ReviewInsights into GABAAergic system deficits in fragile X syndrome lead to clinical trials
Introduction
The fragile X syndrome is a frequent inherited cause of intellectual disability and syndromic autism (Santoro et al., 2012). The disease is caused by a loss of function mutation in the FMR1 gene at Xq27.3 (Verkerk et al., 1991). Expansion of a CGG repeat in the 5′ untranslated region to more than 200 units, leads to hypermethylation of the FMR1 promoter and the repeat. As a consequence, FMR1 transcription is silenced and no fragile X mental retardation protein (FMRP) is synthesised. FMRP is a selective RNA-binding protein that regulates transport and translation of its target mRNAs (Heulens and Kooy, 2011, Santoro et al., 2012). Male patients present with mild to severe intellectual disability, physical features, including macroorchidism and facial dysmorphisms, and behavioural problems (Hagerman, 2002, Berry-Kravis, 2014). The latter include hyperactivity, impulsivity, attention problems, anxiety, mood lability and autistic features. Specific medical problems are childhood seizures and strabismus. As females still have one active copy of the FMR1 gene, they are typically less severely affected and their clinical presentation is more variable.
GABA, the major inhibitory neurotransmitter in the adult mammalian brain, exerts its action through ionotropic GABAA receptors and metabotropic GABAB receptors (Bettler and Tiao, 2006). Both receptors have been implicated in the fragile X syndrome. Here, we focus on the GABAAergic system as the abnormalities in the GABAB system have been reviewed in detail elsewhere (Berry-Kravis, 2014, Braat and Kooy, 2014). GABAA receptors are heteropentameric ligand-gated chloride channels, as described in more depth elsewhere in this issue. The GABAA receptor is assembled as a non-random combination of receptor subunits; α1–6, β1–3, γ1–3, δ, ε, θ, π, ρ1–3 (Farrant and Nusser, 2005, D'Hulst et al., 2009a). This results in a wide variety of subtypes, with distinct expression patterns, different physiological properties and distinct sensitivity to pharmacological modulation. Moreover, the receptor subtypes have specific subcellular expression patterns. GABAA receptors that contain an α1–3, β2/3 and a γ2 subunit are mainly synaptic, whereas α4–6- and δ-containing receptors are mainly perisynaptically or extrasynaptically located (Farrant and Nusser, 2005). Functionally, two types of GABAA receptor-mediated signalling can be discerned, phasic and tonic inhibition. Phasic inhibition occurs when synaptic GABAA receptors are briefly activated by a high concentration of GABA, released from presynaptic vesicles. Binding of GABA to the postsynaptic receptors results in a chloride influx and hyperpolarisation of the postsynaptic membrane and so reduces the firing probability of action potentials. Tonic inhibition is mediated by extrasynaptic GABAA receptors. These receptor subtypes are relatively insensitive to desensitisation and have a high affinity for GABA as they are activated by low concentrations of GABA, a spill over from the GABA released in the synaptic cleft.
While GABAA receptors are inhibitory during adult life, they act as excitatory receptors during early development (Ben-Ari, 2002). The early depolarising effect is suggested to have a crucial trophic role and contribute to cell proliferation, migration, differentiation and synaptogenesis (Cellot and Cherubini, 2013). The developmental polarity switch from excitatory to inhibitory occurs following changes in intracellular chloride concentration, which are mediated by changes in expression of the ion transporters Na+–K+–2Cl− co-transporter 1 (NKCC1) and K+–Cl− co-transporter 2 (KCC2) (Ben-Ari, 2002, Cellot and Cherubini, 2013). The balance between the amount of the chloride importer NKCC1 and the chloride exporter KCC2 determines the intracellular chloride concentration and, therefore, the nature of GABAA receptor-mediated signalling. In immature neurons, the NKCC1 co-transporter maintains a high intracellular chloride concentration, leading to chloride efflux through GABAA receptors and GABA-mediated depolarisation. As neurons mature, the expression of the co-transporter KCC2 is up-regulated, while at the same time NKCC1 expression is down-regulated. Consequently, intracellular chloride concentrations decrease and GABA signalling becomes hyperpolarising.
An increasing amount of experimental evidence supports the key involvement of the GABAA receptor in the pathophysiology of the fragile X syndrome. The aim of this review is to summarise the defects in the GABAAergic pathway in the fragile X syndrome that have been reported at different levels, including at the expression, the functional, the neurotransmitter and the neuroanatomical level (Table 1).
Section snippets
mRNA and protein level
Using whole-genome expression profiling, we initially identified a twofold reduction in δ subunit mRNA in the hippocampus and cortex of adult Fmr1 knockout mice (Gantois et al., 2006). Subsequently, several studies used quantitative real-time PCR to assess GABAA receptor mRNA expression in adult Fmr1 knockout mice (Table 1). Firstly, mRNA expression levels of 18 GABAA receptor subunits (α1–6, β1–3, γ1–3, δ, ε, θ, π, ρ1–2) were measured in cortical and cerebellar samples (D'Hulst et al., 2006).
The link to FMRP
The mechanism of the global underexpression of the entire GABAAergic system, consistently reported in the majority of studies, remains an enigma. Among the biochemically validated mRNA targets of FMRP is the GABAA receptor δ subunit mRNA (Santoro et al., 2012). The δ subunit mRNA was first identified as a target with antibody-positioned RNA amplification (APRA), an observation that was confirmed using UV-crosslinking and filter binding assays (Miyashiro et al., 2003). Moreover, direct binding
D. melanogaster
dfmr1 Deficient D. melanogaster die before puparium formation when reared on glutamate-rich food (Chang et al., 2008). This feature allowed high-throughput screening of a library of 2000 FDA-approved drugs and natural products, The Spectrum Collection, for their efficacy to rescue glutamate-induced lethality. Three of the nine lead compounds were part of the GABAergic system; GABA, nipecotic acid (a GABA reuptake inhibitor) and creatinine (a potential GABAA receptor activator). The potential of
Clinical trial with ganaxolone
Ganaxolone is an interesting therapeutic candidate for treatment of fragile X patients. Its pharmacological activity is similar to alphaxalone, but it is orally active and lacks hormonal side-effects due to 3β-methyl substitution (Carter et al., 1997, Nohria and Giller, 2007). Ganaxolone has been evaluated in clinical trials for various types of epilepsy (http://www.clinicaltrials.gov/). Recently, additional trials have been initiated to evaluate the effect of ganaxolone on post-traumatic
Concluding remarks
Recent findings in both Fmr1 knockout mice and dfmr1 deficient D. melanogaster add further evidence to the involvement of the GABAAergic system in the fragile X syndrome. Most studies are consistent with a global down-regulation of the GABAAergic system at multiple levels, however, it is clear that the abnormalities vary across different brain regions and are age-dependent. While most intervention strategies are aimed at targeting the glutamatergic system, this review clearly demonstrates the
Acknowledgements
Our research on fragile X syndrome is funded by grants from FRAXA, Research Foundation Flanders (FWO-Vlaanderen), Fondation Jérôme Lejeune and Marguerite-Marie Delacroix to R.F.K. and a PhD grant from the IWT (Agency for Innovation by Science and Technology) to S.B.
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