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

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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

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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

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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

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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.

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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 good friend to me and helped keep me grounded and aiding in the final stages for my thesis defense. Without Saisho and Yee-Hsee’s support, I would have had a very difficult and lonely time in the lab.

Other collaborators outside the lab also made it essential for completion of this body of work. Midori Hitomi helped me in completing the electron microscopy work and teaching me techniques associated with this essential part of studying remyelination. People of the Myelin Repair Foundation, who are one of the laboratory’s major funding sources, also contributed to driving me to completion of CXCR2 inhibitors for therapies for demyelinating diseases.

This would not be complete without acknowledging my thesis committee, Jerry Silver, Ruth Siegel, and Richard Zigmond. Every committee meeting was very eventful and I enjoyed seeing their perspective. They were also essential in keeping me on track and helping me develop my project. I cherish their feedback and thank them for contributing to my development as a scientist. Special thanks to Jerry Silver for aiding in the publishing of my Exp Neurol paper. His door was always open and his expertise is valued.

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I must also thank Dr. Alison Hall for her guidance and support the past five years. She provided a lot of guidance during my completion of my thesis work. She is an admirable woman and she provides a great example as to how one becomes successful in science without compromising family. I hope to follow in her footsteps and if I could be half the woman she is I will be thankful.

My family and friends have also been my support system and helping me complete my studies. My parents, Keith and Theresa Kerstetter, helped drive me and keep me grounded. My brothers, Jake and Jerod, I thank for being a support system and always reminding me to breathe. I thank my sisters-in-laws, Jessica and Chalynne, for always being understanding and good listeners. Kelly, Kim and Joan and Merlin, also family members, also helped guide me along the way and helped me see the light at the end of the tunnel. Although, I know my family and friends did not always comprehend what I was going through, they did their best. I love them and am greatful to them for making me who I am today.

Finally, I must thank my husband, Gary Fogle, for always being there and loving me every step of the way. He always made our home and being with him my respite. He has given up a lot for me to pursue my dream, which became his dream. This thesis is dedicated to him. I am greatful for his support, unconditional love, patience, attention, compassion, and understanding. I could not have wished for a better partner for life. I look forward to our future together and our new life.

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LIST OF ABBREVIATIONS

APC antigen presenting cells BBB blood brain barrier BMP bone morphogenetic protein bp base pairs BrdU bromodeoxyuridine CNS central nervous system CSF cerebral spinal fluid DC dendritic cells DMEM dulbecco’s modified eagle’s medium cDNA complementary DNA CNTF ciliary neurotrphic factor CNP 2’3’-cyclic nucleotide phosphodiesterase CSPG chondroitin sulfate proteoglycan, CS-56 CXCL1/Groα/IL8/CXCL8 Growth related oncogene alpha/CINC CXCL2/MIP2α macrophage inflammatory protein 2 alpha CXCL10/IP10 interferon-gamma induced protein DAB 3’,3’-diaminobenzidine DAPI 4’,6’-diamindino-2-phenylindole EAE experimental autoimmune encephalomyelitis ECM extracellular matrix molecules EDTA ethylenediaminetetraacitic acid ELR glutamate-lysine-arginine EM electron microscopy ERK extracellular signal regulated kinase FBS fetal bovine serum FGF-2 fibroblast growth factor-2 GFAP glial fibrillary acidic protein GPCR G-protein coupled receptor Groα CXCL1/growth related oncogene alpha/CINC HCl hydrochloric acid HRP horseradish peroxidase IACUC Institutional animal care and use committee IFN interferon IGF insulin-like growth factor IL interleukin IP intraperitoneal JAK janus kinase JNK c-Jun N-terminal kinases kDa kilodaltons LFB Luxol fast blue LIF leukemia inhibitory factor LPC lysophosphatidyl choline, lysolecithin mAb monoclonal antibody MAG myelin associated glycoprotein

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MBP myelin basic protein MCP-1/CCL2 monocyte chemotactic protein MEM minimum essential medium MIG/CXCL9 monokine induced by gamma interferon MMP matrix metalloproteinases MOG myelin oligodendrocyte glycoprotein MS Multiple Sclerosis NCAM neural cell adhesion molecule NGS normal goat serum OPC oligodendrocyte precursor/progenitor cell OsO4 osmium tetraoxide PBS phosphate buffered saline PBST PBS with Triton X-100 PDGF platelet derived growth factor PDGFRα platelet derived growth factor receptor alpha PFA paraformaldehyde PI3K phosphatidylinositol-3-kinase PLC phospholipase C PLP proteolipid protein PNS peripheral nervous system PLL poly-L-lysine PPMS primary progressive MS PT pertusssis toxin RANTES/CXCL5 regulated upon activation, normal T cell expressed, and secreted RRMS relapsing remitting MS RT-PCR reverse transcriptase polymerase chain reaction SDF-1/CXCL12 stromal cell derived factor-1 SEM standard error of the mean sICAM soluble intercellular adhesion molecule SPMS secondary progressive MS STAT signal transducers and activators of transcription TBS tris-buffered saline TBST tris-buffered saline with tween 20 TGFβ transforming growth factor beta TIMP-1 tissue inhibitor of metalloproteinase-1 TNF tumor necrosis factor VCAM-1 vascular cell adhesion molecule-1

