REGULATION OF GLIOSIS IN THE MOUSE RETINA A Dissertation Submitted to the Faculty of Purdue University by Subramanian Dharmarajan In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2017 Purdue University Indianapolis, Indiana ii THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Teri Belecky-Adams Department of Biology Dr. Jason S. Meyer Department of Biology Dr. Stephen Randall Department of Biology Dr. AJ Baucum Department of Biology Dr. Yuk Fai Leung Department of Biological Sciences Approved by: Dr. Theodore R. Cummins Head of the Graduate Program iii To my wife Akshaya and my daughter Ananya. iv ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Teri Belecky-Adams, for all of her support, encouragement and mentorship. You have helped me become the researcher I am today. In addition to my advisor, I would also like to thank my committee members Dr. AJ Baucum, Dr. Yuk Fei Leung, Dr. Jason Meyer and Dr. Stephen Randall. I appreciate your insightful comments and assistance throughout my project which have helped develop my skills. To my fellow graduate students and staff of the department of Biology, I would like to thank you for lending a helping hand whenever I needed it. I would like to also thank the faculty of the department of Biology for all their help with my projects and career advise. I am grateful to my friends Nilesh, Chetan, Rishi, Shrikant and Amrit. The game nights, dinners, road trips and general help and friendship helped me feel at home and was a welcome distraction from my lab work. Finally, this work would not be complete without the unwavering support of my wife Akshaya. She has been a great companion, loved and encouraged me throughout this challenging period, to make them the best years of my life. I would also like to thank my parents for their unconditional love and support in allowing me to follow my ambitions. v TABLE OF CONTENTS Page LIST OF TABLES :::::::::::::::::::::::::::::::::: ix LIST OF FIGURES ::::::::::::::::::::::::::::::::: x ABBREVIATIONS :::::::::::::::::::::::::::::::::: xii ABSTRACT ::::::::::::::::::::::::::::::::::::: xiv 1 INTRODUCTION :::::::::::::::::::::::::::::::: 1 1.1 Mammalian retina ::::::::::::::::::::::::::::: 1 1.2 Development of the retina ::::::::::::::::::::::::: 4 1.3 Retinal glia ::::::::::::::::::::::::::::::::: 5 1.4 Retinal glial functions ::::::::::::::::::::::::::: 7 1.4.1 Regulating glucose metabolism :::::::::::::::::: 7 1.4.2 Regulation of glutamate metabolism ::::::::::::::: 9 1.4.3 Synaptic function :::::::::::::::::::::::::: 9 1.4.4 Development and maintenance of blood retinal barrier ::::: 10 1.4.5 Maintenance of ion homeostasis in the retina ::::::::::: 10 1.5 Microglia ::::::::::::::::::::::::::::::::::: 11 1.6 Reactive gliosis ::::::::::::::::::::::::::::::: 13 1.7 Microglia activation ::::::::::::::::::::::::::::: 16 1.8 M¨ullerglia and microglia interaction in the retina :::::::::::: 17 1.9 Factors in gliosis :::::::::::::::::::::::::::::: 18 1.10 Bone morphogenetic proteins (BMP) in the retina :::::::::::: 20 1.11 Hippo pathway and role in gliosis ::::::::::::::::::::: 21 2 BONE MORPHOGENETIC PROTEIN 7 REGULATES REACTIVE GLIO- SIS IN RETINA