The Role of Pycr1 in the Pathomechanism of Autosomal Recessive Cutis Laxa vorgelegt von Saniye Sprenger, geborene Yumlu aus Fritzlar von der Fakultät III – Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Ingenieurwissenschaften — Dr.-Ing. — genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Leif-Alexander Garbe, TU-Berlin Gutachter: Prof. Dr. Roland Lauster, TU-Berlin Prof. Dr. Uwe Kornak, Charité, MPI-MG Prof. Dr. Jens Kurreck, TU-Berlin Tag der wissenschaftlichen Aussprache: 18. Juli 2014 Berlin 2014 D 83 Die vorliegende Arbeit wurde in der Zeit von Januar 2009 bis Juni 2014 am Max-Planck- Institut für Molekulare Genetik sowie am Institut für Medizinische und Humangenetik der Charité Universitätsmedizin unter der Leitung von Prof. Dr. Uwe Kornak und Prof. Dr. Stefan Mundlos angefertigt. Hiermit erkläre ich an Eides Statt, dass die vorliegende Dissertation in allen Teilen von mir selbständig angefertigt wurde und die benutzen Hilfsmittel und Literaturquellen voll- ständig angegeben worden sind. Weiter erkläre ich, dass ich nicht schon anderweitig ein- mal die Promotionsabsicht angemeldet oder ein Promotionseröffnungsverfahren beantragt habe. Berlin, den 10.06.2014 ...................................... Saniye Sprenger Contents 1 Introduction 8 1.1 Definition of cutis laxa ........................... 8 1.1.1 Acquired cutis laxa (ACL) ..................... 8 1.1.2 Inherited cutis laxa ......................... 9 1.2 PYCR1 ................................... 15 1.3 The proline cycle .............................. 16 1.4 Mitochondria ................................ 17 1.4.1 Mitochondrial energy production ................. 18 1.4.2 Mitochondrial reactive oxygen species ............... 19 1.4.3 Mitochondria and apoptosis .................... 20 1.4.4 Mitochondrial morphology ..................... 21 1.4.5 Mitochondria and aging ...................... 24 1.5 Aims of the study ............................. 26 2 Abstract 27 3 Zusammenfassung 29 4 Material 31 4.1 Instruments ................................ 31 4.2 Chemicals ................................. 32 4.3 Buffers ................................... 32 4.4 Enzymes .................................. 32 4.5 Kits ..................................... 33 4.6 Plasmids .................................. 33 4.7 Antibodies ................................. 34 4.8 Bacteria .................................. 34 4.9 Primer ................................... 35 4.10 Software .................................. 39 4.11 Internet resources ............................. 39 5 Methods 40 5.1 Molecular Biological Methods ....................... 40 5.1.1 DNA Isolation ........................... 40 5.1.2 RNA isolation ........................... 41 4 5.1.3 Generation of cDNA ........................ 42 5.1.4 Polymerase chain reaction (PCR) ................. 42 5.1.5 Sanger sequencing ......................... 46 5.1.6 Cloning ............................... 46 5.1.7 Southern blot with radioactively labeled probes .......... 48 5.2 Microbiological Methods .......................... 49 5.2.1 Preparation of electro-competent E. coli ............. 49 5.2.2 Electroporation of E. coli ..................... 50 5.2.3 Bacterial recombination ...................... 50 5.3 General Cell Culture Methods ....................... 50 5.3.1 Thawing of cells .......................... 50 5.3.2 Splitting of cells .......................... 50 5.3.3 Cryopreservation of cells ...................... 51 5.3.4 Cell number determination ..................... 51 5.3.5 Cell transfection .......................... 51 5.3.6 Immunocytochemistry (ICC) .................... 51 5.4 ES Cell Culture Methods .......................... 52 5.4.1 Preparation of murine embryonic fibroblasts (MEF) ........ 52 5.4.2 Mitotically inactivation of MEFs .................. 52 5.4.3 Cultivation of embryonic stem cells ................ 53 5.4.4 Transfection of ES cells ...................... 53 5.4.5 Selection of transfected ES cells .................. 54 5.4.6 Isolation of ES cell colonies .................... 54 5.4.7 Expansion of ES cell colonies ................... 54 5.4.8 Cryopreservation of ES cells in 96-well plates ........... 54 5.4.9 Thawing and expanding ES cells .................. 55 5.4.10 Screening of targeted ES cells ................... 55 5.4.11 Tetraploid aggregation ....................... 55 5.5 Biochemical Methods ........................... 55 5.5.1 Protein extraction from cultured cells ............... 55 5.5.2 Protein extraction from tissue ................... 56 5.5.3 Determination of protein concentration .............. 56 5.5.4 SDS PAGE ............................. 56 5.5.5 Western blot (WB) ........................ 57 5.5.6 Determination of ATP concentration ............... 