Yeast as a model and tool to study mitochondrial diseases (NARP)
Mitochondrial diseases represent a class of diseases which are individually rare but non negligible considering that there are more than one hundred known mitochondrial diseases. No efficient therapy is available to cure these disorders. NARP (Neuropathy, Ataxia and Retinitis Pigmentosa) and MILS (Maternally Inherited Leigh’s Syndrome) are mitochondrial disorders associated with point mutations in MTATP6, a mitochondrial gene encoding the subunit 6 of the F1F0-ATPase. The budding yeast Saccharomyces cerevisiae is a relevant model to characterize mitochondrial function and to study human pathologies (Mager and Winderickx, 2005; Schwimmer et al., 2006). Some yeast mutants are model of human diseases. Therefore, using a yeast model for the mitochondrial disease NARP (Neuropathy, Ataxia and Retinitis Pigmentosa) (Rak et al., 2007), we recently set up a screening assay to select into chemical libraries molecules able to compensate the mutant deficiencies. This method permits to isolate drugs that can then be tested and validated on animal models, in particular mammalian models for further development. This NARP screening is a proof of principle and other pharmacological screening on different mitochondria diseases are possible depending on the availability of the corresponding yeast mutant models. Furthermore, the molecules can also help to find the targeted cellular pathways and to identify pathogenic mechanisms.
The most common and studied mutation of these NARP/MILS mutations is T8993G converting a highly conserved leucine 156 into arginine. Jean-Paul di Rago’s team has introduced this mutation at the corresponding position of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. Due to a severe deficiency of ATP production by the F1F0-ATPase, the T8993G NARP yeast model grew very slowly on respiratory substrates (glycerol- or ethanol-based media). In our laboratory, we recently set up a screening assay to select molecules able to compensate the mutant deficiencies in order to isolate potential pharmacological drugs active against the NARP disorder.
After the screening of more than 10,000 molecules from chemical libraries available in our laboratory, about 20 molecules were able to increase the growth rate of the T8993G NARP yeast mutant on non-fermentable (respiratory) substrates. These active compounds isolated as efficient to restore the respiratory growth of the T8993G NARP yeast mutant have also been validated on other respiratory-deficient yeast mutants (the T8851C and T9176G NARP mutants and the fmc1∆ mutant, a thermosensitive mutant of the F1F0-ATPase assembly).
We measured bioenergetic parameters on entire yeast mutant cells and on isolated mitochondria after their treatment or not by the various active compounds (measurements of the mitochondrial membrane potential, the respiration, the ATP production and the ATP hydrolysis). Secondly, the main issue is to validate the efficiency of the active molecules (isolated on yeast model) in other models like animal (drosophila), or mammalian cells for the considered diseases (NARP/MILS). NARP fibroblasts are currently tested in our laboratory and, in a near future, collaborating processes will allow us to use NARP cybrids (cells lines derived from patient’s cells).
This NARP screening is a proof of principle to find active molecule against the mitochondrial disorders. Indeed, yeast has been extensively used for the study of mitochondrial diseases and several other yeast models for mitochondrial diseases are available or possible to create, therefore the systematic application of our approach to the discovery of active molecules against these disorders seems to be promising. Moreover, after the screening and the validation of the active drugs on other models, the most promising molecules will be modified by chemists to synthesize several chemical derivatives in order to improve their efficiency and possibly reduce their side effects. Furthermore, a “reverse screening” approach will be developed in order to identify cellular factors that interact with these chemical compounds.
Therefore, considering that no causal therapy is available today to treat mitochondrial diseases, our project appears to be based on a promising screening method for the discovery of therapies for some of these diseases. Finally, the aim of this project, in addition to find molecules rescuing mitochondrial deficiencies, is to identify the targets of the drugs in order to unravel cellular and mitochondrial pathways involved in the considered mitochondrial pathologies.
Mager, W.H. and Winderickx, J. (2005) Yeast as a model for medical and medicinal research. Trends Pharmacol Sci, 26, 265-273.
Rak, M., Tetaud, E., Duvezin-Caubet, S., Ezkurdia, N., Bietenhader, M., Rytka, J. and di Rago, J.P. (2007) A yeast model of the neurogenic ataxia retinitis pigmentosa (NARP) T8993G mutation in the mitochondrial ATP synthase-6 gene. J Biol Chem, 282, 34039-34047.
Schwimmer, C., Rak, M., Lefebvre-Legendre, L., Duvezin-Caubet, S., Plane, G. and di Rago, J.P. (2006) Yeast models of human mitochondrial diseases: from molecular mechanisms to drug screening. Biotechnol J, 1, 270-281.