Courting a cure for fragile X

Fragile X syndrome is the most common heritable cause of mental retardation. Previous work has suggested that overactive signaling by group I metabotropic glutamate receptors (mGluRs) may be a mechanism underlying many of the disease symptoms. As a test of this theory, McBride et al. show that in a Drosophila model for Fragile X syndrome, treatment with mGluR antagonists can rescue short-term memory, courtship, and mushroom body defects.     

Identification of small molecules rescuing fragile X syndrome phenotypes in Drosophila

Fragile X syndrome is caused by the functional loss of the fragile X mental retardation 1 (FMR1) gene. Deletion of the FMR1 ortholog in Drosophila melanogaster (Fmr1) recapitulates many phenotypes associated with fragile X syndrome. We have discovered that Fmr1 mutant Drosophila die during development when reared on food containing increased levels of glutamate, which is consistent with the theory that FMR1 loss results in excess glutamate signaling. Using this lethal phenotype, we screened a chemical library of 2,000 compounds and identified nine molecules that rescued the lethality, including three that implicate the GABAergic inhibitory pathway. Indeed, GABA treatment rescued several known Fmr1 mutant phenotypes in flies, including mushroom bodies defects, excess Futsch translation and abnormal male courtship behavior. These data are consistent with GABAergic inhibition of the enhanced excitatory pathway in fragile X syndrome. In addition, our screen reveals that the muscarinic cholinergic receptors may have a role in fragile X syndrome in parallel to the GABAergic pathway. These results point to potential therapeutic approaches for treating fragile X syndrome.     

Drosophila lacking dfmr1 activity show defects in circadian output and fail to maintain courtship interest

Fragile X mental retardation is a prominent genetic disorder caused by the lack of the FMR1 gene product, a known RNA binding protein. Specific physiologic pathways regulated by FMR1 function have yet to be identified. Adult dfmr1 (also called dfxr) mutant flies display arrhythmic circadian activity and have erratic patterns of locomotor activity, whereas overexpression of dFMR1 leads to a lengthened period. dfmr1 mutant males also display reduced courtship activity which appears to result from their inability to maintain courtship interest. Molecular analysis fails to reveal any defects in the expression of clock components; however, the CREB output is affected. Morphological analysis of neurons required for normal circadian behavior reveals subtle abnormalities, suggesting that defects in axonal pathfinding or synapse formation may cause the observed behavioral defects.     

An assay for social interaction in Drosophila fragile X mutants

We developed a novel assay to examine social interactions in Drosophila and, as a first attempt, apply it here at examining the behavior of Drosophila Fragile X Mental Retardation gene (dfmr1) mutants. Fragile X syndrome is the most common cause of single gene intellectual disability (ID) and is frequently associated with autism. Our results suggest that dfmr1 mutants are less active than wild-type flies and interact with each other less often. In addition, mutants for one allele of dfmr1, dfmr1^B55, are more likely to come in close contact with a wild-type fly than another dfmr1^B55 mutant. Our results raise the possibility of defective social expression with preserved receptive abilities. We further suggest that the assay may be applied in a general strategy of examining endophenoypes of complex human neurological disorders in Drosophila, and specifically in order to understand the genetic basis of social interaction defects linked with ID.Key words: Drosophila, Fragile X, autism, social behavior, novel assay     

Protein expression profiling of the drosophila fragile X mutant brain reveals up-regulation of monoamine synthesis

Fragile X syndrome is the most common form of inherited mental retardation, associated with both cognitive and behavioral anomalies. The disease is caused by silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the mRNA-binding, translational regulator FMRP. Previously we established a disease model through mutation of Drosophila fmr1 (dfmr1) and showed that loss of dFMRP causes defects in neuronal structure, function, and behavioral output similar to the human disease state. To uncover molecular targets of dFMRP in the brain, we use here a proteomic approach involving two-dimensional difference gel electrophoresis analyses followed by mass spectrometry identification of proteins with significantly altered expression in dfmr1 null mutants. We then focus on two misregulated enzymes, phenylalanine hydroxylase (Henna) and GTP cyclohydrolase (Punch), both of which mediate in concert the synthetic pathways of two key monoamine neuromodulators, dopamine and serotonin. Brain enzymatic assays show a nearly 2-fold elevation of Punch activity in dfmr1 null mutants. Consistently brain neurochemical assays show that both dopamine and serotonin are significantly increased in dfmr1 null mutants. At a cellular level, dfmr1 null mutant neurons display a highly significant elevation of the dense core vesicles that package these monoamine neuromodulators for secretion. Taken together, these data indicate that dFMRP normally down-regulates the monoamine pathway, which is consequently up-regulated in the mutant condition. Elevated brain levels of dopamine and serotonin provide a plausible mechanistic explanation for aspects of cognitive and behavioral deficits in human patients.     