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Role of Chemokines in Regulating Oligodendrocyte Development,

Astrogliosis and Demyelinating Disorders

Abstract

by

AMBER E. KERSTETTER-FOGLE

Oligodendrocyte development and maturation is crucial for efficient axonal transduction in the central nervous system. When perturbations in myelination occur such as with Multiple Sclerosis functional deficits rapidly follow. Previous studies demonstrated that local expression of CXCL1 in astrocytes of the spinal cord promotes oligodendrocyte proliferation and inhibits PDGF mediated migration by signaling through CXCR2 on oligodendrocyte precursor cells

(OPCs). Inhibiting CXCL1/CXCR2 signaling in EAE and after lysolecithin injection, models of demyelination, promotes functional recovery, remyelination, and decreases in astrogliosis and microglial activation. CXCL1 treatment of astrocytes enhances secretion of proinflammatory cytokines and chondroitin sulfate proteoglycans that likely retard the process of repair. We hypothesis that decreasing CXCR2 signaling decreases immune cell infiltration, decreases astrogliosis and factors secreted downstream of the astrogliosis response, releasing OPCs from migratory arrest, enhancing endogenous OPC differentiation, leading to remyelination after injury. This enhancement in remyelination promotes functional recovery in EAE and increased MBP

10 expression and myelin thickness consistent with remyelination. Thus the inhibition of CXCR2 enhances CNS repair suggestin this receptor is a therapeutic target for the treatment of demyelinating disorders, such as Multiple Sclerosis

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CHAPTER 1: BACKGROUND AND INTRODUCTION

Cellular composition of the Central Nervous System

The vertebrate central nervous system is comprised of two major classes of cells- neurons and glia. Glia make up the supportive structure of the brain and spinal cord. The term glia refers primarily to astrocytes and oligodendrocytes that are instrumental in regulating aspects of homeostasis within the brain and spinal cord and make up the preponderance of the cells in the nervous system.

Neurons of the nervous system are essential for sending electrical signals throughout the brain to help us move and think in everyday life. Neurons are highly specialized cells that have processes and channels essential for signal propagation from neuron to neuron. Neurons have processes called dendrites that in general receive input from other neurons and convey messages across other processes called axons. The axons and dendrites allow for electrical signals to transmit from neuron to neuron through synapses formed by neighboring neurons, such that axons end presynaptically and dendrites post synaptically to transmit signals. There are variations in the populations of neurons in the brain, and they differ in morphology and their ability to transmit signals via electrical activity and release of different chemicals called neurotransmitters. Neuronal populations also vary in the functions they serve.

Neurons may be excitatory or inhibitory depending upon the type of neurotransmitter they release upon excitation. The rate of conduction of axons depends, in part, on the diameter of the axon. For example, the giant squid axon, formed through the fusion of many axons, allowed for ease of study of the

12 neuronal signaling and an essential model utilized in early neuroscience investigations. In the mammalian nervous system, neurons would have to be very large cells with very long and large diameter axons if there weren’t supportive glia. Giant squid axons must be large for the production of slow undulating movements through the water conversely; vertebrates must have small and fast neuronaltransmission to allow for quick propagation and signaling from neuron to neuron.

The term glia refers to four distinct cells- oligodendrocytes in the CNS,

Schwann cells in the PNS, astrocytes, and microglia. Oligodendrocytes allow for insulation along the axon producing myelin and one oligodendrocyte can wrap many axons in the CNS. Schwann cells are also insulators but only insulate one portion of an axon in the PNS. Astrocytes are supportive glia and maintain homeostasis in the nervous system. Microglia, which are derived from macrophages, act as immune cells in the CNS (Aloisi, 2001). Glia cells arise from the neuroepithelium (Soula et al., 1990). Gliogenesis, the development and maturation of glia, occurs within the first two weeks after birth. Glial progenitors migrate extensively through the brain and spinal cord parenchyma where they eventually differentiate into oligodendrocytes and astrocytes. Migration, proliferation and eventual differentiation take place using a closely monitored program, although it is not fully understood.

Astrocytes role in CNS function

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Astrocytes have been proposed to perform many functions in the developing and adult CNS. Astrocytes are classically defined as cells that express the intermediate filament glial fibrillary acidic protein (GFAP) (Fig 1.1)

(Chiu et al., 1981; Bullon et al., 1984; Jessen et al., 1984; Eng, 1985). Stellate- shaped astrocytes are important for the formation and support of the blood brain barrier via their end feet enveloping endothelium (Abbott et al., 1992; Xu and

Ling, 1994; Nakazawa and Ishikawa, 1998; Abbott, 2002). They also have recently been characterized as not only supportive glial cells but also have the ability to propagate calcium signals allowing glia-glia communication (Kang et al.,

1998; Blomstrand et al., 1999; Parri et al., 2001). The many fine processes of astrocytes are found to envelope the synapses of neurons and form many networks within the CNS (Butt et al., 1994; Gallo and Chittajallu, 2001).

