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1.
Exp Neurol ; 357: 114170, 2022 11.
Article in English | MEDLINE | ID: mdl-35863501

ABSTRACT

Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability and is characterized by autistic behaviors, childhood seizures, and deficits in learning and memory. FXS has a loss of function of the FMR1 gene that leads to a lack of Fragile X Mental Retardation Protein (FMRP) expression. FMRP is critical for synaptic plasticity, spatial learning, and memory. Reelin is a large extracellular glycoprotein essential for synaptic plasticity and numerous neurodevelopmental processes. Reduction in Reelin signaling is implicated as a contributing factor in disease etiology in several neurological disorders, including schizophrenia, and autism. However, the role of Reelin in FXS is poorly understood. We demonstrate a reduction in Reelin in Fmr1 knock-out (KO) mice, suggesting that a loss of Reelin activity may contribute to FXS. We demonstrate here that Reelin signaling enhancement via a single intracerebroventricular injection of the Reelin central fragment into Fmr1 KO mice can profoundly rescue cognitive deficits in hidden platform water maze and fear conditioning, as well as hyperactivity during the open field. Improvements in behavior were associated with rescued levels of post synaptic marker in Fmr1 KO mice when compared to controls. These data suggest that increasing Reelin signaling in FXS could offer a novel therapeutic for improving cognition in FXS.


Subject(s)
Fragile X Syndrome , Animals , Cognition , Dietary Supplements , Disease Models, Animal , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/complications , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Mice , Mice, Knockout
2.
Mol Brain ; 14(1): 84, 2021 05 25.
Article in English | MEDLINE | ID: mdl-34034796

ABSTRACT

Down syndrome (DS) is the most frequent genetic cause of intellectual disability including hippocampal-dependent memory deficits. We have previously reported hippocampal mTOR (mammalian target of rapamycin) hyperactivation, and related plasticity as well as memory deficits in Ts1Cje mice, a DS experimental model. Here we characterize the proteome of hippocampal synaptoneurosomes (SNs) from these mice, and found a predicted alteration of synaptic plasticity pathways, including long term depression (LTD). Accordingly, mGluR-LTD (metabotropic Glutamate Receptor-LTD) is enhanced in the hippocampus of Ts1Cje mice and this is correlated with an increased proportion of a particular category of mushroom spines in hippocampal pyramidal neurons. Remarkably, prenatal treatment of these mice with rapamycin has a positive pharmacological effect on both phenotypes, supporting the therapeutic potential of rapamycin/rapalogs for DS intellectual disability.


Subject(s)
Dendritic Spines/metabolism , Dendritic Spines/pathology , Down Syndrome/pathology , Down Syndrome/physiopathology , Long-Term Synaptic Depression , Receptors, Metabotropic Glutamate/metabolism , Sirolimus/pharmacology , Animals , Dendritic Spines/drug effects , Disease Models, Animal , Fragile X Mental Retardation Protein/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/pathology , Hippocampus/physiopathology , Long-Term Synaptic Depression/drug effects , Mice, Transgenic , Mitochondrial Proteins/metabolism , Neuronal Plasticity/drug effects , Proteomics , Pyramidal Cells/drug effects , Pyramidal Cells/metabolism , Pyramidal Cells/pathology , Synapses/drug effects , Synapses/metabolism
3.
Cells ; 11(1)2021 12 27.
Article in English | MEDLINE | ID: mdl-35011630

ABSTRACT

Fragile X syndrome (FXS) is the most common inherited cause of autism and intellectual disability. The majority of FXS cases are caused by transcriptional repression of the FMR1 gene due to epigenetic changes that are not recapitulated in current animal disease models. FXS patient induced pluripotent stem cell (iPSC)-derived gene edited reporter cell lines enable novel strategies to discover reactivators of FMR1 expression in human cells on a much larger scale than previously possible. Here, we describe the workflow using FXS iPSC-derived neural cell lines to conduct a massive, unbiased screen for small molecule activators of the FMR1 gene. The proof-of-principle methodology demonstrates the utility of human stem-cell-based methodology for the untargeted discovery of reactivators of the human FMR1 gene that can be applied to other diseases.


Subject(s)
Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/pathology , High-Throughput Screening Assays , Neurons/metabolism , Small Molecule Libraries/pharmacology , Drug Evaluation, Preclinical , Genetic Loci , Humans , Neural Stem Cells/drug effects , Neural Stem Cells/metabolism , Neurons/drug effects , Reproducibility of Results
4.
Neurochem Int ; 140: 104847, 2020 11.
Article in English | MEDLINE | ID: mdl-32927026

