MMMolecularMolecular Effects of Idebenone Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Roman Hans Haefeli aus Wetzikon, ZH Winterthur, 2012 Molecular Effects of Idebenone 1 Table of Content Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Markus Rüegg PD Dr. Thomas Meier PD Dr. Dirk Fischer Basel, den 20. September 2011 Prof. Dr. Martin Spiess Dekan 2 Molecular Effects of Idebenone Table of Contents Table of Contents Table of Contents .................................................................................................................................................................................................. 3 1 Abstract ................................................................................................................................................................................................... 7 2 Introduction .......................................................................................................................................................................................... 9 2.1 Mitochondria-Related Diseases ............................................................................................................................................... 9 2.1.1 Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like Syndrome (MELAS) ................. 10 2.1.2 Leber’s Hereditary Optic Neuropathy (LHON) .......................................................................................................... 10 2.1.3 Duchenne Muscular Dystrophy ............................................................................................................................................ 11 2.2 Mitochondria and Energy-Generating Systems .............................................................................................................. 13 2.2.1 Glycolysis Produces ATP Independent of Mitochondria ......................................................................................... 14 2.2.2 Electron Transport Chain and Oxidative Phosphorylation ......................................................................................15 2.2.3 Other Relevant Electron Transport Systems ................................................................................................................. 18 2.2.4 Janus-faced Mitochondria: The Rise of Reactive Oxygen Species during Respiration ............................... 20 2.3 The Importance of the Redox Environment .................................................................................................................... 23 2.3.1 Oxidative Stress ........................................................................................................................................................................... 23 2.3.2 Redox Environment and ROS Signaling............................................................................................................................ 25 2.4 Idebenone ........................................................................................................................................................................................ 26 2.4.1 Quinones ........................................................................................................................................................................................ 26 2.4.2 Idebenone: An Overview.........................................................................................................................................................27 2.4.3 Idebenone: Preclinical Data of the Brain .......................................................................................................................... 28 2.4.4 Idebenone: Interaction with the Electron Transport Chain .................................................................................... 28 2.4.5 Idebenone: Preclinical Evidence for an Antioxidant Effect ..................................................................................... 29 2.4.6 Idebenone: Additional Findings ........................................................................................................................................... 29 2.4.7 Idebenone: Clinical Data ......................................................................................................................................................... 30 2.5 Perspectives .................................................................................................................................................................................... 30 3 Results ................................................................................................................................................................................................... 31 3.1 Confirmation of Duchenne Muscular Dystrophy Model Systems ........................................................................ 31 3.2 Influence of Idebenone on Mitochondrial Activity ...................................................................................................... 37 3.3 Idebenone Rescues ATP Levels under Conditions of Dysfunctional Complex I ........................................... 39 3.4 Investigating the Mode of Quinone-Dependent ATP Rescue ................................................................................. 43 3.5 Can Idebenone Ameliorate ATP Levels Via AMPK? ................................................................................................. 45 3.6 Quinone-Dependent ATP Rescue Is Dependent on NQO1 and Complex III ................................................ 46 3.7 The Influence of Cell Medium on ATP Rescue Experiments .................................................................................51 3.8 Idebenone Reduced ATP Levels in Cells Deficient of NQO1 ................................................................................. 52 3.9 Interactions of Idebenone with Additional Oxidoreductases .................................................................................. 54 3.10 Potential Benefits from ATP Rescue in Disorders with Deficient Complex I ................................................. 59 3.11 Toxicological Analyses of Idebenone and Related Quinones .................................................................................. 61 3.12 Idebenone Influences Akt-1 Status ...................................................................................................................................... 62 3.13 Idebenone Protects Cells from Oxidative Stress-Induced Cell Death ................................................................. 65 3.14 Alteration in Gene Expression as a Mode of Action of Idebenone?..................................................................... 70 3.15 Metabolic Effects of Idebenone in Vivo ............................................................................................................................. 74 3.16 Characterization of Idebenone Analogs ............................................................................................................................. 79 4 Discussion ............................................................................................................................................................................................ 86 4.1 Choice of Suitable Model Systems....................................................................................................................................... 86 4.2 Influence of Idebenone on Mitochondrial Parameters in Healthy Mitochondria ........................................... 88 4.3 Idebenone Is an Inhibitor of Complex I ............................................................................................................................ 88 4.4 Idebenone Restores ATP Levels under Conditions of Impaired Complex I .................................................... 91 4.5 ATP Restoration in Presence of Dysfunctional Complex I Is Carried Out by Cytosolic-Mitochondrial Electron Shuttling .......................................................................................................................................................................................... 92 4.6 Idebenone Is Reduced by Other Oxidoreductases than NQO1 as Well ............................................................ 98 4.7 Idebenone Inhibits Complex I in Vivo and Thereby Changes Metabolism....................................................... 98 4.8 Idebenone Is a Mild Pro-Oxidant ....................................................................................................................................... 101 4.9 Idebenone Analogs Exhibit a High Disparity in Biological Function ................................................................. 102 4.10 Idebenone Protects From Oxidative Stress-Induced Cell Death .......................................................................... 103 4.11 Implications of this Thesis on Mitochondrial Diseases: DMD, MELAS, and LHON ................................. 105 4.11.1 Duchenne Muscular Dystrophy ......................................................................................................................................... 105 4.11.2 Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Syndrome ..................................... 106 4.11.3 Leber’s Hereditary Optic