New perspectives on the biology of fragile X syndrome

Fragile X syndrome (FXS) is a trinucleotide repeat disorder caused by a CGG repeat expansion in FMR1, and loss of its protein product FMRP. Recent studies have provided increased support for the role of FMRP in translational repression via ribosomal stalling and the microRNA pathway. In neurons, particular focus has been placed on identifying the signaling pathways such as PI3K and mTOR downstream of group 1 metabotropic glutamate receptors (mGluR1/5) that regulate FMRP. New evidence also suggests that loss of FMRP causes presynaptic dysfunction and abnormal adult neurogenesis. In addition, studies on FXS stem cells especially induced pluripotent stem (iPS) cells and new sequencing efforts hold out promise for deeper understanding of the silencing process and mutation spectrum of FMR1.     

From FMRP Function to Potential Therapies for Fragile X Syndrome

Fragile X syndrome (FXS) is caused by mutations in the fragile X mental retardation 1 (FMR1) gene. Most FXS cases occur due to the expansion of the CGG trinucleotide repeats in the 5′ un-translated region of FMR1, which leads to hypermethylation and in turn silences the expression of FMRP (fragile X mental retardation protein). Numerous studies have demonstrated that FMRP interacts with both coding and non-coding RNAs and represses protein synthesis at dendritic and synaptic locations. In the absence of FMRP, the basal protein translation is enhanced and not responsive to neuronal stimulation. The altered protein translation may contribute to functional abnormalities in certain aspects of synaptic plasticity and intracellular signaling triggered by Gq-coupled receptors. This review focuses on the current understanding of FMRP function and potential therapeutic strategies that are mainly based on the manipulation of FMRP targets and knowledge gained from FXS pathophysiology.     

Bidirectional regulation of dendritic voltage-gated potassium channels by the fragile X mental retardation protein

How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel expression remains an open question. Here we report dendritic localization of mRNA of Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local translational regulation by fragile X mental retardation protein (FMRP) linked to fragile X syndrome (FXS), the most common heritable mental retardation. FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout (KO) mice and in neurons expressing Kv4.2-3'UTR that binds FMRP. Moreover, treating hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP regulation of Kv4.2 deepens our knowledge of NMDAR signaling and reveals a FMRP target of potential relevance to FXS.     

Excessive Astrocyte-Derived Neurotrophin-3 Contributes to the Abnormal Neuronal Dendritic Development in a Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS) is a form of inherited mental retardation in humans that results from expansion of a CGG repeat in the Fmr1 gene. Recent studies suggest a role of astrocytes in neuronal development. However, the mechanisms involved in the regulation process of astrocytes from FXS remain unclear. In this study, we found that astrocytes derived from a Fragile X model, the Fmr1 knockout (KO) mouse which lacks FMRP expression, inhibited the proper elaboration of dendritic processes of neurons in vitro. Furthermore, astrocytic conditioned medium (ACM) from KO astrocytes inhibited proper dendritic growth of both wild-type (WT) and KO neurons. Inducing expression of FMRP by transfection of FMRP vectors in KO astrocytes restored dendritic morphology and levels of synaptic proteins. Further experiments revealed elevated levels of the neurotrophin-3 (NT-3) in KO ACM and the prefrontal cortex of Fmr1 KO mice. However, the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) were normal. FMRP has multiple RNA–binding motifs and is involved in translational regulation. RNA–binding protein immunoprecipitation (RIP) showed the NT-3 mRNA interacted with FMRP in WT astrocytes. Addition of high concentrations of exogenous NT-3 to culture medium reduced the dendrites of neurons and synaptic protein levels, whereas these measures were ameliorated by neutralizing antibody to NT-3 or knockdown of NT-3 expression in KO astrocytes through short hairpin RNAs (shRNAs). Prefrontal cortex microinjection of WT astrocytes or NT-3 shRNA infected KO astrocytes rescued the deficit of trace fear memory in KO mice, concomitantly decreased the NT-3 levels in the prefrontal cortex. This study indicates that excessive NT-3 from astrocytes contributes to the abnormal neuronal dendritic development and that astrocytes could be a potential therapeutic target for FXS.     

G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3′-untranslated region of the FMR1 gene on the X chromosome, that 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 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3′ UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson–Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site.     