ABSTRACT

Fragile X mental retardation protein (FMRP), strongly associated with fragile X syndrome, plays important roles by regulating gene expression via interacting with other RNA binding proteins in the brain. However, the role of FMRP in hypothalamus, a central part responsible for metabolic control, is poorly known. Our study shows that FMRP is primarily located in the hypothalamic arcuate nucleus (ARC). Using proteomic analysis, we identified 56 up-regulated and 22 down-regulated proteins in the hypothalamus of Map1b KO mice, with microtubule-associated protein 1 B (MAP1B) being the most outstanding increased protein (more than 10 folds). Immunofluorescent assays showed that MAP1B significantly increased in the Map1b-KO ARC, in which the number of agouti-related peptide (AgRP)-staining neurons significantly reduced, but not altered for pro-opiomelanocortin (POMC) neurons. We further showed an age-dependent reduces in food intake and body weight of the KO mice, along with the decreases of MAP1B and AgRP at the same time points. In hypothalamic GT1-7 cells, the AgRP expression decreased upon knockdown of FMRP or overexpression of MAP1B, and increased in response to overexpression of FMRP or knockdown of MAP1B. Co-knockdown or co-overexpression of FMRP and MAP1B led to a reverse expression of AgRP compared to overexpression of knockdown of FMRP alone, demonstrating that MAP1B is essential for the regulatory effect of FMRP on AgRP expression. Taken together, these data suggest that FMRP-deficiency-induced increase of hypothalamic MAP1B and decrease of AgRP might be associated with reduces in food intake and body weight.


Subject(s)
Agouti-Related Protein/biosynthesis , Body Weight/physiology , Eating/physiology , Fragile X Mental Retardation Protein/metabolism , Hypothalamus/metabolism , Microtubule-Associated Proteins/biosynthesis , Agouti-Related Protein/antagonists & inhibitors , Agouti-Related Protein/genetics , Animals , Fragile X Mental Retardation Protein/genetics , Gene Expression , Male , Mice , Mice, Knockout , Microtubule-Associated Proteins/genetics , Up-Regulation/physiology
5.
J Neurochem ; 155(5): 538-558, 2020 12.
Article in English | MEDLINE | ID: mdl-32374912

ABSTRACT

Individuals with Fragile X Syndrome (FXS) and autism spectrum disorder (ASD) exhibit cognitive impairments, social deficits, increased anxiety, and sensory hyperexcitability. Previously, we showed that elevated levels of matrix metalloproteinase-9 (MMP-9) may contribute to abnormal development of parvalbumin (PV) interneurons and perineuronal nets (PNNs) in the developing auditory cortex (AC) of Fmr1 knock-out (KO) mice, which likely underlie auditory hypersensitivity. Thus, MMP-9 may serve as a potential target for treatment of auditory hypersensitivity in FXS. Here, we used the MMP-2/9 inhibitor, SB-3CT, to pharmacologically inhibit MMP-9 activity during a specific developmental period and to test whether inhibition of MMP-9 activity reverses neural oscillation deficits and behavioral impairments by enhancing PNN formation around PV cells in Fmr1 KO mice. Electroencephalography (EEG) was used to measure resting state and sound-evoked electrocortical activity in auditory and frontal cortices of postnatal day (P)22-23 male mice before and one-day after treatment with SB-3CT (25 mg/kg) or vehicle. At P27-28, animal behaviors were tested to measure the effects of the treatment on anxiety and hyperactivity. Results show that acute inhibition of MMP-9 activity improved evoked synchronization to auditory stimuli and ameliorated mouse behavioral deficits. MMP-9 inhibition enhanced PNN formation, increased PV levels and TrkB phosphorylation yet reduced Akt phosphorylation in the AC of Fmr1 KO mice. Our results show that MMP-9 inhibition during early postnatal development is beneficial in reducing some auditory processing deficits in the FXS mouse model and may serve as a candidate therapeutic for reversing sensory hypersensitivity in FXS and possibly other ASDs.


Subject(s)
Acoustic Stimulation/methods , Auditory Perception/physiology , Fragile X Mental Retardation Protein/metabolism , Heterocyclic Compounds, 1-Ring/pharmacology , Matrix Metalloproteinase 9/metabolism , Nerve Net/metabolism , Sulfones/pharmacology , Animals , Animals, Newborn , Auditory Cortex/drug effects , Auditory Cortex/metabolism , Auditory Perception/drug effects , Electroencephalography/drug effects , Electroencephalography/methods , Enzyme Inhibitors/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Net/drug effects , Peripheral Nerves/growth & development , Peripheral Nerves/metabolism
6.
G3 (Bethesda) ; 9(9): 3009-3022, 2019 09 04.
Article in English | MEDLINE | ID: mdl-31324746

