DISCOVERING NOVEL MECHANISMS OF HUMAN CORTICAL DEVELOPMENT & DISEASE USING IN VIVO MOUSE MODEL AND IN VITRO HUMAN-DERIVED CEREBRAL ORGANOIDS Isabel Yasmin Buchsbaum Dissertation at the Graduate School of Systemic Neurosciences Ludwig‐Maximilians‐Universität München April 2019 Supervisor and first Reviewer: Dr. Silvia Cappello Developmental Neurobiology Max Planck Institute of Psychiatry Second Reviewer: Prof. Dr. Elisabeth Binder External Reviewer: Dr. Felipe Ortega de la O Date of Submission: 10th April 2019 Date of Defense: 22nd July 2019 ABSTRACT ABSTRACT This thesis combines three research studies with the common interest of identifying novel mechanisms underlying human cortical development. This aim is pursued from different angles, always basing the investigations on human induced pluripotent stem cell-derived 2D and 3D in vitro model systems that are partly combined with in vivo studies in the developing mouse cortex. Namely, in the pieces of work combined here, we 1) bring to light a neurodevelopmental role of a gene already implicated in adult nervous system function, 2) discover a novel mechanism that fine-tunes human neurogenesis, and 3) identify a novel gene whose mutations lead to a malformation of cortical development. The entirety of this work thus adds several aspects to the existing knowledge. In the first study, we identified a neurodevelopmental function of a gene mutated in patients with the progressive gait disorder hereditary spastic paraplegia (HSP). In this group of inherited neurodegenerative diseases, mutations in lipid, mitochondrial, cytoskeletal or transport proteins lead to degeneration of primary motor neurons, which, due to the length of their axons, are particularly sensitive to disruption of these processes. Here, were generated cerebral organoids (COs) derived from HSP patients with mutations in SPG11 coding for spatacsin. Previous work had shown impaired proliferation of SPG11 patient-derived neural progenitor cells (NPCs). We found a proliferation defect also in CO NPCs, leading to a thinner progenitor zone and premature neurogenesis due to increased asymmetric progenitor divisions, along with smaller size of patient-derived COs. Molecularly, we found a decrease in deactivated GSK3β and increase in P-βcatenin at the basis of the observed proliferation/neurogenesis imbalance. We thus confirmed the neurodevelopmental role of SPG11 that had previously been suggested from 2D human in vitro findings. Both the observed reduction in proliferating progenitors and in organoid size were rescued through inhibition of GSK3β, with the Food and Drug Administration (FDA) approved compound tideglusib only affecting patient COs. These rescue experiments thus stressed the opportunity that COs represent for drug testing and translation of findings to precision medicine. In the second study, we investigated the role of a novel posttranslational modification (PTM) termed AMPylation in neurogenesis. Using a novel probe for the detection of AMPylated proteins and a combination of mass spectrometry-based proteomics, immunohistochemistry, and acute interference with the expression of the AMPylating enzyme, we made several interesting findings: AMPylation takes place on a cell type- specific set of proteins, is responsive to the predominant environmental condition, and both AMPylator and targets localize to cell type-specific intracellular localizations. During the process of neuronal differentiation, the set of AMPylated proteins is completely remodeled, with a very high number of unique targets in neurons. These include metabolic enzymes as in all analyzed cell types and, additionally and specifically, cytoskeletal and motor proteins. Cytoskeletal and motor proteins in neural progenitors and neurons are known to be differentially modified by several PTMs whose correct establishment is highly important during neurodevelopment; AMPylation may thus be an additional one. To assess the role of AMPylation in neurodevelopment, we manipulated the expression of the AMPylating enzyme FICD in COs. Downregulation kept cells in a proliferating progenitor state, whereas overexpression increased neurogenesis. We thus suggest AMPylation as a novel PTM fine-tuning neurogenesis. i ABSTRACT The third study focused on the identification of new mechanisms underlying cortical malformations, aiming at a better understanding of how the human brain develops. In patients with periventricular heterotopia (PH), a neuronal migration disorder in which a subset of neurons fail to migrate to the developing cortical plate and instead form nodules of grey matter lining the lateral ventricles as their site of production, biallelic mutations in endothelin converting enzyme 2 (ECE2) were identified as candidate causative. Combining in vitro