Proteomic analysis reveals CCT is a target of Fragile X mental retardation protein regulation in Drosophila

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that is required for the translational regulation of specific target mRNAs. Loss of FMRP causes Fragile X syndrome (FXS), the most common form of inherited mental retardation in humans. Understanding the basis for FXS has been limited because few in vivo targets of FMRP have been identified and mechanisms for how FMRP regulates physiological targets are unclear. We have previously demonstrated that Drosophila FMRP (dFMRP) is required in early embryos for cleavage furrow formation. In an effort to identify new targets of dFMRP-dependent regulation and new effectors of cleavage furrow formation, we used two-dimensional difference gel electrophoresis and mass spectrometry to identify proteins that are misexpressed in dfmr1 mutant embryos. Of the 28 proteins identified, we have identified three subunits of the Chaperonin containing TCP-1 (CCT) complex as new direct targets of dFMRP-dependent regulation. Furthermore, we found that the septin Peanut, a known effector of cleavage, is a likely conserved substrate of fly CCT and is mislocalized in both cct and in dfmr1 mutant embryos. Based on these results we propose that dFMRP-dependent regulation of CCT subunits is required for cleavage furrow formation and that at least one of its substrates is affected in dfmr1− embryos suggesting that dFMRP-dependent regulation of CCT contributes to the cleavage furrow formation phenotype.     

GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.     

FXS causing missense mutations disrupt FMRP granule formation,dynamics, and function

Fragile X Syndrome (FXS) is the most prevalent cause of inherited mental deficiency and is the most common monogenetic cause of autism spectral disorder (ASD). Here, we demonstrate that disease-causing missense mutations in the conserved K homology (KH) RNA binding domains (RBDs) of FMRP cause defects in its ability to form RNA transport granules in neurons. Using molecular, genetic, and imaging approaches in the Drosophila FXS model system, we show that the KH1 and KH2 domains of FMRP regulate distinct aspects of neuronal FMRP granule formation, dynamics, and transport. Furthermore, mutations in the KH domains disrupt translational repression in cells and the localization of known FMRP target mRNAs in neurons. These results suggest that the KH domains play an essential role in neuronal FMRP granule formation and function which may be linked to the molecular pathogenesis of FXS.     

Altered mTOR signaling and enhanced CYFIP2 expression levels in subjects with fragile X syndrome

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism. The protein (FMRP) encoded by the fragile X mental retardation gene (FMR1), is an RNA-binding protein linked to translational control. Recently, in the Fmr1 knockout mouse model of FXS, dysregulated translation initiation signaling was observed. To investigate whether an altered signaling was also a feature of subjects with FXS compared to typical developing controls, we isolated total RNA and translational control proteins from lymphocytes of subjects from both groups (38 FXS and 14 TD). Although we did not observe any difference in the expression level of messenger RNAs (mRNAs) for translational initiation control proteins isolated from participant with FXS, we found increased phosphorylation of the mammalian target of rapamycin (mTOR) substrate, p70 ribosomal subunit 6 kinase1 (S6K1) and of the mTOR regulator, the serine/threonine protein kinase (Akt), in their protein lysates. In addition, we observed increased phosphorylation of the cap binding protein eukaryotic initiation factor 4E (eIF4E) suggesting that protein synthesis is upregulated in FXS. Similar to the findings in lymphocytes, we observed increased phosphorylation of S6K1 in brain tissue from patients with FXS (n = 4) compared to normal age-matched controls (n = 4). Finally, we detected increased expression of the cytoplasmic FMR1-interacting protein 2 (CYFIP2), a known FMRP interactor. This data verify and extend previous findings using lymphocytes for studies of neuropsychiatric disorders and provide evidence that misregulation of mTOR signaling observed in the FXS mouse model also occurs in human FXS and may provide useful biomarkers for designing targeted treatments in FXS.     

Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms

Fragile X syndrome, the most common form of inherited mental impairment in humans, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5′-untranslated region (UTR) and subsequent translational silencing of the fragile x mental retardation-1 (FMR1) gene. FMRP, which is proposed to be involved in the translational regulation of specific neuronal messenger RNA (mRNA) targets, contains an arginine-glycine-glycine (RGG) box RNA binding domain that has been shown to bind with high affinity to G-quadruplex forming mRNA structures. FMRP undergoes alternative splicing, and the binding of FMRP to a proposed G-quadruplex structure in the coding region of its mRNA (named FBS) has been proposed to affect the mRNA splicing events at exon 15. In this study, we used biophysical methods to directly demonstrate the folding of FMR1 FBS into a secondary structure that contains two specific G-quadruplexes and analyze its interactions with several FMRP isoforms. Our results show that minor splice isoforms, ISO2 and ISO3, created by the usage of the second and third acceptor sites at exon 15, bind with higher affinity to FBS than FMRP ISO1, which is created by the usage of the first acceptor site. FMRP ISO2 and ISO3 cannot undergo phosphorylation, an FMRP post-translational modification shown to modulate the protein translation regulation. Thus, their expression has to be tightly regulated, and this might be accomplished by a feedback mechanism involving the FMRP interactions with the G-quadruplex structures formed within FMR1 mRNA.     