ASTROCYTES AND MULLER¨ GLIA ::::::::::: 26 2.1 Introduction ::::::::::::::::::::::::::::::::: 26 vi Page 2.2 Materials and methods ::::::::::::::::::::::::::: 29 2.2.1 Tissue Processing and Fluorescent Immunohistochemistry ::: 29 2.2.2 Isolation of RNA and protein ::::::::::::::::::: 32 2.2.3 Cell Isolation, Cytospin, and Immunocytochemistry ::::::: 33 2.2.4 Cell culture ::::::::::::::::::::::::::::: 34 2.2.5 BMP7 injections in vivo :::::::::::::::::::::: 35 2.2.6 Western blot analysis :::::::::::::::::::::::: 36 2.2.7 Real Time-Quantitative PCR (RT-qPCR) :::::::::::: 36 2.2.8 Statistical Analysis ::::::::::::::::::::::::: 37 2.3 Results :::::::::::::::::::::::::::::::::::: 42 2.3.1 M¨ullerglia express bone morphogenetic protein type IA, IB and activin receptor like kinase 2 receptors :::::::::::::: 42 2.3.2 Bone morphogenetic protein 7 signaling components in the reti- nal glia ::::::::::::::::::::::::::::::: 45 2.3.3 Bone morphogenetic protein 7 can trigger changes in retinal astrocytes and MIO-M1 M¨ullercells resembling mild reactive gliosis :::::::::::::::::::::::::::::::: 45 2.3.4 Intravitreal injection of bone morphogenetic protein 7 induces reactive gliosis :::::::::::::::::::::::::::: 51 2.4 Discussion :::::::::::::::::::::::::::::::::: 60 2.4.1 Regulation of glutamine synthetase during reactive gliosis :::: 60 2.4.2 Bone morphogenetic protein 7 triggers reactive gliosis via the SMAD and the transforming growth factor-β activated kinase pathway ::::::::::::::::::::::::::::::: 61 2.4.3 Differences in response patterns :::::::::::::::::: 63 2.4.4 Extracellular matrix and reactive gliosis ::::::::::::: 65 3 MICROGLIA ACTIVATION IS ESSENTIAL FOR BMP7-MEDIATED RETI- NAL REACTIVE GLIOSIS ::::::::::::::::::::::::::: 67 3.1 Introduction ::::::::::::::::::::::::::::::::: 67 3.2 Methods ::::::::::::::::::::::::::::::::::: 70 3.2.1 Cell culture ::::::::::::::::::::::::::::: 70 vii Page 3.2.2 Experimental groups :::::::::::::::::::::::: 71 3.2.3 Intraocular Injections :::::::::::::::::::::::: 72 3.2.4 Microglia Ablation ::::::::::::::::::::::::: 72 3.2.5 Tissue Processing :::::::::::::::::::::::::: 73 3.2.6 RT-qPCR :::::::::::::::::::::::::::::: 73 3.2.7 Immunofluorescence :::::::::::::::::::::::: 75 3.2.8 Western Blotting :::::::::::::::::::::::::: 76 3.2.9 ELISA :::::::::::::::::::::::::::::::: 78 3.2.10 Retinal Flatmounts ::::::::::::::::::::::::: 78 3.2.11 Statistical Analysis ::::::::::::::::::::::::: 79 3.3 Results :::::::::::::::::::::::::::::::::::: 79 3.3.1 BMP signaling in retinal microglia :::::::::::::::: 79 3.3.2 BMP7 induces inflammatory changes in vivo ::::::::::: 85 3.3.3 Activated microglia secrete factors that induce gliosis :::::: 88 3.3.4 PLX ablates retinal microglia ::::::::::::::::::: 90 3.3.5 Microglial ablation reduces BMP7-mediated gliosis ::::::: 95 3.4 Discussion :::::::::::::::::::::::::::::::::: 97 3.4.1 BMP pathway in retinal disease :::::::::::::::::: 97 3.4.2 Activated microglia drive retinal gliosis ::::::::::::: 100 3.4.3 BMP and inflammation ::::::::::::::::::::: 104 3.4.4 Microglia release inflammatory factors prior to formation of gliosis106 3.4.5 Potential factors regulating microglia mediated