iPSCs-derived and Mouse Friedreich's Ataxia Models to test Calcitriol Therapeutic Approach

Friedreich’s Ataxia (FRDA) is an autosomal recessive neurodegenerative disease. It is Europe's most common form of recessive ataxia, with an incidence of 1 affected individual every 40,000. This disease is caused by low Frataxin (FXN), a protein encoded in the FXN gene (chr. 9q13). It exerts its action mainly in the mitochondria and the presence of low levels of it in patients ranges from 5% to 30% compared to healthy individuals. Most patients (96%) display a homozygous expansion of a GAA trinucleotide repeat in the gene's first intron in this locus. This mutation leads to transcriptional defects and a lack of FXN protein. The remaining patients (4%) are compound heterozygous presenting the trinucleotide repeat expansion on one allele, and a point mutation or deletion on the second allele. There are no differences between homozygous and compound heterozygous patients at the phenotypic level. The most affected cell types are identified as the ones displaying higher energy consumption levels, such as cardiomyocytes, neurons, and beta-pancreatic cells. Among the most prevalent clinical manifestations, we can observe neurodegeneration, ataxia, development of diabetes mellitus in around 30% of patients, and cardiomyopathy, being also the leading cause of death. However, the detrimental mechanisms behind the clinical picture are still unclear and a matter of debate. This thesis project has two main objectives. The first one consists of the characterization of sensory neurons originating from the differentiation of Induced Pluripotent Stem Cells (iPSCs) derived from a patient affected by FRDA, and from a healthy donor. In this context, the aim was also the establishment of an optimized differentiation protocol that could be faster and easier to perform. The second main objective consisted of the assessment of a potential treatment for FRDA by administration of Calcitriol. Possible implications concerning Calcitriol administration in a primary cell culture model were already explored by the research group. To study the possible beneficial effect of drug administration two main models were exploited, the differentiated iPSC lines and the FXNI151F mouse model, to observe and correlate results both in the in vitro and in vivo contexts. To pursue these objectives different cell culture-based and molecular biology-based techniques have been exploited to assess the differentiation status of cells and the changes determined by Calcitriol administration both at the phenotypic and molecular levels. While the differentiation process showed that even if cells start to differentiate, they are not able to overcome a specific time-point (determined to be around day 10), Calcitriol administration in the mouse model showed not only an increase in FXN level in mutant mice after drug administration but also the amelioration of levels of various relevant mitochondrial proteins, indicating a potential therapeutic effect of this drug.