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.#

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 since the reversal potential for Cl⁻ is more negative than the threshold for neuronal firing. Like other types of ionotropic receptors, mammalian GABAA receptors are pentamers assembled from a combination of individual subunits from 8 families with multiple isoforms: α1–6, β1–4, γ1–4, δ, ε, θ, π and ρ1–2. The expression pattern of individual subunits generates a high diversity of GABAA receptor subtypes in a spatio-temporal dependent manner with a major functional and pharmacological diversity between GABAA receptor subtypes mutually (Barnard et al. 1998; Kneussel 2002; Korpi et al. 2002). GABAA receptors are modulated by many drugs, including ethanol, benzodiazepines, various anesthetics and neuroactive steroids.#

D. melanogaster has a much simpler GABA receptor system, consisting of three genes only, namely Rdl (resistance to dieldrin), Grd (GABA and glycine-like receptor of Drosophila) and Lcch3 (ligand-gated chloride channel homologue 3) (Hosie et al., 1997). As in their vertebrate counterparts, the binding of GABA to the receptor causes a fast, temporary opening of anion-selective ion channels with an inhibitory response as a consequence. The subunits encoded by Rdl display 30%–38% homology with vertebrate GABA receptor subunits, about the same percentage of identity as seen between the different classes of vertebrate subunits (Ffrench-Constant et al., 1991). Grd displays 33–44% identity with vertebrate GABAA and glycine receptor α subunits (Harvey et al., 1994), and Lcch3 shows 47% identity with the vertebrate GABAA receptor β subunit isoforms (Henderson et al., 1993) (Fig. 2). Lcch3 can function as a subunit with Rdl in Cl⁻ channels (Zhang et al., 1995) and with Grd in cation channels (Gisselmann et al., 2004). It is the subunit composition which determines the picrotoxin and bicuculline sensitivity of the receptor.#

Using real-time PCR, we found significant under expression of 8 out of 18 known subunits of the GABAA receptor, namely α1, α3 and α4, β1 and β2 and γ1 and γ2 and δ, in cortex of fragile X mice, a validated model for fragile X syndrome, compared to their control littermates. The under expression of the δ subunit is the most significant, thereby offering a possible explanation why only this subunit was picked up in our initial genome wide expression profiling study. Our results suggest under expression of both the most frequent subtype of the GABAA receptor, α1β2γ2, that makes up 60% of the GABAA receptors in brain and which is sensitive to benzodiazepines (e.g., Diazepam), and the δ-containing subtype of the GABAA receptor, α4βnδ, which is sensitive to neuroactive steroids such as alphaxalone, a synthetic analogue of allopregnanolone (a natural occurring metabolite of progesterone).#

In addition, we found a nearly 50% reduction in expression of all 3 GABA receptor subunits, Grd, Rdl and Lcch3, in the fragile X fruit fly compared with wild-type strains, suggesting that under expression of specific subunits of the GABAA receptor is an evolutionary conserved hallmark of fragile X syndrome. Moreover, mRNA expression of subunits Rdl and Grd was shown to be dependent on the number of dFmr1 copies, indicating a direct correlation between the amount of dFmrp and the expression of the GABA receptor.#

We can only speculate the cause of the under expression of the GABAA receptor subunits in the fragile X animal models. Miyashiro et al. (2003) demonstrated a direct binding between FMRP and the mRNA of the δ subunit of the GABAA receptor using the Antibody Positioned RNA Amplification (APRA) technique, suggesting a direct effect of FMRP on transport and/or localization of GABAA receptor subunits. As FMRP plays an important role in transport and translation of mRNA, it could be speculated that in the absence of FMRP RNAs, normally bound to FMRP, are misregulated and/or degraded.#

Although we demonstrate under expression of various subunits at the mRNA level, results by others indicate that the protein level is also affected. Firstly, El Idrissi et al. (2005) have detected a reduced expression of the β subunit of the GABAA receptor on protein level in cortex, hippocampus, diencephalon and brainstem of fragile X mice using Western blot analysis. Nevertheless, additional protein expression studies are necessary to validate our results on protein level. Secondly, Gruss and Braun (2004) found that the ratio between inhibitory (taurine and GABA) and excitatory (aspartate and glutamate) amino acids in fragile X mouse brainstem, hippocampus and caudal cortex was decreased. Thirdly, electrophysiological recordings suggest a decreased GABAergic system efficiency in fragile X knock out mice that in turn may interfere with cholinergic mechanisms (D'Antuono et al., 2003). Moreover, preliminary results of our group predict mRNA under expression of glutamic acid decarboxylase (GAD), the limiting enzyme responsible for GABA synthesis in the presynaptic terminal of inhibiting synapses, in brain of fragile X mutant fruit flies (D'Hulst, Hassan and Kooy, unpublished results).#

In conclusion, we present evidence that decreased expression of the GABAA receptor is an evolutionary conserved hallmark of fragile X syndrome. As GABAA receptors are involved in anxiety, depression, epilepsy, insomnia and learning and memory (Mihalek et al., 1999), processes also disturbed in patients (Table 3), we might have identified a new target for rational drug therapy of the behavioral abnormalities and epilepsy associated with fragile X syndrome.#