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.     

Altered anxiety-related and social behaviors in the Fmr1 knockout mouse model of fragile X syndrome

The loss of fragile X mental retardation (FMR1) gene function causes fragile X syndrome (FXS), a common mental retardation syndrome. Anxiety and abnormal social behaviors are prominent features of FXS in humans. To better understand the role of FMR1 in these behaviors, we analyzed anxiety-related and social behaviors in Fmr1 knockout (KO) mice. In the mirrored chamber test, Fmr1 KO mice showed greater aversion to the central mirrored chamber than wild-type (WT) littermates, suggesting increased anxiety-like responses to reflected images of mice. Fmr1 KO mice exhibited abnormal social interactions in a tube test of social dominance, winning fewer matches than WT littermates. In a partition test, Fmr1 KO mice had normal levels of social interest and social recognition. However, during direct interaction tests, Fmr1 KO mice showed significant increases in sniffing behaviors. We further tested the influence of environmental familiarity on the social responses of Fmr1 KO mice to unfamiliar partners. In unfamiliar partitioned cages, Fmr1 KO mice did not differ from WT mice in investigation of unfamiliar partners. However, in familiar partitioned cages, Fmr1 KO mice showed less investigation of a newly introduced partner during the first 5 min and more investigation during the last 5 min of a 20-min partition test, behaviors consistent with initial social anxiety followed by enhanced social investigation. Our findings indicate that the loss of Fmr1 gene function results in altered anxiety and social behavior in mice and demonstrate that the Fmr1 KO mouse is a relevant animal model for the abnormal social responses seen in FXS.     

Control of dendritic development by the Drosophila fragile X-related gene involves the small GTPase Rac1

Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 gene. How these mutations affect neuronal development and function remains largely elusive. We generated specific point mutations or small deletions in the Drosophila fragile X-related (Fmr1) gene and examined the roles of Fmr1 in dendritic development of dendritic arborization (DA) neurons in Drosophila larvae. We found that Fmr1 could be detected in the cell bodies and proximal dendrites of DA neurons and that Fmr1 loss-of-function mutations increased the number of higher-order dendritic branches. Conversely, overexpression of Fmr1 in DA neurons dramatically decreased dendritic branching. In dissecting the mechanisms underlying Fmr1 function in dendrite development, we found that the mRNA encoding small GTPase Rac1 was present in the Fmr1-messenger ribonucleoprotein complexes in vivo. Mosaic analysis with a repressor cell marker (MARCM) and overexpression studies revealed that Rac1 has a cell-autonomous function in promoting dendritic branching of DA neurons. Furthermore, Fmr1 and Rac1 genetically interact with each other in controlling the formation of fine dendritic branches. These findings demonstrate that Fmr1 affects dendritic development and that Rac1 is partially responsible for mediating this effect.     

Deficits in Tactile Learning in a Mouse Model of Fragile X Syndrome

The fragile X mental retardation 1 mutant mouse (Fmr1 KO) recapitulates several of the neurologic deficits associated with Fragile X syndrome (FXS). As tactile hypersensitivity is a hallmark of FXS, we examined the sensory representation of individual whiskers in somatosensory barrel cortex of Fmr1 KO and wild-type (WT) mice and compared their performance in a whisker-dependent learning paradigm, the gap cross assay. Fmr1 KO mice exhibited elevated responses to stimulation of individual whiskers as measured by optical imaging of intrinsic signals. In the gap cross task, initial performance of Fmr1 KO mice was indistinguishable from WT controls. However, while WT mice improved significantly with experience at all gap distances, Fmr1 KO mice displayed significant and specific deficits in improvement at longer distances which rely solely on tactile information from whiskers. Thus, Fmr1 KO mice possess altered cortical responses to sensory input that correlates with a deficit in tactile learning.     

Comprehensive Analysis of Ultrasonic Vocalizations in a Mouse Model of Fragile X Syndrome Reveals Limited, Call Type Specific Deficits

Fragile X syndrome (FXS) is a well-recognized form of inherited mental retardation, caused by a mutation in the fragile X mental retardation 1 (Fmr1) gene. The gene is located on the long arm of the X chromosome and encodes fragile X mental retardation protein (FMRP). Absence of FMRP in fragile X patients as well as in Fmr1 knockout (KO) mice results, among other changes, in abnormal dendritic spine formation and altered synaptic plasticity in the neocortex and hippocampus. Clinical features of FXS include cognitive impairment, anxiety, abnormal social interaction, mental retardation, motor coordination and speech articulation deficits. Mouse pups generate ultrasonic vocalizations (USVs) when isolated from their mothers. Whether those social ultrasonic vocalizations are deficient in mouse models of FXS is unknown. Here we compared isolation-induced USVs generated by pups of Fmr1-KO mice with those of their wild type (WT) littermates. Though the total number of calls was not significantly different between genotypes, a detailed analysis of 10 different categories of calls revealed that loss of Fmr1 expression in mice causes limited and call-type specific deficits in ultrasonic vocalization: the carrier frequency of flat calls was higher, the percentage of downward calls was lower and that the frequency range of complex calls was wider in Fmr1-KO mice compared to their WT littermates.     

