ROLE OF CHEMOKINES IN REGULATING OLIGODENDROCYTE DEVELOPMENT, ASTROGLIOSIS, AND DEMYELINATING DISEASES by AMBER E. KERSTETTER-FOGLE Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. Robert H. Miller Department of Neuroscience CASE WESTERN RESERVE UNIVERSITY January, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Amber E. Kerstetter-Fogle________________________ candidate for the _________Ph.D.__________degree *. (signed)______Jerry Silver________________________ (chair of the committee) ___________ Robert H. Miller______________________ __________ Ruth Siegel_________________________ ___________Richard Zigmond_____________________ ______________________________________________ ______________________________________________ (date) __October 26, 2009_____________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright © 2010 by Amber E. Kerstetter-Fogle All rights reserved This work is dedicated to my husband, Gary D. Fogle Jr. This is as much an accomplishment of his as it is mine. He has given up so much for me to get where I am today. I am thankful and greatful for the support and love he has given me all these years. TABLE OF CONTENTS List of Figures 3 Preface 5 Acknowledgements 6 List of Abbreviations 8 Abstract 10 Chapter 1 Background and Introduction I. Cellular composition of the nervous system 12 a. Astrocytes role in central nervous system function 13 b. Microglia: the primary immune defense in the CNS 15 c. Oligodendrocyte development and unique features 17 II. Inflammatory reaction and pathology in the CNS 21 a. Cytokines and their responsibility in inflammatory response 22 b. Chemokines and cytokines in development and disease 23 c. CXCR2 function and role in oligodendrocyte development and pathology 25 III. Demyelinating disorders and Multiple Sclerosis 27 a. Current MS treatments modulate symptoms and inflammation 31 b. Experimental models of Multiple Sclerosis 32 c. Remyelination success and failure in MS 36 IV. Primary hypothsis of this dissertation 38 Chapter 2 Inhibition of CXCR2 signaling promotes recovery in models of Multiple Sclerosis I. Abstract 46 II. Introduction 47 III. Materials and methods 50 IV. Results 55 V. Discussion 62 Chapter 3 Regulation of astrogliosis by the chemokine CXCL1 I. Abstract 82 1 II. Introduction 84 III. Materials and methods 87 IV. Results 94 V. Discussion 100 Chapter 4 Discussion and Future Directions I. Overview 112 II. Astrogliosis may confer reason for recovery in animal models of demyelination 114 III. Neuroprotection elicited by inhibition of CXCR2 in demyelinating disorders 115 IV. Chemokine receptor/ligand promiscuity of binding 117 V. The contribution of the immune system to models of demyelination and potential targets for therapeutics 118 VI. Modulation of blood brain barrier and inflammation 121 VII. Expression of CXCL1/CXCR2 and signaling in vivo 122 VIII. Downstream signaling components of CXCR2 123 IX. Stability of antagonists 124 X. Alternate chemokine targets alone or in combination with CXCR2 inhibitors 126 XI. Ex vivo application of brain lesions may be helpful in understanding role of T cells and other cells outside the CNS 127 XII. Other demyelinating disease models and the efficacy for CXCR2 inhibitors 128 XIII. Conclusions and potential of CXCR2 inhibitors for the treatment of demyelinating disorders 130 Bibliography 133 2 LIST OF FIGURES Figure 1.1 Cell lineage of astrocytes and oligodendrocytes in the central nervous system 40 Figure 1.2 Saltatory conduction associated with myelination and clustering of sodium channels promoting efficient axonal transduction 41 Figure 1.3 Oligodendrocyte specification occurs in the ventral spinal cord and cell proliferation is conducted by locally derived signals by astrocytes 42 Figure 1.4 Canonical CXCL1/CXCR2 signaling resulting in modulation of proliferation, differentiation, and migration in a number of cell types 43 Figure 1.5 Disability progression in different types of Multiple Sclerosis 44 Figure 1.6 Model of CXCL/CXCR2 modulation in reducing immune mediated pathology and enhancement of migration and differentiation of oligodendrocyte progenitor cells 45 Figure 2.1 Paradigm for local injection of antibody or small molecule inhibitor after lysolecithin lesion 67 Figure 2.2 Structure of small molecule inhibitor against CXCR2 68 Figure 2.3 Lesion volume quantification of lysolecithin lesions 69 Figure 2.4 Myelin thickness/axonal diameter measurements in EAE and lysolecithin lesions 70 Figure 2.5 Local delivery of anti-CXCR2 antibodies reduces the size of LPC induced demyelinating lesions 71 Figure 2.6 Local delivery of CXCR2 antagonists enhances remyelination in LPC lesions 73 Figure 2.7 Systemic