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Fragile X–associated tremor/ataxia syndrome (FXTAS) is a debilitating late-onset neurodegenerative disease in premutation carriers of the expanded CGG repeat in FMR1 that presents with a spectrum of neurological manifestations, such as gait ataxia, intention tremor, and parkinsonism [P. J. Hagerman, R. J. Hagerman, Ann. N. Y. Acad. Sci. 1338, 58–70 (2015); S. Jacquemont et al., JAMA 291, 460–469 (2004)]. Here, we performed whole-genome sequencing (WGS) on male premutation carriers (CGG55–200) and prioritized candidate variants to screen for candidate genetic modifiers using a Drosophila model of FXTAS. We found 18 genes that genetically modulate CGG-associated neurotoxicity in Drosophila, such as Prosbeta5 (PSMB5), pAbp (PABPC1L), e(y)1 (TAF9), and CG14231 (OSGEPL1). Among them, knockdown of Prosbeta5 (PSMB5) suppressed CGG-associated neurodegeneration in the fly as well as in N2A cells. Interestingly, an expression quantitative trait locus variant in PSMB5, PSMB5rs11543947-A, was found to be associated with decreased expression of PSMB5 and delayed onset of FXTAS in human FMR1 premutation carriers. Finally, we demonstrate evidence that PSMB5 knockdown results in suppression of CGG neurotoxicity via both the RAN translation and RNA-mediated toxicity mechanisms, thereby presenting a therapeutic strategy for FXTAS.