Astrocytes are also important for the structuring of the brain, maintaining efficient synaptic function, metabolism and , responding to pathology or disease to maintain homeostasis in the nervous system by sequestering areas of injury

(Berry et al., 1995; Araque et al., 1999; Bacci et al., 1999; Benarroch, 2005).

Astrocytes also react to injury via upregulation of GFAP, proliferation, process expansion and a set of reactions that may or may not be detrimental to repair termed astrogliosis (Nieto-Sampedro et al., 1985; Eng and Ghirnikar, 1994;

Guenard et al., 1996; Sykova et al., 1999; Little and O'Callagha, 2001).

Astrocytes form a fibrous scar that may impede regeneration (Fitch and Silver,

2008).

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Reactive gliosis or astrogliosis involves the hypertrophy of cellular processes of astrocytes, proliferation, upregulation of glial fibrillary acidic protein

(GFAP) and vimentin, re-expression of nestin and formation of scars (Eng and

Ghirnikar, 1994; Conrad et al., 2005). These astrocytic scars form a dense network of hypertrophic cells which is accompanied by a number of genetic changes in cell adhesion molecules, antigen presentation, cytokines, growth factors, proteases inhibitors and enzymes(Canning et al., 1996; Fitch and Silver,

2008). There are a number of heterogeneous cellular events in an astrogliotic scar that are not well understood. Astrocyte activation has been suggested to be both beneficial and detrimental to CNS recovery after pathology or insult.

Microglia: the primary immune defense cells in the CNS

Microglia cells are smaller star shaped cells which act as resident immune cells in the CNS (Gehrmann et al., 1995; Aloisi, 2001). Microglia are CNS resident bone marrow derived cells (Lassmann et al., 1993; Rinner et al., 1995;

Eglitis and Mezey, 1997; Ono et al., 1999). The macrophage/microglia lineage is the known mediator of CNS inflammation. Microglia survey and move around the

CNS analyzing areas for damage, infection, or plaques (Gehrmann, 1996). In cases when inflammation occurs, the resident immune cells, in addition to dendritic cells (DC), must respond rapidly and recognize a potential detrimental agent. Blood borne dendritic cells occupy a unique role in recognizing antigens and acting as antigen-presenting cells and are one of the first defenses in inflammation. Recently it has been described that the state of

15 maturation/activation of dendritic cells and their ability to act as antigen presenting cells in inhibiting or activating a T cell response is essential for autoimmunity (Weiner, 2008). DC activation and migration are known to be controlled by chemokines and other inflammatory mediators (Gottenberg and

Chiocchia, 2007). Activation of microglia can occur via a number of modalities, such as inflammation or damage to cells, and microglia display different responses dependent upon the inflammatory mediator. Microglia represent 5-

20% of the adult CNS cells and are the equivalent of tissue macrophages (Aloisi,

2001). Functional states of microglia can vary consisting of a resting state and an activated state. Microglial cells in a resting state have small cell bodies but weakly express epitopes of the hematopoetic cell linage and activated microglia have epitopes that are highly upregulated and cells exhibit shorter processes and proliferate rapidly (Rezaie and Male, 1999; Stoll and Jander, 1999). Microglia are sensitive to identifying infectious and damaging agents and remove pathogens via endocytosis (Kadiu et al., 2005; Ponomarev et al., 2005;

Wirenfeldt et al., 2005). Microglial cells also respond to autoimmune inflammation by either acting as an antigen presenting cell and/or through secretion of chemokines or cytokines (Benveniste, 1997; Carson et al., 1998).

Macrophage and microglia may seem detrimental to remyelination or axonal regeneration however, they also play an essential role. Macrophage depletion in experimental models of disease or injury resulted in reduced oligodendrocyte mediated repair and also alterations in growth factors essential for recovery

(Kotter et al., 2005).

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Oligodendrocytes: development and unique features

Oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system are specialized cells important for myelination and the modulation of fast axonal signaling. Myelin is insulation around axons important for efficient axonal conduction. Successful myelination involves many wrappings of the glial cell membrane around the axon with little or no cytoplasm between each wrap (Remahl and Hildebrand, 1990). The insulation is placed around axons in a ordered manner which areas lacking specialized insulation by myelin known as the Nodes of Ranvier (Hildebrand et al., 1993; Sherman and Brophy,

2005; Rasband, 2006). Nodes of Ranvier allow for a clustering of sodium channels which causes a leaping of action potentials along the length of axon known as saltatory conduction (Joe and Angelides, 1993; Kaplan et al., 2001;

Nashmi and Fehlings, 2001). Unmyelinated axons have uniform distribution of sodium channels which must open in response to depolarization in the axon and the signal must be propagated along the length of the axon (Fig 1.2). The interaction between oligodendrocytes and the axons they myelinate is thought to be activity dependent. Activity in axons may assist in stimulating the release or production of mitogens from the neurons themselves or other surrounding cells, such as astrocytes (Barres et al., 1993; Barres and Raff, 1993, , 1999; Bjartmar et al., 2003)

For myelination to be successful, the progenitors must proceed through a number of sequential steps of maturation. Differentiation of oligodendrocytes has been extensively studied. These studies rely upon oligodendrocytes proliferative

17 capacity and cell morphology, in addition to the expression of antigens which mark discreet phases of development. Central nervous system myelination is generated during the first few postnatal weeks in rodents (Miller, 2002).

