UNIVERSITY COLLEGE LONDON UCL INSTITUTE OF NEUROLOGY Department of Molecular Neuroscience FULL GENOME ANALYSIS OF MICROGLIAL ACTIVATION; RAMIFICATIONS OF TREM2 Thesis submitted for the degree of Doctor of Philosophy By, Claudio N Villegas Llerena 2017 Supervisors, Prof John Hardy Dr Jennifer Pocock DECLARATION I, Claudio Nicolas Villegas Llerena, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 ABSTRACT Neuroinflammation is a pathological hallmark of Alzheimer's disease (AD) and it is well established that microglia, the brain's resident phagocytes, are pivotal for the immune response observed in AD. In the healthy brain, microglia attack and remove pathogens and cell debris, but have been shown to become reactive in AD. An apparent link between microglia and AD is Amyloid β (Aβ), which accumulates in the plaques observed in the brains of AD patients and has been reported as a microglia activator. Genome Wide Association Studies (GWAS) have allowed the identification of more than 20 genetic risk associations to AD. Many of these associations highlight the importance of immune pathways (and others) in AD. More recently, the identification of mutations in TREM2 (Triggering Receptor Expressed on Myeloid Cells 2), a gene exclusively expressed by microglia in the brain, has brought microglial activation and dysfunction back to the attention of the AD community. The main focus of this study is to understand microglial activation elicited by different stimuli -including Aβ1-42 monomers, oligomers and fibrils- with regards to their inflammatory activation status (M1, M2 or other) and whole-genome expression profile. To this end, the mouse-derived BV2 cell line was used to assess gene expression changes during microglial activation. Data shows that M1 and M2 activators alter gene expression of AD-associated genes in a manner that is potentially detrimental for AD progression. A second objective of this thesis was to use the CRISPR/Cas9 gene editing technology for the generation of Trem2-deficient BV2 cell lines. As a result, Trem2 +/- (haploinsufficient) and Trem2 -/- (knockout) BV2 cell lines were generated. Subsequently, these cell lines were characterised in terms of their phagocytic, proliferation, migration, cytokine release capacities and whole genome expression. In consequence, this study provides new and well- characterised in vitro models for the study of Trem2 function. 3 ACKNOWLEDGEMENTS I would like to wholeheartedly thank my supervisors, Prof John Hardy and Dr Jennifer Pocock, for giving me the opportunity to work in their groups. I am very grateful for their support and guidance throughout the course of my PhD. I would also like to thank the Pocock lab team; ‘Dr Piers’, Pablo, Kat, Matt, Anna and Alex. Many thanks for your help, scientific advice and for making the lab a fun place to work in. Alex, you have been a great PhD colleague/friend and I am terribly sorry about ever creating the B5 cells! My sincere gratitude to all people who do science at 1 Wakefield Street and go to the Harrison’s pub; including the Selina’s, Guerreiro-Bras’, Rohan’s, Warner’s and Patani’s groups. Particular thanks go to Ellie, Bimali, Celia and Charlie. Their advice and friendship (and sometimes, reagents) have made my PhD a lot less stressful. I would also like to thank Prof Smith’s lab for making the 2nd floor a friendly place, particularly Dimitra, Mario, Kim, Charlotte and Sharmeen. In addition, I would like to thank Mar and Seb, from the Molecular Neuroscience Dept., for their help with gene expression experiments and general advice. My PhD, and my life, has been greatly enriched by the wonderful people, I was fortunate to meet during these past 4 years. Thank you, Alice, Vitaly, Mattia, Chris, Natalia, Martha, Khaled, Alastair and the ION’s 5-a-side postgraduate team (Matt, Diego, Seb and all the others). A big thank you also to Sara, Eva and Leyla, your foodie-ness and friendship have made of London a familiar place. A collective thank you to all members of the University of London Archery Club, you have been pivotal in maintaining my sanity levels just about right (or maybe not); especially Bridget, Lifei, Dom, Richard, Aylish, Shabnam, Julie, Dr O’connell and Jay. Friends are the family that we get to choose and in that I have chosen 12 brothers (you know who you are). I could not have finished my PhD without your constant cheering me on through difficult days and without the laughs we share. Gracias hermanos! 