From the beginning to the end: the story of the mitochondrial disease associated with the ADCK2 gene, going through CoQ10 supplementation and caloric restriction

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Biotecnología, Biomedicina y Ciencias de la SaludClave Programa: DBICódigo Línea: 110Mitochondria are enclosed membrane organelles that burn nutrient molecules to produce adenosine triphosphate (ATP) by oxidative phosphorylation (OXPHOS) and are present in cells of animals, plants and fungi. Mitochondria also carry out other important processes such as urea cycle, fatty acids ß-oxidation and the Krebs cycle. A crucial element in mitochondrial function is Coenzyme Q10 (CoQ10) is a unique lipid endogenously produced, a crucial element in the mitochondrial electron transport chain (ETC) and antioxidant found in all cell membranes. The main function of CoQ is to send electrons that receive from complex I and complex II to complex III at the ETC resulting in ATP production. But, CoQ10 could be also reduced by different hydrogenases, showing the high number of pathways where CoQ10 participates. CoQ is composed of a benzoquinone ring and a polyisoprenoid chain which inserts CoQ into the phospholipid bilayer. CoQ is produced at mitochondria by a nuclear-encoded CoQ proteins cluster. The biosynthetic pathway is not clear yet and it is carried by at least 13 proteins. The ADCK2 gene has been recently associated with CoQ pool regulation and fatty acids metabolism. ADCK2 haploinsufficiency in humans is related to mitochondrial myopathy, muscle wasting, reduction of CoQ levels and impairment of fatty acids ß-oxidation. We developed a heterozygous Adck2 knockout mouse that reproduces the mutation. The four chapters of the thesis are focused on this mutant mouse model to better understand the mitochondrial syndrome associated with the ADCK2 gene. Firstly, we present a detailed study of the embryonic development of mutant mice to elucidate if damages could start during this stage. We analysed the transcriptomic profile during early and late development, finding tissue-specific deregulation during late embryonic development. Heterozygous Adck2 knockout mouse embryos presented an abnormal somatogenesis analysed with two different Myogenic Regulatory Factors. We examined a prenatal CoQ10 administration obtaining that mutant embryos under CoQ10 supplementation showed a reversion to wild type profile in the transcriptomic profile and skeletal muscle formation. Secondly, we decided to focus on postnatal myogenesis based on the results from the first chapter. For that purpose, adult muscle stem cells were isolated from wild type and mutant mice under standard conditions and under CoQ10 administration and differentiated. Mutant cells presented a defect in differentiation that leads to smaller myotubes and delayed differentiation that was associated with a reduction in oxygen consumption and fatty acids ß-oxidation. Mutant cells isolated from mutant mice supplemented with CoQ10 showed an improvement in differentiation and also on mitochondrial respiration. An in vivo analysis of satellite cells was performed to support our in vitro data inducing muscle damage and examining skeletal muscle regeneration. Mutant mice presented an impaired skeletal muscle regeneration and were more susceptible to muscle damage, CoQ10 administration improved skeletal muscle regeneration. We performed the experiments in cells from young and old mice, the differences were bigger in cells from old mice suggesting that ageing could act as a modulator in the phenotype of mutant mice. As summary of this chapter, mutant mice suffer a defect in postnatal myogenesis, particularly during myogenic differentiation. We decided to study the decline associated with the ageing process in our mutant mice in the third chapter. Old mutant mice presented a decrease in myofiber size and showed different myofiber composition while old mutant mice longitudinally supplemented with CoQ10 exhibited a myofiber size and composition more similar to wild type mice. Skeletal muscle performance was assessed through ageing, mutant mice presented a more severe decline in physical capacity compared to wild type mice. CoQ10 administration ameliorated skeletal muscle function decline in mutant mice. We developed a method to examine respiration in isolated mitochondria from skeletal muscle. Mitochondria from skeletal muscle of mutant mice presented a reduction in oxygen consumption while isolated mitochondria from mutant mice on CoQ10 administration showed an increase in respiration with both glucose and fatty acids substrates. Heterozygous Adck2 knockout mice suffer a more severe decline through ageing and CoQ10 administration could improve the phenotype of mutant mice. Finally, we studied the metabolic modulation of the phenotype of mutant mice by caloric restriction (CR). CR restored glucose and insulin homeostasis in mutant mice to wild type levels. CR modulated skeletal muscle metabolic profile, resulting in a more oxidative myofiber composition and increased oxygen consumption using glucose and fatty acids substrates in isolated mitochondria. To further explore the effect of CR on muscle stem cells, satellite cells were cultured with serum obtained from mice on ab libitum and CR. Mutant cells cultivated with CR serum showed an increase in differentiation and oxygen consumption. CR intervention improved age-associated decline and defects associated with Adck2 haploinsufficiency. The four chapters of the project are focus on the study of Adck2 mutant mice but under different conditions and methods including CoQ10 administration or under CR condition, resulting in a completed phenotype analysis of the model studied.Universidad Pablo de Olavide de Sevilla. Departamento de Fisiología, Anatomía y Biología Celula