Reversal of activity-mediated spine dynamics and learning impairment in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is characterized by intellectual disability and autistic traits, and results from the silencing of the FMR1 gene coding for a protein implicated in the regulation of protein synthesis at synapses. The lack of functional Fragile X mental retardation protein has been proposed to result in an excessive signaling of synaptic metabotropic glutamate receptors, leading to alterations of synapse maturation and plasticity. It remains, however, unclear how mechanisms of activity-dependent spine dynamics are affected in Fmr knockout (Fmr1-KO) mice and whether they can be reversed. Here we used a repetitive imaging approach in hippocampal slice cultures to investigate properties of structural plasticity and their modulation by signaling pathways. We found that basal spine turnover was significantly reduced in Fmr1-KO mice, but markedly enhanced by activity. Additionally, activity-mediated spine stabilization was lost in Fmr1-KO mice. Application of the metabotropic glutamate receptor antagonist α-Methyl-4-carboxyphenylglycine (MCPG) enhanced basal turnover, improved spine stability, but failed to reinstate activity-mediated spine stabilization. In contrast, enhancing phosphoinositide-3 kinase (PI3K) signaling, a pathway implicated in various aspects of synaptic plasticity, reversed both basal turnover and activity-mediated spine stabilization. It also restored defective long-term potentiation mechanisms in slices and improved reversal learning in Fmr1-KO mice. These results suggest that modulation of PI3K signaling could contribute to improve the cognitive deficits associated with FXS.

Translating molecular advances in fragile X syndrome into therapy: a review.

Fragile X syndrome is an inherited disease with cognitive, behavioral, and neurologic manifestations, resulting from a single genetic mutation. A variety of treatments that target individual symptoms of fragile X syndrome are currently utilized with limited efficacy. Research in animal models has resulted in the development of potential novel pharmacologic treatments that target the underlying molecular defect in fragile X syndrome, rather than the resultant symptoms. This review describes recent advances in our understanding of the molecular basis of fragile X syndrome and summarizes the ongoing clinical research programs.

Fragile X-syndrome: literature review and report of two cases

Fragile X-syndrome is caused by a mutation in chromosome X. It is one of the most frequent causes of learning disability. The most frequent manifestations of fragile X-syndrome are learning disability, different orofacial morphological alterations and an increase in testicle size. The disease is associated with cardiac malformations, joint hyperextension and behavioural alterations. We present two male patients aged 17 and 10 years, treated in our Service due to severe gingivitis. Both showed the typical facial and dental characteristics of the syndrome. In addition, we detected the presence of root anomalies such as taurodontism and root bifurcation, which had not been associated with fragile X-syndrome in the literature. In some cases these root malformations have been associated with other sex-linked congenital syndromes, though in none of the studies published in the literature have they been related with fragile X-syndrome. This syndrome is relevant due to its high prevalence, the presentation of certain oral and facial characteristics that can facilitate the diagnosis, and the few cases published to date

Dysregulated ADAM10-Mediated Processing of APP during a Critical Time Window Leads to Synaptic Deficits in Fragile X Syndrome.

The Fragile X mental retardation protein (FMRP) regulates neuronal RNA metabolism, and its absence or mutations leads to the Fragile X syndrome (FXS). The β-amyloid precursor protein (APP) is involved in Alzheimer's disease, plays a role in synapse formation, and is upregulated in intellectual disabilities. Here, we show that during mouse synaptogenesis and in human FXS fibroblasts, a dual dysregulation of APP and the α-secretase ADAM10 leads to the production of an excess of soluble APPα (sAPPα). In FXS, sAPPα signals through the metabotropic receptor that, activating the MAP kinase pathway, leads to synaptic and behavioral deficits. Modulation of ADAM10 activity in FXS reduces sAPPα levels, restoring translational control, synaptic morphology, and behavioral plasticity. Thus, proper control of ADAM10-mediated APP processing during a specific developmental postnatal stage is crucial for healthy spine formation and function(s). Downregulation of ADAM10 activity at synapses may be an effective strategy for ameliorating FXS phenotypes.

