Search

Aims. Mitochondrial complex I assembly is a multi-step process which necessitates the involvement of a variety of assembly factors and chaperones to ensure the final active enzyme is correctly assembled. The role of the assembly factor ECSIT was studied across various murine tissues to determine its role in this process and how this varied between tissues of varying energetic demands. We hypothesised that many of the known functions of ECSIT were unhindered by the introduction of an ENU induced mutation, whilst it’s role in complex I assembly was affected on a tissue specific basis. Methods and Results. Here we describe a mutation in the mitochondrial complex I assembly factor ECSIT which reveals tissue specific requirements for ECSIT in complex I assembly. Mitochondrial complex I assembly is a multi-step process dependent on assembly factors that organise and arrange the individual subunits, allowing for their incorporation into the complete enzyme complex. We have identified an ENU induced mutation in ECSIT (N209I) that exhibits a profound effect on complex I component expression and assembly in heart tissue, resulting in hypertrophic cardiomyopathy in the absence of other phenotypes. The dysfunction of complex I appears to be cardiac specific, leading to a loss of mitochondrial output as measured by Seahorse extracellular flux and various biochemical assays in heart tissue, whilst mitochondria from other tissues were unaffected. Conclusions. These data suggest that the mechanisms underlying complex I assembly and activity may have tissue specific elements tailored to the specific demands of cells and tissues. Our data suggest that tissues with high energy demands, such as the heart, may utilise assembly factors in different ways to low energy tissues in order to improve mitochondrial output. This data have implications for the diagnosis and treatment of various disorders of mitochondrial function as well as cardiac hypertrophy with no identifiable underlying gene

Mitochondrial disorders are a group of highly invalidating human conditions due to defective oxidative phosphorylation, for which no effective treatment is nowadays available. In order to develop effective therapies for these disorders, I focused on an experimental approach based on the manipulation of mitochondrial morphology. Opa1 is a GTPase of the inner mitochondrial membrane involved in both mitochondrial fusion and cristae shaping. The role of OPA1 in mitodynamics has also a documented impact on controlling the assembly of the respiratory supercomplexes and respiratory proficiency. Based on these considerations, I tested whether Opa1 overexpression could mitigate the effects of a severe mitochondrial respiratory chain deficit in vivo. In this thesis, the effects of mild transgenic overexpression of Opa1 on two mouse models of defective mitochondrial bioenergetics, a constitutive knockout for Ndufs4 (Ndufs4-/-), encoding a structural component of complex I, and a muscle-specific conditional knockout for Cox15, (Cox15sm/sm), encoding a heme-a biosynthetic enzyme involved in complex IV hemylation and assembly are shown. Crossing of both models with an Opa1 transgenic mouse line (Opa1tg) was associated with clinical and biochemical improvement, but whilst the effect was limited in Ndufs4-/-::Opa1tg mice, the Cox15sm/sm::Opa1tg mice showed relevant amelioration of motor performance, remarkable prolongation of survival, and significant correction of mitochondrial respiration. This effect was associated with the increased amount of active cIV holocomplex and cIV-containing supercomplexes.

Mutations in human MPV17 cause a hepatocerebral form of mitochondrial DNA depletion syndrome (MDS) hallmarked by early-onset liver failure, leading to premature death. Liver transplantation and frequent feeding using slow-release carbohydrates are the only available therapies, although surviving patients eventually develop slowly progressive peripheral and central neuropathy. The physiological role of Mpv17, including its functional link to mitochondrial DNA (mtDNA) maintenance, is still unclear. We show here that Mpv17 is part of a high molecular weight complex of unknown composition, which is essential for mtDNA maintenance in critical tissues, i.e. liver, of a Mpv17 knockout mouse model. On a standard diet, Mpv17 -/- mouse shows hardly any symptom of liver dysfunction, but a ketogenic diet (KD) leads these animals to liver cirrhosis and failure. However, when expression of human MPV17 is carried out by adeno-associated virus (AAV)-mediated gene replacement, the Mpv17 knockout mice are able to reconstitute the Mpv17-containing supramolecular complex, restore liver mtDNA copy number and oxidative phosphorylation (OXPHOS) proficiency, and prevent liver failure induced by the KD. These results open new therapeutic perspectives for the treatment of MPV17-related liver-specific MDS. © The American Society of Gene & Cell Therapy.