Rab35-regulated lipid turnover by myotubularins represses mTORC1 activity and controls myelin growth Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of the Freie Universität Berlin by Linda Sawade from Berlin, Germany 2019 Period of doctorate studies: November 2013 to August 2019 Supervisor: Prof. Dr. Volker Haucke Institute: Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin 1st Reviewer: Prof. Dr. Volker Haucke 2nd Reviewer: Prof. Dr. Stephan Sigrist Date of defense: 29.10.2019 Affidavit I declare that my PhD thesis at hand has been written independently and with no other sources and aids than quoted. Berlin, August the 13th, 2019 Acknowledgements First of all, I would like to express my deep gratitude to my supervisor, Prof. Dr. Volker Haucke, for his guidance, support and constant motivation throughout my PhD studies, which were crucial for the success of this project. I owe special thanks to our collaboration partners Prof. Dr. Alessandra Bolino (INSPE, Milan, Italy), and her team Federica Grandi, Marianna Mignanelli, and Roberta Di Guardo who performed great work with the Schwann cell-specific Rab35 knockout mice and provided us the MTMR2 shRNA. I am also very grateful to Prof. Dr. Arnaud Echard (Pasteur-Institut, Paris, France) who provided us with the GFP-Rab35endo KI HeLa cells, and especially the Rab35Fl/Fl mice for conditional gene knockout. Many thanks also to his team, Dr. Kerstin Klinkert and Dr. Francina Langa Vives, as well as to Prof. Dr. Stephen Shaw (formerly: NIH, Bethesda, USA) and Dr. Genaro Patiño-López (formerly: NIH; now: Hospital Infantil de México, Ciudad de México, México), who generated these animals. Furthermore, I would like to thank Prof. Dr. Cesare Montecucco and his team (University of Padova, Italy), especially Dr. Samuele Negro, for the training in preparation of sciatic nerves and Schwann cell cultures in vitro. Special thanks also to Dr. Eberhard Krause, the former head of the mass spectrometry facility at the FMP-Berlin (Germany), and his team, especially Heike Stephanowitz, for the BioID-mass spectrometry. Many thanks to Dr. Dmytro Puchkov, the head of the electron microscopy facility, and his team for the ultrastructural analysis of the conditional Rab35 knockout neurons in culture. I would also like to thank Dr. Burkhard Wiesner, Jenny Eichhorst and Dr. Martin Lehmann from the Imaging facility (FMP Berlin), who provided us with terrific microscopes, technical knowledge and support. I am also very grateful for financial funding by the Grant SFB 958 (Deutsche Forschungsgemeinschaft). I would like to thank the secretary office, Heidemarie Petschick and Alexandra Chylla, who were always helpful and supportive, as well as to the FMP-IT, especially Alexander Heyne. Special thanks to Prof. Dr. Tanja Maritzen for the management and supervision of animal experimental administration, and to the animal house facility, Dr. Natali Wisbrun and her team Eva Lojek, Sina Scholz and Jeanette Unasch. Furthermore, I would like to thank the technicians of the Haucke lab: Delia Löwe, especially for the management of the common cell and neuron culture. Uwe Fink for his skills and advices regarding protein purifications. Claudia Schmidt for providing help whenever required. Silke Zillmann for the ordering management and genotypings, as well as Sophia Griese. Many thanks to Sabine Hahn, for the support with astrocytic culture preparations, and 3 especially for the time when I joined the lab as an unexperienced undergraduate student. And special thanks to Maria Mühlbauer for the technical assistance and her great support in this project. I owe special thanks to Dr. Natalie Kaempf and Dr. Domenico Azarnia Tehran for fruitful discussions, advices and especially their kind support in many ways. I thank Dr. Natalia Kononenko, who supervised me as an undergraduate and during my first year as a PhD, and Dr. Tania Lopez Hernandez and Dennis Vollweiter for introducing me into the wonderful world of astrocytes and “R”, respectively. Thanks to Dr. Wen-Ting Lo for his advices regarding affinity purifications. Thanks also to our former lab member Katarina Ketel, who worked on MTM1, and thus, already obtained the basic constructs which made the work with this protein family a bit easier. And thanks to all other past and present members of the Haucke lab I’ve got to know over the years and who contributed to a joyful atmosphere in the lab: Jelena Bacetic, Marietta Bergmann, Svenja Bolz, Caroline Bruns, Gabrielle Capin, Gala Claßen, Katrin Diesenberg, Michael Ebner, Marielle Eichhorn-Grünig, Fabian Feutlinske, Paula Samsó Ferre, Niclas Gimber, Marine Gil, Hannes Gonschoir, Claudia Gras, Manuel Hessenberger, Lennart Hoffmann, Burkhard Jakob, Wonyul