Yeast, worm, patient’s fibroblasts and zebrafish as models for drug repositioning of molecules targeting POLG-related diseases

The mitochondrial DNA (mtDNA) is replicated by the DNA polymerase gamma (POLG) and its accessory unit POLG2, both encoded by nuclear genes. To date, more than 300 mutations in POLG have been identified as a major cause of mitochondrial disorders with a spectrum of clinical presentations, ranging from infantile-onset epilepsies, liver failure, polyneuropathy, ataxia, dilated/hypertrophic cardiomyopathy to late-onset ophthalmoplegia and muscle weakness, all associated with multiple deletions and/or depletion of the mitochondrial DNA (mtDNA). The therapeutic treatment of POLG diseases is currently limited to symptom management. Based on the conservation of mitochondrial function from yeast to human, we used Saccharomyces cerevisiae harboring mutations in MIP1, the yeast POLG orthologous gene, as a tool to identify drugs already approved by the FDA for other pathologies, for their ability to suppress mtDNA instability. Twelve molecules were found in such drug repurposing analysis. Seven of them, called MRS (MIP1 rescuing substance) 1,8-13, have been characterized in this study. We characterized the MRS effects on the extended mtDNA mutability, the respiratory activity, the MIP1 mRNA levels and the Mip1 protein levels. We found that four MRS molecules decreased significantly the petite frequency and increased the respiratory activity of the yeast mutant strains. Treatment with these drugs strongly increased the levels of Mip1 protein, without increasing the levels of MIP1 mRNA, suggesting that these drugs stabilize Mip1. In addition, some drugs had an additive effect, indicating that they likely suppress the mtDNA defects acting on different pathways. The effects of the positive drugs have been further tested on a Caenorhabditis elegans models deleted for polg-1, in which a rescue of the detrimental phenotype was observed for several of them. A positive effect for one of such molecules, MRS8, was also observed in patients’ fibroblasts in which mtDNA depletion was induced by treatment with EtBr. One of the twelve drugs, clofilium tosylate (CLO), has been already previously characterized in yeast, worm and patients’ fibroblasts. However, a potential use in human therapy needs a validation in a vertebrate model. To reach this aim, we used a zebrafish-based model harboring homozygous Polg mutations associated to mtDNA depletion and complex I deficiency. We found that CLO is able to increase the mtDNA levels in such model as well as in a wild type zebrafish line with EtBr-induced mtDNA depletion.