Background: Fragile X syndrome (FXS) is a leading genetic cause of autism and intellectual disability with cortical hyperexcitability and sensory hypersensitivity attributed to loss and hypofunction of inhibitory parvalbumin-expressing (PV) cells. Our studies provide novel insights into the role of excitatory neurons in abnormal development of PV cells during a postnatal period of inhibitory circuit refinement., Methods: To achieve Fragile X mental retardation gene (Fmr1) deletion and re-expression in excitatory neurons during the postnatal day (P)14-P21 period, we generated Cre CaMKIIa /Fmr1 Flox/y (cOFF) and Cre CaMKIIa /Fmr1 FloxNeo/y (cON) mice, respectively. Cortical phenotypes were evaluated in adult mice using biochemical, cellular, clinically relevant electroencephalogram (EEG) and behavioral tests., Results: We found that similar to global Fmr1 KO mice, the density of PV-expressing cells, their activation, and sound-evoked gamma synchronization were impaired in cOFF mice, but the phenotypes were improved in cON mice. cOFF mice also showed enhanced cortical gelatinase activity and baseline EEG gamma power, which were reduced in cON mice. In addition, TrkB phosphorylation and PV levels were lower in cOFF mice, which also showed increased locomotor activity and anxiety-like behaviors. Remarkably, when FMRP levels were restored in only excitatory neurons during the P14-P21 period, TrkB phosphorylation and mouse behaviors were also improved., Conclusions: These results indicate that postnatal deletion or re-expression of FMRP in excitatory neurons is sufficient to elicit or ameliorate structural and functional cortical deficits, and abnormal behaviors in mice, informing future studies about appropriate treatment windows and providing fundamental insights into the cellular mechanisms of cortical circuit dysfunction in FXS., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Moderate to hyper-expansion of trinucleotide repeats at the FRAXA and FRAXE fragile sites, with or without concurrent hypermethylation, has been associated with intellectual disability and other conditions. Unlike molecular diagnosis of FMR1 CGG repeat expansions in FRAXA, current detection of AFF2 CCG repeat expansions in FRAXE relies on low-throughput and otherwise inefficient techniques combining Southern blot analysis and PCR. A novel triplet-primed PCR assay was developed for simultaneous screening for trinucleotide repeat expansions at the FRAXA and FRAXE fragile sites, and was validated using archived clinical samples of known FMR1 and AFF2 genotypes. Population samples and FRAXE-affected samples were sequenced for the evaluation of variations in the AFF2 CCG repeat structure. The duplex assay accurately identified expansions at the FMR1 and AFF2 trinucleotide repeat loci. On Sanger sequencing of the AFF2 CCG repeat, the single-nucleotide polymorphism variant rs868914124(C) that effectively adds two CCG repeats at the 5'-end, was enriched in the Malay population and with short repeats (<11 CCGs), and was present in all six expanded AFF2 alleles of this study. All expanded AFF2 alleles contained multiple non-CCG interruptions toward the 5'-end of the repeat. A sensitive, robust, and rapid assay has been developed for the simultaneous detection of expansion mutations at the FMR1 and AFF2 trinucleotide repeat loci, simplifying screening for FRAXA- and FRAXE-associated disorders., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Women heterozygous for an expansion of CGG repeats in the 5'UTR of FMR1 risk developing fragile X-associated primary ovarian insufficiency (FXPOI) and/or tremor and ataxia syndrome (FXTAS). We show that expanded CGGs, independent of FMR1, are sufficient to drive ovarian insufficiency and that expression of CGG-containing mRNAs alone or in conjunction with a polyglycine-containing peptide translated from these RNAs contribute to dysfunction. Heterozygous females from two mouse lines expressing either CGG RNA-only (RNA-only) or CGG RNA and the polyglycine product FMRpolyG (FMRpolyG+RNA) were used to assess ovarian function in aging animals. The expression of FMRpolyG+RNA led to early cessation of breeding, ovulation and transcriptomic changes affecting cholesterol and steroid hormone biosynthesis. Females expressing CGG RNA-only did not exhibit decreased progeny during natural breeding, but their ovarian transcriptomes were enriched for alterations in cholesterol and lipid biosynthesis. The enrichment of CGG RNA-only ovaries for differentially expressed genes related to cholesterol processing provided a link to the ovarian cysts observed in both CGG-expressing lines. Early changes in transcriptome profiles led us to measure ovarian function in prepubertal females that revealed deficiencies in ovulatory responses to gonadotropins. These include impairments in cumulus expansion and resumption of oocyte meiosis, as well as reduced ovulated oocyte number. Cumulatively, we demonstrated the sufficiency of ectopically expressed CGG repeats to lead to ovarian insufficiency and that co-expression of CGG-RNA and FMRpolyG lead to premature cessation of breeding. However, the expression of CGG RNA-alone was sufficient to lead to ovarian dysfunction by impairing responses to hormonal stimulation., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Fragile X syndrome (FXS) is a leading genetic cause of autism with symptoms that include sensory processing deficits. In both humans with FXS and a mouse model [Fmr1 knockout (KO) mouse], electroencephalographic (EEG) recordings show enhanced resting state gamma power and reduced sound-evoked gamma synchrony. We previously showed that elevated levels of matrix metalloproteinase-9 (MMP-9) may contribute to these phenotypes by affecting perineuronal nets (PNNs) around parvalbumin (PV) interneurons in the auditory cortex of Fmr1 KO mice. However, how different cell types within local cortical circuits contribute to these deficits is not known. Here, we examined whether Fmr1 deletion in forebrain excitatory neurons affects neural oscillations, MMP-9 activity, and PV/PNN expression in the auditory cortex. We found that cortical MMP-9 gelatinase activity, mTOR/Akt phosphorylation, and resting EEG gamma power were enhanced in CreNex1/Fmr1Flox/y conditional KO (cKO) mice, whereas the density of PV/PNN cells was reduced. The CreNex1/Fmr1Flox/y cKO mice also show increased locomotor activity, but not the anxiety-like behaviors. These results indicate that fragile X mental retardation protein changes in excitatory neurons in the cortex are sufficient to elicit cellular, electrophysiological, and behavioral phenotypes in Fmr1 KO mice. More broadly, these results indicate that local cortical circuit abnormalities contribute to sensory processing deficits in autism spectrum disorders., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects premutation carriers (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Much remains unknown regarding the metabolic alterations associated with FXTAS, especially in the brain, and the most affected region, the cerebellum. Investigating the metabolic changes in FXTAS will aid in the identification of biomarkers as well as in understanding the pathogenesis of disease. To identify the metabolic alterations associated with FXTAS, we took advantage of our FXTAS mouse model that expresses 90 CGG repeats in cerebellar Purkinje neurons and exhibits the key phenotypic features of FXTAS. We performed untargeted global metabolic profiling of age-matched control and FXTAS mice cerebella at 16-20 weeks and 55 weeks. Out of 506 metabolites measured in cerebellum, we identified 186 metabolites that demonstrate significant perturbations due to the (CGG)90 repeat (P<0.05) and found that these differences increase dramatically with age. To identify key metabolic changes in FXTAS pathogenesis, we performed a genetic screen using a Drosophila model of FXTAS. Out of 28 genes that we tested in the fly, 8 genes showed significant enhanced neuronal toxicity associated with CGG repeats, such as Schlank (ceramide synthase), Sk2 (sphingosine kinase) and Ras (IMP dehydrogenase). By combining metabolic profiling with a Drosophila genetic screen to identify genetic modifiers of FXTAS, we demonstrate an effective method for functional validation of high-throughput metabolic data and show that sphingolipid and purine metabolism are significantly perturbed in FXTAS pathogenesis., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Unlabelled: How the loss of fragile X mental retardation protein (FMRP) in different brain cell types, especially in non-neuron glial cells, induces fragile X syndrome (FXS) phenotypes has just begun to be understood. In the current study, we generated inducible astrocyte-specific Fmr1 conditional knock-out mice (i-astro-Fmr1-cKO) and restoration mice (i-astro-Fmr1-cON) to study the in vivo modulation of FXS synaptic phenotypes by astroglial FMRP. We found that functional expression of glutamate transporter GLT1 is 40% decreased in i-astro-Fmr1-cKO somatosensory cortical astrocytes in vivo, which can be fully rescued by the selective re-expression of FMRP in astrocytes in i-astro-Fmr1-cON mice. Although the selective loss of astroglial FMRP only modestly increases spine density and length in cortical pyramidal neurons, selective re-expression of FMRP in astrocytes significantly attenuates abnormal spine morphology in these neurons of i-astro-Fmr1-cON mice. Moreover, we found that basal protein synthesis levels and immunoreactivity of phosphorylated S6 ribosomal protein (p-s6P) is significantly increased in i-astro-Fmr1-cKO mice, while the enhanced cortical protein synthesis observed in Fmr1 KO mice is mitigated in i-astro-Fmr1-cON mice. Furthermore, ceftriaxone-mediated upregulation of surface GLT1 expression restores functional glutamate uptake and attenuates enhanced neuronal excitability in Fmr1 KO mice. In particular, ceftriaxone significantly decreases the growth rate of abnormally accelerated body weight and completely corrects spine abnormality in Fmr1 KO mice. Together, these results show that the selective loss of astroglial FMRP contributes to cortical synaptic deficits in FXS, presumably through dysregulated astroglial glutamate transporter GLT1 and impaired glutamate uptake. These results suggest the involvement of astrocyte-mediated mechanisms in the pathogenesis of FXS., Significance Statement: Previous studies to understand how the loss of function of fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) have largely focused on neurons; whether the selective loss of astroglial FMRP in vivo alters astrocyte functions and contributes to the pathogenesis of FXS remain essentially unknown. This has become a long-standing unanswered question in the fragile X field, which is also relevant to autism pathogenesis. Our current study generated astrocyte-specific Fmr1 conditional knock-out and restoration mice, and provided compelling evidence that the selective loss of astroglial FMRP contributes to cortical synaptic deficits in FXS, likely through the dysregulated astroglial glutamate transporter GLT1 expression and impaired glutamate uptake. These results demonstrate previously undescribed astrocyte-mediated mechanisms in the pathogenesis of FXS., (Copyright © 2016 the authors 0270-6474/16/367080-16$15.00/0.)

