Elsevier

Neurobiology of Disease

Volume 21, Issue 2, February 2006, Pages 346-357
Neurobiology of Disease

Expression profiling suggests underexpression of the GABAA receptor subunit δ in the fragile X knockout mouse model

https://doi.org/10.1016/j.nbd.2005.07.017Get rights and content

Abstract

It is still unclear why absence of the fragile X protein (FMRP) leads to mental retardation and specific behavioral problems. In neurons, the protein transports specific mRNAs towards the actively translating ribosomes near the synapses.

To unravel the mechanism leading to the disorder, we performed global gene expression analysis by means of the differential display method using the fragile X mouse model. To verify differential expression, we used microarray technology and real-time PCR. Three differentially expressed cDNAs showed consistent underexpression in the fragile X knockout mouse, including a GABAA receptor subunit δ, a Rho guanine exchange factor 12 and an EST BU563433. In addition, we identified 5 genes that showed differential expression dependent on the sample of RNA analysis. We consider their differential expression as provisional. It is possible that these differentially expressed genes play an important role in the cognitive and behavioral problems observed in the fragile X syndrome.

Introduction

Fragile X syndrome, the most common cause of hereditary mental retardation, is usually caused by a dynamic expansion of a CGG trinucleotide repeat in the 5′ UTR of the causative gene, FMR1 (Gantois and Kooy, 2002, Jin and Warren, 2003, Verkerk et al., 1991). The syndrome has an estimated prevalence of one in 4000 males and one in 6000 females (de Vries et al., 1997, Turner et al., 1996). Patients suffer from mild to severe mental retardation, macroorchidism (enlarged testis), facial dysmorphologies and behavioral problems including hyperactivity and autistiform features (Cornish et al., 2004, Hagerman, 2002). Twenty-two percent of patients suffer from epilepsy (Musumeci et al., 1999).

The fragile X syndrome is caused by the absence of the FMR1 protein, FMRP. FMRP is a nearly ubiquitously expressed protein with abundant expression in neurons and spermatogonia. It is an RNA binding protein containing two KH domains and an RGG box (Ashley et al., 1993, Siomi et al., 1993). It binds to RNA homopolymers as well as a subset of brain transcripts in vitro. The presence of both a nuclear export signal (NES) and nuclear localization signal (NLS) suggested that FMRP is able to shuttle between the nucleus and cytoplasm (Eberhart et al., 1996). Though FMRP is predominantly localized in the cytoplasm, some FMRP is observed in the nucleus and in nuclear pores (Feng et al., 1997, Willemsen et al., 1996). In the cytoplasm, FMRP is associated with actively translating ribosomes via messenger ribonucleoprotein particles (mRNP) (Feng et al., 1997). This FMRP–mRNP complex contains multiple proteins and mRNAs. Proteins shown to be part of the FMRP–mRNP complex include FXR1P and FXR2P (paralogues of FMRP), nucleolin, YB1/p50, NUFIP, CYFIP1 and CYFIP2 (Bardoni et al., 2003, Ceman et al., 1999, Ceman et al., 2000, Schenck et al., 2003, Zhang et al., 1995).

The identity of the mRNAs in the FMRP–mRNA complex remained elusive. Recently, it was demonstrated that FMRP preferentially binds to mRNAs containing a novel three-dimensional structure called a G-quartet (Darnell et al., 2001, Schaeffer et al., 2001). Comparing the precipitated mRNA of FMRP–mRNP particles between fragile X knockout and control mouse brains and mRNA profiles in polyribosomal fractions of cell lines from fragile X patients and controls, revealed that G-quartet containing mRNAs are also part of the FMRP–mRNP complex in vivo (Brown et al., 2001). The region overlapping with the RGG-box of FMRP was shown to be responsible for the G-quartet binding. Interestingly, the part of the FMRP–mRNA encoding the RGG-box also forms a G-quartet structure itself (Schaeffer et al., 2001). Some of the identified mRNAs in the FMRP–mRNP complex have a function in synaptic growth and maturation including NAP-22, which is present in axon terminals and dendritic spines and plays a role in maturation and maintenance of synapses. Others, including MAP1B, a microtubule-associated protein, play a role in transport within neurons. Interestingly, Futsch, the Drosophila ortholog of MAP1B, is also bound by Drosophila dFMR1, and the level of futsch mRNA is inversely related to the dFMR1 expression level (Zhang et al., 2001).

