Reference:
1
Abstract:
The central hypothesis that there is a tendency to evaluate this project is that the mechanisms that regulate actin polymerization-dependent proteins of the ENA / VASP are able to modulate the effects of the mutation of MeCP2. In the case of positive results, then you might investigate whether a similar phenomenon also occurs with the CDKL5 mutation. This hypothesis will be studied by evaluating in the first place if the mutation alters the expression of MeCP2 and the correct localization of actin and a battery of factors related to the polymerization of actin. The study will be carried out in vivo in mice with deletion of MeCP2 in murine neuronal cultures with reduced expression of MeCP2 (knockdown) in lymphoblasts obtained from tissue banks, and in cells with mutations in MECP2 obtained by reprogramming of fibroblasts into iPSCs to differentiate patients then in neurons. Secondly, it will examine whether, as suggested by genetic studies, variations of the expression of the protein (ENA murine ortholog of ENAH) can modulate the phenotype in cells with mutations for MeCP2. In particular, the study will test whether an increase or a decrease in the levels of ENA in cultured neurons obtained from mice with deletion of MeCP2 or iPSCs derived from patients, may negatively or positively modulate synaptic alterations induced by mutations in MECP2. The analysis of the synapses will be carried out with both morphological techniques (visualization of dendritic spines and their motility imaging, immunocytochemistry for markers of pre-and postsynaptic terminals), and functional (electrophysiological recordings of synaptic currents from neurons in culture). The multiplicity of approaches necessary for the performance of the project is guaranteed by the differentiation of the centers involved. The laboratory of Pisa (Prof. Pizzorusso), which will act as coordinator, has experience in electrophysiological and imaging of dendritic spines, also home to a colony of mice with deletion of MeCP2 (Jaenisch model). The laboratory of Siena (Prof. Renieri) has identified the gene duplication ENAH in the child with Z-RTT and continue genetic studies on these aspects; Prof. Renieri also directs the biobank Italian RTT ( www.sienabiobanknetwork.org ) to which requests will be the lymphoblastoid cell lines and primary fibroblasts necessary for the production of iPSC. The unit of Siena evaluate the expression of factors associated with actin remodeling in these cells and iPSCs will provide to the other units of the project. The laboratory of Turin (Prof. Giustetto) has extensive experience in the study of the organization of synapses by confocal and electron microscopy. Its role will be to evaluate by immunohistochemical analysis of protein subcellular localization of the actin cytoskeleton, will also morphological and molecular analysis of synapses in vivo and in vitro preparations described above. All three units have extensive experience in previous studies on Rett syndrome documented by publications, carried out in part in collaboration, high-impact international journals. Prof. Renieri and Prof. Pizzorusso cooperate with the AIRETT as part of its Scientific Council. If the concept behind this project was tested, the results would be of great importance for the RTT as it would allow the identification of new biochemical pathways altered due to the mutation of MeCP2. The factors involved in these alterations could therefore serve as a potential molecular targets for drug therapies. Moreover, the analysis of changes in these factors could contribute to the understanding of the genetic mechanisms capable of modifying the clinical picture of the girls with. Finally, the reprogramming of fibroblasts obtained from patients in iPSC and their differentiation into neuronal cells is an added value in the study of synaptic alterations present in this model could validate for the screening of treatments aimed at improving these defects induced by mutations of MeCP2 in cells of patients.
PROJECT DETAILS
beginning: 2010.
end: 2011.
Country of research: Italy
Counry of funding source: Italy
Funding organization: AIRETT
Financing: PRIVATE FUNDERS – 80 000 €