ABSTRACT

Binge eating (BE) is a heritable trait associated with eating disorders and involves episodes of rapid, large amounts of food consumption. We previously identified cytoplasmic FMR1-interacting protein 2 (Cyfip2) as a genetic factor underlying compulsive-like BE in mice. CYFIP2 is a homolog of CYFIP1 which is one of four paternally-deleted genes in patients with Type I Prader-Willi Syndrome (PWS), a neurodevelopmental disorder whereby 70% of cases involve paternal 15q11-q13 deletion. PWS symptoms include hyperphagia, obesity (if untreated), cognitive deficits, and obsessive-compulsive behaviors. We tested whether Cyfip1 haploinsufficiency (+/-) would enhance compulsive-like behavior and palatable food (PF) intake in a parental origin- and sex-dependent manner on two Cyfip2 genetic backgrounds, including the BE-prone C57BL/6N (Cyfip2N/N) background and the BE-resistant C57BL/6J (Cyfip2J/J) background. Cyfip1+/- mice showed increased compulsive-like behavior on both backgrounds and increased PF intake on the Cyfip2N/N background. In contrast, maternal Cyfip1 haploinsufficiency on the BE-resistant Cyfip2J/J background induced a robust escalation in PF intake in wild-type Cyfip1J/J males while having no effect in Cyfip1J/- males. Notably, induction of behavioral phenotypes in wild-type males following maternal Fmr1+/- has previously been reported. In the hypothalamus, there was a paternally-enhanced reduction in CYFIP1 protein whereas in the nucleus accumbens, there was a maternally-enhanced reduction in CYFIP1 protein. Nochange in FMR1 protein (FMRP) was observed in Cyfip1+/- mice, regardless of parental origin. To summarize, Cyfip1 haploinsufficiency increased compulsive-like behavior and induced genetic background-dependent, sex-dependent, and parent-of-origin-dependent effects on PF consumption and CYFIP1 expression that could have relevance for neurodevelopmental and neuropsychiatric disorders.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Appetite Regulation/genetics , Compulsive Behavior/genetics , Haploinsufficiency , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Behavior, Animal/physiology , Female , Fragile X Mental Retardation Protein/metabolism , Hypothalamus/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Proteins/genetics , Proteins/metabolism , Reward
7.
Brain Res ; 1719: 24-29, 2019 09 15.
Article in English | MEDLINE | ID: mdl-31128097

ABSTRACT

Sensory processing abnormalities are consistently associated with autism, but the underlying mechanisms and treatment options are unclear. Fragile X Syndrome (FXS) is the leading known genetic cause of intellectual disabilities and autism. One debilitating symptom of FXS is hypersensitivity to sensory stimuli. Sensory hypersensitivity is seen in both humans with FXS and FXS mouse model, the Fmr1 knock out (Fmr1 KO) mouse. Abnormal sensorimotor gating may play a role in the hypersensitivity to sensory stimuli. Humans with FXS and Fmr1 KO mice show abnormalities in acoustic startle response (ASR) and prepulse inhibition (PPI) of startle, responses commonly used to quantify sensorimotor gating. Recent studies have suggested high levels of matrix metalloproteinase-9 (MMP-9) as a potential mechanism of sensory abnormalities in FXS. Here we tested the hypothesis that genetic reduction of MMP-9 in Fmr1 KO mice rescues ASR and PPI phenotypes in adult Fmr1 KO mice. We measured MMP-9 levels in the inferior colliculus (IC), an integral region of the PPI circuit, of WT and Fmr1 KO mice at P7, P12, P18, and P40. MMP-9 levels were higher in the IC of Fmr1 KO mice during early development (P7, P12), but not in adults. We compared ASR and PPI responses in young (P23-25) and adult (P50-80) Fmr1 KO mice to their age-matched wildtype (WT) controls. We found that both ASR and PPI were reduced in the young Fmr1 KO mice compared to age-matched WT mice. There was no genotype difference for ASR in the adult mice, but PPI was significantly reduced in the adult Fmr1 KO mice. The adult mouse data are similar to those observed in humans with FXS. Genetic reduction of MMP-9 in the Fmr1 KO mice resulted in a rescue of adult PPI responses to WT levels. Taken together, these results show sensorimotor gating abnormalities in Fmr1 KO mice, and suggest the potential for MMP-9 regulation as a therapeutic target to reduce sensory hypersensitivity.


Subject(s)
Fragile X Mental Retardation Protein/genetics , Matrix Metalloproteinase 9/genetics , Prepulse Inhibition/physiology , Reflex, Startle/genetics , Acoustic Stimulation/methods , Animals , Disease Models, Animal , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/genetics , Fragile X Syndrome/physiopathology , Genotype , Male , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Knockout , Phenotype , Prepulse Inhibition/genetics , Sensory Gating/genetics
8.
Cell Rep ; 23(3): 878-887, 2018 Apr 17.
Article in English | MEDLINE | ID: mdl-29669291