Oligodendrocytes arise from the ventral ventricular zone of the developing neural tube where they migrate and proliferate in presumptive white matter regions of the spinal cord (Fig 1.3) (Baumann and Pham-Dinh, 2001; Miller, 2002). In more rostral regions of the CNS, the oligodendrocytes arise from the ventricular and subventricular zones, with the exception of the telencephalonic regions where oligodendrocytes arise from the lateral and medial ganglion eminence.

Oligodendrocytes are particularly vulnerable to injury and disease and loss will lead to demyelination of axons and decrease in conduction velocity.

Oligodendrocytes are easily identifiable by their small round, sometimes oval, nuclei and location within white matter tracts and gray matter neurons in the

CNS. Ramon y Cajal first identified this neuroglia cell with multiple processes as oligodendrocytes based upon metal impregnation techniques. Lucifer yellow intracellular injection further identified these multiple process cells as oligodendrocytes (Butt and Ransom, 1989). Electron micrography identified variable clumped chromatin, dense cytoplasm and round/oval nuclei to be that of oligodendrocytes. Oligodendrocytes contain many mitochondria and no intermediate filaments (characteristic of astrocytes)(Peters, 2004). The most widely used method for labeling and identifying oligodendrocytes is immunohistochemistry, using a range of antibodies distinguishing surface and intracellular markers to recognize developmental stage specific markers.

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Classifying glia cells in culture using oligodendrocyte markers in a stage specific manner is beneficial as is the identification of oligodendrocytes in an injury or pathological setting. The oligodendrocyte markers most influential in recongnizing oligodendrocytes specifically in culture are the monoclonal antibodies A2B5, O4, and O1 (Fig 1.1). Oligodendrocytes are first known to be

O2A progenitor cells and are identifiable by their expression of A2B5 and

PDGFRα. Late and postmitotic oligodendrocyte progenitor markers are identifiable by cell surface expression of O4 and O1, respectively (Baracskay et al., 2007). The chondroitin sulfate proteoglycan NG2 (Baracskay et al., 2007) detects early and adult oligodendrocyte progenitors. Mature oligodendrocytes and myelin sheets can be identified with antibodies against myelin basic protein

(MBP), proteolipoprotein (PLP), myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) (Yamamura et al., 1991).

Identification of mature oligodendrocytes intracellularly involves antibodies for

RIP, CC1 and 2’,3’- cyclic nucleotide 3’phosphodiesterase (CNPase) (Ness et al.,

2005). Classification of particular stages of oligodendrocyte development allows for cell purification as well as identification.

Migration of oligodendrocytes is controlled by extracellular matrix in addition to metalloproteinases (MMPs) (Uhm et al., 1998). Elevated levels of

MMPs coincide with tissue rich in oligodendrocytes and myelination (Larsen et al., 2003; Larsen et al., 2006; Werner et al., 2008). Migration of oligodendrocytes is also controlled by polysialylated forms of NCAM (PSA-NCAM), an extracellular matrix protein (Decker et al., 2000). Knockdown of PSA-NCAM retards dispersal

19 of oligodendrocytes to proper locations (Charles et al., 2000). A number of growth factors are involved in the initial stages of myelination in development and steps may be recalled as a result of injury or disease. These factors include mitogens, chemokines, cytokines, and extracellular matrix molecules and some are reviewed below. Oligodendrocyte precursor cells will proliferate in response to PDGF-AA and basic FGF, as demonstrated by a number of in vitro studies

(Fok-Seang et al., 1998; Miller, 2002; Kadi et al., 2006). PDGF-AA production during development occurs in neurons and astrocytes and is a survival and mitogenic factor to oligodendrocyte precursor cells (Miller, 2002). Basic FGF or

FGF-2 has been found to work synergistically with PDGF-AA for the modulation of migration and proliferation of oligodendrocyte progenitors (Fok-Seang et al.,

1998; Woodruff et al., 2004). Understanding recruitment and differentiation of oligodendrocytes is to key to modulating pathological events. Insulin-like growth factor 1 (IGF1) has also been found to have involvement in oligodendrocyte precursor cell migration and proliferation (Hinks and Franklin, 1999). IGF1 is found in the subventricular zone, an area where oligdendrocyte specification occurs. However, there may be a context-dependent manner of modulation of oligodendrocyte proliferation and subsequent myelination. Additionally, neuregulin has been observed to inhibit the differentiation of oligodendrocyte progenitor cells due to lack of expression of mature markers of differentiated cells

(Barres and Raff, 1999; Canoll et al., 1999). When myelinating oligodendendrocytes come within proximity to an unmyelinated axon, the axonal surface expression of the ligand Jagged and oligodendrocytes expression of the

20 receptor Notch will inhibit differentiation of oligodendrocytes (Barres and Raff,

1999; Dubois-Dalcq and Murray, 2000; Hu et al., 2004). Downregulation of

Jagged must be achieved prior to wrapping of myelin and formation of sheaths around axons however, this is not entirely clear as to how and when this occurs

(Rogister et al., 1999).