and in vivo models, we found a role for ECE2 in neuronal migration and cortical development. In the absence of ECE2, several processes of general importance to proper neuronal migration were disrupted. Namely, changes in progenitor cell polarity and morphology and in apical adherens junctions led to their delamination, restricting their use as a scaffold for neuronal migration. This resulted in ectopic neurons reminiscent of nodules in PH. Besides a deregulation of cytoskeletal, polarity, and apical adhesion proteins, extracellular matrix (ECM) proteins were reduced in absence of ECE2, suggesting its role in ECM production and underlining the necessity of ECM components for proper neuronal migration during cortical development. Moreover, we detected differential phosphorylation of several cytoskeletal, motor and adhesion proteins in the absence of ECE2, which is functionally in line with the former findings and suggests an additional involvement of ECE2 in the regulation of PTMs. Altogether, the studies presented here underline the heterogeneity and complexity of pathways and mechanisms that contribute to human cortical development and its disorders, converging on the regulation of cytoskeleton and transport within the involved cells and of the ECM on their outside. ii ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Before starting this PhD, I never considered alternative routes and not do a PhD. Not for the sake of the title: I took it for the natural way to go if you want to do science, which is what I have wanted since my childhood. But then, facing the reality of failures and ups and downs of the PhD life, I more than once thought about quitting it. It was thanks to many people that I overcame these hurdles and now hopefully finish this big chapter of my life with success. I would like to express my heartfelt gratitude… to Silvia, for being the most enthusiastic and positive supervisor I could have imagined, for supporting my New Zealand experience and attendance of numerous conferences, for enabling me to work on several different projects and sharing my fascination of diversity, for your scientific advice and encouragement, and for believing in me and my work. Thank you for your always open door and ear and for readily sharing your own experiences – also outside the sciences. to Stephen and Adam, for guiding me into the PhD and enriching me with an invaluable experience on the other side of the world. to my TAC members Elisabeth, Magdalena and Jürgen, for supporting and steering my work with fruitful discussions and to Felipe for reviewing this thesis – thanks for your time and support. to the GSN and the GSN team, for selecting me for the Fast-track PhD program, for their friendly support both with advice and financially, which enabled me to make my New Zealand experience and to visit international conferences, and for this scientific and cultural inspiration and supportive network that the GSN represents. to my collaborators in all projects, especially to Pavel and Stephan, for the great teamwork and constructive discussions and for broadening my angle of view. to Magdalena and her group at the Helmholtz, for housing us while our lab at the MPI was being renovated. to Ane, for many hours and meters of height with precious talking, a huge backpack and a camera in our Alps and the Taiwanese mountains, and for proofreading my discussion. ¡Muchas gracias mi amiga! to Johannes, for being great company in the beginning of my PhD, for teaching me the in vitro methods and for proofreading my discussion – danke; to Grazia, for your support in the hardest phase of the ECE2 project – mille grazie; to Christina, for sharing your in vivo expertise and always knowing advice – ευχαριστώ; to Timo and Barbara for your technical support. And thanks to all yet unmentioned members of the C-lab, the A-, Z- and Binder-labs, and Marisa and her group for sharing and enriching this section of my life. to Antonietta – without your coffee and smile, life at the MPI would not have been the same! to Sara, for introducing me to Lindy Hop, which became a great source of positive energy for me; to my roomies Ines, Patrick, and Grandma Hilde, and to my Munich iii ACKNOWLEDGEMENTS family of friends, for believing in me and being there for me with uplifting chats, good coffees and food, handicraft sessions, game nights, and innumerable hugs. to Babaganouch, for teaching me not to let the failures close to me and that “time will graduate everyone”. to my parents and my sisters, for your steadfast support and love over this far too far distance, for enduring my fluctuations and complaints, and for believing in me and my dreams not only during these last years but throughout my whole life. an Andrej, mein Lieblingsmensch. Danke, dass Du an meiner