Thesis submitted to University College London for the degree of Doctor of Philosophy Role of Sonic Hedgehog signalling in regulating neural stem cell activity in the adult and aged murine hippocampus Marco Antonio Ortiz-Sanchez The Francis Crick Institute University College London Neural Stem Cell Biology Laboratory Department of Cell and Developmental Biology 1 Midland Road, London, UK. NW1 1AT Gower St, Bloomsbury, London WC1E 6BT June 2019 Abstract Abstract The homeostatic behaviour of neural stem cells (NSCs) in the adult hippocampus is controlled by signals from the niche. In young mice, the activation of dormant NSCs is typically followed by consecutive divisions producing neuronal progenitors followed by astrocyte differentiation, resulting in a rapid depletion of the NSC population. In a subset of active NSCs, however, the proactivation factor ASCL1 is degraded by the ubiquitin- ligase HUWE1, resulting in the return of these NSCs to a transient state of quiescence. These resting NSCs show a high propensity to reactivate and are essential for the maintenance of neurogenesis. How niche signals control the generation of the different NSC populations in appropriate ratios to ensure the long-term maintenance of neurogenesis is unknown. In my thesis, I have studied the regulation of NSC activity in the adult and mature mouse hippocampus by Sonic hedgehog (Shh) signalling. Firstly, I show that NSCs are able to respond to Shh signalling and that in young mice, this promotes activation of dormant NSCs and suppresses the formation of resting NSCs by inhibiting HUWE1-mediated degradation of ASCL1. In older mice, however, I show that Shh signalling activity diminishes progressively, which slows down the rate of NSC depletion. Finally, I identify oligodendrocyte cells as a cellular source of Shh in the adult hippocampus. My results provide cellular and molecular insights into the preservation of the hippocampal NSC pool in mature mice. 2 Declaration of authenticity Declaration of authenticity The work presented in this thesis has been completed at the Neural Stem Cell Biology Laboratory in The Francis Crick Institute, formerly the Division of Molecular Neurobiology at the National Institute for Medical Research, Mill Hill Laboratory. I, Marco Ortiz, confirm that this work is my own, and where information has been derived from other sources, this has been indicated in the thesis. Sectioning of mouse brains was entirely performed by Lan Chen (Senior laboratory research scientist in the laboratory). Immunostainings where SHH protein has been detected, were performed by Lan Chen. Initial processing of raw data from RNA- sequencing was performed by Stefania Vaga (former post-doc in the laboratory). Finally, adult hippocampus-derived neural stem cell cultures were created by Noelia Urbán (former post-doc in the laboratory). 3 Acknowledgements Acknowledgements I cannot be more grateful to my advisor, Francois Guillemot, who from the beginning showed dedication to my scientific formation. I also would like to specially thank Lachlan Harris, who has been an astounding colleague with whom I am fortunate to collaborate. Undoubtedly, without the many scientific discussions that we had, this thesis would have suffered in content and impact. I thank the Guillemot group, past and present members for their support and patience through all these years. In particular to Noelia Urban for introducing me to the field of adult neurogenesis and to Isabelle Blomfield for advising me on cell culture techniques. I thank my thesis committee, specially James Briscoe and Claudio Stern for providing insightful advises that will resonate for the rest my scientific career. I would like to thank Nicholas Chisholm, from the animal facility for his excellent support in the mice husbandry. I also would like to thank my Mexican friends Gaby, Pam and Omar, without whom it would have been harder to complete the work presented in this thesis. Also, I would like to thank my Mexican friends spread in Europe, Yuvia, Daniela, Sama, Mayela and Ahmed for being supportive despite the time and distance. A special thanks to Deri for always being there, and for who being an extremely supportive partner during the last year. Finally, I thank my parents and my sister for always supporting and encouraging me to keep pursuing my goals. 4 Dedicated to my grandmother and to my aunt Eli Had there been more scientific knowledge they could have had far better lives and they would still be here 5 Impact statement Impact statement Neurogenesis in postnatal stages of the human life have been shown in a number of studies. It has been shown that in children, neurogenesis is prominent, but it declines to almost undetectable levels in older humans. This thesis has shown a cellular and molecular mechanisms by which this process might occur, where sonic hedgehog signalling plays a major role in these mechanisms. Additionally, this thesis has found a novel source of SHH. These findings and a methodological approach designed in this thesis can be applied. For instance, it has been shown that adult neurogenesis plays a critical role in memory formation and mood regulation. It is possible that researchers design approaches to enhance memory processes or to alleviate mood related conditions such as depression. Alternatively, therapeutic approaches that impact on Shh signalling in the brain would have an impact in adult neurogenesis. This thesis found that a type of cells called oligodendrocytes express SHH. Oligodendrocytes and Shh signalling have been implicated in several types of cancer. It is possible that dysregulation of SHH expression by oligodendrocytes lead to tumour formation. This thesis might impact on research relating to brain cancer. Finally, this thesis designed a method to identify the major component of the adult neural stem cell pool in the adult dentate gyrus called dormant neural stem cells. This method has been already implemented in another project within the laboratory where this thesis was carried out. It is possible that more researchers implement this method to address other functions of neural stem cells not addressed here. 6 Table of Contents Table of Contents ABSTRACT 2 DECLARATION OF AUTHENTICITY ................................................................................................................ 3 ACKNOWLEDGEMENTS ................................................................................................................................... 4 IMPACT STATEMENT ........................................................................................................................................ 6 TABLE OF CONTENTS ...................................................................................................................................... 7 LIST OF FIGURES AND TABLES ................................................................................................................... 10 ABBREVIATIONS ............................................................................................................................................. 12 CHAPTER 1. INTRODUCTION .................................................................................................................... 15 1.1 INTRODUCTION TO THE FIELD ............................................................................................................. 16 1.1.1 Historical overview ....................................................................................................................... 17 1.1.2 Adult neural stem cells, adult neural stem cell niches and adult neurogenesis ............... 20 1.1.3 Adult neurogenesis in vertebrates ............................................................................................. 21 1.1.4 Adult neurogenesis in humans................................................................................................... 26 1.1.5 The mouse brain as a model system for adult neurogenesis ............................................... 27 1.2 NEUROGENIC NICHES IN THE ADULT BRAIN ................................................................................................ 28 1.2.1 The V-SVZ ....................................................................................................................................... 28 1.2.2 Dentate Gyrus ................................................................................................................................ 30 1.3 ADULT NEUROGENESIS IN THE DG ............................................................................................................ 31 1.3.1 Organization and connectivity of the DG .................................................................................. 31 1.3.1.1 Oligodendrocytes ................................................................................................................................... 33 1.3.2 Development of the DG ................................................................................................................ 36 1.4 ADULT NEURAL STEM CELLS ..................................................................................................................... 38 1.4.1 Adult NSC heterogeneity ............................................................................................................. 38 1.4.2 aNSC quiescence .......................................................................................................................... 39 1.5 MECHANISMS REGULATING QUIESCENCE, ACTIVATION AND