The pathophysiology of respiratory chain dysfunction

Abstract: Mutations of mitochondrial DNA cause a variety of clinical syndromes. It is unclear whether impaired oxidative phosphorylation on its own is the main cause of pathology or whether other factors such as secondary metabolic alterations, enhanced formation of reactive oxygen species (ROS) and induction of apoptosis also may contribute to the clinical phenotype. This thesis focuses on several topics relevant to the pathophysiology of mitochondrial disease, i.e. a mouse model for mitochondrial diabetes, analyses of ROS formation and cell death in mouse strains with tissuespecific respiratory chain (RC) deficiency and regulation of uncoupling protein (UCP) activity and RC function by superoxide. We studied the pathogenesis of mitochondrial diabetes by tissue-specific (Cre-loxP mediated) inactivation of the gene encoding the mitochondrial transcription factor A (Tfam) in insulin producing pancreatic Pcells. Inactivation of Tfam resulted in mtDNA depletion, severe RC deficiency and abnormally appearing mitochondria in mutant islets. The beta-cell specific Tfam knockout (KO) mice were diabetic with lowered pancreatic insulin release. Studies of isolated islets showed the lowered insulin release was caused by altered Ca2+ signaling. The RC deficient pancreatic beta-cells eventually died as concluded from histological studies. This study thus demonstrates 2 phases in the pathogenesis of mitochondrial diabetes. Impaired glucose stimulated insulin release is observed at an early stage and is later followed by P-cell loss. This study provides the first genetic in vivo evidence for a critical role of the RC in glucose-stimulated insulin release (Paper I). We next examined whether RC deficiency caused ROS formation and cell death. We found increased cell death in homozygous germline Tfam KO embryos and in mice with tissuespecific inactivation of Tfam in cardiomyocytes and pyramidal forebrain neurons. Tfam KO embryos showed massive induction of apoptosis at embryonic day 9.5, Tfam KO cardiomyocytes showed a moderate increase in apoptosis and Tfam KO in forebrain neurons caused massive cell death. The majority of the neurons affected by the knockout died by necrosis as reflected by absence of apoptosis markers and presence of an inflammatory reaction in brain sections. We found only a moderate induction of antioxidant defenses in the Tfam KO cardiomyocytes and almost no induction in Tfam KO neurons. The activities of several iron-sulphurcluster enzymes sensitive to ROS damage were essentially normal in mtDNA-depleted heart and brain cortex. This result suggests that mitochondrial ROS production is normal or only moderately increased in tissues with RC deficiency and that any increase in ROS formation is fully compensated for by induction of antioxidant defenses (Paper II and III). Superoxide has been reported to activate mitochondrial UCPs thus providing a feedback control to lower generation of superoxide by uncoupling respiration. The superoxide-effect is expected to significantly affect energy metabolism due to widespread tissue distribution of UCPs. We generated transgenic mice harboring P1 artificial chromosomes with the human SOD2 gene to investigate whether superoxide regulates UCP activity and energy expenditure in vivo. The human SOD2 protein was ubiquitously expressed and SOD2 enzyme activities showed an overall increase in transgenic mice. There was a linear correlation between SOD2 enzyme activity and mitochondrial oxidative capacity. Mitochondria with increased SOD2 activity were also resistant to induction of mitochondrial permeability. However, despite these obvious effects on mitochondria, SOD2 overexpressing mice exhibited normal UCP activities and adapted normally to cold. They also displayed normal metabolic rates and no change in mitochondrial mass or mitochondrial gene expression. These results suggest that superoxide does not regulate UCP activity and energy expenditure in vivo (Paper IV).