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Functional Characterization of Novel Rhot1 Variants, Which Are Associated with Parkinson’S Disease

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Functional Characterization of Novel Rhot1 Variants, Which Are Associated with Parkinson’S Disease FUNCTIONAL CHARACTERIZATION OF NOVEL RHOT1 VARIANTS, WHICH ARE ASSOCIATED WITH PARKINSON’S DISEASE DISSERTATION zur Erlangung des Grades eines DOKTORS DER NATURWISSENSCHAFTEN DER MATHEMATISCH-NATURWISSENSCHAFTLICHEN FAKULTÄT und DER MEDIZINISCHEN FAKULTÄT DER EBERHARD-KARLS-UNIVERSITÄT TÜBINGEN vorgelegt von DAJANA GROßMANN aus Wismar, Deutschland Mai 2016 II PhD-FSTC-2016-15 The Faculty of Sciences, Technology and Communication The Faculty of Science and Medicine and The Graduate Training Centre of Neuroscience DISSERTATION Defense held on 13/05/2016 in Luxembourg to obtain the degree of DOCTEUR DE L’UNIVERSITÉ DU LUXEMBOURG EN BIOLOGIE AND DOKTOR DER EBERHARD-KARLS-UNIVERISTÄT TÜBINGEN IN NATURWISSENSCHAFTEN by Dajana GROßMANN Born on 14 August 1985 in Wismar (Germany) FUNCTIONAL CHARACTERIZATION OF NOVEL RHOT1 VARIANTS, WHICH ARE ASSOCIATED WITH PARKINSON’S DISEASE. III IV Date of oral exam: 13th of May 2016 President of the University of Tübingen: Prof. Dr. Bernd Engler …………………………………… Chairmen of the Doctorate Board of the University of Tübingen: Prof. Dr. Bernd Wissinger …………………………………… Dekan der Math.-Nat. Fakultät: Prof. Dr. W. Rosenstiel …………………………………… Dekan der Medizinischen Fakultät: Prof. Dr. I. B. Autenrieth .................................................. President of the University of Luxembourg: Prof. Dr. Rainer Klump …………………………………… Supervisor from Luxembourg: Prof. Dr. Rejko Krüger …………………………………… Supervisor from Tübingen: Prof. Dr. Olaf Rieß …………………………………… Dissertation Defence Committee: Committee members: Dr. Alexander Skupin ……………………………………... Prof. Dr. Olaf Rieß ………………………………………………. Prof. Dr. Rejko Krüger …………………………………………... Prof. Dr. Enza Maria Valente …………………………………… Prof. Dr. Daniela Vogt-Weisenhorn ……………………………. Prof. Dr. Bernd Wissinger ………………………………………. V VI Affidavit / Declaration I hereby declare that I have produced the work entitled “Miro1 mutations cause mitochondrial dysfunction and are associated with Parkinson’s disease ”, submitted for the award of a doctorate, on my own (without external help), have used only the sources and aids indicated and have marked passages included from other works, whether verbatim or in content, as such. I swear upon oath that these statements are true and that I have not concealed anything. I am aware that making a false declaration under oath is punishable by a term of imprisonment of up to three years or by a fine. Luxembourg, the 10.06.2016 …………………………………… Signature VII Acknowledgements The work for this dissertation was carried out in the “Functional Neurogenomics group” at the Hertie Institute for Clinical Brain Research (HIH) at the University of Tübingen and in the “Clinical and Experimental Neuroscience group” at the Luxembourg Centre for Systems Biomedicine (LCSB) at the University of Luxembourg between June 2012 and May 2016. In the first place, I would like to thank the supervisor of my thesis, Prof. Dr. Rejko Krüger, for giving me the chance to work on this exciting project within his group. Furthermore, I would like to thank you for all the advice and supervision throughout the recent years and for the challenging tasks that helped me to learn and improve skills. Thank you for the everything-is-possible-spirit and for the support and encouragement for starting a career in science. Furthermore, I thank the members of my advisory board, Prof. Dr. Olaf Rieß and Prof. Dr. Bernd Wissinger, for taking time and for the profound advice and the interest in this project. I would like to acknowledge the funding source for this work. The project was funded by a Fortüne Grant awarded to Dr. Lena Brubulla by the Fortune program of the University of Tübingen. I also want to acknowledge the Graduate School for Cellular and Molecular Neuroscience of the University of Tübingen for offering an excellent doctoral training program with a manifold of diverse courses. In the present project I had the opportunity to work in collaboration with many very helpful and supporting people. Thank you Prof. Dr. Doron Rapaport and Dr. Kai-Stefan Dimmer (Interfakultäres Institut für Biochemie, IFIB, University of Tübingen, Germany), Dr. Peter Lichtner (Helmholtz Centre, Munich, Germany), Dr. Patrick May (LCSB, University of Luxembourg, Belvaux, Luxembourg), Dr. Enrico Glaab (LCSB, University of Luxembourg, Belvaux, Luxembourg), Dr. Simon Lehle (DNA Damage & Repair Unit Tübingen, Tübingen, Germany) and Dr. Manu Sharma (Hertie-Institute for Brain Research, University of Tübingen, Tübingen) for the prolific collaboration and the interest in the project topic. Also thank you very much to Dr. Alexander Skupin and Dr. Aymeric Fouquier D'Herouël (LCSB, University of Luxembourg, Belvaux, Luxembourg) for