Résumé :
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Mitochondrial disorders belong, as a group, to the most common types of genetic disorders. Many of the patients carry either a mutation in a large fraction of theirmitochondrial DNA (mtDNA) molecules or show a depletion of mtDNA. In both cases, the molecular basis of the disease appears to be a deficiency of mitochondrialwild-type gene levels. This deficiency leads to a shortage of the enzyme complexes of the oxidative phosphorylation system, resulting in a decrease of aerobic ATPsynthesis. The decrease in ATP synthesis will hamper the cell's ability to function properly and may, ultimately, cause cell death.In this project, we intend to define theinfluence of mtDNA levels on mitochondrial energy metabolism in cultured myoblasts and neuronal cells. For this study, we will use the human LHCN-M2 myogenic cellline and the human ReNell® VM neuronal stem cell line to create cultures with adjustable mtDNA copy numbers. In order to be able to vary mtDNA copy numbers, stablecell lines will be generated with doxycycline-inducible, ectopic expression of single hairpin RNA sequences that target the mtDNA packaging protein gene TFAM. As themtDNA copy number is directly proportional to TFAM protein levels, the transfected cells will show a doxycycline dose-dependent depletion of mtDNA. We propose touse these cell lines to identify the limiting step(s) of the pathway from mtDNA to cellular respiration and mitochondrial ATP production by determining the steady-statecorrelations between: (1) mtDNA levels, (2) mitochondrial mRNA levels, (3) mitochondrially expressed protein levels, (4) assembled oxidative phosphorylation enzymecomplex levels, (5) respiratory chain enzyme activities, (6) respiratory rate and (7) ATP synthesis. In addition, we propose to study the correlation between mtDNA copynumber, cell viability and the cell's ability to differentiate. The creation of the cell models will provide the first stable, diploid cell lines with adjustable copy numbers ofmtDNA. Our study of these cell lines will offer a better understanding of the pathophysiology of mtDNA disorders. The new insights gained from these studies may beused to devise pharmacological interventions that may be tested using the cell culture models. As such, the cell culture models will provide an important resource forfurther studies aimed at treatment of mtDNA disorders.
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