Fragile X syndrome results from loss of FMR1 expression. Individuals with the disorder exhibit not only intellectual disability, but also an array of physical and behavioral abnormalities, including sleep difficulties. Studies in mice demonstrated that Fmr1, along with its paralog Fxr2, regulate circadian behavior, and that their absence disrupts expression and cycling of essential clock mRNAs in the liver. Recent reports have identified circadian genes to be essential for normal metabolism. Here we describe the metabolic defects that arise in mice mutated for both Fmr1 and Fxr2. These mice have reduced fat deposits compared with age- and weight-matched controls. Several metabolic markers show either low levels in plasma or abnormal circadian cycling (or both). Insulin levels are consistently low regardless of light exposure and feeding conditions, and the animals are extremely sensitive to injected insulin. Glucose production from introduced pyruvate and glucagon is impaired and the mice quickly clear injected glucose. These mice also have higher food intake and higher VO2 and VCO2 levels. We analyzed liver expression of genes involved in glucose homeostasis and found several that are expressed differentially in the mutant mice. These results point to the involvement of Fmr1 and Fxr2 in maintaining the normal metabolic state in mice., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Determining the molecular mechanism(s) leading to Purkinje neuron loss in the neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS) is limited by the complex morphology of this cell type. Purkinje neurons are notoriously difficult to isolate and maintain in culture presenting considerable difficultly to identify molecular changes in response to expanded CGG repeat (rCGG)-containing mRNA that induces neurotoxicity in FXTAS. Several studies have uncovered a number of RNA-binding proteins involved in translation that aberrantly interact with the CGG-containing RNA; however, whether these interactions alter the translational profile of cells has not been investigated. Here we employ bacTRAP translational profiling to demonstrate that Purkinje neurons ectopically expressing 90 CGG repeats exhibit a dramatic change in their translational profile even prior to the onset of rCGG-induced phenotypes. This approach identified ∼500 transcripts that are differentially associated with ribosomes in r(CGG)₉₀-expressing mice. Functional annotation cluster analysis revealed broad ontologies enriched in the r(CGG)₉₀ list, including RNA binding and response to stress. Intriguingly, a transcript for the Tardbp gene, implicated in a number of other neurodegenerative disorders, exhibits altered association with ribosomes in the presence of r(CGG)₉₀ repeats. We therefore tested and showed that reduced association of Tardbp mRNA with the ribosomes results in a loss of TDP-43 protein expression in r(CGG)₉₀-expressing Purkinje neurons. Furthermore, we showed that TDP-43 could modulate the rCGG repeat-mediated toxicity in a Drosophila model that we developed previously. These findings together suggest that translational dysregulation may be an underlying mechanism of rCGG-induced neurotoxicity in FXTAS., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder in which patients carry premutation alleles of 55-200 CGG repeats in the FMR1 gene. To date, whether alterations in epigenetic regulation modulate FXTAS has gone unexplored. 5-Hydroxymethylcytosine (5hmC) converted from 5-methylcytosine (5mC) by the ten-eleven translocation (TET) family of proteins has been found recently to play key roles in neuronal functions. Here, we undertook genome-wide profiling of cerebellar 5hmC in a FXTAS mouse model (rCGG mice) and found that rCGG mice at 16 weeks showed overall reduced 5hmC levels genome-wide compared with age-matched wild-type littermates. However, we also observed gain-of-5hmC regions in repetitive elements, as well as in cerebellum-specific enhancers, but not in general enhancers. Genomic annotation and motif prediction of wild-type- and rCGG-specific differential 5-hydroxymethylated regions (DhMRs) revealed their high correlation with genes and transcription factors that are important in neuronal developmental and functional pathways. DhMR-associated genes partially overlapped with genes that were differentially associated with ribosomes in CGG mice identified by bacTRAP ribosomal profiling. Taken together, our data strongly indicate a functional role for 5hmC-mediated epigenetic modulation in the etiology of FXTAS, possibly through the regulation of transcription.