57 5.5.7 Determination of lactate concentration .............. 58 5.5.8 PicoGreen dsDNA Quantitation .................. 58 5 5.5.9 Sample preparation for OXPHOS complex activity determination 58 5.6 Histological Methods ............................ 59 5.6.1 Tissue preparation for electron microscopy ............ 59 5.6.2 Paraffin embedding and sectioning ................ 59 5.6.3 Cryo embedding and sectioning .................. 59 5.6.4 Methylmethacrylat (MMA) embedding and sectioning ...... 59 5.6.5 Immunhistochemistry (IHC) on paraffin embedded tissue ..... 60 5.6.6 Immunhistochemistry on cryo embedded tissue .......... 60 5.6.7 Histological stainings ........................ 61 5.7 Radiological Methods ........................... 62 5.7.1 MicroCT Analysis ......................... 62 6 Results 63 6.1 Expression analysis of Pycr1 in mice .................... 63 6.2 Human and murine PYCR proteins are highly conserved ......... 63 6.3 Generation of the Pycr1 conditional knock out mouse model ....... 66 6.3.1 Strategy for targeting Pycr1 .................... 66 6.3.2 Identification of targeted stem cells ................ 66 6.3.3 Generation of transgenic mice ................... 67 6.3.4 Pycr1 knock out has an efficiency of 100 % ........... 69 6.4 The Pycr1 mouse model reflects major aspects of the human disease . 71 6.4.1 Pycr1 −/− mice present a skin phenotype ............. 72 6.4.2 Pycr1 −/− mice present a bone phenotype. ............ 73 6.4.3 Pycr1 −/− mice present a muscular phenotype ........... 78 6.5 Pycr1 deficiency causes mitochondrial dysfunction ............ 86 6.5.1 Pycr1 deficiency causes metabolic changes ............ 86 6.6 Expression and biochemical comparison of PYCR paralogues in men and mice .................................... 90 6.6.1 Human and murine PYCR paralogues are similarly expressed in adult tissues ............................ 90 6.6.2 Human and murine PYCR proteins are differently post-translationally modified.............................. 91 6.7 The knock down of Pycr paralogues decreases stress resistance and in- creases apoptosis .............................. 92 7 Discussion 96 7.1 Generation of the Pycr1 knock out model ................. 96 6 7.2 Pycr1 and dermal abnormalities ...................... 96 7.3 Pycr1 and bone abnormalities ....................... 98 7.4 Pycr1 and muscular abnormalities ..................... 100 7.5 Pycr1 and mitochondrial alterations .................... 102 7.6 Pycr1, proline and the mitochondrial redox system ............ 106 7.7 Differences between human and murine PYCRs .............. 107 7.8 Examples for species differences in metabolic diseases .......... 109 8 References 111 9 Appendix 126 Acknowledgements ................................ 126 List of figures .................................. 129 List of tables ................................... 130 Abbreviation ................................... 131 List of publications ................................ 132 7 1 Introduction Aging is defined as a gradual change in an organism that leads to an increased risk of weakness, disease and death. As a process, it occurs at the cellular, organic and at the level of the total organism over the entire adult life span. In the course of aging, biological functions and the ability to adapt to stress decline. Overall effects of aging include: decline in memory and cognition; loss of elasticity of the skin; loss of bone mass and; loss of muscle strength [1]. In the course of a lifetime almost all organs undergo a constant remodeling, including formation and degradation of cells and extra cellular material. In healthy and young individuals this remodeling is in a homeostasis, whereas with age and aging-related disorders degradation overwhelms the new formation. Aging-related, or progeroid, disorders are a group of rare genetic syndromes characterized by premature aging. Several monogenic disorders are known to premature cause the hallmarks of aging. Most of the causative genes are encoding DNA repair factors or are affecting the nuclear stability and chromatin structure [2]. In addition, in several animal models of premature aging, mitochondrial dysfunction was reported [2]. Cutis laxa (CL) describes a group of disorders reflecting cardinal symptoms of aging. Understanding the pathomechanism of CL disorders could help towards a better understanding of the mechanisms of aging. 1.1 Definition of cutis laxa Cutis laxa, from the Latin meaning lax or loose skin, is an umbrella term describing a heterogeneous group of rare connective tissue disorders associated with abnormalities of elastic fibers, which are important components of the extra cellular matrix (ECM). The connective tissue provides structure and strength to the skin, muscles,