delivery of CXCR2 antagonists has limited effect on repair of LPC lesions 74 Figure 2.8 Inhibition of CXCR2 promotes the differentiation of spinal cord OPCs in vitro 75 Figure 2.9 Systemic inhibition of CXCR2 results in functional 3 improvement in MOG35-55 peptide induced EAE 76 Figure 2.10 Systemic inhibition of CXCR2 results in decreased cell infiltration and increased remyelination in MOG35-55 induced EAE 78 Figure 2.11 Systemic treatment with CXCR2 antagonists results in increased MBP and decreased Iba1 expression in EAE animals 80 Figure 2.12 Long term but not short term treatment with CXCR2 antagonist results in sustained remyelination 81 Figure 3.1 CXCR2 and CXCL1 mRNA is expressed by purified astrocytes in vitro 104 Figure 3.2 CSPG protein is secreted and expressed by astrocytes in response to CXCL1 treatment 105 Figure 3.3 CXCL1 treatment increases the number and protein levels of GFAP in astrocyte cultures 106 Figure 3.4 Cytokine profile in astrocyte conditioned media treated with CXCL1 demonstrate an upregulation of inflammatory mediators and migratory signals 107 Figure 3.5 CXCR2 protein is expressed in the periphery 3 days post LPC demyelination 109 Figure 3.6 The chemokine CXCL1 is upregulated around LPC lesions 3 days post demyelination and CXCR2 is expressed by GFAP expressing cells within the lesion 110 Figure 3.7 Constant intrathecal delivery of CXCL1 enhances GFAP immunohistochemistry suggesting an astrogliotic response in the spinal cord 111 Figure 4.1 Lysolecithin lesion in nude rats are decreased compared to control animals as indicated by histology 132 4 PREFACE The goal of the research described here is to define the role of CXCR2 chemokine receptor signaling in respect to development and repair of demyelinating disorders of the central nervous system. The focus of this work is specifically on the role of glia, astrocytes and oligodendrocytes, on processes involving myelin generation during development and disease. This thesis characterizes the methods of development of myelination, demyelination, and remyelination and addresses the roles of the chemokine receptor, CXCR2 and its primary ligand CXCL1 in these processes. The studies utilize a CXCR2 inhibitor in demyelinating disorder similar to Multiple Sclerois in which I demonstrate that repair is enhanced. Further, I show that CXCR2 modulates functions related to oligodendrocyte differentiation, microglial activation, and astrogliosis. Treatment of multiple models of demyelination with CXCR2 inhibitors promotes function recovery and remyelination and reduces immunological attacks. Additionally, treatment of astrocytes with the ligand, CXCL1, promotes astroliosis and may be impeding repair in demyelination. The results of this research suggest that inhibitors to CXCR2 are attractive candidates for therapeutic tools for the treatment of demyelinating disorders as they modulate astrogliosis, microgliosis, lymphoid cell entry and most importantly, oligodendrocyte maturation. 5 ACKNOWLEDGEMENTS This work was conducted in the Department of Neurosciences at Case Western Reserve University in Dr. Robert H. Miller’s laboratory. Support of this work was by the NIH and the Myelin Repair Foundation. The pioneers of this work include Dr. Shenandoah Robinson, Dr. Hui-Hsin Tsai, Dr. Dolly Padovani- Claudio and Dr. Robert Miller. Without their contributions and the Miller laboratory I would not have been able to complete this body of work. I must thank my current mentor, Dr. Robert Miller, for the freedom he allowed me in pursuing this project and allowing me to develop into an independent researcher. I envy his knowledge and expertise in a number of fields. His balance between work and family are something I hope to emulate in the future. The Miller cohort of people also aided me in development of my research and I value the friendships I have made. I must specifically thank, Sara Vandomelen for helping me with scheduling and being a support system for me. Additionally, Anne Dechant for also helping me with my development as a researcher. Lianhua Bai for helping me learn immunology, against my will, and being a great lab mate in the afterhours times, when it really matters. Anita Zaremba was a great person to work with and helped me iron out some of the issues that go along with tissue culture. I thank Molly Fuller for being a friend and great ally in the laboratory. I thank Steve Selkirk for being a mentor and helping guide me in my decisions as a researcher. Saisho Mangla was instrumental in helping me complete some experiments and aiding in my psychological outlook on the politics associated with science. He was also a great friend to me. Yee-Hsee Hsieh was a very