Recently, in vivo binding of additional mRNAs to FMRP in hippocampal neurons was determined using antibody-positioned RNA amplification, a novel technique that allows to identify RNAs that are directly associated with FMRP in the cell (Miyashiro et al., 2003). Predominantly non-G-quartet containing mRNAs were identified including the glucocorticoid receptor α (GRα), a low affinity receptor for corticosteroid hormones involved in the electrical activity in the hippocampus in response to mineralocorticoid receptor stimulation. In addition, the receptor was shown to have an altered dendritical distribution in the hippocampus of the fragile X knockout mouse. Diminished responsiveness of the receptor is compatible with learning problems observed in fragile X patient. Analyzing translation at the synapse, Zalfa et al. (2003) identified BC1 RNA as part of the FMR1–mRNP. They revealed that BC1 determines the binding specificity and is required in the FMRP–mRNP complex for binding of FMRP to other RNAs, including MAP1B.

Several in vitro and in vivo studies revealed that the translation of bound mRNAs is influenced by FMRP (Laggerbauer et al., 2001, Li et al., 2001, Sung et al., 2003, Zalfa et al., 2003). In the cytoplasm, the FMRP–mRNP complex can be transported through dendrites to the synapses regulating local protein synthesis of specific mRNAs in response to synaptic stimuli. In synaptoneurosomes, local translation of FMRP is increased in response to metabotropic glutamate receptor stimulation (Greenough et al., 2001, Weiler et al., 1997). When FMRP is absent, the mRNAs normally associated in the FMRP–mRNP complex might be translationally misregulated leading to impaired synaptic plasticity potentially related to cognitive deficits.

A fragile X knockout mouse has been constructed using gene targeting (Bakker et al., 1994). Absence of the protein induces macroorchidism, impaired cognitive behavior, hyperactivity, anxiety and increased sensitivity to audiogenic epileptic seizures, compatible with the phenotype of fragile X patients (Bakker and Oostra, 2003, Kooy, 2003). Pathological studies revealed the presence of long, tortuous, immature dendritic spines being denser along the dendrites, as observed in human patients (Braun and Segal, 2000, Comery et al., 1997, Irwin et al., 2002, Nimchinski et al., 2001). In the fragile X knockout mouse, the protein synthesis dependent long term depression (LTD) is increased (Bear et al., 2004, Huber et al., 2002), consistent with FMRP being a negative regulator of translation (Laggerbauer et al., 2001, Li et al., 2001).

However, despite our increased insights in the function of FMRP in the cell and in the RNAs and proteins bound by FMRP, the pathways leading to the observed cognitive and behavioral deficits remain elusive. Therefore, we searched for genes that are differentially expressed in neurons. Identification of under- or overexpressed mRNAs can be speculated to play a role in the fragile X syndrome and may lead to a better understanding of the pathways causing mental retardation and behavioral problems observed in fragile X patients. Differential display was selected, as this is an extremely sensitive technique that unlike microarray-based technologies is not dependent on a preselection of cDNAs. It is able to detect expression differences in the majority of all expressed mRNAs. We revealed a limited amount of genes of which the expression profile is influenced by the absence of FMR1 in neurons. These identified genes can be speculated to play a role in the pathogenesis of the fragile X syndrome.

Section snippets

Animals and tissue preparation

Male fragile X knockout mice (Bakker et al., 1994) and male control littermates inbred for more than 10 generations (e.g., congenic) in a C57Bl/6J and FVBS/Ant background were used (V. Errijgers et al., unpublished data). The mice were between 9–13 weeks old. After sacrificing mice in ether, the brain was immediately removed and hippocampus dissected and frozen in liquid nitrogen.

RNA

Total cellular RNA (pools of 4 hippocampi, from knockout and control littermates for the two mouse strains) was

Differential display

Expression profiling was performed using a modification of the original differential display technique (Liang and Pardee, 1992). In our experimental setup, we used 26 (20 bp long) semi-arbitrary primers which enable comparison of >90% (676 different primer combinations) of all genes expressed (Table 1) (Bauer et al., 1994, Chang et al., 1997, Liang and Pardee, 1992, Welsh et al., 1992). Hippocampal tissue of adult fragile X knockout mice and control littermates from 9–13 weeks of age was

Discussion

Using differential display followed by microarray analysis and real-time PCR, we did not observe gross differences in gene expression in the hippocampus between knockout and wild type mice. This suggests that the expression profile of only a small number of genes is disturbed in the fragile X knockout mouse, but we cannot exclude the possibility that the expression of a higher number of genes is mildly disturbed as a result of the absence of FMR1 and that the sensitivity of the applied

Acknowledgments

We thank Leen J. Blok for help with the differential display protocol, Jeltje Van Baren for help with analysis of sequence data and thank Werner Sieghart for kindly providing the antibody against the δ subunit. This study was supported, in part, through grants of the Belgian National Fund for Scientific Research-Flanders (FWO), an Interuniversity Attraction Pole (IUAP-V) and the FRAXA Research Foundation.

