Characterising Gene Regulation during Epileptogenesis in Different Models of Epilepsy Bao-Luen Chang A thesis submitted for the degree of Doctor of Philosophy University College London Department of Clinical and Experimental Epilepsy Institute of Neurology Queen Square 2018 Declaration I, Bao-Luen Chang, confirm that the work presented in this thesis is my original research work. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. 1 Acknowledgements I would like to express my deepest appreciation to my supervisor Professor Stephanie Schorge for providing me the opportunity to study in the Institute of Neurology, Queen Square. Sincere thanks for her guidance, encouragement and fully support on my research. I am fortunate to meet such a scholar as my role model of a genuine scientist in the research field. I am especially grateful to Professor Matthew Walker and Professor Dimitri Michael Kullmann for their academical expertise, scientific guidance and suggestions on my study. I specially thank Dr. Marife Cano-Jaimez, Dr. Elodie Chabrol, Dr. Gabriele Lignani, and Dr. Pishan Chang for teaching me scientific methods and providing necessary expertise and advice. I am very appreciative to the senior clinical scientist, Dr. James Polke, for his kindly assistance in showing me how to use the gradient PCR and qRT-PCR machines and sharing his experience of experiments using qRT-PCR. I am thankful to all my colleagues in the department of clinical and experimental epilepsy, especially Andrianos Liavas, Albert Snowball, Janosch Heller, Kotaro Sugao, Gareth Morris, Andreas Lieb and Jenna Carpenter, for all their help and friendship. Thank you also to my dear friends in London, in particular to Qiang Gang, Chiawei Wu, and Yenwen Cheng with whom I have shared the ups and downs of our PhD life, and made it an unforgettable period in my life. Specific thanks to Mr. David Blundred at ION Education unit for always providing very helpful support and information. I also thank the IT officer Mr. Mat Shaffi for the professional IT support. An immense gratitude to my dear family, in particular to my parents for tolerating me to be unable to accompany them during these years, and giving me endless love, bravery and support; also to my brother, Chih-Jung, and sister, Pao-Ting, for their excellent care to all my family members that allows me to complete and concentrate on my PhD study abroad. Finally, I would like extend my thanks to Chang Gung Memorial Hospital, Taiwan for funding me this Doctor of Philosophy programme. 2 Abstract As epilepsy develops an enormous range of changes occurs in neurons. This process, epileptogenesis, involves complex spatiotemporal alterations of neuronal homeostasis and neural networks. The molecular mechanism of epileptogenesis remains obscure and gene regulation during the epileptogenic process dynamically controls various signalling and functional pathways which play an important role in defining the mechanisms of epilepsy. This thesis explores gene regulation in different in vitro models of seizure like activity, and further focuses on the temporal profiles of molecular changes during an in vivo model of epilepsy. We seek to identify important regulators of epileptogenesis which may be the targets for further study of the mechanism of epilepsy in human. The High-K+, Low-Mg2+, Kainic acid, and Pentylenetetrazole models were used to elicit seizure like activity in cortical neuronal cultures. The tetanus toxin (TeNT) model of focal neocortical epilepsy in rats was utilised to characterise gene regulation in different time points: acute, subacute and chronic stages (48-72 hours, 2 weeks, and 30 days after first spontaneous seizure, respectively). A set of candidate genes relevant to epilepsy was selected to analyse changes in mRNA expression during these in vitro and in vivo models. The mRNA expression of the different candidate genes reveals diverse regulatory behaviours in different models, as well as at different time points during the process of epileptogenesis. The cell culture model treated with Low-Mg2+ for 72 hours displayed the most similar mRNA expression profile to the in vivo model of TeNT neocortical epilepsy during subacute to chronic stages. Furthermore, in the in vivo model, GFAP, mTOR, REST, and SNAP-25 are all temporarily apparently up-regulated during epileptogenesis, while CCL2 is strongly up-regulated later when epilepsy is established. Transient down-regulation of BDNF in the acute stage, and the distinctly lower expression of GABRA5 in late stage suggest that this GABAergic signalling pathway may be down-regulated in the late phase of epileptogenesis. Our work highlights how different candidate genes are differentially regulated during epileptogenesis, and how the regulation of individual genes changes as epileptogenesis progresses. 3 Table of Contents Table of Contents Declaration .......................................................................................................... 1 Acknowledgements ............................................................................................. 2 Abstract ............................................................................................................... 3 Table of Contents ................................................................................................ 4 List of Figures .................................................................................................... 11 List of Tables ...................................................................................................... 14 Abbreviations .................................................................................................... 15 Chapter 1 Introduction ................................................................................... 18 1.1 Seizures, Epilepsy, Refractory Epilepsy and Status Epilepticus ................... 18 1.1.1 Introduction ........................................................................................... 18 1.1.2 Definitions .............................................................................................. 19 1.1.3 Classification of Seizures and Epilepsies ................................................ 21 1.1.4 Current treatment strategies for epilepsy ............................................. 25 1.2 Mechanisms of Epileptogenesis................................................................... 27 1.2.1 Introduction ........................................................................................... 27 1.2.2 Definitions .............................................................................................. 28 1.2.3 Circuit dysfunction in epileptogenesis ................................................... 30 1.2.4 Cellular biology of epileptogenesis ........................................................ 34 1.2.4.1 Neurodegeneration and Cell death ............................................................ 34 1.2.4.2 Neurogenesis and Neural reorganisation .................................................. 35 1.2.4.3 Gliosis ......................................................................................................... 36 1.2.4.4 Angiogenesis and BBB dysfunction ............................................................ 38 1.2.5 Molecular mechanisms of epileptogenesis............................................ 39 4 Table of Contents 1.2.5.1 Dysfunctional ion channels and receptors ................................................. 39 1.2.5.2 Molecular signalling pathways and related genes .................................... 40 1.2.5.3 Inflammatory and Immune pathways ....................................................... 44 1.2.5.4 Synapse regulation .................................................................................... 45 1.3 Experimental Models of Seizure and Epilepsy ............................................. 47 1.3.1 Introduction ........................................................................................... 47 1.3.2 In Vitro Models for Studying Seizure and Epilepsy ................................ 47 1.3.2.1 Introduction ............................................................................................... 47 1.3.2.2 High potassium model ............................................................................... 50 1.3.2.3 Low magnesium model .............................................................................. 50 1.3.2.4 Kainic acid model ....................................................................................... 53 1.3.2.5 Pentylenetetrazole (PTZ) model ................................................................. 53 1.3.3 In vivo epilepsy models .......................................................................... 56 1.3.3.1 Introduction ............................................................................................... 56 1.3.3.2 Tetanus toxin Model of Focal Epilepsy ....................................................... 65 1.3.3.3 Kainic Acid Model of Chronic Epilepsy ....................................................... 67 1.4 Selection of Candidate Genes ...................................................................... 71 Chapter 2 Thesis Aims .................................................................................... 73