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The REST/NRSF Pathway As a Central Mechanism in CNS Dysfunction

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The REST/NRSF Pathway as a Central Mechanism in CNS Dysfunction Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Alix Warburton April 2015 Disclaimer The data in this thesis is a result of my own work. The material collected for this thesis has not been presented, nor is currently being presented, either wholly or in part for any other degree or other qualification. All of the research, unless otherwise stated, was performed in the Department of Physiology and Department of Pharmacology, Institute of Translational Medicine, University of Liverpool. All other parties involved in the research presented here, and the nature of their contribution, are listed in the Acknowledgements section of this thesis. i Acknowledgements First and foremost, I would like to express my upmost gratitude to my primary and secondary supervisors Professor John Quinn (a.k.a Prof. Quinny) and Dr Jill Bubb for all of their support, guidance, wisdom (thank you Jill) and encouragement throughout my PhD; I could not have wished for a better pair. I am also extremely grateful to the BBSRC for funding my PhD project. I would also like to extend my thanks to Dr Graeme Sills for providing samples and assistance with my work on the SANAD epilepsy project, Dr Fabio Miyajima for offering his knowledge and knowhow on many occasions, Dr Gerome Breen for being a bioinformatics wizard and providing support on several projects, Dr Minyan Wang’s lab for their help and hospitality during my 3 month visit to Xi'an Jiaotong-Liverpool University, Dr Roshan Koron for assisting with the breast cancer study, Dr Chris Murgatroyd for his invaluable advice on ChIP and Professor Dan Rujescu’s lab for providing clinical samples and support with statistical analyses on the schizophrenia project. A massive thank you to Abigail Savage (Savbot), Kate Haddley (Katie Bear), Paul Myers (Curly), Veridiana Miyajima (Master of the Sushi), Maurizio Manca, Olympia Gianfrancesco, Kejhal Khursheed and all other members of White Block, past and present, for making my time in the lab an absolute ball and for keeping me sane on the science-fail days and celebrating the success days. To the Pub Friday peeps (you know who you are), thank you for drinking wine/beer/lemonade with me and for being beautiful and fabulous individuals. To my Mum, Poppy (Dad) and beautiful sisters Donna and Gina, thank you for being the most amazing family EVER and for supporting me throughout my studies and for making me smile every single day - I love you all to bits! And a huge thanks to all my other family and friends for being such a fabulous bunch. I would also like to say a special thank you to Big Dave and Tina-Bean for welcoming me into the Peeney residence and for supporting me during my thesis write-up; it means a lot to me. Last but not least, thank you to David Peeney for being the best; encouraging me, keeping me well fed and watered, putting-up with my write-up tantrums, saving me from spiders and giving me lots of cuddles. ii Contents Disclaimer ............................................................................................................................................................................... i Acknowledgements ............................................................................................................................................................ ii Publications ....................................................................................................................................................................... viii Abbreviations ...................................................................................................................................................................... ix List of Figures ....................................................................................................................................................................xiv List of Tables ......................................................................................................................................................................xvi Abstract .............................................................................................................................................................................. xvii Chapter 1 ................................................................................................................................................................................ 1 General Introduction ......................................................................................................................................................... 1 1.1 Overview ........................................................................................................................................................... 2 1.2 Understanding CNS disorders ................................................................................................................. 7 1.3 Regions of the brain important in mental health ............................................................................ 9 1.4 The effects of mood modifying drugs on the brain ..................................................................... 15 1.4.1 Mood stabilisers ............................................................................................................................... 16 1.4.2 Psychostimulants ............................................................................................................................. 19 1.5 Polymorphic variation as a biomarker for CNS disease ............................................................ 22 1.6 Gene-environment interactions in the CNS .................................................................................... 27 1.7 Epigenetics: Bridging the GxE response in neurological disease .......................................... 29 1.8 The NRSF signalling pathway ............................................................................................................... 32 1.8.1 The NRSE ............................................................................................................................................. 33 1.8.2 Regulation of target genes by NRSF ........................................................................................ 37 1.8.3 Regulation of NRSF expression ................................................................................................. 44 1.8.4 The NRSF pathway in disease .................................................................................................... 46 1.8.5 NRSF and the brain-expressed miRNAs ................................................................................ 49 1.9 MicroRNAs in the CNS .............................................................................................................................. 54 1.9.1 MicroRNA Biogenesis .................................................................................................................... 54 1.9.2 Mode of action of mature miRNAs in gene regulation .................................................... 57 1.9.3 Expression and function of miRNAs in the brain .............................................................. 60 1.10 Project aims .................................................................................................................................................. 65 Chapter 2 ............................................................................................................................................................................. 66 Materials and Methods .................................................................................................................................................. 66 2.1 Materials ......................................................................................................................................................... 67 2.1.1 Commonly used Buffers and Reagents................................................................................... 67 2.1.2 Chromatin Immunoprecipitation (ChIP) buffers ............................................................... 67 iii 2.1.3 Drug Treatment Solutions ........................................................................................................... 68 2.1.4 Human DNA Samples ..................................................................................................................... 69 2.1.5 Human cell lines ............................................................................................................................... 72 2.1.6 Cell culture media ............................................................................................................................ 72 2.1.7 Rat DNA Samples ............................................................................................................................. 73 2.1.8 PCR primers ....................................................................................................................................... 74 2.1.9 DNA constructs and commercial vectors .............................................................................. 76 2.1.10 ChIP grade antibodies .................................................................................................................... 77 2.1.11 StellARrayTM Mood disorder genes .........................................................................................
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