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Mitochondrial Copper Overload in a Rat Model for Wilson Disease Is Paralleled by Upregulated COX17 and Can Be Restored Using the Bacterial Peptide Methanobactin

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Mitochondrial Copper Overload in a Rat Model for Wilson Disease Is Paralleled by Upregulated COX17 and Can Be Restored Using the Bacterial Peptide Methanobactin Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Lehrstuhl Experimentelle Genetik Mitochondrial copper overload in a rat model for Wilson disease is paralleled by upregulated COX17 and can be restored using the bacterial peptide methanobactin Christin Leitzinger Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzende: Univ.-Prof. Dr. Dr. Hannelore Daniel Prüfer der Dissertation: 1. apl. Prof. Dr. Dr. Jerzy Adamski 2. Univ.-Prof. Dr. Martin Klingenspor Die Dissertation wurde am 26.10.2016 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 03.02.2017 angenommen. Table of contents List of figures ............................................................................................................. XI List of supplementary figures ................................................................................. XIII List of tables............................................................................................................ XIV List of supplementary tables .................................................................................. XV Summary ..................................................................................................................... 1 Zusammenfassung ..................................................................................................... 3 1 Introduction ................................................................................................... 7 1.1 Wilson disease: a copper-storage disease with progressive hepatolenticular degeneration ......................................................................... 7 1.2 Mitochondria – essential copper-dependent cell organelles that are affected in Wilson disease .............................................................................. 8 1.3 Treatment of Wilson disease using copper chelators .................................... 12 1.4 Methanobactin – a peptide with a very high affinity for copper ...................... 13 1.5 The LPP rat as an animal model for studying mitochondrial alterations in Wilson disease ............................................................................................. 14 1.6 Aim of this study ........................................................................................... 15 2 Material and Methods ................................................................................. 17 2.1 Material ......................................................................................................... 17 2.1.1 Cell line ......................................................................................................... 17 2.1.2 Bacteria ........................................................................................................ 17 2.1.3 Plasmid and enzymes ................................................................................... 17 2.1.4 Oligonucleotides ........................................................................................... 17 2.1.5 Media ............................................................................................................ 18 2.1.6 Chemicals ..................................................................................................... 19 2.1.7 Solutions and buffers .................................................................................... 22 2.1.8 Antibodies ..................................................................................................... 23 2.1.9 Kits ............................................................................................................... 24 2.1.10 Equipment and consumables ........................................................................ 24 2.1.11 Software ....................................................................................................... 28 2.2 Methods ........................................................................................................ 29 2.2.1 Wilson disease patient liver samples ............................................................ 29 2.2.2 Animal experiments ...................................................................................... 29 2.2.2.1 Animals ......................................................................................................... 29 2.2.2.2 Genotyping of LPP rats ................................................................................. 30 2.2.2.3 Examination of serum and plasma parameters and definition of animal disease states ............................................................................................... 30 2.2.2.4 Animal treatments ......................................................................................... 31 2.2.3 Cell culture experiments ............................................................................... 31 2.2.3.1 Cell cultivation .............................................................................................. 31 2.2.3.2 Copper-histidine solution .............................................................................. 34 2.2.4 Isolation and analysis of mitochondria from liver tissue and HepG2 cells ...... 35 III Table of contents 2.2.4.1 Subfractionation of human and rat liver tissue and isolation of liver mitochondria ................................................................................................. 35 2.2.4.2 Isolation of mitochondria from HepG2 cells ................................................... 35 2.2.4.3 Measurement of the mitochondrial membrane fluidity ................................... 35 2.2.4.4 Simultaneous measurement of MMP-loss and MPT-induction and curve sketching ...................................................................................................... 36 2.2.4.5 ATP synthesis ............................................................................................... 37 2.2.5 Protein analyses ........................................................................................... 37 2.2.5.1 Determination of mitochondrial and cellular protein concentration ................ 37 2.2.5.2 Immunoblot analyses .................................................................................... 37 2.2.5.3 Proteomic analyses of Atp7b rat liver mitochondria ....................................... 38 2.2.6 Molecular biological methods ........................................................................ 39 2.2.6.1 Knockdown of COX17 in HepG2 cells using siRNA ...................................... 39 2.2.6.2 Overexpression of COX17 in HepG2 cells .................................................... 39 2.2.6.3 Reverse transfection of HepG2 cells with COX17 ......................................... 42 2.2.6.4 Quantitative realtime polymerase chain reaction (qRT-PCR) ........................ 42 2.2.7 Chromatography of mitochondrial proteins.................................................... 42 2.2.7.1 Gel filtration .................................................................................................. 43 2.2.7.2 Anion exchange chromatography ................................................................. 43 2.2.8 Miscellaneous ............................................................................................... 43 2.2.8.1 Immunoprecipitation ..................................................................................... 43 2.2.8.2 Metal content determination .......................................................................... 44 2.2.8.3 Electron microscopy ..................................................................................... 44 2.2.8.4 Immunohistochemistry .................................................................................. 45 2.2.9 Statistics ....................................................................................................... 45 3 Results ........................................................................................................ 47 3.1 The LPP Atp7b-/- rat – an animal model for liver failure in Wilson disease ..... 47 3.1.1 Progressing disease states in the Wilson disease LPP Atp7b-/- rat model ..... 47 3.1.2 Copper increasingly accumulates in liver homogenate and mitochondria from Wilson disease patients and Atp7b-/- rats with disease progression ...... 48 3.1.3 Structural alterations are comparable among mitochondria from Wilson disease patients and diseased Atp7b-/- rats ................................................... 49 3.1.4 Increasing mitochondrial copper load is paralleled by structural alterations of liver mitochondria from Atp7b-/- rats ......................................... 50 3.1.5 Mitochondrial membrane fluidity and swelling is reduced in Atp7b-/- rats with disease progression .............................................................................. 53 3.1.6 Mitochondrial function is increasingly harmed in Atp7b-/- rats with disease progression ...................................................................................... 56 3.2 Methanobactin – a highly efficient agent to decopper liver mitochondria in a Wilson disease rat model ....................................................................... 58 3.2.1 Methanobactin efficiently decoppers HepG2 cells without
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