4 I am very grateful to my family, especially Tío Danny, Tía Dora, Tío Frank, Tía Luz and my grandparents as well. I am also super thankful to my ever-expanding nuclear family; Cuchi (that’s my Mom), Dad, Javier, Martin, Mehnaz, Maria, Lyby, Abi, Laila and Alex. Thank you for your love and faith in me. I am also thankful to the Paredes-Moscosso family for their support and encouragement, especially to Anna-Lu. The biggest of thank-yous goes to my wonderful girlfriend, Sol, whose love, patience, support and scientific advice have been fundamental in completing my PhD. Thank you for inspiring me to be a better person and laughing at my jokes. Finally, I thank God for the opportunities I have been given, for looking after my family and me, and for what comes next. 5 TABLE OF CONTENTS TABLE OF CONTENTS TABLE OF CONTENTS ................................................................................................ 6 LIST OF FIGURES ..................................................................................................... 11 LIST OF TABLES ....................................................................................................... 14 ABBREVIATIONS ....................................................................................................... 15 1 CHAPTER 1: Introduction .................................................................................... 17 1.1 Alzheimer’s disease ...................................................................................... 17 1.1.1 Clinical features of AD ........................................................................... 17 1.1.2 Pathological features of AD ................................................................... 18 1.1.3 AD diagnosis ......................................................................................... 19 1.2 AD risk .......................................................................................................... 20 1.2.1 Genetic AD risk ..................................................................................... 20 1.2.2 Non-genetic AD risk ............................................................................... 22 1.3 Neuroinflammation in AD .............................................................................. 23 1.3.1 Cellular mediators of Neuroinflammation in AD ..................................... 24 1.3.2 Non-cellular mediators and modulators of neuroinflammation ............... 27 1.4 Microglia diversity and modulation of activation states: M1 vs M2, are there any boundaries? ..................................................................................................... 30 1.4.1 M1 state ................................................................................................ 33 1.4.2 M2 state ................................................................................................ 33 1.4.3 Microglial phenotypes in AD .................................................................. 33 1.4.4 Aging and microglial phenotypes ........................................................... 35 1.5 Microglial genes in AD risk ........................................................................... 35 1.5.1 TREM2 .................................................................................................. 36 1.5.2 CR1 (Complement Receptor 1) ............................................................. 40 1.5.3 CD33 ..................................................................................................... 41 1.5.4 MS4A cluster ......................................................................................... 41 1.5.5 HLA-DRB5/HLA-DRB1 (Major Histocompatibility Complex, Class II, DR Beta 1/Major Histocompatibility Complex, Class II, DR Beta 5) ............................ 42 1.5.6 APOE (Apolipoprotein E) ....................................................................... 42 1.5.7 CLU (Clusterin or APOJ) ....................................................................... 43 1.5.8 ABCA7 (ATP-binding cassette transporter A7) ...................................... 43 1.5.9 INPP5D, MEF2C and EPHA1 ................................................................ 43 1.6 Genome editing – CRISPR/Cas9 tools ......................................................... 44 1.6.1 Genome editing: Hijacking the endogenous DNA repair systems. ......... 44 6 TABLE OF CONTENTS 1.6.2 Programmable genome editing tools: ZFNs, TALENS and CRISPRs .... 46 1.6.3 Zinc-finger nucleases (ZFNs) ................................................................ 46 1.6.4 Harnessing the power of the CRISPR/Cas9 system – More tools in the kit ………………………………………………………………………………….50 2 CHAPTER 2: Materials and Methods ................................................................... 52 2.1 Cell lines and culture .................................................................................... 52 2.2 Generation of Trem2 and Dap12 Knockdown BV2 cell lines