Inflammatory reaction and pathology in the CNS

Inflammation in the CNS is characterized by a number of events and involves the activation of microglia/macrophages, astrocytes, and lymphocytes and phagocytosing debris within the CNS. In the case of demyelinating diseases, myelin becomes degraded in response to inflammation(Becher et al.,

2006; McFarland and Martin, 2007). T lymphocyte activation is present in all multiple sclerosis patients and recapitulated in a number of animal models of multiple sclerosis. Development of T cells results in a change in T cell receptor gene expression and CD3+ T cells express coreceptor complexes, either CD4 or

CD8, on mature T cell surfaces, which exhibit different functions upon activation.

Destruction of damaged tissue has been proposed to protect the propagation of disease. Secondary inflammation has been found to promote single cells survival or tissue reconstruction and repair and this may also be mediated by scar formation (Frohman et al., 2008). Consequently, activation of resident immune cells in the CNS does have beneficial effects to remyelination promotion

(Bauer et al., 1995; Benveniste, 1997; Aloisi, 2001). There are at least three sites of immune cell infiltration into the CNS, despite the thought of the CNS as

21 being immunopriviledged. These are migration from the blood into cerebral spinal fluid via meningeal vessels or choroid plexus or from blood to perivascular space of the brain or spinal cord (Cayrol et al., 2008). Inflammation has beneficial and detrimental effects depending on the context and persistence of inflammatory reactions.

Cytokines and their responsibility in the inflammatory response

Cytokines regulate a number of events in response to inflammation in numerous systems. Cellular responses, such as migration, proliferation, and differentiation, are mediated via the secretion of cytokines by both immune and non-immune cells. Upon stimulation by inflammation mediated by injury, autoimmunity or infection, cytokines become upregulated in the primary cells in the immune system, including macrophages and lymphocytes (Bartholdi and

Schwab, 1997). Inflammation mediates secretion of pro-inflammatory cytokines such as TNFα, IFNγ, and interleukin-23 (Juedes et al., 2000). This secretion may directly affect myelin structure or activate macrophages to release nitric oxide, reactive oxygen species, or matrix metalloproteinases (MMPs) (Merrill and

Benveniste, 1996; Tran et al., 2000; Ben-Hur et al., 2006). B lymphocytes may also play a role by terminally differentiating in response to cytokines and then releasing myelin specific antibodies, inducing demyelination by complement

(Williams et al., 1994; Cudrici et al., 2006; Klawiter and Cross, 2007).

A number of cytokines are important for CNS function and development.

Astrocytes, microglia, and OPCs express IL1β constitutively, and it is expressed

22 highly in the developing CNS and downregulated in adulthood (Peterson et al.,

1997; Neumann, 2001; Ferrari et al., 2004). After brain injury in IL1β knockout animals, the brain failed to remyelinate over time (Mason et al., 2001). TNFα is a known proinflammatory mediator; however, it is highly expressed in the embryonic CNS. TNFα mutant mice have impaired remyelination in a toxin- induced demyelination model(Bachmann et al., 1999). Astrocytes and oligodendrocytes are known expressers of TNFα-R I(Barnes et al., 1998).

Chemokines and cytokines in development and disease

Migration and proliferation of oligodendrocytes and maturation into myelinating cells may be controlled by certain specified growth factors and differentiation by another set of growth factors mentioned above, however, this remains to be proven. In vitro studies have shown there are a number of cytokines that may modulate survival. Leukemia inhibitory factor (LIF)

(Butzkueven et al., 2002; Azari et al., 2006; Butzkueven et al., 2006) and ciliary neurotrophic factor (CNTF) (Barres et al., 1996; Vos et al., 1996) promote oligodendrocyte survival, and LIF may enhance myelination as well (Mayer et al.,

1994; Park et al., 2001). Knockout animals for the heterotrimeric receptor complexes of LIF and CNTF have found a worsening of pathology in animal models of inflammatory demyelination (Bugga et al., 1998; Giess et al., 2002).