Fragile X syndrome (FXS) is a debilitating neurodevelopmental disorder thought to arise from disrupted synaptic communication in several key brain regions, including the amygdala, a central processing center for information with emotional and social relevance. Recent studies reveal defects in both excitatory and inhibitory neurotransmission in mature amygdala circuits in Fmr1(-/y) mutants, the animal model of FXS. However, whether these defects are the result of altered synaptic development or simply faulty mature circuits remains unknown. Using a combination of electrophysiological and genetic approaches, we show the development of both presynaptic and postsynaptic components of inhibitory neurotransmission in the FXS amygdala is dynamically altered during critical stages of neural circuit formation. Surprisingly, we observe that there is a homeostatic correction of defective inhibition, which, despite transiently restoring inhibitory synaptic efficacy to levels at or beyond those of control, ultimately fails to be maintained. Using inhibitory interneuron-specific conditional knock-out and rescue mice, we further reveal that fragile X mental retardation protein function in amygdala inhibitory microcircuits can be segregated into distinct presynaptic and postsynaptic components. Collectively, these studies reveal a previously unrecognized complexity of disrupted neuronal development in FXS and therefore have direct implications for establishing novel temporal and region-specific targeted therapies to ameliorate core amygdala-based behavioral symptoms.

Purpose: The ability to accurately predict the likelihood of expansion of the CGG repeats in the FMR1 gene to a full mutation is of critical importance for genetic counseling of women who are carriers of premutation alleles (55-200 CGG repeats) and who are weighing the risk of having a child with fragile X syndrome. The presence of AGG interruptions within the CGG repeat tract is thought to decrease the likelihood of expansion to a full mutation during transmission, thereby reducing risk, although their contribution has not been quantified., Methods: We retrospectively analyzed 267 premutation alleles for number and position of AGG interruptions, length of pure CGG repeats, and CGG repeat lengths present in the offspring of the maternal transmissions. In addition, we determined the haplotypes of four markers flanking the 5'-UTR locus in the premutation mothers., Results: We found that the presence of AGG interruptions significantly increased genetic stability, whereas specific haplotypes had a marginal association with transmission instability., Conclusion: The presence of AGG interruptions reduced the risk of transmission of a full mutation for all maternal (premutation) repeat lengths below ~100 CGG repeats, with a differential risk (0 vs. 2 AGG) exceeding 60% for alleles in the 70- to 80-CGG repeat range.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder recognized in fragile X premutation carriers. Using Drosophila, we previously identified elongated non-coding CGG repeats in FMR1 allele as the pathogenic cause of FXTAS. Here, we use this same FXTAS Drosophila model to conduct a chemical screen that reveals small molecules that can ameliorate the toxic effects of fragile X premutation ribo-CGG (rCGG) repeats, among them several known phospholipase A(2) (PLA(2)) inhibitors. We show that specific inhibition of PLA(2) activity could mitigate the neuronal deficits caused by fragile X premutation rCGG repeats, including lethality and locomotion deficits. Furthermore, through a genetic screen, we identified a PLA(2) Drosophila ortholog that specifically modulates rCGG repeat-mediated neuronal toxicity. Our results demonstrate the utility of Drosophila models for unbiased small molecule screens and point to PLA(2) as a possible therapeutic target to treat FXTAS.

Deficiency in fragile X mental retardation protein (FMRP) results in fragile X syndrome (FXS), an inherited form of intellectual disability. Despite extensive research, it is unclear how FMRP deficiency contributes to the cognitive deficits in FXS. Fmrp-null mice show reduced adult hippocampal neurogenesis. As Fmrp is also enriched in mature neurons, we investigated the function of Fmrp expression in neural stem and progenitor cells (aNSCs) and its role in adult neurogenesis. Here we show that ablation of Fmrp in aNSCs by inducible gene recombination leads to reduced hippocampal neurogenesis in vitro and in vivo, as well as markedly impairing hippocampus-dependent learning in mice. Conversely, restoration of Fmrp expression specifically in aNSCs rescues these learning deficits in Fmrp-deficient mice. These data suggest that defective adult neurogenesis may contribute to the learning impairment seen in FXS, and these learning deficits can be rectified by delayed restoration of Fmrp specifically in aNSCs.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a progressive neurodegenerative disorder that has been diagnosed in a substantial fraction of older male fragile X premutation carriers. Patients affected by FXTAS have elevated levels of ribo-rCGG repeat containing FMR1 mRNA with normal to slightly reduced levels of FMRP in blood leukocytes. Coupled with the absence of FXTAS in fragile X syndrome patients, this suggests premutation-sized elongated rCGG repeats in the FMR1 transcript rather than alterations in the levels of FMRP are responsible for the FXTAS pathology. Mice expressing rCGG in the context of Fmr1 or the enhanced green fluorescent protein specifically in Purkinje neurons were generated to segregate the effects of rCGG from alterations in Fmr1 and to provide evidence that rCGG is necessary and sufficient to cause pathology similar to human FXTAS. The models exhibit the presence of intranuclear inclusions in Purkinje neurons, Purkinje neuron cell death and behavioral deficits. These results demonstrate that rCGG expressed in Purkinje neurons outside the context of Fmr1 mRNA can result in neuronal pathology in a mammalian system and demonstrate that expanded CGG repeats in RNA are the likely cause of the neurodegeneration in FXTAS.