ABSTRACT

Sensory experiences dynamically modify whether animals respond to a given stimulus, but it is unclear how innate behavioral thresholds are established. Here, we identify molecular and circuit-level mechanisms underlying the innate threshold of the zebrafish startle response. From a forward genetic screen, we isolated five mutant lines with reduced innate startle thresholds. Using whole-genome sequencing, we identify the causative mutation for one line to be in the fragile X mental retardation protein (FMRP)-interacting protein cyfip2. We show that cyfip2 acts independently of FMRP and that reactivation of cyfip2 restores the baseline threshold after phenotype onset. Finally, we show that cyfip2 regulates the innate startle threshold by reducing neural activity in a small group of excitatory hindbrain interneurons. Thus, we identify a selective set of genes critical to establishing an innate behavioral threshold and uncover a circuit-level role for cyfip2 in this process.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Interneurons/metabolism , Zebrafish Proteins/metabolism , Acoustic Stimulation , Adaptor Proteins, Signal Transducing/genetics , Animals , Axons/metabolism , Behavior, Animal , Calcium/metabolism , Cytoskeleton/metabolism , Excitatory Postsynaptic Potentials , Fragile X Mental Retardation Protein/metabolism , Hypersensitivity/metabolism , Hypersensitivity/pathology , Larva/metabolism , Mutagenesis , Reflex, Startle/physiology , Zebrafish/growth & development , Zebrafish/metabolism , Zebrafish Proteins/genetics
9.
Cell Physiol Biochem ; 44(4): 1651-1664, 2017.
Article in English | MEDLINE | ID: mdl-29216638

ABSTRACT

BACKGROUND/AIMS: An increase in intracellular lipid droplet formation and hepatic triglyceride (TG) content usually results in nonalcoholic fatty liver disease. However, the mechanisms underlying the regulation of hepatic TG homeostasis remain unclear. METHODS: Oil red O staining and TG measurement were performed to determine the lipid content. miRNA expression was evaluated by quantitative PCR. A luciferase assay was performed to validate the regulation of Yin Yang 1 (YY1) by microRNA (miR)-122. The effects of miR-122 expression on YY1 and its mechanisms involving the farnesoid X receptor and small heterodimer partner (FXR-SHP) pathway were evaluated by quantitative PCR and Western blot analyses. RESULTS: miR-122 was downregulated in free fatty acid (FFA)-induced steatotic hepatocytes, and streptozotocin and high-fat diet (STZ-HFD) induced nonalcoholic steatohepatitis (NASH) in mice. Transfection of hepatocytes with miR-122 mimics before FFA induction inhibited lipid droplet formation and TG accumulation in vitro. These results were verified by overexpressing miR-122 in the livers of STZ-HFD-induced NASH mice. The 3'-untranslated region (3'UTR) of YY1 mRNA is predicted to contain an evolutionarily conserved miR-122 binding site. In silico searches, a luciferase reporter assay and quantitative PCR analysis confirmed that miR-122 directly bound to the YY1 3'UTR to negatively regulate YY1 mRNA in HepG2 and Huh7 cells. The (FXR-SHP) signaling axis, which is downstream of YY1, may play a key role in the mechanism of miR-122-regulated lipid homeostasis. YY1-FXR-SHP signaling, which is negatively regulated by FFA, was enhanced by miR-122 overexpression. This finding was also confirmed by overexpression of miR-122 in the livers of NASH mice. CONCLUSIONS: The present results indicate that miR-122 plays an important role in lipid (particularly TG) accumulation in the liver by reducing YY1 mRNA stability to upregulate FXR-SHP signaling.


Subject(s)
Lipid Droplets/metabolism , MicroRNAs/metabolism , Triglycerides/metabolism , YY1 Transcription Factor/metabolism , 3' Untranslated Regions , Animals , Antagomirs/metabolism , Base Sequence , Cell Line, Tumor , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diet, High-Fat , Disease Models, Animal , Down-Regulation/drug effects , Fatty Acids, Nonesterified/pharmacology , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Hep G2 Cells , Humans , Lipid Droplets/drug effects , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred C57BL , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Sequence Alignment , YY1 Transcription Factor/chemistry , YY1 Transcription Factor/genetics
10.
eNeuro ; 4(1)2017.
Article in English | MEDLINE | ID: mdl-28451631

ABSTRACT

Fragile X syndrome is a genetic condition resulting from FMR1 gene mutation that leads to intellectual disability, autism-like symptoms, and sensory hypersensitivity. Arbaclofen, a GABA-B agonist, has shown efficacy in some individuals with FXS but has become unavailable after unsuccessful clinical trials, prompting interest in publicly available, racemic baclofen. The present study investigated whether racemic baclofen can remediate abnormalities of neural circuit function, sensory processing, and behavior in Fmr1 knockout mice, a rodent model of fragile X syndrome. Fmr1 knockout mice showed increased baseline and auditory-evoked high-frequency gamma (30-80 Hz) power relative to C57BL/6 controls, as measured by electroencephalography. These deficits were accompanied by decreased T maze spontaneous alternation, decreased social interactions, and increased open field center time, suggestive of diminished working memory, sociability, and anxiety-like behavior, respectively. Abnormal auditory-evoked gamma oscillations, working memory, and anxiety-related behavior were normalized by treatment with baclofen, but impaired sociability was not. Improvements in working memory were evident predominantly in mice whose auditory-evoked gamma oscillations were dampened by baclofen. These findings suggest that racemic baclofen may be useful for targeting sensory and cognitive disturbances in fragile X syndrome.