Auditory and visual event-related potential alterations in fragile X syndrome

Le syndrome du X fragile (SXF) est la première cause héréditaire de déficience intellectuelle et également la première cause monogénique d’autisme. Le SXF est causé par l'expansion de la répétition du nucléotide CGG sur le gène FMR1, ce qui empêche l’expression de la protéine FMRP. L’absence du FMRP mène à une altération du développement structurel et fonctionnel de la synapse, ce qui empêche la maturation des synapses induite par l’activité et l’élagage synaptique, qui sont essentiels pour le développement cérébral et cognitif. Nous avons investigué les potentiels reliés aux événements (PRE) évoqués par des stimulations fondamentales auditives et visuelles dans douze adolescents et jeunes adultes (10-22) atteints du SXF, ainsi que des participants contrôles appariés en âge chronologique et développemental. Les résultats indiquent un profil des PRE altéré, notamment l’augmentation de l’amplitude de N1 auditive, par rapport aux deux groupes contrôle, ainsi que l’augmentation des amplitudes de P2 et N2 auditifs et de la latence de N2 auditif. Chez les patients SXF, le traitement sensoriel semble être davantage perturbé qu’immature. En outre, la modalité auditive semble être plus perturbée que la modalité visuelle. En combinaison avec des résultats anatomique du cerveau, des mécanismes biochimiques et du comportement, nos résultats suggèrent une hyperexcitabilité du système nerveux dans le SXF.

Prefrontal social cognition network dysfunction underlying face encoding and social anxiety in fragile X syndrome

Individuals with fragile X syndrome (FXS) commonly display characteristics of social anxiety, including gaze aversion, increased time to initiate social interaction, and difficulty forming meaningful peer relationships. While neural correlates of face processing, an important component of social interaction, are altered in FXS, studies have not examined whether social anxiety in this population is related to higher cognitive processes, such as memory. This study aimed to determine whether the neural circuitry involved in face encoding was disrupted in individuals with FXS, and whether brain activity during face encoding was related to levels of social anxiety. A group of 11 individuals with FXS (5 M) and 11 age-and gender-matched control participants underwent fMRI scanning while performing a face encoding task with onlineeye-tracking. Results indicate that compared to the control group, individuals with FXS exhibited decreased activation of prefrontal regions associated with complex social cognition, including the medial and superior frontal cortex, during successful face encoding. Further, the FXS and control groups showed significantly different relationships between measures of social anxiety (including gaze-fixation) and brain activity during face encoding. These data indicate that social anxiety in FXS may be related to the inability to successfully recruit higher level social cognition regions during the initial phases of memory formation. (C) 2008 Elsevier Inc. All rights reserved.

Copy number variants on the X chromosome in women with primary ovarian insufficiency

Objective: To investigate whether submicroscopic copy number variants (CNVs) on the X chromosome can be identified in women with primary ovarian insufficiency (POI), defined as spontaneous secondary amenorrhea before 40 years of age accompanied by follicle-stimulating hormone levels above 40 IU/L on at least two occasions. Design: Analysis of intensity data of single nucleotide polymorphism (SNP) probes generated by genomewide Illumina 370k CNV BeadChips, followed by the validation of identified loci using a custom designed ultra-high-density comparative genomic hybridization array containing 48,325 probes evenly distributed over the X chromosome. Setting: Multicenter genetic cohort study in the Netherlands. Patient(s): 108 Dutch Caucasian women with POI, 97 of whom passed quality control, who had a normal karyogram and absent fragile X premutation, and 235 healthy Dutch Caucasian women as controls. Intervention(s): None. Main Outcome Measure(s): Amount and locus of X chromosomal microdeletions or duplications. Result(s): Intensity differences between SNP probes identify microdeletions and duplications. The initial analysis identified an overrepresentation of deletions in POI patients. Moreover, CNVs in two genes on the Xq21.3 locus (i.e., PCDH11X and TGIF2LX) were statistically significantly associated with the POI phenotype. Mean size of identified CNVs was 262 kb. However, in the validation study the identified putative Xq21.3 deletions samples did not show deviations in intensities in consecutive probes. Conclusion(s): X chromosomal submicroscopic CNVs do not play a major role in Caucasian POI patients. We provide guidelines on how submicroscopic cytogenetic POI research should be conducted. (Fertil Steril (R) 2011;95:1584-8. (C) 2011 by American Society for Reproductive Medicine.)

