Elsevier

Brain Research

Volume 1253, 9 February 2009, Pages 176-183
Brain Research

Research Report
Expression of the GABAergic system in animal models for fragile X syndrome and fragile X associated tremor/ataxia syndrome (FXTAS)

https://doi.org/10.1016/j.brainres.2008.11.075Get rights and content

Abstract

After our initial discovery of reduced expression of several subunits of the GABAA receptor in two different animal models for fragile X syndrome, a frequent form of inherited mental retardation, we analyzed further components of the GABAergic pathway. Interestingly, we found a down regulation of many additional elements of the GABA signalling system, strengthening our hypothesis of involvement of the GABAergic pathway in the pathophysiology of fragile X syndrome. This is of special interest with regard to new therapeutic opportunities for treatment of this disorder. Remarkably, under expression was predominantly observed in cortex, although some elements of the GABAergic system that are expressed presynaptically or in the glial cells were also down regulated in the cerebellum. Additionally, we assessed the GABAergic system in expanded CGG-repeat mice, a model for fragile X associated tremor/ataxia syndrome (FXTAS). This late onset neurodegenerative disorder occurs in carriers of the fragile X premutation (55–200 CGG repeats) and is completely distinct (from both clinical and molecular pathogenic perspectives) from the neurodevelopmental disorder fragile X syndrome. Here we found upregulation of many components of the GABAergic system in cerebellum, but not in cortex. This finding is consistent with the cerebellar phenotype of FXTAS patients and has implications for the mechanism causative of differential gene expression.

Introduction

Loss of the fragile X mental retardation gene (FMR1) causes a neurodevelopmental and behavioural disorder called fragile X syndrome, which affects 1/2500 individuals (Verkerk et al., 1991, Hagerman, 2008). Patients present with mental retardation, autistic features, anxiety, hyperactivity and mood instability and display various physical abnormalities, such as enlarged testes (macroorchidism) and craniofacial anomalies (Hagerman, 2002). In contrast, over expression of FMR1 mRNA causes fragile X associated tremor/ataxia syndrome (FXTAS). This late-onset neurodegenerative disorder is characterized by progressive intention tremor and gait ataxia, with more variable associated features including parkinsonism, dysautonomia, peripheral neuropathy, and dementia (Jacquemont et al., 2003). Thus, the same FMR1 gene presents two opposing faces: a childhood-onset disorder (fragile X syndrome), caused by the absence of gene activity and a late-onset neurodegenerative syndrome (FXTAS), caused by toxic RNA gain of function (Jacquemont et al., 2007).

The 5′ untranslated region of the FMR1 gene contains a CGG repeat sequence that is stably transmitted in the normal range (5–44/55 repeats). Expansion of this repeat is associated with changes in the amount of FMR1 mRNA and protein with varied phenotypes [reviewed by Jacquemont et al., 2007]. Full mutation alleles (> 200 repeats) are associated with gene methylation and transcriptional silencing, which lead to the absence of the FMR1 gene product FMRP and cause fragile X syndrome. Premutation alleles (55–200 repeats) are unstable and may expand to full mutation alleles when transmitted maternally. The prevalence of the premutation has been estimated to be 1/813 males and 1/259 females in one study (Rousseau et al., 1995), but recent studies challenge this view (Hagerman, 2008). The pathogenic basis of FXTAS is (over) expression of this ‘toxic’ expanded CGG repeat FMR1 RNA which leads to neural cell dysregulation, formation of intranuclear inclusions in neurons and astrocytes, and disruption of the nuclear lamin architecture (Greco et al., 2002, Garcia Arocena et al., 2005).

Interruption of the murine fmr1 gene generated a mouse model for fragile X syndrome (Bakker et al., 1994). Fragile X knockout (KO) mice show mild cognitive deficits, hyperactivity, macroorchidism, immature dendritic spines and increased sensitivity to epileptic seizures, features comparable with symptoms observed in fragile X patients (Bakker and Oostra, 2003, Kooy, 2003). The invertebrate homologue of fmr1 in fruit flies, namely Drosophila melanogaster fragile X mental retardation gene 1 (dfmr1), displays considerable amino acid sequence identity/similarity with the vertebrate fmrp, especially within the functional domains. It possesses similar RNA-binding capacity as well as the ability to interact with human FMR1 (Wan et al., 2000). Dfmr1 deficient fly models have been generated (Morales et al., 2002, Dockendorff et al., 2002, Michel et al., 2004, Zhang and Broadie, 2005) and loss of dfmrp causes behavioural defects like abnormal eclosion and circadian rhythm behaviour and anomalies in the morphology of several central nervous system neuronal populations.

To better understand the timing and mechanism involved in the FMR1 CGG repeat instability and methylation, a mouse model was generated in which the endogenous mouse CGG repeat was replaced by a human CGG repeat carrying 98 CGG units, which is in the premutation range (Bontekoe et al., 2001). The inheritance of the CGG repeat is only moderately unstable, upon both maternal and paternal transmission, indicating differences between the behaviour of the fmr1 premutation CGG expanded-repeat in mouse and in human transmissions. The model displays biochemical, phenotypic and neuropathological characteristics of FXTAS (Willemsen et al., 2003). As in humans, the expanded CGG repeat model shows 2–3.5 fold elevated fmr1 mRNA levels in brain tissue compared with control. Protein levels are mildly decreased. Immunohistochemical studies provide significant evidence for the presence of ubiquitin-positive intranuclear inclusions in neurons of this mouse model. Brouwer et al. (2007) reported further repeat instability up to 230 CGG repeats. In humans, this would be considered as a full mutation and result in hypermethylation and gene silencing. However, mice carrying long repeats do not show any signs of abnormal methylation, suggesting that modelling the fragile X full mutation requires additional repeats or other genetic manipulation.