Drosophila fragile X protein,DFXR, regulates neuronal morphology and function in the brain

Mental retardation is a pervasive societal problem, 25 times more common than blindness for example. Fragile X syndrome, the most common form of inherited mental retardation, is caused by mutations in the FMR1 gene. Fragile X patients display neurite morphology defects in the brain, suggesting that this may be the basis of their mental retardation. Drosophila contains a single homolog of FMR1, dfxr (also called dfmr1). We analyzed the role of dfxr in neurite development in three distinct neuronal classes. We find that DFXR is required for normal neurite extension, guidance, and branching. dfxr mutants also display strong eclosion failure and circadian rhythm defects. Interestingly, distinct neuronal cell types show different phenotypes, suggesting that dfxr differentially regulates diverse targets in the brain.     

The Drosophila fragile X mental retardation protein controls actin dynamics by directly regulating profilin in the brain

Loss of Fragile X mental retardation protein (FMRP) function causes the highly prevalent Fragile X syndrome [1 and 2]. Identifying targets for the RNA binding FMRP is a major challenge and an important goal of research into the pathology of the disease. Perturbations in neuronal development and circadian behavior are seen in Drosophila dfmr1 mutants. Here we show that regulation of the actin cytoskeleton is under dFMRP control. dFMRP binds the mRNA of the Drosophila profilin homolog and negatively regulates Profilin protein expression. An increase in Profilin mimics the phenotype of dfmr1 mutants. Conversely, decreasing Profilin levels suppresses dfmr1 phenotypes. These data place a new emphasis on actin misregulation as a major problem in fmr1 mutant neurons.     

Elevated levels of the vesicular monoamine transporter and a novel repetitive behavior in the Drosophila model of fragile X syndrome

Fragile X Syndrome (FXS) is characterized by mental impairment and autism in humans, and it often features hyperactivity and repetitive behaviors. The mechanisms for the disease, however, remain poorly understood. Here we report that the dfmr1 mutant in the Drosophila model of FXS grooms excessively, which may be regulated differentially by two signaling pathways. Blocking metabotropic glutamate receptor signaling enhances grooming in dfmr1 mutant flies, whereas blocking the vesicular monoamine transporter (VMAT) suppresses excessive grooming. dfmr1 mutant flies also exhibit elevated levels of VMAT mRNA and protein. These results suggest that enhanced monoamine signaling correlates with repetitive behaviors and hyperactivity associated with FXS.     

An assay for social interaction in Drosophila fragile X mutants

We developed a novel assay to examine social interactions in Drosophila and, as a first attempt, apply it here at examining the behavior of Drosophila Fragile X Mental Retardation gene (dfmr1) mutants. Fragile X syndrome is the most common cause of single gene intellectual disability (ID) and is frequently associated with autism. Our results suggest that dfmr1 mutants are less active than wild-type flies and interact with each other less often. In addition, mutants for one allele of dfmr1, dfmr1^B55, are more likely to come in close contact with a wild-type fly than another dfmr1^B55 mutant. Our results raise the possibility of defective social expression with preserved receptive abilities. We further suggest that the assay may be applied in a general strategy of examining endophenoypes of complex human neurological disorders in Drosophila, and specifically in order to understand the genetic basis of social interaction defects linked with ID.     

The cyclic AMP cascade is altered in the fragile X nervous system

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.     