Subject(s)
Baclofen/pharmacology , Evoked Potentials, Auditory/drug effects , Fragile X Syndrome/complications , GABA-B Receptor Agonists/pharmacology , Mental Disorders/etiology , Mental Disorders/pathology , Acoustic Stimulation , Animals , Disease Models, Animal , Electroencephalography , Evoked Potentials, Auditory/genetics , Exploratory Behavior/drug effects , Exploratory Behavior/physiology , Female , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/genetics , Interpersonal Relations , Male , Maze Learning/drug effects , Memory, Short-Term/drug effects , Memory, Short-Term/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Spectrum Analysis
11.
J Biomol Screen ; 20(9): 1101-11, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26024946

ABSTRACT

Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused in most of cases by epigenetic silencing of the Fmr1 gene. Today, no specific therapy exists for FXS, and current treatments are only directed to improve behavioral symptoms. Neuronal progenitors derived from FXS patient induced pluripotent stem cells (iPSCs) represent a unique model to study the disease and develop assays for large-scale drug discovery screens since they conserve the Fmr1 gene silenced within the disease context. We have established a high-content imaging assay to run a large-scale phenotypic screen aimed to identify compounds that reactivate the silenced Fmr1 gene. A set of 50,000 compounds was tested, including modulators of several epigenetic targets. We describe an integrated drug discovery model comprising iPSC generation, culture scale-up, and quality control and screening with a very sensitive high-content imaging assay assisted by single-cell image analysis and multiparametric data analysis based on machine learning algorithms. The screening identified several compounds that induced a weak expression of fragile X mental retardation protein (FMRP) and thus sets the basis for further large-scale screens to find candidate drugs or targets tackling the underlying mechanism of FXS with potential for therapeutic intervention.


Subject(s)
Fragile X Syndrome/drug therapy , Gene Silencing/drug effects , Induced Pluripotent Stem Cells/drug effects , Neural Stem Cells/drug effects , Cells, Cultured , Drug Evaluation, Preclinical , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/genetics , High-Throughput Screening Assays , Humans , Induced Pluripotent Stem Cells/physiology , Neural Stem Cells/physiology , Trinucleotide Repeats
12.
Psychopharmacology (Berl) ; 231(6): 1217-26, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24232444

ABSTRACT

RATIONALE: Fragile X syndrome (FXS) is considered the leading inherited cause of intellectual disability and autism. In FXS, the fragile X mental retardation 1 (FMR1) gene is silenced and the fragile X mental retardation protein (FMRP) is not expressed, resulting in the characteristic features of the syndrome. Despite recent advances in understanding the pathophysiology of FXS, there is still no cure for this condition; current treatment is symptomatic. Preclinical research is essential in the development of potential therapeutic agents. OBJECTIVES: This review provides an overview of the preclinical evidence supporting metabotropic glutamate receptor 5 (mGluR5) antagonists as therapeutic agents for FXS. RESULTS: According to the mGluR theory of FXS, the absence of FMRP leads to enhanced glutamatergic signaling via mGluR5, which leads to increased protein synthesis and defects in synaptic plasticity including enhanced long-term depression. As such, efforts to develop agents that target the underlying pathophysiology of FXS have focused on mGluR5 modulation. Animal models, particularly the Fmr1 knockout mouse model, have become invaluable in exploring therapeutic approaches on an electrophysiological, behavioral, biochemical, and neuroanatomical level. Two direct approaches are currently being investigated for FXS treatment: reactivating the FMR1 gene and compensating for the lack of FMRP. The latter approach has yielded promising results, with mGluR5 antagonists showing efficacy in clinical trials. CONCLUSIONS: Targeting mGluR5 is a valid approach for the development of therapeutic agents that target the underlying pathophysiology of FXS. Several compounds are currently in development, with encouraging results.


Subject(s)
Excitatory Amino Acid Antagonists/pharmacology , Fragile X Syndrome/drug therapy , Receptor, Metabotropic Glutamate 5/antagonists & inhibitors , Animals , Drug Evaluation, Preclinical , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Humans , Receptor, Metabotropic Glutamate 5/metabolism
13.
Cereb Cortex ; 23(11): 2657-66, 2013 Nov.
Article in English | MEDLINE | ID: mdl-22918986

ABSTRACT

Executive dysfunction in fragile X-associated tremor/ataxia syndrome (FXTAS) has been suggested to mediate other cognitive impairments. In the present study, event-related potentials and neuropsychological testing were combined to investigate the brain mechanisms underlying the executive dysfunction in FXTAS. Thirty-two-channel electroencephalography was recorded during an auditory "oddball" task requiring dual responses. FXTAS patients (N= 41, mean age= 62) displayed prolonged latencies of N1 and P3 and reduced amplitudes of P2 and P3, whereas their N2 measures remained within the normal range, indicating relatively preserved early-stage auditory attention but markedly impaired late-stage attention and working memory updating processes (as indexed by P3). Topographical mapping revealed a typical parietal P3 peak preceded by a prominent fronto-central P3 in normal control subjects (N= 32), whereas FXTAS patients had decreased parietal P3 amplitude and diminished fronto-central positivities with a delayed onset (∼50 ms later than controls, P < 0.002). The P3 abnormalities were associated with lower executive function test (e.g., BDS-2) scores. Smaller P3 amplitudes also correlated with increased CGG repeat length of fragile X mental retardation 1 (FMR1) gene and higher FMR1 mRNA levels. These results indicate that abnormal fronto-parietal attentional network dynamics underlie executive dysfunction, the cardinal feature of cognitive impairment in FXTAS.


