GENERAZIONE DI IPSC COME MODELLO IN VITRO E SVILUPPO DI UN POSSIBILE APPROCCIO TERAPEUTICO PER LA MALATTIA DI CHARCHOT-MARIE-TOOTH DI TIPO 2A(CMT2A)

Charcot-Marie-Tooth disease type 2A (CMT2A) is a sensory-motor polyneuropathy, characterized by the involvement of motor and sensitive neurons, resulting in progressive weakness, limb muscle atrophy and loss of sensitivity. Mitofusin2 (MFN2) gene has been identified as causative of the disease. The MFN2 protein, located in the outer mitochondrial membrane, is involved in the mitochondrial network. Defects in this network are features of several neurodegenerative diseases. A treatment for CMT2A is not currently available. The development of an effective therapy needs a better understanding of the molecular mechanisms of the disease, not only to identify new therapeutic targets, but also to define biomarkers of the disease phenotype. The first aim of this study was the generation of an in vitro disease model, not currently available. The reprogramming of mature somatic cells into induced pluripotent stem cells (iPSCs) provides the derivation of disease-specific cell types, such as motor and sensitive neurons, affected in the disease. Based on this method, we successfully generated human iPSCs from a CMT2A patient and demonstrated their differentiation into motor neurons. We analyzed mitochondrial phenotypes (i.e mitochondrial localization, content and stability of mitochondrial DNA, and respiratory capacity) associated with the disease in CMT2A motor neurons as well as in fibroblasts. In particular, we observed an alteration in mitochondria localization, a reduction in the amount of mitochondrial DNA and a dysfunction of the mitochondrial respiratory chain, identifying specific hallmarks of the disease phenotype. These defects are more evident in neuronal cells compared to fibroblasts, in agreement with neuronal specificity of the disease. In addition in vitro models, the analysis of these aspects has been conducted in the only currently available murine model of CMT2A (MitoCharc 1) to extend its characterization, searching for biomarkers of disease phenotype. The second aim of this work was the development of a therapeutic approach for this disorder. We silenced endogenous MFN2 gene by short harpin RNA (shRNA) in CMT2A fibroblasts. At the same time, in order to restore correct MFN2 protein levels, we transfected a MFN2 c-DNA modified to be resistant to shRNA-mediated silencing. The results of this strategy were very promising in CMT2A fibroblasts, and preliminary data were also obtained in the CMT2A mouse model. In conclusion, this study contributed to deepen the knowledge about disease molecular mechanisms, generating an in vitro model of CMT2A by patient-specific iPSCs, and to identify a possible therapeutic strategy for CMT2A.