introducing me to calcium imaging as well as to Prof. Dr. Daniela Vogt-Weisenhorn and Annerose Kurz-Drexler VIII (Institute of Developmental Genetics, Helmholtz Zentrum München, Germany) for help and advice on the immortalization of fibroblasts. I would like to thank the members of the laboratory of “Functional Neurogenomics” (HIH, University of Tübingen): Daniela, Katharina, David, Lena and especially Brigitte (I will always miss our lunch conversations). I also want to thank Dr. Lena Burbulla for supervising. A very warm thank you to the whole LCSB team for the nice welcome and the support of our move from Tübingen to Luxembourg, in particular Nicolas and Christophe. Thank you to the CEN group: Mary, Gérald, Francois, Yvonne, Marie, Zoé, Bruno, Pete and Nicole. Thank you very much Caro for being the best fellow PhD student (CEN group is not the same without you) and Jill for treating my fibroblasts with so much care (you are the best fibroblast technician). I very much appreciated the spirit of helping and having fun at work. I owe special thanks to Julia and Dr. Ibo, who provided very valuable support and advice for my project. Your interest in the project and your patience allowed me to learn and improve so much. Thanks to your guidance I didn’t felt lost during my work. I also want to thank my mum and my grandparents Manfred and Wanda. You always believed in me and supported my academic ambitions. Although you do not fully understand what I am doing you understand that this work is so important for me. Special thanks to Maja, who is the best sister ever, for always listening and cheering me up. Finally I cannot express how grateful I am to my husband Alex. During the recent years you showed more patience and support than one could ask for. Your strength and support is most precious to me. I really owe this work to you. IX Table of Contents 1 Introduction ..................................................................................................................... 1 1.1 Parkinson’s disease (PD)............................................................................................. 1 1.2 Aetiology of PD.......................................................................................................... 2 1.2.1 α-synuclein – PARK1 ........................................................................................... 2 1.2.2 Parkin – PARK2 and PINK1 – PARK6 ..................................................................... 3 1.2.3 DJ-1 – PARK7...................................................................................................... 3 1.2.4 LRRK2 – PARK8 ................................................................................................... 4 1.2.5 Omi/HtrA2 – PARK13 .......................................................................................... 5 1.3 Mitochondria and PD ................................................................................................. 5 1.3.1 Mitochondrial electron transport chain ............................................................... 6 1.3.2 Oxidative stress .................................................................................................. 7 1.3.3 Ubiquitin-proteasome system (UPS) .................................................................... 9 1.3.4 Mitochondrial quality control via PINK1 and Parkin ............................................ 10 1.3.5 Mitochondrial transport ................................................................................... 11 1.3.6 Mitochondrial membrane potential and calcium homeostasis control cell death.. 12 1.4 Miro – more than just an adaptor for mitochondrial transport ................................... 13 1.4.1 The structure of Miro proteins .......................................................................... 15 1.4.2 Miro as adaptor for mitochondrial transport ...................................................... 17 1.4.3 Miro as calcium binding protein ........................................................................ 21 1.4.4 Miro is involved in ER-mitochondrial interaction ................................................ 22 1.4.5 Miro is targeted by the PINK1/Parkin pathway for mitochondrial quality control . 23 1.4.6 BDNF and Miro – Synaptic maintenance and plasticity........................................ 24 1.4.7 VopE and Miro – Mitochondria-mediated immune response .............................. 24 1.5 Patient-derived fibroblasts as cell model ................................................................... 26 1.6 Aim of the study ...................................................................................................... 27 2 Materials and Methods ................................................................................................... 29 2.1 Chemicals, Kits, Equipment and Software.................................................................
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