References (83)

  • M. Gruss et al.

    Age- and region-specific imbalances of basal amino acids and monoamine metabolism in limbic regions of female Fmr1 knock-out mice

    Neurochem. Int.

    (2004)
  • M. Hirotani et al.

    Interaction of plexin-B1 with PDZ domain-containing Rho guanine nucleotide exchange factors

    Biochem. Biophys. Res. Commun.

    (2002)
  • P. Jin et al.

    New insights into fragile X syndrome: from molecules to neurobehaviors

    Trends Biochem. Sci.

    (2003)
  • S. Kaneko et al.

    Genetics of epilepsy: current status and perspectives

    Neurosci. Res.

    (2002)
  • M. Kneussel

    Dynamic regulation of GABAA receptors at synaptic sites

    Brain Res. Rev.

    (2002)
  • E.R. Korpi et al.

    Drug interactions at GABA(A) receptors

    Prog. Neurobiol.

    (2002)
  • K.Y. Miyashiro et al.

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

    Neuron

    (2003)
  • V. Perrot et al.

    Plexin B regulates Rho through the guanine nucleotide exchange factors leukemia-associated Rho GEF (LARG) and PDZ-RhoGEF

    J. Biol. Chem.

    (2002)
  • A. Schenck et al.

    CYFIP/Sra-1 controls neuronal connectivity in Drosophila and links the Rac1 GTPase pathway to the fragile X protein

    Neuron

    (2003)
  • H. Siomi et al.

    The protein product of the fragile X gene, FMR1, has characteristics of an RNA binding protein

    Cell

    (1993)
  • Y.J. Sung et al.

    The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo

    J. Biol. Chem.

    (2003)
  • J.M. Swiercz et al.

    Plexin-B1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology

    Neuron

    (2002)
  • V. Tretter et al.

    Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells

    J. Biol. Chem.

    (2001)
  • J. Vandesompele et al.

    Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR

    Anal. Biochem.

    (2002)
  • A.J.M.H. Verkerk 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)
  • R. Willemsen et al.

    Association of FMRP with ribosomal precursor particles in the nucleolus

    Biochem. Biophys. Res. Commun.

    (1996)
  • C. Windpassinger et al.

    The human gamma-aminobutyric acid A receptor delta (GABRD) gene: molecular characterisation and tissue-specific expression

    Gene

    (2002)
  • W. Wisden et al.

    GABAA receptor channels: from subunits to functional entities

    Curr. Opin. Neurobiol.

    (1992)
  • F. Zalfa et al.

    The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses

    Cell

    (2003)
  • Y.Q. Zhang et al.

    Drosophila fragile X-related gene regulates the MAP1B homolog futsch to control synaptic structure and function

    Cell

    (2001)
  • C.T. Ashley et al.

    FMR-1 protein: conserved RNP family domains and selective RNA binding

    Science

    (1993)
  • J. Aurandt et al.

    The semaphorin receptor plexin-B1 signals through a direct interaction with the Rho-specific nucleotide exchange factor, LARG

    Proc. Natl. Acad. Sci. U. S. A.

    (2002)
  • C.E. Bakker et al.

    Understanding fragile X syndrome: insights from animal models

    Cytogenet. Genome Res.

    (2003)
  • C.E. Bakker et al.

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

    Cell

    (1994)
  • C.E. Bakker et al.

    Introduction of a FMR1 transgene in the fragile X knockout mouse

    Neurosci. Res. Commun.

    (2000)
  • E.A. Barnard et al.

    International Union of Pharmacology: XV. Subtypes of γ-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function

    Pharmacol. Rev.

    (1998)
  • T. Bast et al.

    Hyperactivity, decreased startle reactivity, and disrupted prepulse inhibition following disinhibition of the rat ventral hippocampus by the GABAA receptor antagonist picrotoxin

    Psychopharmacology

    (2001)
  • D. Bauer et al.

    Detection and differential display of expressed genes by DDRT-PCR

    PCR Methods Appl.

    (1994)
  • A. Beckel-Mitchener et al.

    Correlates across the structural, functional, and molecular phenotypes of fragile X syndrome

    Ment. Retard. Dev. Disabil. Res. Rev.

    (2004)
  • E.A. Benardete et al.

    Increased excitability and decreased sensitivity to GABA in an animal model of dysplastic cortex

    Epilepsia

    (2002)
  • K. Braun et al.

    FMRP involvement in formation of synapses among cultured hippocampal neurons

    Cereb. Cortex

    (2000)
  • Cited by (0)

    1

    Present address: Department of Biological Psychology, University of Leuven, Leuven, Belgium.

    View full text