Reverse Engineering Glaucoma Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor of Philosophy by Karen Leah Lester April 2018 Abstract Primary open angle glaucoma (POAG) is a leading cause of irreversible blindness worldwide, and the only modifiable risk factor is intraocular pressure (IOP). Glaucoma is a complex disease with specific endophenotypes, and disease pathogenesis is likely to involve multiple pathways linking genetic and environmental interactions. Growth factors present in the aqueous humour in POAG increase outflow resistance and elevate IOP. TGF-β2 alters ECM production and turnover in the trabecular meshwork (TM) and has been shown in numerous studies to play a role in the pathogenesis of POAG. No current pharmacological interventions target the deleterious effects of TGF-β2 of the TM which produced elevated IOP. In addition to TGF-β another well characterised glaucoma stimulus is corticosteroids. Corticosteroids are used in ophthalmology to decrease inflammation and preserve ocular function. However side effects including cataract, enhanced infection, and glaucoma are associated with their use. Small, naturally occurring regulatory genes, micro RNAs (miRNAs), target many genes downstream of TGF-β2 and are expressed in response to corticosteroids. The current work set out to identify key differentially expressed genes by RNA-Seq in the human trabecular meshwork (TM) in response to two glaucoma stimuli; TGF- β2 and dexamethasone; and investigate the ability of miRNAs to manipulate gene expression within the TM to reduce pathological insults central to glaucoma. Investigating the influence of TGF-β2 on gene expression in primary human TM cells demonstrated that the majority of the significantly differentially expressed genes were involved in extracellular matrix remodelling and actin cytoskeletal re- organisation likely via the RhoA signalling pathway. The influence of dexamethasone on gene expression in primary human TM cells identified genes involved in extracellular matrix remodelling and genes required for glucocorticoid receptor nuclear translocation. Differentially expressed miRNAs in healthy and glaucomatous human TM cells were identified by a miRNA microarray. Manipulation of validated mRNA targets by identified miRNAs indicated a complex regulatory network and in vitro functional analyses further identified regulation of actin cytoskeleton remodelling via miRNA inhibition. The findings of this study indicate that TGF-β2 and dexamethasone have significant effects on extracellular matrix remodelling and actin cytoskeletal re-organisation in human TM cells. The RNA-Seq and miRNA array have identified potential novel therapeutic targets for glaucoma. ii Acknowledgments First and foremost I would like to thank my supervisors Prof. Colin Willoughby (Bob), Dr. Carl Sheridan, and Mrs. Anshoo Choudhary. Thank you firstly for affording me the wonderful opportunity and for your continued help and support over the last three years. Carlos, thanks for keeping me going over the last few months! Bob, I understand now why you wanted to drag me from the lab and encourage my reading! I have learned so much under your guidance and everything you taught me will stick with me throughout my scientific career. You have encouraged me every step of the way and for this I will always be grateful. Thank you to Dr. Kevin Hamill for being an honorary supervisor, an everyday llama expert and a teacher extraordinaire! Your enthusiasm for science is admirable. Thank you to Dr. S. Senthilkumari (Aravind Medical Research Foundation, Madurai, India) for providing training on the H.O.C.A.S setup. Thank you to Dr. Abbot Clark (UNTHSC, Texas, USA) for providing glaucomatous TM samples for this research. Thank you to Dr. David Simpson and his team (Queens University Belfast, UK) for RNA-Sequencing services. Thank you to Dr. Natalie Pollock and Dr. Stephnie Kennedy for your help with primary cell culture. Thank you Neeru for you advice and collection of corneal rims. Thank you NC3R, International Glaucoma Association/UK and Eire Glaucoma, International Glaucoma Association and Fight for Sight for funding this work. In addition I would like to thank everyone in E.V.S who have made my time in U.of.L such an enjoyable experience, and have been so helpful throughout. Miss. Jessica Eyre, thank you for dragging me to yoga even when I protested and being there over the last year to provide the lols. Thank you to my girls back home, your support has been phenomenal and I apologise for only knowing how to talk about one thing for the past three years. To my best friend and partner in crime, Leeburt, thank you for being with me every step of the way since the very beginning. You have cheered me up no end