Another variety of inflammatory mediators are known as chemokines or chemotactic cytokines. These small molecular weight (8-14kD) secreted molecules also are triggered by proinflammatory cytokines in response to

23 inflammation (Huang et al., 2000). The tertiary structure of chemokines is highly conserved in spite of their low sequence homology. The originally described role of chemokines was to direct leukocytes to areas of inflammation (Charo and

Ransohoff, 2006). They also have important roles in controlling cell migration within tissue during development, angiogenesis and tissue repair. It has additionally been found a number of chemokines are expressed in neurons

(Horuk et al., 1997) and glia (Jiang et al., 1998) in the central nervous system

(Hesselgesser and Horuk, 1999). Chemokines are generally classified according to the sequence organization of their four cysteine residues near the N-terminus which form two disulfide bonds (Fernandez and Lolis, 2002). These subfamilies consist of CC, CXC, CX3C and C (Laing and Secombes, 2004). A chemokine ligand is denoted with the “L” nomenclature, and the receptor is indicated with an

“R”. Signaling is mediated through G-protein coupled receptors that have seven transmembrane domains (Fig1.4) (Laing and Secombes, 2004). Each receptor has a relative affinity for different chemokines within its subfamily (Laing and

Secombes, 2004). However stimulation may occur more readily with one chemokine versus another. This promiscuity of receptor-ligand interaction may be protective mechanism and also for a larger variation in downstream signaling events. There is additional evidence for chemokine involvement in the modulation of CNS disease (Bajetto et al., 2002). Chemokine function in the nervous system is to modulate proliferation, differentiation, chemotaxis, angiogenesis, and cell adhesion events of a number of cell populations (Brown,

2002).

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Chemokine receptor function in physiology and pathology by receptors such as CXCR2, CXCR3, CCR3, and CXCR4 has recently been studied.

Guidance of growth cones in the retina, motor axon pathfinding, sensory neuron progenitor migration, and limb innervation are known to be modulated via CXCR4 signaling (Chalasani et al., 2007). Cells expressing CXCR4 can give rise to neurons, astrocytes and oligodendrocytes (Ni et al., 2004). CXCL12 signaling via

CXCR4 is crucial for CNS development, since in knockout animals brain anomalies are present due to disruption in cortical neuron migration (Bonavia et al., 2003; Calderon et al., 2006)

Chemokines such as CXCL9 and CXCL10, the ligands for CXCR3, are known to be upregulated after traumatic brain injury (Huang et al., 2000).

CXCR4/SDF1 guides neural precursors to sites of injury in the CNS

(Dziembowska et al., 2005). CXCL12 has been found to be upregulated in astrocytes and blood vessels in demyelination in MS (Krumbholz et al., 2006;

McCandless et al., 2008). In totality, CXC chemokines play a vital role in development of white matter and reactivity of inflammatory mediators and are important for maintenance of normal physiological conditions in the CNS.

CXCR2 function and role in oligodendrocyte development and pathology

CXCR2 signaling has been studied using leukocyte trafficking as a basis for investigation in the central nervous system. CXCR2 is linked to pertussis toxin sensitive Gi proteins (Fig1.4) (Fernandez and Lolis, 2002). The N-terminal domain of the CXCR2 receptor is essential for ligand binding and signaling

25 specificity. CXCR2 gene maps to chromosome 2 in the human and the mouse homolog maps to chromosome 1 (NCBI GenBank no. 3579 and 12765, respectively). The CXCR2 gene consists of three exons separated by introns of sizes 3 and 5.4 kb (Bajetto et al., 2002). CXCR2 is known to bind to CXCL3,

CXCL5, CXCL6, and CXCL8 in addition to CXCL1.

The major ligand for CXCR2 is CXCL1, due to CXCL1 high affinity for

CXCR2, a soluble secreted factor known to be chemoattractive. Signaling events downstream of CXCL1/CXCR2 binding are known to mediate proliferation, differentiation and migration (Fig 1.4) (Davis et al., 2003). These function are mediated by actin dependent mechanisms and adhesion molecules on the surface of cells (Cross and Woodroofe, 1999) which has been described in leukocyte modulation via their arrest from rolling and infiltration into tissues

(Wu et al., 2000a). It is known CXCR2 is expressed highly in neutrophils and monocytes, and these cells are guided into inflammatory sites due to chemokine signaling (De Groot and Woodroofe, 2001). It has additionally been discovered that CXCL1 is upregulated around neuritic plaques in Alzheimer’s disease

(Johnstone et al., 1999), experimental closed head injury (Kossmann et al., 1997;

Otto et al., 2001), ischemic injury (Bona et al., 1999), surrounding contusion spinal cord lesions (McTigue et al., 1998) and around MS plaques (Omari et al.,

2006). CXCL1 is secreted by microglia and a subset of astrocytes (Robinson et al., 1998; Omari et al., 2006). CXCR2 is expressed in projection neurons in the brain and spinal cord and known to enhance survival of hippocampal neurons

(Horuk et al., 1997). CXCR2 is expressed in oligodendrocyte progentitor cells

26 and modulate downstream events via binding of CXCL1 (Robinson et al., 1998).

