MAB2160 Sigma-AldrichAnti-Fragile X Mental Retardation Protein Antibody, clone 1C3

Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

Recent advances in techniques for manipulating genomes have allowed the generation of transgenic animals other than mice. These new models enable cross-mammalian comparison of neurological disease from core cellular pathophysiology to circuit and behavioural endophenotypes. Moreover they will enable us to directly test whether common cellular dysfunction or behavioural outcomes of a genetic mutation are more conserved across species. Using a new rat model of Fragile X Syndrome, we report that Fmr1 knockout (KO) rats exhibit elevated basal protein synthesis and an increase in mGluR-dependent long-term depression in CA1 of the hippocampus that is independent of new protein synthesis. These defects in plasticity are accompanied by an increase in dendritic spine density selectively in apical dendrites and subtle changes in dendritic spine morphology of CA1 pyramidal neurons. Behaviourally, Fmr1 KO rats show deficits in hippocampal-dependent, but not hippocampal-independent, forms of associative recognition memory indicating that the loss of fragile X mental retardation protein (FMRP) causes defects in episodic-like memory. In contrast to previous reports from mice, Fmr1 KO rats show no deficits in spatial reference memory reversal learning. One-trial spatial learning in a delayed matching to place water maze task was also not affected by the loss of FMRP in rats. This is the first evidence for conservation across mammalian species of cellular and physiological hippocampal phenotypes associated with the loss of FMRP. Furthermore, while key cellular phenotypes are conserved they manifest in distinct behavioural dysfunction. Finally, our data reveal novel information about the selective role of FMRP in hippocampus-dependent associative memory.

Hyperactive mammalian target of rapamycin (mTOR) is associated with cognitive deficits in several neurological disorders including tuberous sclerosis complex (TSC). The phosphorylation of the mRNA-binding protein FMRP reportedly depends on mTOR complex 1 (mTORC1) activity via p70 S6 kinase 1 (S6K1). Because this phosphorylation is thought to regulate the translation of messages important for synaptic plasticity, we explored whether FMRP phosphorylation of the S6K1-dependent residue (S499) is altered in TSC and states of dysregulated TSC-mTORC1 signaling. Surprisingly, we found that FMRP S499 phosphorylation was unchanged in heterozygous and conditional Tsc1 knockout mice despite significantly elevated mTORC1-S6K1 activity. Neither up- nor down-regulation of the mTORC1-S6K1 axis in vivo or in vitro had any effect on phospho-FMRP S499 levels. In addition, FMRP S499 phosphorylation was unaltered in S6K1-knockout mice. Collectively, these data strongly suggest that FMRP S499 phosphorylation is independent of mTORC1-S6K1 activity and is not altered in TSC.

Large expansions of a CGG-repeat element (greater than 200 repeats; full mutation) in the fragile X mental retardation 1 (FMR1) gene cause fragile X syndrome (FXS), the leading single-gene form of intellectual disability and of autism spectrum disorder. Smaller expansions (55-200 CGG repeats; premutation) result in the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Whereas FXS is caused by gene silencing and insufficient FMR1 protein (FMRP), FXTAS is thought to be caused by 'toxicity' of expanded-CGG-repeat mRNA. However, as FMRP expression levels decrease with increasing CGG-repeat length, lowered protein may contribute to premutation-associated clinical involvement. To address this issue, we measured brain Fmr1 mRNA and FMRP levels as a function of CGG-repeat length in a congenic (CGG-repeat knock-in) mouse model using 57 wild-type and 97 expanded-CGG-repeat mice carrying up to ~250 CGG repeats. While Fmr1 message levels increased with repeat length, FMRP levels trended downward over the same range, subject to significant inter-subject variation. Human comparisons of protein levels in the frontal cortex of 7 normal and 17 FXTAS individuals revealed that the mild FMRP decrease in mice mirrored the more limited data for FMRP expression in the human samples. In addition, FMRP expression levels varied in a subset of mice across the cerebellum, frontal cortex, and hippocampus, as well as at different ages. These results provide a foundation for understanding both the CGG-repeat-dependence of FMRP expression and for interpreting clinical phenotypes in premutation carriers in terms of the balance between elevated mRNA and lowered FMRP expression levels.

Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (Ca(V)) channels. Here we show that the functional expression of neuronal N-type Ca(V) channels (Ca(V)2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases Ca(V) channel density in somata and in presynaptic terminals. We then show that FMRP controls Ca(V)2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and Ca(V)2.2 occurs between the carboxy-terminal domain of FMRP and domains of Ca(V)2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via Ca(V)2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

Topoisomerases are crucial for solving DNA topological problems, but they have not been linked to RNA metabolism. Here we show that human topoisomerase 3β (Top3β) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein that is deficient in fragile X syndrome and is known to regulate the translation of mRNAs that are important for neuronal function, abnormalities of which are linked to autism. Notably, the FMRP-Top3β interaction is abolished by a disease-associated mutation of FMRP, suggesting that Top3β may contribute to the pathogenesis of mental disorders. Top3β binds multiple mRNAs encoded by genes with neuronal functions linked to schizophrenia and autism. Expression of one such gene, that encoding protein tyrosine kinase 2 (ptk2, also known as focal adhesion kinase or FAK), is reduced in the neuromuscular junctions of Top3β mutant flies. Synapse formation is defective in Top3β mutant flies and mice, as well as in FMRP mutant flies and mice. Our findings suggest that Top3β acts as an RNA topoisomerase and works with FMRP to promote the expression of mRNAs that are crucial for neurodevelopment and mental health.

Hypermethylation of the fragile X mental retardation 1 gene FMR1 results in decreased expression of FMR1 protein FMRP, which is the underlying cause of Fragile X syndrome - an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism. Disease-modifying therapies for Fragile X syndrome are thus aimed at treatments that increase the FMRP expression levels in the brain. We describe the development and characterization of two assays for simple and quantitative detection of FMRP protein.Antibodies coupled to fluorophores that can be employed for time-resolved Förster's resonance energy transfer were used for the development of homogeneous, one-step immunodetection. Purified recombinant human FMRP and patient cells were used as control samples for assay development.The assays require small sample amounts, display high stability and reproducibility and can be used to quantify endogenous FMRP in human fibroblasts and peripheral blood mononuclear cells. Application of the assays to FXS patient cells showed that the methods can be used both for the characterization of clinical FXS patient samples as well as primary readouts in drug-discovery screens aimed at increasing endogenous FMRP levels in human cells.This study provides novel quantitative detection methods for FMRP in FXS patient cells. Importantly, due to the simplicity of the assay protocol, the method is suited to be used in screening applications to identify compounds or genetic interventions that result in increased FMRP levels in human cells.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that targets ∼5% of all mRNAs expressed in the brain. Previous work by our laboratory demonstrated significantly lower protein levels for FMRP in lateral cerebella of subjects with schizophrenia, bipolar disorder and major depression when compared with controls. Absence of FMRP expression in animal models of fragile X syndrome (FXS) has been shown to reduce expression of gamma-aminobutyric acid A (GABAA) receptor mRNAs. Previous work by our laboratory has found reduced expression of FMRP, as well as multiple GABAA and GABAB receptor subunits in subjects with autism. Less is known about levels for GABAA subunit protein expression in brains of subjects with schizophrenia and mood disorders. In the current study, we have expanded our previous studies to examine the protein and mRNA expression of two novel GABAA receptors, theta (GABRθ) and rho 2 (GABRρ2) as well as FMRP, and metabotropic glutamate receptor 5 (mGluR5) in lateral cerebella of subjects with schizophrenia, bipolar disorder, major depression and healthy controls, and in superior frontal cortex (Brodmann Area 9 (BA9)) of subjects with schizophrenia, bipolar disorder and healthy controls. We observed multiple statistically significant mRNA and protein changes in levels of GABRθ, GABRρ2, mGluR5 and FMRP molecules including concordant reductions in mRNA and proteins for GABRθ and mGluR5 in lateral cerebella of subjects with schizophrenia; for increased mRNA and protein for GABRρ2 in lateral cerebella of subjects with bipolar disorder; and for reduced mRNA and protein for mGluR5 in BA9 of subjects with bipolar disorder. There were no significant effects of confounds on any of the results.

Fragile X syndrome (FXS), a common inherited form of intellectual disability with learning deficits, results from a loss of fragile X mental retardation protein (FMRP). Despite extensive research, treatment options for FXS remain limited. Since FMRP is known to play an important role in adult hippocampal neurogenesis and hippocampus-dependent learning and FMRP regulates the adult neural stem cell fate through the translational regulation of glycogen synthase kinase 3β (GSK3β), we investigated the effects of a GSK3β inhibitor, SB216763, on Fmr1 knockout mice (Fmr1 KO). We found that the inhibition of GSK3β could reverse the hippocampus-dependent learning deficits and rescue adult hippocampal neurogenesis at multiple stages in Fmr1 KO mice. Our results point to GSK3β inhibition as a potential treatment for the learning deficits seen in FXS.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism-spectrum disorders sharing common pathophysiology.