Fmr1 knockout mice are impaired in a leverpress escape/avoidance task

Fragile X syndrome (FXS) is the most common form of inherited mental retardation (MR). FXS is typically caused by a mutation of the Fmr1 gene (Verkerk et al. 1991, Cell 65, 905-914). To better understand the role of Fmr1 and its gene product fragile X mental-retardation protein (FMRP) in central nervous system function, researchers have turned to the use of animal model systems to generate an Fmr1 knockout (KO) mouse that is deficient in FMRP (Bakker et al. 1994, Cell 78, 23-33). Unfortunately, a number of studies have found no consistent, robust learning and memory impairment in the Fmr1 KO mice. We conducted a study to assess the performance of Fmr1 KO and wildtype (WT) animals in a leverpress escape/avoidance paradigm. Fmr1 KO and WT littermates were studied in four daily 1-h sessions. The Fmr1 KO mice performed fewer avoidance and total responses than WT mice. The KO animals were not simply deficient in avoidance, but a within-factor ANOVA revealed that they did not acquire the leverpress response to any appreciable degree. Observation during the sessions indicated that the Fmr1 KO animals clearly responded to the shock, eliminating an obvious sensory explanation for the deficit. The fact that other studies have found that the KO mice displayed increased exploratory and locomotor activity compared with WT controls argues against a motoric deficit. Future studies will attempt to delineate the nature of the behavioral deficit as well as attempt to rescue the response with glutamatergic or dopaminergic agents.     

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.     

Evidence of reactive astrocytes but not peripheral immune system activation in a mouse model of Fragile X syndrome

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is one of the few known genetic causes of autism. FXS results from the loss of Fmr1 gene function; thus, Fmr1 knockout mice provide a model to study impairments associated with FXS and autism and to test potential therapeutic interventions. The inhibitory serine phosphorylation of glycogen synthase kinase-3 (GSK3) is lower in brain regions of Fmr1 knockout mice than wild-type mice and the GSK3 inhibitor lithium rescues several behavioral impairments in Fmr1 knockout mice. Therefore, we examined if the serine phosphorylation of GSK3 in Fmr1 knockout mice also was altered outside the brain and if administration of lithium ameliorated the macroorchidism phenotype. Additionally, since GSK3 regulates numerous functions of the immune system and immune alterations have been associated with autism, we tested if immune function is altered in Fmr1 knockout mice. The inhibitory serine phosphorylation of GSK3 was significantly lower in the testis and liver of Fmr1 knockout mice than wild-type mice, and chronic lithium treatment reduced macroorchidism in Fmr1 knockout mice. No alterations in peripheral immune function were identified in Fmr1 knockout mice. However, examination of glia, the immune cells of the brain, revealed reactive astrocytes in several brain regions of Fmr1 knockout mice and treatment with lithium reduced this in the striatum and cerebellum. These results provide further evidence of the involvement of dysregulated GSK3 in FXS, and demonstrate that lithium administration reduces macroorchidism and reactive astrocytes in Fmr1 knockout mice.     

Failed Stabilization for Long-Term Potentiation in the Auditory Cortex of Fmr1 Knockout Mice

Fragile X syndrome is a developmental disorder that affects sensory systems. A null mutation of the Fragile X Mental Retardation protein 1 (Fmr1) gene in mice has varied effects on developmental plasticity in different sensory systems, including normal barrel cortical plasticity, altered ocular dominance plasticity and grossly impaired auditory frequency map plasticity. The mutation also has different effects on long-term synaptic plasticity in somatosensory and visual cortical neurons, providing insights on how it may differentially affect the sensory systems. Here we present evidence that long-term potentiation (LTP) is impaired in the developing auditory cortex of the Fmr1 knockout (KO) mice. This impairment of synaptic plasticity is consistent with impaired frequency map plasticity in the Fmr1 KO mouse. Together, these results suggest a potential role of LTP in sensory map plasticity during early sensory development.     

