Expression of the GABAergic system in animal models for fragile X syndrome and fragile X associated tremor/ataxia syndrome (FXTAS).

After our initial discovery of reduced expression of several subunits of the GABA(A) receptor in two different animal models for fragile X syndrome, a frequent form of inherited mental retardation, we analyzed further components of the GABAergic pathway. Interestingly, we found a down regulation of many additional elements of the GABA signalling system, strengthening our hypothesis of involvement of the GABAergic pathway in the pathophysiology of fragile X syndrome. This is of special interest with regard to new therapeutic opportunities for treatment of this disorder. Remarkably, under expression was predominantly observed in cortex, although some elements of the GABAergic system that are expressed presynaptically or in the glial cells were also down regulated in the cerebellum. Additionally, we assessed the GABAergic system in expanded CGG-repeat mice, a model for fragile X associated tremor/ataxia syndrome (FXTAS). This late onset neurodegenerative disorder occurs in carriers of the fragile X premutation (55-200 CGG repeats) and is completely distinct (from both clinical and molecular pathogenic perspectives) from the neurodevelopmental disorder fragile X syndrome. Here we found upregulation of many components of the GABAergic system in cerebellum, but not in cortex. This finding is consistent with the cerebellar phenotype of FXTAS patients and has implications for the mechanism causative of differential gene expression.

Mutational analysis establishes a critical role for the N terminus of fragile X mental retardation protein FMRP.

Fragile X syndrome is the most common form of heritable mental retardation caused by the loss of function of the fragile X mental retardation protein FMRP. FMRP is a multidomain, RNA-binding protein involved in RNA transport and/or translational regulation. However, the binding specificity between FMRP and its various partners including interacting proteins and mRNA targets is essentially unknown. Previous work demonstrated that dFMRP, the Drosophila homolog of human FMRP, is structurally and functionally conserved with its mammalian counterparts. Here, we perform a forward genetic screen and isolate 26 missense mutations at 13 amino acid residues in the dFMRP coding dfmr1. Interestingly, all missense mutations identified affect highly conserved residues in the N terminal of dFMRP. Loss- and gain-of-function analyses reveal altered axonal and synaptic elaborations in mutants. Yeast two-hybrid assays and in vivo analyses of interaction with CYFIP (cytoplasmic FMR1 interacting protein) in the nervous system demonstrate that some of the mutations disrupt specific protein-protein interactions. Thus, our mutational analyses establish that the N terminus of FMRP is critical for its neuronal function.

Decreased expression of the GABAA receptor in fragile X syndrome.

After our initial discovery of under expression of the GABA(A) receptor delta subunit in a genome wide screening for differentially expressed mRNAs in brain of fragile X mice, a validated model for fragile X mental retardation syndrome, we analyzed expression of the 17 remaining subunits of the GABA(A) receptor using real-time PCR. We confirmed nearly 50% under expression of the delta subunit and found a significant 35%-50% reduction in expression of 7 additional subunit mRNAs, namely alpha(1), alpha(3), and alpha(4), beta(1) and beta(2) and gamma(1) and gamma(2), in fragile X mice compared to wild-type littermates. In concordance with previous results, under expression was found in cortex, but not in cerebellum. Moreover, decreased expression of specific GABA(A) receptor subunits in fragile X syndrome seems to be an evolutionary conserved hallmark since in the fragile X fly (Drosophila melanogaster) model we also found almost 50% under expression of all 3 subunits which make up the invertebrate GABA receptor, namely Grd, Rdl and Lcch3. In addition, we demonstrated a direct correlation between the amount of dFmrp and the expression of the GABA receptor subunits Rdl and Grd. Our results add evidence to previous observations of an altered GABAergic system in fragile X syndrome. Because GABA(A) receptors are the major inhibitory receptors in brain, involved in anxiety, depression, insomnia, learning and memory and epilepsy, processes also disturbed in fragile X patients, the well described GABA(A) receptor pharmacology might open new powerful opportunities for treatment of the behavioral and epileptic phenotype associated with fragile X syndrome.

The Drosophila fragile X mental retardation protein controls actin dynamics by directly regulating profilin in the brain.

Loss of Fragile X mental retardation protein (FMRP) function causes the highly prevalent Fragile X syndrome [1 and 2]. Identifying targets for the RNA binding FMRP is a major challenge and an important goal of research into the pathology of the disease. Perturbations in neuronal development and circadian behavior are seen in Drosophila dfmr1 mutants. Here we show that regulation of the actin cytoskeleton is under dFMRP control. dFMRP binds the mRNA of the Drosophila profilin homolog and negatively regulates Profilin protein expression. An increase in Profilin mimics the phenotype of dfmr1 mutants. Conversely, decreasing Profilin levels suppresses dfmr1 phenotypes. These data place a new emphasis on actin misregulation as a major problem in fmr1 mutant neurons.