Extinction of an instrumental response: a cognitive behavioral assay in Fmr1 knockout mice

Fragile X (FX) is the most common genetic cause of intellectual disability and autism. Previous studies have shown that partial inhibition of metabotropic glutamate receptor signaling is sufficient to correct behavioral phenotypes in a mouse model of FX, including audiogenic seizures, open‐field hyperactivity and social behavior. These phenotypes model well the epilepsy (15%), hyperactivity (20%) and autism (30%) that are comorbid with FX in human patients. Identifying reliable and robust mouse phenotypes to model cognitive impairments is critical considering the 90% comorbidity of FX and intellectual disability. Recent work characterized a five‐choice visuospatial discrimination assay testing cognitive flexibility, in which FX model mice show impairments associated with decreases in synaptic proteins in prefrontal cortex (PFC). In this study, we sought to determine whether instrumental extinction, another process requiring PFC, is altered in FX model mice, and whether downregulation of metabotropic glutamate receptor signaling pathways is sufficient to correct both visuospatial discrimination and extinction phenotypes. We report that instrumental extinction is consistently exaggerated in FX model mice. However, neither the extinction phenotype nor the visuospatial discrimination phenotype is corrected by approaches targeting metabotropic glutamate receptor signaling. This work describes a novel behavioral extinction assay to model impaired cognition in mouse models of neurodevelopmental disorders, provides evidence that extinction is exaggerated in the FX mouse model and suggests possible limitations of metabotropic glutamate receptor‐based pharmacotherapy.     

Substitution of critical isoleucines in the KH domains of Drosophila fragile X protein results in partial loss-of-function phenotypes

Fragile X mental retardation proteins (FMRP) are RNA-binding proteins that interact with a subset of cellular RNAs. Several RNA-binding domains have been identified in FMRP, but the contribution of these individual domains to FMRP function in an animal model is not well understood. In this study, we have generated flies with point mutations in the KH domains of the Drosophila melanogaster fragile X gene (dfmr1) in the context of a genomic rescue fragment. The substitutions of conserved isoleucine residues within the KH domains with asparagine are thought to impair binding of RNA substrates and perhaps the ability of FMRP to assemble into mRNP complexes. The mutants were analyzed for defects in development and behavior that are associated with deletion null alleles of dfmr1. We find that these KH domain mutations result in partial loss of function or no significant loss of function for the phenotypes assayed. The phenotypes resulting from these KH domain mutants imply that the capacities of the mutant proteins to bind RNA and form functional mRNP complexes are not wholly disrupted and are consistent with biochemical models suggesting that RNA-binding domains of FMRP can function independently.     

Argonaute2 Suppresses Drosophila Fragile X Expression Preventing Neurogenesis and Oogenesis Defects

Fragile X Syndrome is caused by the silencing of the Fragile X Mental Retardation gene (FMR1). Regulating dosage of FMR1 levels is critical for proper development and function of the nervous system and germ line, but the pathways responsible for maintaining normal expression levels are less clearly defined. Loss of Drosophila Fragile X protein (dFMR1) causes several behavioral and developmental defects in the fly, many of which are analogous to those seen in Fragile X patients. Over-expression of dFMR1 also causes specific neuronal and behavioral abnormalities. We have found that Argonaute2 (Ago2), the core component of the small interfering RNA (siRNA) pathway, regulates dfmr1 expression. Previously, the relationship between dFMR1 and Ago2 was defined by their physical interaction and co-regulation of downstream targets. We have found that Ago2 and dFMR1 are also connected through a regulatory relationship. Ago2 mediated repression of dFMR1 prevents axon growth and branching defects of the Drosophila neuromuscular junction (NMJ). Consequently, the neurogenesis defects in larvae mutant for both dfmr1 and Ago2 mirror those in dfmr1 null mutants. The Ago2 null phenotype at the NMJ is rescued in animals carrying an Ago2 genomic rescue construct. However, animals carrying a mutant Ago2 allele that produces Ago2 with significantly reduced endoribonuclease catalytic activity are normal with respect to the NMJ phenotypes examined. dFMR1 regulation by Ago2 is also observed in the germ line causing a multiple oocyte in a single egg chamber mutant phenotype. We have identified Ago2 as a regulator of dfmr1 expression and have clarified an important developmental role for Ago2 in the nervous system and germ line that requires dfmr1 function.     

