Research reportPharmacological treatment of fragile X syndrome with GABAergic drugs in a knockout mouse model
Highlights
► The GABAA receptor is still functional in fragile X mice. ► Treatment with neuroactive steroids can rescue the audiogenic seizures in fragile X mice. ► We propose the GABAA receptor as a novel therapeutic target for fragile X syndrome.
Introduction
Fragile X syndrome is the most common form of intellectual disability. Besides cognitive impairment, patients suffer from several behavioural problems including hyperactivity, sleep problems and autistic-like behaviour [1]. Epileptic seizures are also commonly observed in patients [2]. The syndrome is caused by a dynamic expansion of a CGG triplet located within the 5′ untranslated region of the fragile X mental retardation 1 (FMR1) gene [3]. Due to the dynamic mutation, the CGG repeat and the surrounding CpG island located in the promoter region of the gene become hypermethylated, leading to transcriptional silencing of FMR1 and consequently absence of the FMR1 protein product, FMRP [4]. FMRP is an RNA-binding protein that interacts with various neuronal mRNAs and is involved in the regulation of mRNA translation, transport and stability [5], [6], [7], [8], [9]. Absence of FMRP might lead to deregulation of many neuronal mRNAs eventually cumulating in the fragile X phenotype. One of the main pathways affected in the fragile X syndrome is the GABAA receptor pathway. We have found an altered expression of several components of the GABAergic system in the Fmr1 knockout mouse, including 8 subunits of the GABAA receptor (α1, 3 and 4, β1 and 2, γ1 and 2 and δ), proteins and enzymes involved in synthesis (Gad 1), transport (Gat 1 and Gat 4) and degradation (Ssadh) of GABA and in the clustering and targeting of the GABAA receptors at the post-synaptic membrane (Gephyrin) [10], [11]. Underexpression was also found for all three GABAA receptor subunits (Grd, Rdl and Lcch3) in the fragile X fly [10]. Other groups demonstrated decreased protein levels of several GABAA receptor subunits and abnormal GABA-mediated transmission in the fragile X mouse [12], [13], [14], [15]. The combination of all these molecular and electrophysiological findings together with the fact that GABAA receptors are implicated in anxiety, depression, learning and memory, epilepsy and insomnia, all presenting in the fragile X syndrome, led us to propose the GABAA receptor as a novel target for treatment of the fragile X syndrome [16].
As the GABAergic system is compromised in the fragile X syndrome, it cannot a priori be excluded that the sensitivity of the receptor for GABAA receptor agonists is reduced in fragile X patients. In order to investigate the therapeutic potential of this type of drugs, we first wanted to investigate whether the GABAA receptors in the knockout mouse are amendable to treatment. By administering equal doses of GABAergic drugs to Fmr1 knockout and control animals and comparing the performance in selected tests, we can determine the potential difference in drug sensitivity between both genotypes.
For our experiments, we selected two types of drugs that act on two different prevalent subtypes of the receptor. Diazepam, a classical benzodiazepine, binds GABAA receptors containing a β, γ2 and either an α1, α2, α3 or α5 subunit [17]. Diazepam enhances the affinity of this most frequently present subtype of the receptor for GABA, resulting in an increased inhibition and thus a sedative effect. Neuroactive steroids are an entirely different class of agonists. Examples are the endogenous neurosteroid allopregnanolone, a metabolite of the steroid hormone progesterone and the synthetic drugs alphaxalone and ganaxolone [18]. These compounds bind predominantly to the δ-containing extrasynaptic GABAA receptor subtypes and regulate anxiety, stress and neuronal excitability by increasing both channel-open frequency and open duration [19], [20]. At high concentrations, neurosteroids can even directly activate GABAA receptor channels [21]. Ganaxolone has a similar pharmacological activity as alphaxalone but due to its 3β-methyl substituent, ganaxolone is orally active and lacks hormonal side effects [22]. It was especially developed for its improved bioavailability and potential anxiolytic and anticonvulsant activity.
As sedation and anxiety are modulated through the GABAA receptor, we performed a motor-coordination test (rotarod) and an anxiety-related test (elevated plus maze) to determine the functionality of the GABAA receptor in fragile X mice. With a rotarod test, the motor-coordination and balance of a mouse is tested by placing the mouse on a rotating rod with accelerating speed. The elevated plus maze is commonly used to assess anxiety-like behaviour in mice models. The task is based on the naturalistic conflict between the tendency of mice to explore a novel environment and the aversive properties of a brightly lit, open area [23]. When anxious, the natural tendency of rodents is to prefer enclosed dark spaces over open brightly lit spaces. In addition, as several studies have shown that the GABAA receptor is implicated in epilepsy [24], we investigated the effect of GABAergic drugs on the audiogenic seizure phenotype. We did find that the GABAA receptor is a suitable target for treatment of at least some behavioural symptoms of the fragile X syndrome.
Section snippets
Animals
Male Fmr1 knockout mice and their control littermates (C57BL/6J background) were housed, bred and genotyped as described previously [10]. 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.
Rotarod
We used an automated accelerating rotarod (Ugo Basile, Comerio, Varese, Italy; accelerating model 7650 for mice). Mice, 10 weeks old, were placed on a rotating drum and the latency to
GABAA receptors in fragile X mice are still sensitive to benzodiazepines
To investigate potential differences in the drug sensitivity of the most common GABAA receptor subtypes, we performed a rotarod test where we compared the performance of fragile X mice with control littermates using different concentrations of diazepam. We found a significant effect for treatment for both genotypes (F(3,488) = 153.412, p < 0.001, two-way ANOVA) (Fig. 1A). Wild-type mice as well as fragile X mice became more sedated with increasing doses of diazepam, as shown with the decreased
Discussion
In this study we investigated the functional potential of the GABAA receptor as a novel target for treatment of the fragile X syndrome. In the first place, it is of utmost importance that the receptor still responds to GABAA receptor agonists. The functionality of the GABAA receptor was demonstrated in fragile X mice in two behavioural tests, a motor-coordination test and an anxiety-related test.
Motor-coordination was tested with an accelerating rotarod. Overall, we did not find a significant
Acknowledgements
We would like to thank Frieda Franck and Ellen Vloeberghs from the Laboratory of Neurochemistry and Behaviour, Institute Born-Bunge, University of Antwerp, Belgium and Sofie Willemen for the help with the behavioural experiments. This work was supported by the Belgian National Fund for Scientific Research – Flanders (FWO), the Fragile X Research Foundation (FRAXA), the Institute for the Promotion of Innovations through Science and Technology in Flanders (IWT Vlaanderen), the Fondation Jerôme
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Present address: Department of Biological Sciences, Hunter College, City University of New York, NY, USA.