Lack of fragile X mental retardation protein (FMRP) causes Fragile X Syndrome, the most common form of inherited mental retardation. FMRP is an RNA-binding protein and is a component of messenger ribonucleoprotein complexes, associated with brain polyribosomes, including dendritic polysomes. FMRP is therefore thought to be involved in translational control of specific mRNAs at synaptic sites. In mice lacking FMRP, protein synthesis-dependent synaptic plasticity is altered and structural malformations of dendritic protrusions occur. One hypothesized cause of the disease mechanism is based on exaggerated group I mGluR receptor activation. In this study, we examined the effect of the mGluR5 antagonist MPEP on Fragile X related behavior in Fmr1 KO mice. Our results demonstrate a clear defect in prepulse inhibition of startle in Fmr1 KO mice, that could be rescued by MPEP. Moreover, we show for the first time a structural rescue of Fragile X related protrusion morphology with two independent mGluR5 antagonists.

Fragile X Syndrome is the most common form of hereditary mental retardation. It is caused by a large expansion of the CGG trinucleotide repeat (>200 repeats) in the 5'-untranslated region (UTR) of the FMR1 gene that leads to silencing of its transcript. Individuals with CGG repeat expansions approximately between 60 and 200 are referred to as premutation carriers. Fragile X-associated tremor and ataxia syndrome (FXTAS), an RNA-mediated neurodegenerative disease has been described in up to 50% of males carrying premutation alleles. FRAXE, the most common form of non-syndromic X-linked mental retardation, is caused by expansion of a CCG trinucleotide repeat (>200) in the 5'-UTR of the FMR2 gene. While the FRAXE premutation length repeat is observed in the general population, there has not yet been a report of a neurodegenerative phenotype associated with these alleles. In this study, we show that the CCG premutation length repeat leads to an RNA-mediated neurodegenerative phenotype in a Drosophila model. Furthermore, we show that co-expression of both the CCG and CGG-containing RNAs suppresses their independent toxicity and is dependent on the RNAi pathway. These data support the concept that RNA toxicity is the mechanism of neuronal toxicity and suggests potential reversal of RNA-mediated phenotypes with complementary RNA molecules.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently described neurodegenerative disorder of older adult carriers of premutation alleles (60-200 CGG repeats) in the fragile X mental retardation gene (FMR1). It has been proposed that FXTAS is an RNA-mediated neurodegenerative disease caused by the titration of RNA-binding proteins by the CGG repeats. To test this hypothesis, we utilize a transgenic Drosophila model of FXTAS that expresses a premutation-length repeat (90 CGG repeats) from the 5' UTR of the human FMR1 gene and displays neuronal degeneration. Here, we show that overexpression of RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppresses the phenotype of the CGG transgenic fly. Furthermore, we show that hnRNP A2/B1 directly interacts with riboCGG repeats and that the CUGBP1 protein interacts with the riboCGG repeats via hnRNP A2/B1.

The Fmr1 knockout (KO) mouse is characterized by an increased audiogenic seizure (AGS) susceptibility and is considered a good animal model for epilepsy and seizures in the human fragile-X (FRAX) syndrome. Here, we tested the hypothesis that the reintroduction of the FMR1 gene is able to revert the AGS susceptibility characterizing Fmr1 KO mice. To this aim, two groups of Fmr1 KO transgenic mice, which have additional copies of the human FMR1 gene (YAC) or FMR1 cDNA (G6) were used. AGS susceptibility of these mice was examined and compared to that of Fmr1 KO, wild type, and wild-type animals in whom the FMR1gene was also introduced (over-expressed). Mice were tested at different ages because AGS susceptibility is age dependent. The intensity of response was scored and the results were analyzed by means of 2-way analysis of variance to evaluate the effects of age and genetic condition. We found that AGS susceptibility rescue is complete in the G6 mice and partial in YAC mice. Our data indicate that the introduction of the human FMR1 gene in Fmr1 KO mice is able to revert the Fmr1 KO epileptic phenotype.

Fragile X syndrome is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is a selective RNA-binding protein which forms a messenger ribonucleoprotein (mRNP) complex that associates with polyribosomes. Recently, mRNA ligands associated with FMRP have been identified. However, the mechanism by which FMRP regulates the translation of its mRNA ligands remains unclear. MicroRNAs are small noncoding RNAs involved in translational control. Here we show that in vivo mammalian FMRP interacts with microRNAs and the components of the microRNA pathways including Dicer and the mammalian ortholog of Argonaute 1 (AGO1). Using two different Drosophila melanogaster models, we show that AGO1 is critical for FMRP function in neural development and synaptogenesis. Our results suggest that FMRP may regulate neuronal translation via microRNAs and links microRNAs with human disease.

Obsessive-compulsive disorder (OCD) is a chronic psychiatric disease characterized by recurrent obsessions, compulsions, or both. The prevalence rate of OCD is 2.1% in the general population. Here we report cytogenetic analysis of 26 patients affected with OCD. In one male patient (OCD-K33), we identified a fragile X chromosome by cytogenetic analysis with 21% of cells demonstrating a fragile site at Xq27-q28. Polymerase chain reaction (PCR) and Southern blot analysis demonstrated that the molecular basis of the OCD-K33 fragile X chromosome was expansion of the CCG repeat at FRAXE. The number of the expanded repeats was estimated to be more than 300 copies, qualifying it as a full FRAXE mutation. Further analysis of the family members of OCD-K33 revealed another member with a full FRAXE mutation (630-1,200 copies of the CCG repeat), who had the clinical phenotype of speech impairment, and two other members with normal phenotypes and no FRAXE expansion. The two FRAXE expansions lead to complete methylation at the CCG repeat. The co-segregation of the full FRAXE mutation with apparent neurologic disorders in the same family provides further support to the notion that FRAXE is a genetic neurologic condition. Our findings expand the spectrum of clinical phenotypes associated with FRAXE mutations., (Copyright 2003 Wiley-Liss, Inc.)