Modulation of oligodendrocytes responses during development has been found via CXCL1 signaling (Robinson et al., 1998; Robinson and Franic, 2001; Tsai et al., 2002). CXCL1 signaling potentiates PDGF induced proliferation and decrease migration of OPCs during early postnatal development of the CNS (Fig

1.3) (Tsai et al., 2002). Further, knockout of CXCR2 is associated with increased oligodendrocyte progenitor cell populations early in development; however, spinal cord white matter, myelin thickness, levels of myelin basic protein (MBP) and GFAP were all reduced (Padovani-Claudio et al., 2006) suggestive of a role in maturation. In organotypic human fetal slice culture,

CXCL1 also mediated proliferation of oligodendrocyte progenitor cells which was mediated via activation of extracellular signal related kinase (ERK1/2) and release of IL-6 (Filipovic et al., 2003; Filipovic and Zecevic, 2008). Astrocytes are the sources of a several cytokines and chemokines (CCL2, CXCL10, CXCL1, and CXCL8) (Chen et al., 2002; Charo and Ransohoff, 2006). Subsets of astrocytes secrete CXCL1 and signaling via CXCR2 positions OPCs to presumptive white matter regions in the CNS (Fig 1.3) (Robinson et al., 1998;

Robinson and Franic, 2001; Tsai et al., 2002) by effecting migration. Myelination is dependent on the precise number of OPCs, and this is thought to be partially dependent on CXCL1/CXCR2 signaling.

Demyelination disorders and Multiple Sclerosis

27

Demyelination disorders involve perturbations in development or damage to oligodendrocytes due to injury of disease. One oligodendrocyte can myelinate multiple axons dependent on the diameter of the axon (Miller, 2002). Death of one oligodendrocyte can cause extensive loss due to this phenomenon since one oligodendrocyte can myelinate many internodes and axons. Functional deficits will eventually arise due to large scale destruction of myelin and loss of fast axonal conduction in areas of the CNS. The damage to myelin on axons also leaves neurons vulnerable to severe insults. This destructive pattern can cause more functional and clinical deficits over time. Redistribution of sodium channels along the axon occurs when there is damage to myelin as a result of pathology or injury and in turn decreases axonal conduction. Function and distribution of myelin is essential for proper signal transduction and damage leads to clinical symptoms and pathology.

A demyelination disorder which is the focus of this thesis work is characterized by myelin damage and subsequent axonal loss. Multiple Sclerosis

(MS) is characterized by multifocal inflammatory induced demyelination that usually presents as optic neuritis (Trapp and Nave, 2008). Lesions in the CNS are spatially and temporally but sporadically produced (Namaka et al., 2008).

The locations of lesions manifest themselves with functional deficits. However, the heterogenicity of MS suggests there is no single phenomenon that intiates pathology. Histology of lesions in MS patients are characterized by indistinct lesions with enormous hypercellularity, perivascular infiltration, edema, loss of myelin and oligodendrocytes, axonal damage, macrophages filled with myelin

28 proteins, and hypertrophic astrocytes (Bjartmar et al., 2003; McFarland and

Martin, 2007). Symptoms of the disease vary from person to person but may include symptoms of vision deficits, paralysis, fatigue, numbness, loss of balance and motor coordination, slurred speech, tremors, bladder problems or stiffness

(Namaka et al., 2008).

Multiple Sclerosis (MS) affects 2.5 million young adults world-wide. The prevalence in females is much higher than in males. However, the etiology of the disease has yet to be explained except for the destruction of myelin due to insult attributed to an autoimmune response (Laing and Secombes, 2004; Becher et al., 2006; Benedict and Bobholz, 2007). MS is considered to be a CD4+ Th1- mediated autoimmune disease (Aarli, 1983; Antel, 1999; McFarland and Martin,

2007; Frohman et al., 2008). Pathology of MS is typified by multiple inflammatory foci, gliogenesis, axonal pathology and demyelination in regions within the brain and spinal cord (Benedict and Bobholz, 2007). The disability in patients varies, and they can range from extremely debilitating to none at all.

From the clinical view, inflammation is also superimposed onto clinical relapses; however, gadolinium-enhanced lesions demonstrated on magnetic resonance imaging seem to be the only mechanism of defining the inflammatory sites (Lee et al., 1998). The propensity for disability increases over time possibly due to the inability of the cells to recover from autoimmune insults and remyelinate (Sim et al., 2002; McFarland and Martin, 2007). The frequency of lesions increases over time to a peak level and then increases more slowly as a result of further damage and inflammation.

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The disease course is typified by the number of relapses of disability or inflammation which leads to characterizating the disease via their relapsing rates

(Fig 1.5). Initially presentation of disease typically occurs in the relapsing- remitting mode (RRMS), characterized by lapses in disability due to recovery of myelination after pathological insult (Compston and Coles, 2002; Benedict and

Bobholz, 2007). A gradual worsening of symptoms typically is called the progressive primary mode of MS (PPMS) (Davis, 1970). The mode of disease is exemplified by worsening of disease symptoms and disability in addition to more lesions and attacks (Franklin, 2002). This is typified by later onset of disease and/or occurs as a secondary genre of RRMS known as secondary progressive

MS (Compston and Coles, 2002; Benedict and Bobholz, 2007).

