, defective synaptic retinoic acid (RA) signaling and subsequent defective homeostatic plasticity have emerged as a major synaptic dysfunction. However, the mechanism underlying the defective synaptic RA signaling in the absence of FMRP is unknown. Here, we show that RARα, the RA receptor critically involved in synaptic RA signaling, directly interacts with FMRP. This interaction is enhanced in the presence of RA. Blocking the interaction between FMRP and RARα with a small peptide corresponding to the critical binding site in RARα abolishes RA-induced increases in excitatory synaptic transmission, recapitulating the phenotype seen in the Fmr1 knockout mouse. Taken together, these data suggest that not only are functional FMRP and RARα necessary for RA-dependent homeostatic synaptic plasticity, but that the interaction between these two proteins is essential for proper transcription-independent RA signaling. Our results may provide further mechanistic understanding into FXS synaptic pathophysiology.

An "Omic" Overview of Fragile X Syndrome.

Simple SummaryFragile X syndrome (FXS) is a neurodevelopmental disorder and remains the most frequent inherited cause of intellectual disability. Fragile X patients are at great risk to develop behavior problems including autism, anxiety, aggressivity and hyperactivity. FXS results from a mutation leading to the absence of a single protein, the fragile X mental retardation protein (FMRP). Most studies aiming to understand the physiopathology of FXS are centered on the effect of its absence on protein synthesis. However, besides protein synthesis regulation, FMRP has several other functions that need to be understood and could play a significant role in FXS. The goal of the present work is to review some of these functions and to put them into perspective in order to get a more comprehensive understanding of FXS physiopathology.Fragile X syndrome (FXS) is a neurodevelopmental disorder associated with a wide range of cognitive, behavioral and medical problems. It arises from the silencing of the fragile X mental retardation 1 (FMR1) gene and, consequently, in the absence of its encoded protein, FMRP (fragile X mental retardation protein). FMRP is a ubiquitously expressed and multifunctional RNA-binding protein, primarily considered as a translational regulator. Pre-clinical studies of the past two decades have therefore focused on this function to relate FMRP’s absence to the molecular mechanisms underlying FXS physiopathology. Based on these data, successful pharmacological strategies were developed to rescue fragile X phenotype in animal models. Unfortunately, these results did not translate into humans as clinical trials using same therapeutic approaches did not reach the expected outcomes. These failures highlight the need to put into perspective the different functions of FMRP in order to get a more comprehensive understanding of FXS pathophysiology. This work presents a review of FMRP’s involvement on noteworthy molecular mechanisms that may ultimately contribute to various biochemical alterations composing the fragile X phenotype.

Genetic removal of p70 S6K1 corrects coding sequence length-dependent alterations in mRNA translation in fragile X syndrome mice.

Loss of the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP is widely thought to repress protein synthesis, but its translational targets and modes of control remain in dispute. We previously showed that genetic removal of p70 S6 kinase 1 (S6K1) corrects altered protein synthesis as well as synaptic and behavioral phenotypes in FXS mice. In this study, we examined the gene specificity of altered messenger RNA (mRNA) translation in FXS and the mechanism of rescue with genetic reduction of S6K1 by carrying out ribosome profiling and RNA sequencing on cortical lysates from wild-type, FXS, S6K1 knockout, and double knockout mice. We observed reduced ribosome footprint (RF) abundance in the majority of differentially translated genes in the cortices of FXS mice. We used molecular assays to discover evidence that the reduction in RF abundance reflects an increased rate of ribosome translocation, which is captured as a decrease in the number of translating ribosomes at steady state and is normalized by inhibition of S6K1. We also found that genetic removal of S6K1 prevented a positive-to-negative gradation of alterations in translation efficiencies (RF/mRNA) with coding sequence length across mRNAs in FXS mouse cortices. Our findings reveal the identities of dysregulated mRNAs and a molecular mechanism by which reduction of S6K1 prevents altered translation in FXS.

Dysregulated CRMP Mediates Circadian Deficits in a Drosophila Model of Fragile X Syndrome

Fragile X syndrome (FXS) is the leading inherited cause of intellectual disability, resulting from the lack of functional fragile X mental retardation protein (FMRP), an mRNA binding protein mainly serving as a translational regulator. Loss of FMRP leads to dysregulation of target mRNAs. The Drosophila model of FXS show an abnormal circadian rhythm with disruption of the output pathway downstream of the clock network. Yet the FMRP targets involved in circadian regulation have not been identified. Here, we identified collapsing response mediator protein (CRMP) mRNA as a target of FMRP. Knockdown of pan-neuronal CRMP expression ameliorated the circadian defects and abnormal axonal structures of clock neurons (ventral lateral neurons) in dfmr1 mutant flies. Furthermore, specific reduction of CRMP in the downstream output insulin-producing cells attenuated the aberrant circadian behaviors. Molecular analyses revealed that FMRP binds with CRMP mRNA and negatively regulates its translation. Our results indicate that CRMP is an FMRP target and establish an essential role for CRMP in the circadian output in FXS Drosophila.

Neuron-Specific FMRP Roles in Experience-Dependent Remodeling of Olfactory Brain Innervation during an Early-Life Critical Period.