Fragile X syndrome results from the massive expansion of a CGG repeat in the 5' untranslated region of the gene FMR1. Data suggest that the hyperexpansion properties of FMR1 CGG repeats may depend on flanking cis-acting elements. We have therefore used homologous recombination in yeast to introduce an in situ CGG expansion corresponding to a premutation-sized allele into a human YAC carrying the FMR1 locus. Several transgenic lines were generated that carried repeats of varying lengths and amounts of flanking sequence. Length-dependent instability in the form of small expansions and contractions was observed in both male and female transmissions over five generations. No parent-of-origin effect or somatic instability was observed. Alterations in tract length were found to occur exclusively in the 3' uninterrupted CGG tract. Large expansion events indicative of a transition from a premutation to a full mutation were not observed. Overall, our results indicate both similarities and differences between the behavior of a premutation-sized repeat in mouse and that in human.

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.

FRAXE mental retardation results from expansion and methylation of a CCG trinucleotide repeat located in exon 1 of the X-linked FMR2 gene, which results in transcriptional silencing. The product of FMR2 is a member of a family of proteins rich in serine and proline, members of which have been associated with transcriptional activation. We have developed a murine Fmr2 gene knock-out model by replacing a fragment containing parts of exon 1 and intron 1 with the Escherichia coli lacZ gene, placing lacZ under control of the Fmr2 promoter. Expression of lacZ in the knock-out animals indicates that Fmr2 is expressed in several tissues, including brain, bone, cartilage, hair follicles, lung, tongue, tendons, salivary glands, and major blood vessels. In the CNS, Fmr2 expression begins at the time that cells in the neuroepithelium differentiate into neuroblasts. Mice lacking Fmr2 showed a delay-dependent conditioned fear impairment. Long-term potentiation (LTP) was found to be enhanced in hippocampal slices of Fmr2 knock-out compared with wild-type littermates. To our knowledge, this mouse knock-out is the first example of an animal model of human mental retardation with impaired learning and memory performance and increased LTP. Thus, although a number of studies have suggested that diminished LTP is associated with memory impairment, our data suggest that increased LTP may be a mechanism that leads to impaired cognitive processing as well.

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

BackgroundFragile X syndrome (FXS) is a leading genetic cause of autism and intellectual disability with cortical hyperexcitability and sensory hypersensitivity attributed to loss and hypofunction of inhibitory parvalbumin-expressing (PV) cells. Our studies provide novel insights into the role of excitatory neurons in abnormal development of PV cells during a postnatal period of inhibitory circuit refinement.MethodsTo achieve Fragile X mental retardation gene (Fmr1) deletion and re-expression in excitatory neurons during the postnatal day (P)14-P21 period, we generated CreCaMKIIa/Fmr1Flox/y (cOFF) and CreCaMKIIa/Fmr1FloxNeo/y (cON) mice, respectively. Cortical phenotypes were evaluated in adult mice using biochemical, cellular, clinically relevant electroencephalogram (EEG) and behavioral tests.ResultsWe found that similar to global Fmr1 KO mice, the density of PV-expressing cells, their activation, and sound-evoked gamma synchronization were impaired in cOFF mice, but the phenotypes were improved in cON mice. cOFF mice also showed enhanced cortical gelatinase activity and baseline EEG gamma power, which were reduced in cON mice. In addition, TrkB phosphorylation and PV levels were lower in cOFF mice, which also showed increased locomotor activity and anxiety-like behaviors. Remarkably, when FMRP levels were restored in only excitatory neurons during the P14-P21 period, TrkB phosphorylation and mouse behaviors were also improved.ConclusionsThese results indicate that postnatal deletion or re-expression of FMRP in excitatory neurons is sufficient to elicit or ameliorate structural and functional cortical deficits, and abnormal behaviors in mice, informing future studies about appropriate treatment windows and providing fundamental insights into the cellular mechanisms of cortical circuit dysfunction in FXS.

Carriers of the fragile X premutation (FPM) have CGG trinucleotide repeat expansions of between 55 and 200 in the 5'-UTR of FMR1, compared to a CGG repeat length of between 5 and 54 for the general population. Carriers were once thought to be without symptoms, but it is now recognized that they can develop a variety of early neurological symptoms as well as being at risk for developing the late onset neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS). Several mouse models have contributed to our understanding of FPM and FXTAS, and findings from studies using these models are summarized here. This review also discusses how this information is improving our understanding of the molecular and cellular abnormalities that contribute to neurobehavioral features seen in some FPM carriers and in patients with FXTAS. Mouse models show much of the pathology seen in FPM carriers and in individuals with FXTAS, including the presence of elevated levels of Fmr1 mRNA, decreased levels of fragile X mental retardation protein, and ubiquitin-positive intranuclear inclusions. Abnormalities in dendritic spine morphology in several brain regions are associated with neurocognitive deficits in spatial and temporal memory processes, impaired motor performance, and altered anxiety. In vitro studies have identified altered dendritic and synaptic architecture associated with abnormal Ca(2+) dynamics and electrical network activity. FPM mice have been particularly useful in understanding the roles of Fmr1 mRNA, fragile X mental retardation protein, and translation of a potentially toxic polyglycine peptide in pathology. Finally, the potential for using these and emerging mouse models for preclinical development of therapies to improve neurological function in FXTAS is considered.