Subject(s)
Cerebral Cortex/physiopathology , Evoked Potentials, Auditory , Executive Function/physiology , Acoustic Stimulation , Attention/physiology , Cerebellar Diseases/physiopathology , Electroencephalography , Female , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Humans , Male , Memory, Short-Term/physiology , Middle Aged , Neuropsychological Tests
14.
Hum Reprod ; 27(11): 3287-93, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22811313

ABSTRACT

BACKGROUND: For decades androgens have been considered detrimental to follicle maturation. Animal studies now suggest that they are essential for normal folliculogenesis. Especially in women with premature ovarian aging (POA), recent IVF data in humans are supportive. The literature also suggests an association between recently reported ovarian genotypes of the FMR1 gene and ovarian aging patterns. We, therefore, attempted to determine a potential difference in androgen concentrations and androgen interactions in women with POA who do or do not become pregnant while undergoing androgen supplementation, and whether androgen concentrations and pregnancy chances are affected by FMR1 genotypes. METHODS: We longitudinally assessed androgen metabolism in 91 women with POA, following pre-supplementation with micronized dehydroepiandrosterone (DHEA) prior to IVF. IVF outcomes were assessed based on androgen levels and ovarian FMR1 genotypes. RESULTS: The mean age of the women was 39.8 ± 4.4 years; the clinical pregnancy rate was 25.3%. Total androgen concentrations were not associated with pregnancy; however, in women with abnormal FMR1 genotypes, but not those with the normal genotype, free testosterone significantly affected clinical pregnancy potential (ß = 1.101, SE ± 0.508, P = 0.03). At the start of the IVF cycle, interactions of DHEA with total and free testosterone also significantly affected subsequent pregnancy rates (ß = -0.058, SE ± 0.023, P = 0.01 and ß = -0.496, SE ± 0.197, P = 0.012). CONCLUSIONS: Androgen interactions significantly influence IVF pregnancy rates in women with POA, with the impact of total androgens on cycle outcomes varying according to FMR1 genotypes. These observations suggest that the effectiveness of androgen supplementation in women with POA varies based on FMR1 genotypes, and defines androgen deficiency as a subset of diminished ovarian reserve.


Subject(s)
Androgens/therapeutic use , Dehydroepiandrosterone/therapeutic use , Dietary Supplements , Fragile X Mental Retardation Protein/genetics , Oogenesis , Polymorphism, Genetic , Primary Ovarian Insufficiency/diet therapy , Adult , Androgens/chemistry , Androgens/deficiency , Androgens/metabolism , Cohort Studies , Dehydroepiandrosterone/chemistry , Female , Fertilization in Vitro , Fragile X Mental Retardation Protein/metabolism , Genetic Association Studies , Humans , Infertility, Female/etiology , Infertility, Female/therapy , Longitudinal Studies , New York City , Ovarian Follicle/metabolism , Ovarian Follicle/physiopathology , Pregnancy , Pregnancy Rate , Primary Ovarian Insufficiency/genetics , Primary Ovarian Insufficiency/metabolism , Primary Ovarian Insufficiency/physiopathology , Retrospective Studies
15.
PLoS One ; 7(5): e36981, 2012.
Article in English | MEDLINE | ID: mdl-22615862

ABSTRACT

Although the pathogenic mechanisms that underlie autism are not well understood, there is evidence showing that metabotropic and ionotropic glutamate receptors are hyper-stimulated and the GABAergic system is hypo-stimulated in autism. Memantine is an uncompetitive antagonist of NMDA receptors and is widely prescribed for treatment of Alzheimer's disease treatment. Recently, it has been shown to improve language function, social behavior, and self-stimulatory behaviors of some autistic subjects. However the mechanism by which memantine exerts its effect remains to be elucidated. In this study, we used cultured cerebellar granule cells (CGCs) from Fmr1 knockout (KO) mice, a mouse model for fragile X syndrome (FXS) and syndromic autism, to examine the effects of memantine on dendritic spine development and synapse formation. Our results show that the maturation of dendritic spines is delayed in Fmr1-KO CGCs. We also detected reduced excitatory synapse formation in Fmr1-KO CGCs. Memantine treatment of Fmr1-KO CGCs promoted cell adhesion properties. Memantine also stimulated the development of mushroom-shaped mature dendritic spines and restored dendritic spine to normal levels in Fmr1-KO CGCs. Furthermore, we demonstrated that memantine treatment promoted synapse formation and restored the excitatory synapses to a normal range in Fmr1-KO CGCs. These findings suggest that memantine may exert its therapeutic capacity through a stimulatory effect on dendritic spine maturation and excitatory synapse formation, as well as promoting adhesion of CGCs.


