Reference:
ANRn.a.6-MRARn.a.003
Abstract:
Rett syndrome (RTT) is a neurodevelopmentale dominant X-linked disease affecting almost exclusively girls. Significant progress was the discovery of mutations in the MECP2 gene in over 90% of RTT patients. In recent years, we have developed a French consortium and determined the spectrum of mutations MECP2. We have shown that the four most common MECP2 mutations are nonsense mutations (50% mutated alleles). Recently, studies have shown that aminoglycosides can induce aves very low efficiency a readthrough in vitro and in vivo stop mutations. In our project, we propose to develop a model of stable human cells bearing stop premature MECP2 mutations and screening a chemical library to identify new molecules effective and validate our results on fibroblasts of patients and RTT neurons culture and MECP2 mouse CNS 308 / Y. ******************* In a first step, we propose to develop cellular models of fibroblasts to measure the effectiveness readthrough of the stop codon. In these models, the natural stop codon is deleted and the GFP cDNA is inserted in line with MeCP2 cDNAs containing the 4 most frequent stop mutations in patients with RTT. Chimeric cADNcs are under the control of either the CMV promoter or beta-actin. Models / most sensitive promoters will be used to test the effectiveness of aminoglycosides on nonsense mutations of the MECP2 gene and to identify new drugs by screening a chemical library consisting of more from 30 000 to 40 000 molecules. In the absence of readthrough, no fluorescence will be observed. If a molecule is effective, fluorescence is detected. In a second step, we analyze the effectiveness of selected readthrough in human fibroblasts RTT patients carrying nonsense mutations molecules. To limit the influence of X chromosome inactivation, we use clonal cultures of fibroblasts expressing only the mutated allele. Fibroblasts are cultured with various concentrations of chemical molecules and the qualitative and quantitative evaluation (No. of cells) the expression of the protein MeCP2 full length will be achieved by using immunocytochime directed against the N-and C-ter antibody protein. In a third step (which may extend beyond the two years of the project), we study the efficiency of readthrough induced by carrier selected chemical molecules in neurons in culture and in vivo mouse mutation non- direction (MeCP2 308 / y). In this project we propose to use as fibroblasts Rett patients to assess potential functional interactions between CDKL5 and MeCP2 and their involvement in a common signaling pathway. **************** *** develop a pharmacological strategy for Rett syndrome The project aims. The rationale for this approach is based on the increasing number of arguments that suggest that Rett syndrome is a “post-natal illness” with a therapeutic window of 12 to 18 months. Thus, it is reasonable to assume that molecules that restore partially the expression of MeCP2 in some neurons could reduce the severity of symptoms. The proposed cellular models should allow us to identify some new molecules of interest in the chemical library and test restoring the synthesis of wild MeCP2 protein in fibroblasts of patients RTT. The results of this project will provide a basis for preclinical studies in mice carrying the stop mutation at position 308 (MeCP2 308 / y).
PROJECT DETAILS
beginning: 2006.
end: 2008.
Country of research: France
Counry of funding source: France
Funding organization: The French National Research Agency
Financing: NATIONAL FUNDINGS – 190 000 €