As genes that are over or under expressed might indicate which pathways are involved in fragile X syndrome, our group previously performed expression analyses on different brain regions of fmr1 KO mice compared to wild type (WT) littermates (Gantois et al., 2006, D'Hulst et al., 2006). We found decreased expression of 8 out of 18 known subunits of the GABAA receptor in fragile X mouse cortex, but not in cerebellum. Additionally, we found under expression of all three subunits which make up the GABA receptor in the fragile X fruit fly model. Thus, we consider down regulation of the GABAA receptor as an evolutionary conserved hallmark of fragile X syndrome. As GABAA receptors are involved in hyperactivity, anxiety, epilepsy and learning and memory (Mihalek et al., 1999), we hypothesized that a dysfunction of the GABAA receptor has neurophysiologic and functional consequences that might relate to the behavioural and neurological phenotype associated with fragile X syndrome, and as such might be a novel target for treatment of the disorder (D'Hulst and Kooy, 2007).

Using animal models for fragile X syndrome and FXTAS, we analyzed the expression of genes encoding enzymes and proteins important in the synthesis, transport and degradation of GABA or in the clustering of GABAA receptors at the postsynaptic terminal. This study gives us a broader image of the involvement of the GABAergic system in both syndromes.

Section snippets

fmr1 KO mice

Cortical and cerebellar tissue of adult fragile X male mice and control littermates was dissected, RNA was isolated and reverse transcribed as indicated in the experimental procedure. These regions were selected because in our initial study (D'Hulst et al., 2006) under expression of different subunits of the GABAA receptor was found in cortex but not in cerebellum. Assays-on-demands® (ABI, Foster city, CA, USA) were selected for several important genes of the GABA signalling pathway including

Discussion

Using real-time PCR, we investigated the expression of several important genes of the GABA metabolism in brain of fragile X mice and we found significant under expression of gad1, gat1 and 4, ssadh and gephyrin compared to wild-type animals. From our results it seems that there is no compensation in GABA synthesis or transport for the decreased expression of certain subunits of the GABAA receptor in fragile X mice, reported in our previous study (D'Hulst et al., 2006). It is remarkable that a

Animal models

All experiments were carried out in compliance to the European Community Council Directive (86/609/EEC) and approved by the Animal Ethics Committee of the University of Antwerp.

Fragile X mice (Mus musculus)

Male fragile X mice and control littermates were obtained by backcrossing females heterozygous for the fmr1 knockout mutation, inbred in the C57BL/6J background (N > 20), with C57BL/6J wildtype males (Charles River Laboratories, Brussels, Belgium). Genotyping of the litters was performed as previously described in D'Hulst

Acknowledgments

This study was supported through grants of the Fragile X Research Foundation (FRAXA), the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen) and the Belgian National Fund for Scientific Research — Flanders (FWO).

References (44)

  • HoglundP.J. et al.

    The repertoire of solute carriers of family 6: identification of new human and rodent genes

    Biochem. Biophys. Res. Commun.

    (2005)
  • JacquemontS. et al.

    Fragile X premutation tremor/ataxia syndrome: molecular, clinical, and neuroimaging correlates

    Am. J. Hum. Genet.

    (2003)
  • JacquemontS. et al.

    Fragile-X syndrome and fragile X-associated tremor/ataxia syndrome: two faces of FMR1

    Lancet Neurol.

    (2007)
  • MiyashiroK.Y. et al.

    RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice

    Neuron

    (2003)
  • MoralesJ. et al.

    Drosophila fragile X protein, DFXR, regulates neuronal morphology and function in the brain

    Neuron

    (2002)
  • NamchukM. et al.

    Phosphorylation of serine residues 3, 6, 10, and 13 distinguishes membrane anchored from soluble glutamic acid decarboxylase 65 and is restricted to glutamic acid decarboxylase 65alpha

    J. Biol. Chem.

    (1997)
  • Van DamD. et al.

    Cognitive decline, neuromotor and behavioural disturbances in a mouse model for Fragile-X-associated tremor/ataxia syndrome (FXTAS)

    Behav. Brain Res.

    (2005)
  • VerkerkA.J.M.H. et al.

    Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome

    Cell

    (1991)
  • ZhangY.Q. et al.

    Fathoming fragile X in fruit flies

    Trends Genet.

    (2005)
  • BakkerC.E. et al.

    Understanding fragile X syndrome: insights from animal models

    Cytogenet. Genome Res.

    (2003)
  • BakkerC.E. et al.

    Fmr1 knockout mice: a model to study fragile X mental retardation

    Cell

    (1994)
  • BontekoeC.J.M. et al.

    Instability of a (CGG)98 repeat in the Fmr1 promoter

    Hum. Mol. Genet.

    (2001)
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