Critical periods are developmental windows during which neural circuits effectively adapt to the new sensory environment. Animal models of fragile X syndrome (FXS), a common monogenic autism spectrum disorder (ASD), exhibit profound impairments of sensory experience-driven critical periods. However, it is not known whether the causative fragile X mental retardation protein (FMRP) acts uniformly across neurons, or instead manifests neuron-specific functions. Here, we use the genetically-tractable Drosophila brain antennal lobe (AL) olfactory circuit of both sexes to investigate neuron-specific FMRP roles in the odorant experience-dependent remodeling of the olfactory sensory neuron (OSN) innervation during an early-life critical period. We find targeted OSN class-specific FMRP RNAi impairs innervation remodeling within AL synaptic glomeruli, whereas global dfmr1 null mutants display relatively normal odorant-driven refinement. We find both OSN cell autonomous and cell non-autonomous FMRP functions mediate odorant experience-dependent remodeling, with AL circuit FMRP imbalance causing defects in overall glomerulus innervation refinement. We find OSN class-specific FMRP levels bidirectionally regulate critical period remodeling, with odorant experience selectively controlling OSN synaptic terminals in AL glomeruli. We find OSN class-specific FMRP loss impairs critical period remodeling by disrupting responses to lateral modulation from other odorant-responsive OSNs mediating overall AL gain control. We find that silencing glutamatergic AL interneurons reduces OSN remodeling, while conversely, interfering with the OSN class-specific GABAA signaling enhances remodeling. These findings reveal control of OSN synaptic remodeling by FMRP with neuron-specific circuit functions, and indicate how neural circuitry can compensate for global FMRP loss to reinstate normal critical period brain circuit remodeling.SIGNIFICANCE STATEMENT Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism spectrum disorder (ASD), manifests severe neurodevelopmental delays. Likewise, FXS disease models display disrupted neurodevelopmental critical periods. In the well-mapped Drosophila olfactory circuit model, perturbing the causative fragile X mental retardation protein (FMRP) within a single olfactory sensory neuron (OSN) class impairs odorant-dependent remodeling during an early-life critical period. Importantly, this impairment requires activation of other OSNs, and the olfactory circuit can compensate when FMRP is removed from all OSNs. Understanding the neuron-specific FMRP requirements within a developing neural circuit, as well as the FMRP loss compensation mechanisms, should help us engineer FXS treatments. This work suggests FXS treatments could use homeostatic mechanisms to alleviate circuit-level deficits.

Carbamazepine Restores Neuronal Signaling, Protein Synthesis, and Cognitive Function in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is a leading genetic disorder of intellectual disability caused by the loss of the functional fragile X mental retardation protein (FMRP). To date, there is no efficacious mechanism-based medication for FXS. With regard to potential disease mechanisms in FXS, it is widely accepted that the lack of FMRP causes elevated protein synthesis and deregulation of neuronal signaling. Abnormal enhancement of the ERK½ (extracellular signal-regulated kinase ½) and PI3K-Akt (Phosphoinositide 3 kinase-protein kinase B) signaling pathways has been identified in both FXS patients and FXS mouse models. In this study, we show that carbamazepine, which is an FDA-approved drug and has been mainly used to treat seizure and neuropathic pain, corrects cognitive deficits including passive avoidance and object location memory in FXS mice. Carbamazepine also rescues hyper locomotion and social deficits. At the cellular level, carbamazepine dampens the elevated level of ERK½ and Akt signaling as well as protein synthesis in FXS mouse neurons. Together, these results advocate repurposing carbamazepine for FXS treatment.

The Fragile X Mental Retardation Protein Regulates Striatal Medium Spiny Neuron Synapse Density and Dendritic Spine Morphology.

The fragile X mental retardation protein (FMRP), an RNA-binding protein that mediates the transport, stability, and translation of hundreds of brain RNAs, is critically involved in regulating synaptic function. Loss of FMRP, as in fragile X syndrome (FXS), is a leading monogenic cause of autism and results in altered structural and functional synaptic plasticity, widely described in the hippocampus and cortex. Though FXS is associated with hyperactivity, impaired social interaction, and the development of repetitive or stereotyped behaviors, all of which are influenced by striatal activity, few studies have investigated the function of FMRP here. Utilizing a cortical-striatal co-culture model, we find that striatal medium spiny neurons (MSNs) lacking FMRP fail to make normal increases in PSD95 expression over a short time period and have significant deficits in dendritic spine density and colocalized synaptic puncta at the later measured time point compared to wildtype (WT) MSNs. Acute expression of wtFMRP plasmid in Fmr1 KO co-cultures results in contrasting outcomes for these measures on MSNs at the more mature time point, reducing spine density across multiple spine types but making no significant changes in colocalized puncta. FMRP’s KH2 and RGG RNA-binding domains are required for normal elimination of PSD95, and interruption of these domains slightly favors elimination of immature spine types. Further, KH2 is required for normal levels of colocalized puncta. Our data are largely consistent with a basal role for FMRP and its RNA-binding domains in striatal synapse stabilization on developing MSNs, and in light of previous findings, suggest distinct regional and/or cell type-specific roles for FMRP in regulating synapse structure.