FRAXA Awards:
  $45,000 in 2010
  $50,000 in 2008
  $50,000 in 2007
  $35,000 in 2000
  $30,000 in 1999

Despite recent progress in the understanding of fragile X syndrome, there is no targeted treatment for this disease. In previous studies we showed that the amount of GABAA receptors is significantly decreased in fragile X animal models and that this receptor is a suitable target for treatment. However, before drugs can be tested on human patients, the abnormalities of the GABAergic system observed in animal models need to be verified in human patients. Therefore, we propose to perform position emission tomography (PET), a functional imaging technology that is able to provide non-invasive in vivo assessment and quantification of GABAA receptor binding through injections of labelled flumazenil. A difference in GABAA receptor distribution between fragile X patients and controls will enforce our hypothesis that a dysfunction of the GABAergic system is responsible for the neurologic and behavioural problems seen in fragile X patients. Our research will thus strongly encourage drug trials on fragile X patients.

by Frank Kooy, 9/2008

Absence of a single protein, FMRP, in fragile X patients leads to a cascade of molecular events in brain cells. To find out which other genes are involved the clinical symptoms, we have been looking for genes that are differentially expressed in fragile X syndrome. One of the genes specifically underexpressed is part of the GABAA receptor. As GABAA receptors are the main inhibitory receptors in the brain, involved in processes like anxiety, mood swings, sleep and cognition, processes also disturbed in fragile X patients, we followed up on this finding. In subsequent studies, we demonstrated abnormalities in expression levels of multiple parts of the GABAA receptor, both in the mouse model and in the fly model, indicating underexpression is an evolutionary conserved hallmark of fragile X syndrome. Our case for involvement of the GABAergic system in fragile X is supported by independent findings from other groups.

The purpose of this grant is twofold: first, we want to understand why and how absence of the fragile X protein results in underexpression of the GABA system. Second, we want to investigate whether drugs that work on the GABA system are able to influence the phenotype of the fragile X mouse model.

During the first year of our study, we analyzed the relative expression of many genes that are responsible for GABA synthesis, transport, clustering and degradation. Essentially all of these genes were under expressed in the fragile X syndrome, strengthening our hypothesis that the entire GABA system is down regulated in the disorder. In current animal experiments, we are now investigating whether drugs that reactivate the GABA system are able to correct some of the symptoms observed in patients, including epilepsy, hyper activity and anxiety. In initial experiments, we already verified that GABAergic drugs such as the neurosteroid alphaxolone are still effective in the knockout mice.

A second breakthrough in the first year of this project was the observation that the GABA receptor mRNAs that are down regulated are a part of the FMRP-mRNP complex. This means that FMRP binds GABA RNAs, possibly explaining why the GABAergic system is down regulated in fragile X syndrome. Planned experiments will follow up on this finding and aim to unravel the precise mechanism of GABAergic down regulation.

The fragile X mouse model, our main research tool, has helped us to understand the nature of the cognitive deficit associated with the fragile X syndrome. We analyzed the effect of the introduction of a transgene capable of generating the fragile X protein (the gene missing in fragile X syndrome) in the fragile X knockout mouse. In initial trials, we observed significant amounts of fragile X protein in the knockout mice, but observed little rescue of the phenotype. Experiments to modify the experimental conditions to allow a full "rescue" the phenotype of the fragile X knockout mouse are ongoing.

We also identified about 40 sequences that are differentially expressed in neurons of fragile X mice, e.g. that are significantly under or overrepresented in the fragile X mouse when compared with a control littermate. As the only difference between the control and the knockout is the fragile X gene, these sequences are potentially related to the pathogenesis of fragile X syndrome. Analysis of these genes is ongoing, and may provide insights in the molecular pathways of the fragile X syndrome. Such pathways might be interferable by drug treatment.