every time I needed it and stood by me through it all. You’ve held my hand every step of the way and encouraged me always. Endless Netflix binges and Lindt chocolate was key. It has been the best experience ever doing this with you by my side every day. I especially want to thank my Mum, Dad, and sister Amy. Mum and Dad, you’ve given me the best of everything, always, and I know without your support throughout my life I would not be where I am today. Amy, thanks for making me smile when all I wanted to do was cry, you’ve dealt with my ups and downs throughout this whole process expertly and I know I wouldn’t have gotten through it without you all by my side. ~“Always look someone in the eye, even if they’re blind, just say “I’m looking you in the eye.”~ iii Table of Figures Figure 1.1: Optic Disc and Schematic Cross-section of the ONH to Show Central Cup……………………………………………………………………………………………………………………… 3 Figure 1.2: Characteristic Visual Field Defects in Glaucoma…………………………………………………………………………………………………………….. 5 Figure 1.3: Gonioscope Drainage Angle Images………………………………………………………………………………………………………………… 7 Figure 1.4: Posterior View of the Iris and Ciliary Body Showing the Ciliary Processes……………………………………………………………………………………………………………. 16 Figure 1.5: Active Secretion of Aqueous Humour………………………………………………. 17 Figure 1.6: Cross Section of the Human Eye ……………………………………………………………………………………………………………………. 20 Figure 1.7: Anatomical Structure of the Human Trabecular Meshowrk……….. 21 Figure 1.8: Structure and Transcriptional Isoforms of the Human GR Gene……………………………………………………………………………………………………………. 27 Figure 1.9: Glucocorticoid Mechanisms of Action……………………………………………………………………………………………………………. 30 Figure 1.10: TGF-β Structure, Latency, Activation and Signalling…………………………………………………………………………………………………………… 36 Figure 1.11: miRNA Biosynthesis- Canonical Pathway……………………………………………………………………………………………………………. 45 Figure 1.12: miRNA Biosynthesis- Non-Canonical Pathway……………………………………………………………………………………………………………. 47 Figure 2.1: Representative Images Depicting Human Anterior Segment Dissection Protocol……………………………………………………………………………………………………………. 55 Figure 3.1: Representative Phase Contrast Microscope Images of Human Cultured TM Cells……………………………………………………………………………………………………………. 88 iv Figure 3.2: Representative Fluorescent Confocal Microscope Images of Human TM Cells…………………………………………………………………………………………………………………. 89 Figure 3.3: Principal Component Analysis of RNA-Seq Data for Control Untreated TM Cells and TGF-β2 Treated TM Cells………………………………………………………….. 94 Figure 3.4: Heat Map and Unsupervised Clustering of Human TM Cells Treated with TGF-β2……………………………………………………………………………………………………………. 96 Figure 3.5: volcano Plot Showing the Relationship Between LogFC and the Average Log Counts-Per-Million (CPM) ……………………………………………………………………… 101 Figure 3.6: Top Canonical Pathways of the Top 1% Differentially Expressed Genes in TGF-β2 Treated Human TM Cells Vs Control TM Cells………………………………… 107 Figure 3.7: Canonical Pathway Map for RhoA Signalling Pathway……………………………………………………………………………………………………….. 108 Figure 3.8 Expression Levels of Candidate Genes in Cultured Normal Human Tm Cells……………………………………………………………………………………………………………. 116 Figure 3.9: Expression Levels of Candidate Genes in Cultured Normal Human TM Cells and Glaucomatous TM Cells…………………………………………………………….. 120 Figure 3.10: Schematic of Rho/ROCK Signalling Pathway……………………………………………………………………………………………………………. 124 Figure 3.11: NOX4 Mediated TGF-β-Induced Pro-Fibrotic Responses………………………………………………………………………………………………… 127 Figure 3.12: Pathological Role of EDN1 in Glaucoma……………………………………………………………………………………………………… 129 Figure 3.13: Localisation of CDKN2B and CDKN2B-AS1 on Chr9p21……………………………………………………………………………………………………… 131 Figure 3.14: Effects of CDKN2B and CDKN2b-AS1 on Cell Cycle via CDK4 Inhibition………………………………………………………………………………………………………… 131 v Figure 4.1: Principal Component Analysis of RNA-Seq Data for Control Untreated TM Cells and DEX Treated TM Cells………………………………………………………………… 151 Figure 4.2: Heat Map and Unsupervised Clustering of RNA-Seq Data from Control Untreated TM Cells and DEX Treated TM Cells……………………………………………………………………………………………………………. 153 Figure 4.3: Volcano Plot of the Relationship Between the LogFC and the Average Log Counts-Per-Million (CPM) in Control Untreated TM Cells Compared to DEX Treated TM Cells……………………………………………………………………………………………………… 160 Figure 4.4: Top Canonical Pathways of the Top 1% Differentially Expressed Genes in Human TM Cells +/- Dexamethasone…………………………………………………………. 164 Figure 4.5: Heat Map of Highest Ranked Over-Lapping Canonical Pathways between TGF-β2 Treated Human TM Cells and DEX Treated Human TM Cells……………………………………………………………………………………………………………. 165 Figure 4.6: Expression Levels of Glucocorticoid Receptor (GRα and