Aminoacyl-tRNA synthetases mutations in human mitochondrial diseases: contribution of yeast to understand the functional significance of novel variants and to approach the identification of therapeutical compounds

To date, genetic analysis with Next Generation Sequencing (NGS) approaches in patients affected by mitochondrial diseases (MD) has made possible the identification of new disease genes or new mutations in known disease genes, thus contributing to improve the diagnosis of mitochondrial diseases and to find the molecular defects causing pathological phenotype. Mitochondrial aminoacyl-tRNA synthetases (mtARSs) are nuclear encoded essential enzymes for mitochondrial translation since they ensure the proper attachment of each amino acids to its cognate tRNA. Mutations in the genes encoding for mtARSs are associated to mitochondrial human disorders that can primarly affect central nervous but also other organs and systems. Recently, in patients affected by MD, many novel variants have been found in mtARS genes and for this reason it becomes essential to assess their pathogenic significance. Over the years, the simple eukaryote Saccharomyces cerevisiae demonstrated to be a good tool to understand the causative role of mutations associated to MD thanks to its ability to grow without a functional respiratory chain, if a fermentable carbon source is made available. Moreover, since most of mutations are found in compound heterozygosity, the yeast ability to exist in haploid or diploid status can help to investigate on the contribution of each mutation to the pathological phenotype. Most of this PhD work has focused on the validation and functional analysis of novel variants, identified by our clinical collaborators, in mtARSs genes using ad hoc yeast models and to approach the identification of therapeutical molecules able to restore, at least partially, the pathological phenotype. Specific yeast models for the different mtARS genes considered in this work (DARS2, NARS2, RARS2, WARS2, VARS2) have been constructed and, on mutant strains expressing the different allelic variants, several phenotypic characterizations have been performed. Moreover, since there is currently no established treatment for pathologies caused by mutations in mtARSs this research has addressed also the identification of beneficial molecules. In particular it focused on the evaluation of the ability of amino acid supplementation to rescue the mitochondrial impairment showed by mutant strains and on the search of chemical compounds able to rescue the OXPHOS defective phenotype associated to mtARS mutations using drug repositioning as startegy. Another part of this work concerned the evaluation of the functional conservation of pantothenate kinase (PanK) activity between human and yeast. PanK are the enzymes involved in the first step of biosynthesis of Coenzime A, a key molecule involved in several metabolic pathways. For this purpose, the ability of the three different isoforms of PanK to rescue the pathological phenotypes of yeast model strains lacking for the unique gene encoding for PanK, CAB1, and carrying pathological mutations associated to PKAN has been evaluated.