WORD-FINDING DIFFICULTIES, VERBAL PARAPHASIAS, AND VERBAL DYSPRAXIA IN 10 INDIVIDUALS WITH FRAGILE-X-SYNDROME

Speech/language disorders are common in the fragile X syndrome. [Howard-Peebles, 1979: Am J Hom Genet 31:214-222; Renier et al., 1983: J Ment Defic Res 27:51-59; Sparks, 1984: Birth Defects and Speech-Language Disorders, pp, 39-43; Hanson et al., 1986: Am J Med Genet 23:195-206]. Verbal paraphasias have been considered a rare feature and word-finding difficulties have seldom been reported. Here we report on ten Brazilian patients who were evaluated for speech/language disturbances and found that word-finding difficulties were present in 50% of the cases, which is a slightly higher frequency than that of clear dyspraxia. We suggest, therefore, that word-finding difficulties and verbal dyspraxia can be a common feature within the spectrum of this syndrome. Additional speech findings are discussed. (C) 1995 Wiley-Liss, Inc.

Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1–2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.

Abnormal dendritic spines in fragile X knockout mice: Maturation and pruning deficits

Fragile X syndrome arises from blocked expression of the fragile X mental retardation protein (FMRP). Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. Here we describe dendritic spines in Golgi-impregnated cerebral cortex of transgenic fragile X gene (Fmr1) knockout mice that lack expression of the protein. Dendritic spines on apical dendrites of layer V pyramidal cells in occipital cortex of fragile X knockout mice were longer than those in wild-type mice and were often thin and tortuous, paralleling the human syndrome and suggesting that FMRP expression is required for normal spine morphological development. Moreover, spine density along the apical dendrite was greater in the knockout mice, which may reflect impaired developmental organizational processes of synapse stabilization and elimination or pruning.

Synaptic regulation of protein synthesis and the fragile X protein

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use “synaptoneurosomes,” a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.

A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P

The absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene, is responsible for pathologic manifestations in the Fragile X Syndrome, the most frequent cause of inherited mental retardation. FMRP is an RNA-binding protein associated with polysomes as part of a messenger ribonucleoprotein (mRNP) complex. Although its function is poorly understood, various observations suggest a role in local protein translation at neuronal dendrites and in dendritic spine maturation. We present here the identification of CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) as FMRP interactors. CYFIP1/2 share 88% amino acid sequence identity and represent the two members in humans of a highly conserved protein family. Remarkably, whereas CYFIP2 also interacts with the FMRP-related proteins FXR1P/2P, CYFIP1 interacts exclusively with FMRP. FMRP–CYFIP interaction involves the domain of FMRP also mediating homo- and heteromerization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has recently been shown to interact with the small GTPase Rac1, which is implicated in development and maintenance of neuronal structures. Consistent with FMRP and Rac1 localization in dendritic fine structures, CYFIP1/2 are present in synaptosomal extracts.

Hairpins are formed by the single DNA strands of the fragile X triplet repeats: structure and biological implications.

Inordinate expansion and hypermethylation of the fragile X DNA triplet repeat, (GGC)n.(GCC)n, are correlated with the ability of the individual G- and C-rich single strands to form hairpin structures. Two-dimensional NMR and gel electrophoresis studies show that both the G- and C-rich single strands form hairpins under physiological conditions. This propensity of hairpin formation is more pronounced for the C-rich strand than for the G-rich strand. This observation suggests that the C-rich strand is more likely to form hairpin or "slippage" structure and show asymmetric strand expansion during replication. NMR data also show that the hairpins formed by the C-rich strands fold in such a way that the cytosine at the CpG step of the stem is C.C paired. The presence of a C.C mismatch at the CpG site generates local flexibility, thereby providing analogs of the transition to the methyltransferase. In other words, the hairpins of the C-rich strand act as better substrates for the human methyltransferase than the Watson-Crick duplex or the G-rich strand. Therefore, hairpin formation could account for the specific methylation of the CpG island in the fragile X repeat that occurs during inactivation of the FMR1 gene during the onset of the disease.