activation of M¨ullerglia ::::::::::::::::::::::::::::: 107 4 ROLE OF AMOT-YAP SIGNALING IN REGULATION OF GLIOSIS :: 108 4.1 Introduction :::::::::::::::::::::::::::::::: 108 4.2 Methods :::::::::::::::::::::::::::::::::: 110 4.2.1 Cell culture :::::::::::::::::::::::::::: 110 4.2.2 Intraocular injections ::::::::::::::::::::::: 110 viii Page 4.2.3 Tissue processing and immunofluorescence ::::::::::: 111 4.2.4 Western blotting ::::::::::::::::::::::::: 111 4.2.5 RT-qPCR ::::::::::::::::::::::::::::: 112 4.3 Results ::::::::::::::::::::::::::::::::::: 113 4.3.1 AMOTs and YAP are expressed in the glial cells in the retina 113 4.3.2 AMOT and YAP upregulated during IFN-γ induces gliosis :: 113 4.3.3 Verteporfin treatment decreases RNA levels of YAP/TEAD downstream targets :::::::::::::::::::::::: 116 4.3.4 Effect of IFN-γ on retinal gliosis in presence of verteporfin :: 116 4.4 Discussion ::::::::::::::::::::::::::::::::: 119 5 DISCUSSION :::::::::::::::::::::::::::::::::: 124 5.1 Future directions ::::::::::::::::::::::::::::: 130 REFERENCES ::::::::::::::::::::::::::::::::::: 133 VITA :::::::::::::::::::::::::::::::::::::::: 157 ix LIST OF TABLES Table Page 2.1 List of antibodies used ::::::::::::::::::::::::::::: 31 2.2 Mouse RT-qPCR primers ::::::::::::::::::::::::::: 38 2.3 Human RT-qPCR primers ::::::::::::::::::::::::::: 40 3.1 List of RT-qPCR primers ::::::::::::::::::::::::::: 74 3.2 List of antibodies used ::::::::::::::::::::::::::::: 77 4.1 List of RT-qPCR primers :::::::::::::::::::::::::: 112 x LIST OF FIGURES Figure Page 1.1 Vertebrate eye and the retina. ::::::::::::::::::::::::: 2 1.2 Photoreceptor cell and light activation. :::::::::::::::::::: 2 1.3 Projections of the retinal ganglion cells. ::::::::::::::::::: 4 1.4 Development of the vertebrate eye. :::::::::::::::::::::: 6 1.5 Functions of the M¨ullerglia. :::::::::::::::::::::::::: 8 1.6 Functions of microglia. ::::::::::::::::::::::::::::: 12 1.7 Reactive gliosis in the M¨ullerglia. ::::::::::::::::::::::: 14 1.8 Changes in microglia following activation. :::::::::::::::::: 17 1.9 Known factors regulating gliosis. ::::::::::::::::::::::: 19 1.10 BMP pathway: canonical and non-canonical signaling. ::::::::::: 22 1.11 Hippo pathway and its regulation. ::::::::::::::::::::::: 24 2.1 Bone morphogenetic protein (BMP) type I receptors in the mature mouse retina. :::::::::::::::::::::::::::::::::::::: 44 2.2 Bone morphogenetic protein (BMP) signaling components in the M¨uller glia of adult mouse retina. ::::::::::::::::::::::::::: 46 2.3 Bone morphogenetic protein 7 (BMP7) treatment of retinal astrocyte cells increases markers of glial scar formation. ::::::::::::::::::: 49 2.4 Bone morphogenetic protein 7 (BMP7) treatment of MIO-M1 M¨ullerglial cell line increases glial fibrillary acidic protein (GFAP) expression. ::::: 53 2.5 Retinal astrocytes and MIO-M1 cells show an attenuated response to bone morphogenetic protein 4 (BMP4). ::::::::::::::::::::::: 54 2.6 Canonical bone morphogenetic protein (BMP) signaling is activated in the M¨ullerglia in BMP7-injected murine eyes. ::::::::::::::::: 56 2.7 Non-canonical bone morphogenetic protein (BMP) signaling mediated via