Novel fragile X syndrome 2D and 3D brain models based on human isogenic FMRP-KO iPSCs

Fragile X syndrome (FXS) is a neurodevelopmental disorder, characterized by intellectual disability and sensory deficits, caused by epigenetic silencing of the FMR1 gene and subsequent loss of its protein product, fragile X mental retardation protein (FMRP). Delays in synaptic and neuronal development in the cortex have been reported in FXS mouse models; however, the main goal of translating lab research into pharmacological treatments in clinical trials has been so far largely unsuccessful, leaving FXS a still incurable disease. Here, we generated 2D and 3D in vitro human FXS model systems based on isogenic FMR1 knock-out mutant and wild-type human induced pluripotent stem cell (hiPSC) lines. Phenotypical and functional characterization of cortical neurons derived from FMRP-deficient hiPSCs display altered gene expression and impaired differentiation when compared with the healthy counterpart. FXS cortical cultures show an increased number of GFAP positive cells, likely astrocytes, increased spontaneous network activity, and depolarizing GABAergic transmission. Cortical brain organoid models show an increased number of glial cells, and bigger organoid size. Our findings demonstrate that FMRP is required to correctly support neuronal and glial cell proliferation, and to set the correct excitation/inhibition ratio in human brain development.Subject terms: Neuronal development, Autism spectrum disorders, Genetics of the nervous system     

A Novel Function for Fragile X Mental Retardation Protein in Translational Activation

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the “kissing complex,” which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 null mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.     

FMRP acts as a key messenger for dopamine modulation in the forebrain

The fragile X mental retardation protein (FMRP) is an RNA-binding protein that controls translational efficiency and regulates synaptic plasticity. Here, we report that FMRP is involved in dopamine (DA) modulation of synaptic potentiation. AMPA glutamate receptor subtype 1 (GluR1) surface expression and phosphorylation in response to D1 receptor stimulation were reduced in cultured Fmr1(-/-) prefrontal cortex (PFC) neurons. Furthermore, D1 receptor signaling was impaired, accompanied by D1 receptor hyperphosphorylation at serine sites and subcellular redistribution of G protein-coupled receptor kinase 2 (GRK2) in both PFC and striatum of Fmr1(-/-) mice. FMRP interacted with GRK2, and pharmacological inhibition of GRK2 rescued D1 receptor signaling in Fmr1(-/-) neurons. Finally, D1 receptor agonist partially rescued hyperactivity and enhanced the motor function of Fmr1(-/-) mice. Our study has identified FMRP as a key messenger for DA modulation in the forebrain and may provide insights into the cellular and molecular mechanisms underlying fragile X syndrome.     

β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent 5‐HT4 receptor signalling

G protein‐coupled receptors (GPCRs) have been found to trigger G protein‐independent signalling. However, the regulation of G protein‐independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein‐independent 5‐HT₄ receptor (5‐HT₄R)‐operated Src/ERK (extracellular signal‐regulated kinase) pathway, but not the G_s pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor’ C‐terminus in both human embryonic kidney (HEK)‐293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C‐t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser⁴¹²) bound to the receptor. Phosphorylated β‐arrestin1 in turn prevented activation of Src constitutively bound to 5‐HT₄Rs, a necessary step in receptor‐stimulated ERK signalling. This is the first demonstration that β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent signalling.     

Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death

Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR) signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2) has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb) α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of G_αs, increase level of cAMP and activation of protein kinase A (PKA). Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89) substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP.     

Multiple functions of G protein-coupled receptor kinases

Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1–GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or β-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in β-arrestin-mediated and G protein-independent signaling pathways.     

Identification of a neuronal calcium sensor (NCS-1) possibly involved in the regulation of receptor phosphorylation

Abstract

Persistent stimulation of G protein-coupled receptors by agonists leads rapidly to reduced responses, a phenomenon described as desensitization. It involves primarily the phosphorylation of receptor sites by specific kinases of the G protein-coupled receptor kinase (GRK) family. The β-adrenergic receptor kinase 1 (GRK2) desensitizes agonist-activated β₂-adrenergic receptors, whereas rhodopsin kinase (GRK1) phosphorylates and inactivates photon-activated rhodopsin. Little is known about the role of calcium in desensitization. Here we report the characterization of a novel neuronal calcium sensor (NCS) named NCS-1 possibly involved in the regulation of receptor phosphorylation. NCS-1 is a new member of the EF-hand superfamily, which includes calmodulin, troponin C, parvalbumin, and recoverins. By Northern analysis and in situ hybridization, we discovered that NCS-1 is specifically expressed in the central and peripheral nervous systems. Chick NCS-1 has 72% of amino acid identity with Drosophila frequenin, a protein found in the nervous system and at the motor nerve terminals of neuromuscular junctions. By analogy with the reported function for two other members of the NCS family, we discuss whether G protein-coupled receptors or GRKs are the targets of neuronal calcium sensors.