Fragile X--associated tremor/ataxia syndrome (FXTAS) is a debilitating late-onset neurodegenerative disease in premutation carriers of the expanded CGG repeat in FMR1 that presents with a spectrum of neurological manifestations, such as gait ataxia, intention tremor, and parkinsonism [P. J. Hagerman, R. J. Hagerman, Ann. N. Y. Acad. Sci. 1338, 58-70 (2015); S. Jacquemont et al., JAMA 291, 460-469 (2004)]. Here, we performed whole-genome sequencing (WGS) on male premutation carriers (CGG55-200) and prioritized candidate variants to screen for candidate genetic modifiers using a Drosophila model of FXTAS. We found 18 genes that genetically modulate CGG-associated neurotoxicity in Drosophila,suchas Prosbeta5 (PSMB5), pAbp (PABPC1L), e(y)1 (TAF9), and CG14231 (OSGEPL1). Among them, knockdown of Prosbeta5 (PSMB5) suppressed CGG-associated neurodegeneration in the flyaswellasinN2A cells. Interestingly, an expression quantitative trait locus variant in PSMB5, PSMB5rs11543947-A, was found to be associated with decreased expression of PSMB5 and delayed onset of FXTAS in human FMR1 premutation carriers. Finally, we demonstrate evidence that PSMB5 knockdown results in suppression of CGG neurotoxicity via both the RAN translation and RNA-mediated toxicity mechanisms, thereby presenting a therapeutic strategy for FXTAS. [ABSTRACT FROM AUTHOR]

Deficiency in fragile X mental retardation protein (FMRP) results in fragile X syndrome (FXS), an inherited form of intellectual disability. Despite extensive research, it is unclear how FMRP deficiency contributes to the cognitive deficits in FXS. Fmrp-null mice show reduced adult hippocampal neurogenesis. As Fmrp is also enriched in mature neurons, we investigated the function of Fmrp expression in neural stem and progenitor cells (aNSCs) and its role in adult neurogenesis. Here we show that ablation of Fmrp in aNSCs by inducible gene recombination leads to reduced hippocampal neurogenesis in vitro and in vivo, as well as markedly impairing hippocampus-dependent learning in mice. Conversely, restoration of Fmrp expression specifically in aNSCs rescues these learning deficits in Fmrp-deficient mice. These data suggest that defective adult neurogenesis may contribute to the learning impairment seen in FXS, and these learning deficits can be rectified by delayed restoration of Fmrp specifically in aNSCs. [ABSTRACT FROM AUTHOR]

Fragile X syndrome (FXS) is the most common form of hereditary mental retardation. FXS patients have a deficit for the fragile X mental retardation protein (FMRP) that results in abnormal neuronal dendritic spine morphology and behavioral phenotypes, including sleep abnormalities. In a Drosophila model of FXS, flies lacking the dfmr1 protein (dFMRP) have abnormal circadian rhythms apparently as a result of altered clock output. In this study, we present biochemical and genetic evidence that dFMRP interacts with a known clock output component, the LARK RNA-binding protein. Our studies demonstrate physical interactions between dFMRP and LARK, that the two proteins are present in a complex in vivo, and that LARK promotes the stability of dFMRP. Furthermore, we show genetic interactions between the corresponding genes indicating that dFMRP and LARK function together to regulate eye development and circadian behavior. [ABSTRACT FROM AUTHOR]

The FMR2 gene is dysregulated by the fragile X E triplet repeat expansion in patients with FRAXE mental retardation syndrome. A CCG triplet, located in the 5′ untranslated region of the FRAXE gene undergoes expansion and methylation in these patients, eliminating detectable gene transcription. FRAXE syndrome is distinct from fragile X syndrome, a more common genetic form of mental retardation caused by expansion and methylation of a similar repeat in the FMR1 gene located 600 kb proximal to FRAXE. FRAXE syndrome is rare, and patients’ phenotypes are highly variable, leading to difficulties with predicting specific FMR2 functions based on the human disease. Recently, Lilliputian(Lilli), a Drosophila FMR2 orthologue, was identified; this gene has been linked with several signal transduction pathways, including the transforming growth factor-β (TGF-β) pathway, the Raf/MEK/MAP kinase (MAPK) pathway, and the P13K/PKB pathway. Mutation of Lilli shows defects in germinal band extension, cytoskeletal structure, cell growth, and organ development. The Lilli gene suggests possible functions for FMR2 (and related genes) in humans and mice, but cannot predict specific functions. Modeling Fmr2 mutation in the mouse will be useful to understand specific functions of this gene in vertebrates. This review presents what has been learned thus far from the Fmr2 knockout mouse model and suggests future studies on this model in order to compare it with the human FRAXE mental retardation disorder, Lilli mutants in Drosophila and other mouse models of genes in this family. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Reports on advances in the understanding of the molecular basis of fragile X syndrome and its consequences for patients and families. Clinical aspects of the syndrome; Trinucleotide repeat expansion and DNA methylation; Diagnostic issues; Professional and public awareness of the hereditary disorder.

