THE ROLE OF INTERLEUKIN-6 SIGNALLING MOLECULES IN MURINE MODELS OF INHERITED PHOTORECEPTOR DEGENERATION by Michael Joseph Szego A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Michael Joseph Szego 2009 The role of Interleukin-6 signalling molecules in murine models of inherited photoreceptor degeneration Doctor of Philosophy, 2009 Michael Joseph Szego Department of Molecular Genetics University of Toronto Abstract We previously reported that in inherited photoreceptor degenerations (IPDs), the mutant photoreceptors (PRs) are at a constant risk of death (Pacione, Szego et al, 2003). Using microarrays to identify genes that may mediate the constant risk, I identified 145 differentially expressed transcripts in the Rds+/- mouse model of IPD at a time when 90% of the PRs were alive. A major finding was the up-regulation, quantified by qPCR, of four components of a putative IL-6 cytokine signaling pathway: Oncostatin M (Osm) (2-fold increased) → Oncostatin M receptor (Osmr)(2.6-fold increased) → Stat-3 (2.3-fold increased) → C/EBPδ (3.2- fold increased). Similarly, I found increases in the cognate proteins Osmr (3-fold), Stat-3 (2.6-fold), and the phosphorylated, transcriptionally active form of Stat-3, pStat-3 (5.8-fold)(all p<0.01). Other Il-6 cytokine signaling molecules were largely unchanged, but the mRNA of leukemia inhibitory factor (Lif), was increased (3.0-fold). Comparable increases of most transcripts were also present in the Rd1-/- and mutant rhodopsin P347S transgenic (P347S) IPD models. The increases in cytokine signaling molecules occurred predominantly in Müller glia, although C/EBPδ transcript was increased in PRs. Because exogenous IL-6 cytokine treatment slows PR death in IPDs, I asked whether the endogenous increases in IL-6 pathway proteins in IPD retinas were a survival response, and generated IPD models with Osmr, Lif or C/EBPδ loss- of-function (LOF) mutations. Osmr LOF decreased PR survival in the retinas of Rds+/-;Osmr-/- mice, which had 12.5% fewer PRs than those of Rds+/-;Osmr+/+ mice (n=9, p<0.05) at 4 month of age, and Tg- RHO(P347S);Osmr-/- mice had 13.5% fewer PRs (n=6, p<0.01) at 31 days of age. Unexpectedly, Osmr LOF had no effect on pStat3 levels in Rds+/-;Osmr-/- retinas, indicating that retinal Stat3 activation may be predominantly regulated by other molecules. In contrast to the Osmr LOF, Lif or C/EBPδ LOF unexpectedly increased mutant PR survival. Rd1-/-;Lif -/- mice at 13 days had 14% more PRs than Rd1-/- ;Lif+/+ mice (n=6, p<0.003) and a 1.7 fold decrease in pStat-3 (n=4, p<.05). Similarly, 8 month-old Rds+/-; C/EBPδ-/- mice had 18% more PRs than Rds+/-; C/EBPδ+/+ mice (n=5, p<0.005). These findings suggest that in mutant PRs: 1) up-regulation of the Osmr receptor is protective; 2) the presence of Lif or C/EBPδ is pathogenic, and therefore 3) Osmr, Lif and C/EBPδ act either in different pathways or different cells, to account for the differing effects of their LOF on PR cell death; and 4) the partial effects of Osmr, Lif and C/EBPδ LOF indicate that other genes also mediate the constant risk of death of mutant PRs in IPDs. i i Acknowledgments I have met some incredible people while performing the work summarized in these pages- many of whom will remain life long friends. I would like to thank Rod for his enthusiasm, ability to ask key questions and his support of my career pursuit. I would also like to thank my committee members, Michael Salter and Brenda Andrews for their guidance over the years. Jayne Danska and Freda Miller were also very generous with their time. A former student once described the lab culture as being temporally organized into dynasties. I have been lucky enough to be apart of two such dynasties. In the first dynasty I am indebted to Jonathan, Rachel, Lynda and Laura, otherwise known as members of the Commie Bay. Jonathan, your strong self-identity and ability to say exactly what was on your mind certainly inspired me. You have my utmost respect and love. Rachel, you were always there to support me and spent many a late night helping me edit papers for my various graduate courses. Finally, to Laura, who kept plugging away at our project and kept things moving even when I was feeling discouraged. I still laugh at the thought of the late night we spend working on the review together. Lynda, you have certainly made the lab a fun place to work. I thank for all of your help particularly over this last year. To the second, most recent, dynasty I am especially thankful to Alexa, who spent many hours helping me complete experiments while I wrote this thesis and entered into fatherhood. It has been fun watching you grow as a scientist and your comments over the years were always insightful and certainly helped shape the direction of this project. However, your penchant for motorcycle racing has always concerned me; please drive safely! My Dad has always described my educational trajectory as being an “S” shaped curve. Thus, he was not surprised when I told him I would be doing a Master’s degree after my PhD. Mom and Dad you have always supported me through all of my endeavors. You are model parents that I attempt to emulate. After living for several months in the frigid, dark, and condemned old Graduate House, I moved into a cozy apartment with Arvin and Pleasie and we quickly became like family. Our kitchen was a communal one and I have fond memories of the time we spent together. The best thing that ever happened to me while in graduate school was meeting my wife, Joby. Her intelligence, confidence and infectious laugh drew me right in and I have never looked back. I am so very lucky we met. Finally, I would like to thank Quincie, who was born April 4, 2008, for enabling me to finally stop doing experiments. She has brought learning back to the basics and has renewed my love of developmental biology. iii Table of Contents Abstract ...............................................................................................................ii Acknowledgments..............................................................................................iii Table of Contents...............................................................................................iv List of Tables....................................................................................................viii List of Figures ....................................................................................................ix List of Abbreviations ...........................................................................................x 1. Introduction..................................................................................................... 1 1.1 Structure of the mammalian retina.......................................................................................................1 1.1.1 Neuronal cell types in the retina............................................................................................................1 1.1.2 Retinal pigment epithelium....................................................................................................................5 1.1.3 Müller Glia .........................................................................................................................................5 1.2 Types of Photoreceptor Degeneration ................................................................................................7 1.3 Genes Implicated in Retinal Degeneration .........................................................................................8 1.3.1 Structural proteins- Rds and Rom1 ......................................................................................................8 1.3.2 Phagocytosis- Mertk .......................................................................................................................... 10 1.3.3 Cilia maintenance/Trafficking of intracellular proteins- BBS genes .................................................... 10 1.3.4 Phototransduction- Rhodopsin and Rd1............................................................................................. 11 1.3.5 Signalling, cell-cell interaction, or synaptic interaction- Sema4A.......................................................... 12 1.3.6 Vitamin A metabolism- Rpe65 ........................................................................................................ 12 1.3.7 Transporters/channels- ABCR (rod photoreceptor ABC transporter) or ABCA4............................. 13 1.3.8 Transcription factors- Beta2/NeuroD1............................................................................................. 14 1.3.9 RNA intron-splicing factors .............................................................................................................. 14 1.3.10 Enzymes- IMPDH1...................................................................................................................... 15 iv 1.4 Common Features of IPD.................................................................................................................. 16 1.4.1 Altered calcium homeostasis............................................................................................................... 16 1.4.2 Cell death by apoptosis ...................................................................................................................... 17 1.4.3 Common kinetics of cell death...........................................................................................................