Activation of the extracellular signal-regulated kinase pathway contributes to the behavioral deficit of fragile x-syndrome

Fragile X syndrome (FXS) is a developmental disorder caused by the loss of Fragile X Mental Retardation 1 (FMR1) gene function because of a CGG repeat expansion (> 200 repeats) in the gene. The molecular mechanism(s) linking loss of FMR1 function to the molecular pathology and cognitive/behavioral disability remain unclear. Given the critical role of extracellular signal-regulated kinase (ERK) in synaptic plasticity and neurodevelopment, a number of recent studies have investigated ERK phosphorylation under basal conditions or upon mGluR-induction using neuronal and peripheral tissues from Fmr1 knockout mice and peripheral tissues from FXS patients. However, these reports have presented conflicting results. The current study is the first to focus on the levels of ERK phosphorylation in brain tissue from human FXS patients. In both human brain tissue and brain tissue from Fmr1 knockout mice there was significantly increased phosphorylation of MEK1/2 and ERK. Indeed, treating Fmr1 knockout mice with the MEK1/2 inhibitor SL327 abrogated audiogenic seizure activity, a feature of the Fmr1 knockout mice that replicates the symptom in patients with FXS. These findings suggest that activation of the ERK pathway results in some cardinal cognitive and clinical features in FXS patients and likely have profound translational implications.     

Age- and region-specific imbalances of basal amino acids and monoamine metabolism in limbic regions of female Fmr1 knock-out mice

The Fragile X syndrome, a common form of mental retardation in humans, originates from the loss of expression of the Fragile X mental retardation gene leading to the absence of the encoded Fragile X mental retardation protein 1 (FMRP). A broad pattern of morphological and behavioral abnormalities is well described for affected humans as well as Fmr1 knock-out mice, a transgenic animal model for the human Fragile X syndrome. In the present study, we examined neurochemical differences between female Fmr1 knock-out and wildtype mice with particular focus on neurotransmission. Significant age- and region-specific differences of basal tissue neurotransmitter and metabolite levels measured by high performance liquid chromatography were found. Those differences were more numerous in juvenile animals (postnatal day (PND) 28-31) compared to adults (postnatal day 209-221). In juvenile female knock-out mice, especially aspartate and taurine were increased in cortical regions, striatum, cerebellum, and brainstem. Furthermore, compared to the wildtype animals, the juvenile knock-out mice displayed an increased level of neuronal inhibition in the hippocampus and brainstem reflected by decreased ratios of (aspartate + glutamate)/(taurine + GABA), as well as an increased dopamine (DA) turnover in cortical regions, striatum, and hippocampus. These results provide the first evidence that the lack of FMRP expression in female Fmr1 knock-out mice is accompanied by age-dependent, region-specific alterations in brain amino acids, and monoamine turnover, which might be related to the reported synaptical and behavioural alterations in these animals.     

Increased Training Intensity Induces Proper Membrane Localization of Actin Remodeling Proteins in the Hippocampus Preventing Cognitive Deficits: Implications for Fragile X Syndrome

Behavioral intervention therapy has proven beneficial in the treatment of autism and intellectual disabilities (ID), raising the possibility of certain changes in molecular mechanisms activated by these interventions that may promote learning. Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by autistic features and intellectual disability and can serve as a model to examine mechanisms that promote learning. FXS results from mutations in the fragile X mental retardation 1 gene (Fmr1) that prevents expression of the Fmr1 protein (FMRP), a messenger RNA (mRNA) translation regulator at synapses. Among many other functions, FMRP organizes a complex with the actin cytoskeleton-regulating small Rho GTPase Rac1. As in humans, Fmr1 KO mice lacking FMRP display autistic-like behaviors and deformities of actin-rich synaptic structures in addition to impaired hippocampal learning and synaptic plasticity. These features have been previously linked to proper function of actin remodeling proteins that includes Rac1. An important step in Rac1 activation and function is its translocation to the membrane, where it can influence synaptic actin cytoskeleton remodeling during hippocampus-dependent learning. Herein, we report that Fmr1 KO mouse hippocampus exhibits increased levels of membrane-bound Rac1, which may prevent proper learning-induced synaptic changes. We also determine that increasing training intensity during fear conditioning (FC) training restores contextual memory in Fmr1 KO mice and reduces membrane-bound Rac1 in Fmr1 KO hippocampus. Increased training intensity also results in normalized long-term potentiation in hippocampal slices taken from Fmr1 KO mice. These results point to interventional treatments providing new therapeutic options for FXS-related cognitive dysfunction.     

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.