Modeling Leigh syndrome using patient-specific induced pluripotent stem cells Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by GIZEM INAK-GIRRBACH From Istanbul July 2019 The work was conducted from April 2015 until May 2019 under supervision of M.D. Dr. Alessandro Prigione at the Max Delbrück Center for Molecular Medicine in the Helmholtz-Association. I hereby confirm that I have written the thesis independently using solely the aids mentioned and without illicit assistance from third parties. 1. Reviewer: Prof. Dr. Erich Wanker 2. Reviewer: Prof. Dr. Helge Ewers Date of defense: 06.12.2019 Acknowledgment First of all, I would like to thank Prof. Dr. Erich E. Wanker for allowing me to conduct this Doctoral Thesis at the Max-Delbrück-Center Berlin-Buch and also for his valuable feedbacks during the lab meetings as well as for reviewing this work. Furthermore, I would like to express my gratitude to Prof. Dr. Helge Ewers from Freie Universität Berlin, who took an interest in my project and kindly agreed to review this doctoral thesis. Special thanks are owed to my supervisor Dr. Alessandro Prigione, for giving me the opportunity to work on this interesting project and guiding me with excellent and patient care. He has always been available and given me valuable advice. His honest feedback certainly helped me to become an independent scientist. I greatly enjoyed our regular scientific discussions, which motivated me to stay focused on the project. I shall not be forgetting to thank Prof. Dr. Markus Schülke-Gerstenfeld for providing patient skin fibroblasts for the project, valuable advice throughout the project, as well as providing his lab space and equipment for the complementation of some critical experiments. Furthermore, I am deeply grateful to our collaboration partners within this project. In particular, I would like to thank Dr. Agnieszka Rybak-Wolf for generating the 3D cerebral organoid models. Special thanks to Peter Glazar for his valuable contribution in analyzing the transcriptomics data and to Dr. Chris Secker for providing the lentivirus encoding SURF1. Moreover, I would like to thank all my colleagues and lab mates in the Prigione Group, who made it possible to work in a supportive and harmonic environment. I would like to thank Dr. Barbara Mlody for her constant availability for answering all kind of questions and for making the lab days more entertaining with her humorous nature. Thanks to Annika Zink for her presence and support. Her constant motivation to keep an eye on the stocks of small molecules needed for differentiation protocols and giving me advice on critical time points was very helpful. Also, I would like to thank Carmen Lorenz, who walked me through cell culture and other important techniques when I started working in the lab. Special thanks to Dr. Pawel Lisowski; without his knowledge and help, generating an isogenic line would not have been possible in such a short time. Thank you to Celina Kassner for her technical assistant. There were several bachelor and master students who worked in our lab. In particular, I would like to thank Tobias Hahn for finding the right corrected clone after months of screening. I want to express special gratitude to Ummi Ciptasari who was a motivated, and dedicated student who helped me get through the last phase of my Ph.D. period. Also would like to thank Group Wanker members for their cooperation and for providing a pleasant working atmosphere. I express my deepest appreciation to my husband Kai for his constant support and confidence at all times and walking me through the hard and good times hand in hand. In addition, I would like to thank my close circle of friends who were great listeners. Especially, I would like to thank Basak Arslan for her unlimited support. Lastly, I cannot thank my lovely family enough. They were there whenever I needed them, supporting me regardless of distance. Summary Leigh syndrome (LS) is a severe early-onset neurodegenerative childhood disorder characterized by bilaterally symmetrical necrotic lesions in the basal ganglia region of the brain. It is a rare disease affecting 1/36,000 newborns, and affected ones display psychomotor regression and also lactic acidosis with peak mortality mostly before the age of three. It is a genetically and biochemically heterogeneous disorder caused by mutations of nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) in more than 75 genes leading to disturbances in the mitochondrial respiratory chain. Mutations in the mitochondrial complex IV assembly factor, Surfeit locus protein 1 (SURF1), represent the most common cause and a severe form of LS. So far, SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering the understanding of the disease mechanisms. Thus, there is no effective therapy available. To investigate the molecular mechanisms underlying the disease-specific neuronal cell death, I generated the first mechanistic human induced pluripotent stem cell (iPSC)-derived neuronal model of LS harboring SURF1 mutations. SURF1 iPSC-derived cultures showed aberrant bioenergetics already at the level of neural progenitors, disrupting their neurogenic potency. A biallelic correction of SURF1 via CRISPR/Cas9 restored the bioenergetics and enhanced neuronal sprouting as well as synaptogenesis in SURF1 iPSC-derived cultures. To study the manifestation of the spatial defects, cerebral organoids were generated. In contrast to corrected iPSC-derived organoids, SURF1 iPSC-derived organoids appeared smaller in size and displayed neurodevelopmental defects. Ultimately, analyzing the transcriptomics data of iPSC-derived 2D and 3D cultures, some novel hints for possible therapeutic strategies were revealed. Different treatment strategies were tested on iPSC-derived neural progenitor cells (NPCs) function to find a potential strategy and two candidates were shown to have beneficial effects on the phenotypes: Coactivator of PGC1-α receptor, Bezafibrate, was shown to rescue bioenergetic defects via PPAR signaling pathway and inhibition of TGF-β1 receptor via SB-431542 was shown to rescue neurite outgrowth defects in SURF1 iPSC-derived NPC cultures. Altogether, this study is the first human iPSC-neuronal model for LS and gives insight into the role of SURF1 mutations in neuronal cells in the context of LS pathogenesis. SURF1 mutation caused aberrant energy metabolism and prevented neuronal maturation. With this study, NPCs were shown to be the first affected cell type in the neurodevelopmental cascade. Moreover, two candidate therapeutic targets have been suggested to improve the bioenergetics, and neuronal differentiation defects seen in patients derived neuronal cell model, which could be helpful for clinical disease research. Zusammenfassung Leigh-Syndrom (LS) ist eine schwere neurodegenerative Erkrankung bei Kindern, die sich durch bilateral symmetrische nekrotische Läsionen in der Basalganglienregion des Gehirns auszeichnet. Es ist eine seltene Krankheit, die 1 von 36.000 Neugeborenen betrifft. Betroffene Personen zeigen eine psychomotorische Regression und auch eine Laktatazidose und haben die höchsten Sterblichkeitsrate meist vor dem dritten Lebensjahr. Es handelt sich um eine genetisch und biochemisch heterogene Störung, die durch Mutationen von Zellkern-DNA (nDNA) oder Mitochondrien-DNA (mtDNA) in mehr als 75 Genen verursacht wird und zu Defekten der Atmungskette in den Mitochondrien führt. Mutationen im mitochondrialen Komplex IV Assemblierungsfaktor SURF1 stellen die häufigste Ursache und eine schwere Form von LS dar. Bisher ist es nicht gelungen die neuronale Pathologie des menschlichen LS in SURF1-defizienten Tieren zu rekapitulieren, was das Verständnis der Krankheitsmechanismen erschwert. Daher ist bisher keine wirksame Therapie verfügbar. Um die molekularen Mechanismen zu untersuchen, die dem krankheitsspezifischen neuronalen Zelltod und (auch der Gliazellenfunktionsstörung) zugrunde liegen, erstellte ich mittels humaner induzierter pluripotenter Stammzellen (iPSCs), das erste mechanistische Modell von LS welches SURF1-Mutationen enthält. Von SURF1-iPSCs abgeleitete Kulturen zeigten bereits auf der Ebene der neuronalen Vorläufer eine abweichende Bioenergetik, die ihre neurogene Potenz störte. Eine biallelische Korrektur von SURF1 über CRISPR / Cas9 stellte die Bioenergetik und das verstärkte Sprießen von Neuronen sowie die Synaptogenese in von SURF1-iPSCs abgeleiteten Kulturen wieder her. Um die Manifestation der räumlichen Defekte zu untersuchen, wurden zerebrale Organoide erzeugt. Im Gegensatz zu den von iPSC-abgeleiteten Organoiden der korrigierten Linie, schienen die von SURF1-iPSC abgeleiteten Organoide kleiner zu sein und zeigten neurologische Entwicklungsstörungen. Letztendlich wurden bei der Analyse der Omics-Daten von iPSC- abgeleiteten neuronalen Kulturen einige neuartige Hinweise für mögliche Therapiestrategien aufgedeckt. An iPSC-abgeleiteten neuronalen Vorläuferzellen (NPCs) wurden verschiedene Behandlungsstrategien getestet und es wurde gezeigt, dass zwei Kandidaten vorteilhafte Auswirkungen auf die Phänotypen haben: Mit dem Coactivator von PGC1-α-Rezeptor Bezafibrate konnten bioenergetische Defekte durch Modifikation des PPAR Signalweg behoben werden. Es wurde gezeigt, dass die Hemmung des TGF-β1-Rezeptors durch SB-431542, Neuritenwachstumsdefekte in SURF1-iPSC-abgeleiteten NPC-Kulturen retten. Zusammengenommen