Saccharomyces cerevisiae as a system for studying molecular basis of mitochondrial diseases and evaluating the effects of potentially beneficial molecules

Abstract

Mitochondrial diseases (MDs), currently affecting 1 in 5000 individuals, are the results of either inherited or spontaneous mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) which lead mainly to an impairment in the process of oxidative phosphorylation responsible for the synthesis of ATP, the main energy substrate in living cells. MDs are clinically heterogeneous as they can affect any organ or system of our body and they can arise at childhood or later in adult life. Despite the remarkable advances in their genetic diagnosis, treatments for MDs remain palliative and do not provide a relevant improvement in life of the subjects affected. The yeast Saccharomyces cerevisiae has proved to be not only a skilled model for the study of the mechanisms underlying mitochondrial pathology but also for the discovery of new potential therapies, thanks to the establishment of a two-step yeast-based screening assay called “drug drop test”, a high throughput screening which allows to analyze in short time an extremely high number of compounds. During my PhD study, I investigated the effects of beneficial molecules previously identified in our laboratory in yeast models of Autosomal Dominant Progressive External Ophthalmoplegia (adPEO) and Hepatocerebral Mitochondrial DNA Depletion Syndrome (MDDS) associated with mutations in human genes ANT1 and MPV17, respectively. ANT1 is a nuclear gene that encodes the mitochondrial ADP/ATP carrier which imports ADP into the mitochondrion and exports ATP into the intermembrane space. The mechanism by which mutations in this gene lead to instability of the mtDNA remains highly unsolved issue. In our laboratory five potentially therapeutic molecules for adPEO were identified and I tested them on several phenotypes (respiratory activity, ROS production, mitochondrial membrane potential, mtDNA instability) of the yeast model of ANT1 human mutations, to assess whether they were able to restore all the defects or only some of them, trying to identify the pathway targeted by these drugs. Interestingly none of the molecules decreased the ROS production and none of them were able to restore the mitochondrial membrane potential but they were able to increase the respiratory activity and to strongly improve the mtDNA stability. This suggests that the ROS overproduction and the depolarization of the membrane cannot be the only mechanisms that lead to the onset of mtDNA instability and that the identified molecules probably exert their beneficial effect acting by another mechanism. MPV17 is a nuclear gene that encodes a protein that forms a non-selective channel in the inner mitochondrial membrane whose physiological role and nature of the cargo remain until now elusive. Although it has been demonstrated that lack of Mpv17 determines mtDNA instability in human, mouse and yeast, the role of this protein in mtDNA maintenance remains unclear. Since the nucleotides decrease was reported in mitochondria of mice and fibroblasts of MPV17-mutant patients, it has been proposed that the mitochondrial dNTP insufficiency can be the cause of mtDNA depletion in MPV17 deficiency. To test this hypothesis in yeast, I had set up an enzymatic assay that enables the quantification of low concentrations of dNTPs such those found in mitochondria. The results showed that deletion of MPV17-ortholog, SYM1, resulted in a general decrease in all four mitochondrial nucleotides, confirming yeast as a valuable model for the study of the molecular mechanisms underlying MPV17-related diseases. In our laboratory ten putative therapeutic molecules for MPV17-related MDS were identified and I demonstrated that all these molecules were able to determine a concomitant increase of both mitochondrial dNTP (mtdNTP) pool and mtDNA stability suggesting that the reduced availability of DNA synthesis precursors is the cause of the mtDNA deletion/depletion in Sym1 deficiency. In order to extend the potential use of these drugs to other MDS patients, I also evaluated the effect of these molecules on mtDNA stability of two MDS yeast models characterized by mutations in MIP1 and RNR2, orthologs of the human genes POLG and RRM2B, respectively, identifying molecules beneficial also for these mitochondrial diseases.