Subject(s)
Autistic Disorder/drug therapy , Dendritic Spines/drug effects , Fragile X Syndrome/drug therapy , Memantine/pharmacology , Synapses/drug effects , Animals , Autistic Disorder/metabolism , Autistic Disorder/physiopathology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Cerebellum/drug effects , Cerebellum/metabolism , Dendritic Spines/metabolism , Dendritic Spines/physiology , Disease Models, Animal , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/metabolism , Fragile X Syndrome/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Synapses/metabolism , Synapses/physiology
16.
Dis Model Mech ; 4(5): 673-85, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21669931

ABSTRACT

Fragile X syndrome (FXS), caused by loss of the fragile X mental retardation 1 (FMR1) product (FMRP), is the most common cause of inherited intellectual disability and autism spectrum disorders. FXS patients suffer multiple behavioral symptoms, including hyperactivity, disrupted circadian cycles, and learning and memory deficits. Recently, a study in the mouse FXS model showed that the tetracycline derivative minocycline effectively remediates the disease state via a proposed matrix metalloproteinase (MMP) inhibition mechanism. Here, we use the well-characterized Drosophila FXS model to assess the effects of minocycline treatment on multiple neural circuit morphological defects and to investigate the MMP hypothesis. We first treat Drosophila Fmr1 (dfmr1) null animals with minocycline to assay the effects on mutant synaptic architecture in three disparate locations: the neuromuscular junction (NMJ), clock neurons in the circadian activity circuit and Kenyon cells in the mushroom body learning and memory center. We find that minocycline effectively restores normal synaptic structure in all three circuits, promising therapeutic potential for FXS treatment. We next tested the MMP hypothesis by assaying the effects of overexpressing the sole Drosophila tissue inhibitor of MMP (TIMP) in dfmr1 null mutants. We find that TIMP overexpression effectively prevents defects in the NMJ synaptic architecture in dfmr1 mutants. Moreover, co-removal of dfmr1 similarly rescues TIMP overexpression phenotypes, including cellular tracheal defects and lethality. To further test the MMP hypothesis, we generated dfmr1;mmp1 double null mutants. Null mmp1 mutants are 100% lethal and display cellular tracheal defects, but co-removal of dfmr1 allows adult viability and prevents tracheal defects. Conversely, co-removal of mmp1 ameliorates the NMJ synaptic architecture defects in dfmr1 null mutants, despite the lack of detectable difference in MMP1 expression or gelatinase activity between the single dfmr1 mutants and controls. These results support minocycline as a promising potential FXS treatment and suggest that it might act via MMP inhibition. We conclude that FMRP and TIMP pathways interact in a reciprocal, bidirectional manner.


Subject(s)
Disease Models, Animal , Drosophila melanogaster/enzymology , Fragile X Syndrome/drug therapy , Fragile X Syndrome/enzymology , Matrix Metalloproteinase 1/deficiency , Minocycline/therapeutic use , Nerve Net/pathology , Animals , Cell Shape/drug effects , Circadian Clocks/drug effects , Drosophila melanogaster/drug effects , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/pathology , Fragile X Syndrome/physiopathology , Gene Deletion , Matrix Metalloproteinase 1/metabolism , Minocycline/pharmacology , Mushroom Bodies/drug effects , Mushroom Bodies/pathology , Mushroom Bodies/physiopathology , Nerve Net/drug effects , Neuromuscular Junction/drug effects , Neuromuscular Junction/pathology , Neurons/drug effects , Neurons/pathology , Phenotype , Synapses/drug effects , Synapses/pathology , Tissue Inhibitor of Metalloproteinases/metabolism
17.
J Neurosci ; 30(31): 10263-71, 2010 Aug 04.
Article in English | MEDLINE | ID: mdl-20685971

ABSTRACT

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates synaptic plasticity by repressing translation of specific mRNAs. We found that FMRP binds mRNA encoding the voltage-gated potassium channel Kv3.1b in brainstem synaptosomes. To explore the regulation of Kv3.1b by FMRP, we investigated Kv3.1b immunoreactivity and potassium currents in the auditory brainstem sound localization circuit of male mice. The unique features of this circuit allowed us to control neuronal activity in vivo by exposing animals to high-frequency, amplitude-modulated stimuli, which elicit predictable and stereotyped patterns of input to the anterior ventral cochlear nucleus (AVCN) and medial nucleus of the trapezoid body (MNTB). In wild-type (WT) animals, Kv3.1b is expressed along a tonotopic gradient in the MNTB, with highest levels in neurons at the medial, high-frequency end. At baseline, Fmr1(-/-) mice, which lack FMRP, displayed dramatically flattened tonotopicity in Kv3.1b immunoreactivity and K(+) currents relative to WT controls. Moreover, after 30 min of acoustic stimulation, levels of Kv3.1b immunoreactivity were significantly elevated in both the MNTB and AVCN of WT, but not Fmr1(-/-), mice. These results suggest that FMRP is necessary for maintenance of the gradient in Kv3.1b protein levels across the tonotopic axis of the MNTB, and are consistent with a role for FMRP as a repressor of protein translation. Using numerical simulations, we demonstrate that Kv3.1b tonotopicity may be required for accurate encoding of stimulus features such as modulation rate, and that disruption of this gradient, as occurs in Fmr1(-/-) animals, degrades processing of this information.


