Research ReportDecreased expression of the GABAA receptor in fragile X syndrome
Introduction
Fragile X syndrome is the most common form of inherited mental retardation, with a prevalence of 1 to 4000 in males and 1 to 6000 in females [reviewed by Bardoni et al., 2006, Gantois et al., 2004, O'Donnell and Warren, 2002]. Patients are characterized by mild to severe impairment of the higher cognitive functions and display various physical abnormalities, e.g., macroorchidism (enlarged testes) and craniofacial anomalies such as a typical long face, prominent jaws and elongated ears (Hagerman, 2002). Associated behavioral problems include hyperactivity and autistic-like features. In addition, 20% of the patients suffer from epileptic seizures. The syndrome is usually caused by a dynamic mutation of a CGG repeat in the 5′ untranslated region of the fragile X mental retardation gene 1 (FMR1) (Verkerk et al., 1991). Elongation of this repeat above a threshold of 200 copies induces hypermethylation of the CpG islands in the promoter region and concomitant transcriptional silencing, preventing synthesis of the FMR1 gene product FMRP (Pieretti et al., 1991).
FMRP is an RNA-binding protein with particular high expression in neurons and gonads. The protein aggregates with multiple mRNAs and proteins to form a messenger ribonucleic protein complex (mRNP), which is transported out of the nucleus through its nuclear export signal (Jin and Warren, 2003). Once in the cytoplasm, the complex can associate with members of the RNA-induced silencing complex (RISC) before associating with ribosomes. The FMRP–mRNP complex can be transported through dendrites to actively translating polyribosomes near the synapses, where it may play a role in local protein synthesis as a translational inhibitor (Laggerbauer et al., 2001, Li et al., 2001, Zalfa et al., 2003). Major mechanisms by which FMRP is thought to exert its repressing activity are through the RNA interference pathway or by acting as a nucleic acid chaperone (Bardoni et al., 2006, Gabus et al., 2004, Jin et al., 2004).
Interruption of the murine Fmr1 gene generated a mouse model for fragile X syndrome (Bakker et al., 1994). Fragile X knockout mice show mild cognitive deficits, hyperactivity, macroorchidism and increased sensitivity to epileptic seizures, features comparable with symptoms observed in fragile X patients (Bakker and Oostra, 2003, Kooy, 2003). Pathological studies revealed the presence of long tortuous, immature dendritic spines being denser along dendrites, as observed in patients (Braun and Segal, 2000, Comery et al., 1997, Irwin et al., 2002, Nimchinski et al., 2001). FMRP may therefore affect synaptic development and maturation in the central nervous system. The invertebrate homologue of Fmr1 in fruit flies, namely ‘Drosophila melanogaster fragile X mental retardation gene 1’ (dFmr1), exhibits high neuronal expression levels. The associated gene product dFmrp 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 (Dockendorff et al., 2002, Michel et al., 2004, Morales et al., 2002, Zhang and Broadie, 2005). dFmrp is required for normal neurite expansion, guidance and branching. Loss of dFmrp causes behavioral defects like abnormal eclosion and circadian rhythm behavior and anomalies in the morphology of several central nervous system neuronal populations.
Despite increased insights in the function of FMRP in the cell, the central question why absence of FMRP causes mental retardation and additional symptoms in fragile X patients remains to be elucidated. In a previous genome wide expression profiling study, our group found differential expression in neurons of specific brain parts from fragile X knockout mice limited to 3 cDNAs only, including the δ subunit of the GABAA receptor (Gantois et al., 2006). To further investigate a possible role of decreased expression of this ion channel in fragile X syndrome, we determined the relative expression of all GABAA receptor subunits in the mouse and fly model using real-time PCR. We found evidence that under expression of multiple subunits of the GABAA receptor is an evolutionary conserved hallmark of fragile X syndrome. As GABAA receptors are the main inhibitory receptors in brain, involved in processes also disturbed in fragile X patients such as anxiety, depression, epilepsy, insomnia and learning and memory (Mihalek et al., 1999), we believe that new powerful therapeutic opportunities for treatment of behavioral problems associated with fragile X syndrome might arise from these observations.
Section snippets
Mus musculus
Cortical and cerebellar tissue of adult fragile X male mice and their control littermates was isolated and reverse transcribed as indicated in the experimental procedure. These regions were selected because in the initial genome wide study under expression of the δ subunit of the GABAA receptor was found in cortex but not in cerebellum. GABAA receptors display an extensive structural heterogeneity based on the differential assembly of a family of at least 18 different subunits (α1–6, β1–3, γ1–3
Discussion
Ionotropic receptors for the neurotransmitter γ-aminobutyric acid (GABA) are widespread mediators of rapid neurotransmission in the nervous systems of both vertebrates and invertebrates. In mammals, 30–50% of all synapses in the central nervous system are GABAergic (Paredes and Agmo, 1992). GABAA receptors mediate fast synaptic inhibition in brain and spinal cord because their associated channels are permeable to Cl− ions; the flow of the negatively charged ions inhibits postsynaptic cells
M. musculus
Male C57BL/6J wild-type mice, purchased from Charles River (Wilmington, MA, USA), were crossed with females heterozygous for the Fmr1 mutation and backcrossed for at least 20 generations in the same genetic background. After DNA isolation from mouse tails, genotypes were determined by polymerase chain reaction (PCR) as described (Bakker et al., 1994). Male Fmr1 knockout mice and male control littermates with an average age of 8–12 weeks were used. Mixed genotype groups of approximately 5
Acknowledgments
We thank Jo Vandesompele for his advice in analyzing the real-time PCR results and Philip Pattyn for his help with the RNA quality analysis. We thank Pierre Codde and Ivan Aerts for the animal care. This study was supported through grants of the National Fragile X Foundation (NFXF), the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen), the Belgian National Fund for Scientific Research-Flanders (FWO) and the Fondation Jerôme Lejeune.
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