Therapeutic implications of the mGluR theory of fragile X mental retardation

Evidence is reviewed that the consequences of group 1 metabotropic glutamate receptor (Gp1 mGluR) activation are exaggerated in the absence of the fragile X mental retardation protein, likely reflecting altered dendritic protein synthesis. Abnormal mGluR signaling could be responsible for remarkably diverse psychiatric and neurological symptoms in fragile X syndrome, including delayed cognitive development, seizures, anxiety, movement disorders and obesity.     

S6K1 phosphorylates and regulates fragile X mental retardation protein (FMRP) with the neuronal protein synthesis-dependent mammalian target of rapamycin (mTOR) signaling cascade

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

Reversal of fragile X phenotypes by manipulation of AβPP/Aβ levels in Fmr1KO mice

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading known genetic cause of autism. Fragile X mental retardation protein (FMRP), which is absent or expressed at substantially reduced levels in FXS, binds to and controls the postsynaptic translation of amyloid β-protein precursor (AβPP) mRNA. Cleavage of AβPP can produce β-amyloid (Aβ), a 39-43 amino acid peptide mis-expressed in Alzheimer's disease (AD) and Down syndrome (DS). Aβ is over-expressed in the brain of Fmr1(KO) mice, suggesting a pathogenic role in FXS. To determine if genetic reduction of AβPP/Aβ rescues characteristic FXS phenotypes, we assessed audiogenic seizures (AGS), anxiety, the ratio of mature versus immature dendritic spines and metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD) in Fmr1(KO) mice after removal of one App allele. All of these phenotypes were partially or completely reverted to normal. Plasma Aβ(1-42) was significantly reduced in full-mutation FXS males compared to age-matched controls while cortical and hippocampal levels were somewhat increased, suggesting that Aβ is sequestered in the brain. Evolving therapies directed at reducing Aβ in AD may be applicable to FXS and Aβ may serve as a plasma-based biomarker to facilitate disease diagnosis or assess therapeutic efficacy.     

Group I mGluR antagonist rescues the deficit of D1-induced LTP in a mouse model of fragile X syndrome

ABSTRACT: BACKGROUND: Fragile X syndrome (FXS) is caused by the absence of the mRNA-binding protein Fragile X mental retardation protein (FMRP), encoded by the Fmr1 gene. Overactive signaling by group 1 metabotropic glutamate receptor (Grp1 mGluR) could contribute to slowed synaptic development and other symptoms of FXS. Our previous study has identified that facilitation of synaptic long-term potentiation (LTP) by D1 receptor is impaired in Fmr1 knockout (KO) mice. However, the contribution of Grp1 mGluR to the facilitation of synaptic plasticity by D1 receptor stimulation in the prefrontal cortex has been less extensively studied. RESULTS: Here we demonstrated that DL-AP3, a Grp1 mGluR antagonist, rescued LTP facilitation by D1 receptor agonist SKF81297 in Fmr1KO mice. Grp1 mGluR inhibition restored the GluR1-subtype AMPA receptors surface insertion by D1 activation in the cultured Fmr1KO neurons. Simultaneous treatment of Grp1 mGluR antagonist with D1 agonist recovered the D1 receptor signaling by reversing the subcellular redistribution of G protein-coupled receptor kinase 2 (GRK2) in the Fmr1KO neurons. Treatment of SKF81297 alone failed to increase the phosphorylation of NR2B-containing N-methyl D-aspartate receptors (NMDARs) at Tyr-1472 (p-NR2B-Tyr1472) in the cultures from KO mice. However, simultaneous treatment of DL-AP3 could rescue the level of p-NR2B-Tyr1472 by SKF81297 in the cultures from KO mice. Furthermore, behavioral tests indicated that simultaneous treatment of Grp1 mGluR antagonist with D1 agonist inhibited hyperactivity and improved the learning ability in the Fmr1KO mice. CONCLUSION: The findings demonstrate that mGluR1 inhibition is a useful strategy to recover D1 receptor signaling in the Fmr1KO mice, and combination of Grp1 mGluR antagonist and D1 agonist is a potential drug therapy for the FXS.     

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.