Fragile X syndrome is caused by a CGG trinucleotide repeat expansion of the FMRI gene. Individuals with fragile X display several behavioral abnormalities including hyperactivity, social anxiety, autistic-like features, impaired cognitive processing, and impaired sensorimotor gating. The FmrIKO mouse model of fragile X exhibits several related behavioral phenotypes such as increased activity and altered social interactions. Individuals with fragile X also have impaired sensorimotor gating as measured using the prepulse inhibition of startle response. The authors have recently shown that FmrlKO mice with a yeast artificial chromosome containing the human FMR I gene have connected or overeorrected abnormal behaviors including hyperactivity and altered social interactions. Here the authors present results from a study examining abnormal sensorimotor gating in FmrlKO mice. Consistent with previous findings, FmrlKO mice have increased prepulse inhibition. The KO mice with the yeast artificial chromosome containing the human FMRJ gene had levels of prepulse inhibition comparable to WT mice, indicating not only a correction of this phenotype, but also clearly demonstrating that in mice levels of the fragile X mental retardation protein regulate sensorimotor gating. [ABSTRACT FROM AUTHOR]

Abstract: Fragile X syndrome, the most common form of inherited intellectual disability, is caused by a lack of FMRP, which is the product of the Fmr1 gene. FMRP is an RNA-binding protein and a component of RNA-granules found in the dendrites of neurons. At the synapse, FMRP is involved in regulation of translation of specific target mRNAs upon stimulation of mGluR5 receptors. In this study, we test the effects of a new mGluR5 antagonist (AFQ056) on the prepulse inhibition of startle response in mice. We show that Fmr1 KO mice have a deficit in inhibition of the startle response after a prepulse and that AFQ056 can rescue this phenotype. We also studied the effect of AFQ056 on cultured Fmr1 KO hippocampal neurons; untreated neurons showed elongated spines and treatment resulted in shortened spines. These results suggest that AFQ056 might be a potent mGluR5 antagonist to rescue various aspects of the fragile X phenotype. [Copyright &y& Elsevier]

Abstract: Fragile X syndrome is caused by lack of the protein FMRP. FMRP mediates mRNA binding, dendritic mRNA transport and translational control at spines. We examined the role of functional domains of FMRP in neuronal RNA-granule formation and dendritic transport using different FMRP variants, including the mutant FMRP_I304N and the splice-variant FMRP_Iso12. Both variants are absent from dendritic RNA-granules in Fmr1 knockout neurons. Co-transfection experiments showed that wild-type FMRP recruits both FMRP variants into dendritic RNA-granules. Co-transfection of FXR2, an FMRP homologue, also resulted in redistribution of both variants into dendritic RNA-granules. Furthermore, the capacity of the variants to transport their mRNAs and the mRNA localization of an FMR1 construct containing silent point-mutations affecting only the G-quartet-structure were investigated. In conclusion, we show that wild-type FMRP and FXR2P are able to recruit FMRP variants into RNA-granules and that the G-quartet-structure in FMR1 mRNA is not essential for its incorporation in RNA-granules. [Copyright &y& Elsevier]

Fragile X syndrome results from the absence of the fragile X mental retardation 1 (FMR1) gene product (FMRP). FMR1 has two paralogs in vertebrates: fragile X related gene 1 and 2 (FXR1 and FXR2). Here we show that Fxr1/Fxr2 double knockout (KO) and Fmr1 KO/Fxr2 heterozygous animals exhibit a loss of rhythmic activity in a light:dark (LD) cycle, and that Foul or Fxr2 KO mice display a shorter free-running period of locomotor activity in total darkness (DD). Molecular analysis and in vitro electrophysiological studies suggest essentially normal function of cells in the suprachiasmatic nucleus (SCN) in Fxr1/Fxr2 double KO mice. However, the cyclical patterns of abundance of several core clock component messenger (m) RNAs are altered in the livers of double KO mice. Furthermore, FXR2P alone or FMRP and FXR2P together can increase PER1- or PER2-mediated BMAL1-Neuronal PAS2 (NPAS2) transcriptional activity in a dose-dependent manner. These data collectively demonstrate that FMR1 and FXR2 are required for the presence of rhythmic circadian behavior in mammals and suggest that this role may be relevant to sleep and other behavioral alterations observed in fragile X patients. [ABSTRACT FROM AUTHOR]

Fragile X syndrome (FXS) results from the loss of expression of the fragile X mental retardation (FMR1) gene. Individuals affected by FXS experience many behavioral problems, including cognitive impairment, hyperactivity, social anxiety, and autistic-like behaviors. A mouse model of Fmr1 deficiency (Fmr1KO) exhibits several similar behavioral phenotypes, including alterations in social behavior. In an earlier study, Fmr1 knockout mice carrying a yeast-artificial chromosome (YAC) transgene that over- expresses normal human FMRP (KOYAC) showed a correction or overcorrection of some behavioral responses, such as hyperactivity and anxiety-related responses. This report presents results from a study examining transgenic rescue of abnormal social responses in Fmr1KO mice. Relative to their wild-type (WT) littermates, Fmr1KO mice made more active social approaches to standard C57BL/6 partner mice in a direct social interaction test, a result consistent with a previous study. KOYAC mice showed fewer active approaches to partners than the WT or Fmr1KO littermates, indicating correction of this phenotype. This finding expands the number of murine behavioral responses caused by Fmr1 deficiency and corrected by overexpression of human FMRP. [ABSTRACT FROM AUTHOR]