Jang, Maria Jäpel, Christina Kath, Philipp Koch, Gaga Kochlamazashvili, Seong Joo Koo, Michael Krauß, Marijn Kuijpers, André Lampe, Gregor Lichtner, Guan-Ting Liu, Fabian Lukas, Albert Mackintosh, Marta Maglione, Charles Malek, Andrea Marat, Kristine Oevel, Christoph Ott, Filiz Sila Rizalar, Giulia Russo, Hannah Schachtner, Christopher Schmied, Jan Schmoranzer, Irene Schütz, Kyungyuen Song, Tolga Soykan, Wiebke Stahlschmidt, Anela Vukoja, Alexander Wallroth, Alexander Walter, Haibin Wang, Anna Wawrzyniak and Mirjana Weimershaus. Finally, and most important, I owe my deepest gratitude to my family for all their support throughout my life, and to Thomas Lenz who especially backed me up over the last years. 4 I Table of content I Table of content……………………………………………………………………………….5 II Summary………………………………………………………………………......................9 III Zusammenfassung…………………………………………………………………………11 1. Introduction………………………………………………………………………………. 13 1.1 Myelination of the vertebrate nervous system……………………………………..13 1.2 Myelinating glial cells……………………………………………………………..13 1.3 The function of myelination…………………………………….............................16 1.4 Myelin structure………………………………………...........................................17 1.5 Cell signaling pathways in myelination……………………………………….......20 1.5.1 mTORC1 is a central regulator of myelination…………………………..23 1.5.1.1 Canonical pathways that regulate mTORC1 activity…………..23 1.5.1.2 Regulation of mTORC1 activity by PI(3)-phosphates………...24 1.5.1.3 mTORC1 controls the metabolic switch of cells……………….26 1.5.1.4 Altered mTORC1 activity leads to impaired myelin homeostasis………………………………………………….. 27 1.6 Myelin in pathological conditions…………………………………………............29 1.6.1 Myotubularins are crucially involved in myelination……………………31 1.6.1.1 The family of myotubularin-related lipid phosphatases…..…...31 1.6.1.2 CMT-associated MTMRs……………………………………...34 1.7 The small GTPase Rab35 is implicated in myelination…………………………....37 1.8 Aims……………………………………………………………………………… 41 2. Material and Methods………………………………………………………………….…...42 2.1 Material……………………………………………………………………….…...42 2.1.1 Chemicals and consumables…………………………..………………... 42 2.1.2 Solutions, media and buffers……………………………….…………....42 2.1.3 Enzymes………………………………………………………………....47 2.1.4 Kits……………………………………………………………………....48 2.1.5 Standards for gel electrophoresis………………………………………...48 2.1.6 DNA oligonucleotides……………………………………………….......48 2.1.7 RNA oligonucleotides…………………………………………………...49 2.1.7.1 siRNAs………………………………………...........................49 2.1.7.2 shRNAs………………………………………..........................50 2.1.8 DNA-plasmids and expression vectors……………………………….....50 2.1.9 Antibodies and probes………………………………………...................52 2.1.9.1 Primary antibodies………………………………………......... 52 2.1.9.2 Secondary antibodies………………………………………..... 53 2.1.9.3 Probes……………………………………….............................54 2.1.10 Inhibitors……………………………………….....................................54 2.1.11 Mammalian cell lines………………………………………..................55 2.1.12 Bacteria strains………………………………………............................55 2.1.13 Mouse strains………………………………………..............................55 2.1.14 Software………………………………………......................................56 5 2.1.15 Suppliers……………………………………….................................... 57 2.2 Methods………………………………………………………….......................... 58 2.2.1 Molecular Biology Methods………………………………………......... 58 2.2.1.1 Preparative and analytical Polymerase chain reaction (PCR)….58 2.2.1.2 Genotyping of transgenic mice ………………………………..60 2.2.1.3 Agarose gel electrophoresis……………………………………62 2.2.1.4 Purification of DNA from agarose gels……………………….. 62 2.2.1.5 DNA-restriction digest.……………………………………..... 62 2.2.1.6 Ligation……………………………………………………......63 2.2.1.7 Transformation of chemically competent E. coli……………....63 2.2.1.8 Preparation of bacterial glycerol stocks……………………….64 2.2.1.9 Cultivation of transformed bacteria…………………………....64 2.2.1.10 Isolation of plasmid DNA…………………………………….64 2.2.1.11 Spectrophotometric determination of DNA concentration…...65 2.2.1.12 Sequencing of DNA………………………………………….65 2.2.1.13 Isolation of genomic DNA…………………………………...65 2.2.2 Cell biology methods……………………………………………………66 2.2.2.1 Cultivation and passaging of mammalian cell lines………….. 66 2.2.2.2 Transfection of mammalian cell lines using JetPrime…………67 2.2.2.3 Transfection of mammalian cell lines using calcium phosphate.68 2.2.2.4 Production of lentiviral particles……………………………… 68 2.2.2.5 Preparation of primary cell cultures from mice………………. 70 2.2.2.5.1 Primary neuronal cultures……………………………71 2.2.2.5.2 Primary astrocytic cultures…………………………..72 2.2.2.5.3 Primary oligodendrocytic (precursor) cell cultures…..73 2.2.2.5.4 Primary Schwann