Subject(s)
Auditory Pathways/physiology , Brain Stem/physiology , Evoked Potentials, Auditory, Brain Stem/physiology , Fragile X Mental Retardation Protein/genetics , Shaw Potassium Channels/metabolism , Acoustic Stimulation , Animals , Blotting, Western , Fragile X Mental Retardation Protein/metabolism , Immunohistochemistry , Male , Mice , Mice, Knockout , Neurons , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sound Localization
18.
Biochemistry ; 47(33): 8491-503, 2008 Aug 19.
Article in English | MEDLINE | ID: mdl-18656952

ABSTRACT

Exon 15 of the fragile X mental retardation protein gene (FMR1) is alternatively spliced into three variants. The amino acids encoded by the 5' end of the exon contain several regulatory determinants including phosphorylation sites and a potential conformational switch. Residues encoded by the 3' end of the exon specify FMRP's RGG box, an RNA binding domain that interacts with G-quartet motifs. Previous studies demonstrated that the exon 15-encoded N-terminal residues influence the extent of arginine methylation, independent of S 500 phosphorylation. In the present study we focus on the role the putative conformational switch plays in arginine methylation. Chemical and structural probing of Ex15 alternatively spliced variant proteins and several mutants leads to the following conclusions: Ex15c resides largely in a conformation that is refractory toward methylation; however, it can be methylated by supplementing extracts with recombinant PRMT1 or PRMT3. Protein modeling studies reveal that the RG-rich region is part of a three to four strand antiparallel beta-sheet, which in other RNA binding proteins functions as a platform for nucleic acid interactions. In the Ex15c variant the first strand of this sheet is truncated, and this significantly perturbs the side-chain conformations of the arginine residues in the RG-rich region. Mutating R 507 in the conformational switch to K also truncates the first strand of the beta-sheet, and corresponding decreases in in vitro methylation were found for this and R 507/R 544 and R 507/R 546 double mutants. These effects are not due to the loss of R 507 methylation as a conformational switch-containing peptide reacted under substrate excess and in methyl donor excess was not significantly methylated. Consistent with this, similar changes in beta-sheet structure and decreases in in vitro methylation were observed with a W 513-K mutant. These data support a novel model for FMRP arginine methylation and a role for conformational switch residues in arginine modification.


Subject(s)
Arginine/metabolism , Fragile X Mental Retardation Protein/chemistry , Fragile X Mental Retardation Protein/metabolism , Amino Acid Sequence , Animals , Arginine/chemistry , Exons , Fragile X Mental Retardation Protein/genetics , Humans , Methylation , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , PC12 Cells , Point Mutation , Protein Conformation , Protein Processing, Post-Translational , Rats , Recombinant Proteins , Sequence Deletion
19.
J Biol Chem ; 283(9): 5598-610, 2008 Feb 29.
Article in English | MEDLINE | ID: mdl-18093976

ABSTRACT

Spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein. Although the SMN complex is essential for assembly of spliceosomal U small nuclear RNPs, it is still not understood why reduced levels of the SMN protein specifically cause motor neuron degeneration. SMN was recently proposed to have specific functions in mRNA transport and translation regulation in neuronal processes. The defective protein in Fragile X mental retardation syndrome (FMRP) also plays a role in transport of mRNPs and in their translation. Therefore, we examined possible relationships of SMN with FMRP. We observed granules containing both transiently expressed red fluorescent protein(RFP)-tagged SMN and green fluorescent protein(GFP)-tagged FMRP in cell bodies and processes of rat primary neurons of hypothalamus in culture. By immunoprecipitation experiments, we detected an association of FMRP with the SMN complex in human neuroblastoma SH-SY5Y cells and in murine motor neuron MN-1 cells. Then, by in vitro experiments, we demonstrated that the SMN protein is essential for this association. We showed that the COOH-terminal region of FMRP, as well as the conserved YG box and the region encoded by exon 7 of SMN, are required for the interaction. Our findings suggest a link between the SMN complex and FMRP in neuronal cells.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Fragile X Mental Retardation Protein/metabolism , Hypothalamus/metabolism , Motor Neurons/metabolism , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins/metabolism , Animals , Cell Line, Tumor , Cyclic AMP Response Element-Binding Protein/genetics , Exons/physiology , Fragile X Mental Retardation Protein/genetics , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Fragile X Syndrome/pathology , Humans , Hypothalamus/pathology , Mice , Motor Neurons/pathology , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/pathology , Nerve Tissue Proteins/genetics , Protein Structure, Tertiary/physiology , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SMN Complex Proteins , Survival of Motor Neuron 1 Protein
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