Multiple Sclerosis is illustrative of an immune-mediated disease and research has been focused on determining the mode of action of immune cells in the pathology of disease (Antel, 1999). Studies in animal models of MS demonstrate there is a highly autoreactive T cell population (CD4+ or CD8+) that results in inflammatory induced demyelination of the central nervous system

(Saxena, 2008). However, individual analysis of people with MS and healthy controls seem to contradict the animal models. Patients have the same number of T cells in peripheral blood carrying antigens for myelin proteins as control patients. In spite of this, the response mediated by the T cells have a substantial difference (Hellings et al., 2001; Frohman et al., 2008). T cells carrying myelin antigens exhibit memory phenotype in addition to alterations in cytokine secretion, and specific chemokine expression patterns are also different from

30

MS patients and those of a healthy individual (Becher et al., 2006; Frohman et al., 2008). The myelin specific CD4+ Th1 cells are activated outside the CNS.

Adhesion molecule interaction on the endothelium of the blood brain barrier activates in response to chemotactic cues (Cayrol et al., 2008). T cells then pass through the barrier via the blood vessel endothelium attachment and astrocytic foot processes (Man et al., 2008). These cells then become reactivated due to reexposure to major histocompatibility class II complex with antigen presenting cells (Penton-Rol et al., 2008). T cells secrete proinflammatory cytokines causing activation of microglia and astrocytes, causing damage to myelin (Merrill and Benveniste, 1996; Ponomarev et al., 2005). In MS, reactive astrocytes are also a component associated with demyelinating lesions and involved in multiple processes of the pathophysiology (Raine and Wu, 1993).

Current MS treatment modulate symptoms and inflammation

Current MS treatment is exemplified by a number of inflammatory regulators. Glatiramer acetate (Copaxone) is the most used therapy and modulates cytokine function (Miller et al., 1998; Steinman and Zamvil, 2006).

Copaxone is thought to modulate the following cytokines in MS disease pathology. TNFα in the CNS is known to be upregulated around MS plaques and is expressed by macrophages, microglia and astrocytes in chronic active lesions

(Murphy et al., 2002). In MS patients, IFNγ and IL10 secretion is also increased

(Tran et al., 2000; Lees et al., 2008). Subcutaneous IFNβ has also been shown to reduce attack frequency, progression of disability is reduced, disease severity,

31 and MRI activity is reduced (Miller et al., 1999). IFNβ was also able to increase time to disease progression. Integrins have also been targeted via natalizumab, an antibody against the α4 subunit of integrin (Steinman and Zamvil, 2005). This blocks the binding of endothelial receptors, VCAM1 and MadCAM1, which impairs lymphocyte migration through the blood brain barrier into the brain. It may also work by interrupting interactions of T lymphocytes with extracellular matrix that usually promotes further inflammation. Methylprednisolone has also been used as a modulatory drug on chemokines production and CCR5 expression and IFNβ (Schmitz and Chew, 2008). However, direct modulation of chemokines and receptors is currently been studied by a number of groups as a therapeutic for MS treatment.

Experimental models of Multiple Sclerosis

The ideal experimental model of MS has been debated over time. A perfect model of MS has yet to exist due to a lack of understanding on how MS is induced. However, models must be employed in order to determine the mechanism of action of a number of molecules and cell types in relation to immune and CNS cells. Therefore, by compiling data from a number of models we may be able to extract information and model a story on how chemokine or other molecules modulate pathology in MS. The models currently available are lysophosphatidyl choline/lysolecithin/LPC induced demyelination, experimental autoimmune encephalomyelitis (EAE), and cuprizone (bis- cyclohexylideneoxaldihydrazione) induced demyelination. None of these models

32 mimics pathophysiology of MS precisely; however each has advantages and disadvantages in determining aspects of disease induction and demyelination/remyelination.

Lysolecitin or LPC produces a chemically induced local demyelination event that involves local dissolution of myelin via injection into the spinal cord.

The blood brain barrier becomes compromised at the site of induction (Ousman and David, 2000). LPC has emulsifying properties that induces focal destruction of lipid-rich myelin at site of induction. This detergent, LPC, is formed via esterification of cholesterol by the transfer of fatty acids from lecithin. LPC is usually delivered via a pulled glass pipette in a 1% solution into the dorsal columns of the rodent spinal cord following laminectomy. Due to local blood brain barrier breakdown, LPC injections are known to cause infiltration of T cell, neutrophils, and macrophages in addition to microglia and astrocyte activation

(Ousman and David, 2000). Myelin breakdown begins to occur within hours of local injection; however, the peak of the lesion is achieved at 7 days after injection, where there is already observable remyelination occurring (Jeffery and

Blakemore, 1995; Blakemore et al., 2000; Blakemore and Franklin, 2008).

Additionally, primary myelin damage is followed by remyelination events triggered by myelin regeneration due to oligodendrocyte migration, proliferation and differentiation. However, this mechanism isn’t understood fully. The length and location of the lesion is variable and precise technical skills are required in order to reproduce lesions to compare different treatment populations.

Peripheral areas of LPC lesions have astrogliotic tissue typical of an injury

33 environment. However, in LPC lesions there are also spontaneuous remyelination events occurring peripheral to the lesion core. (Sheikh et al., 2009).

Demyelination is characterized using histological staining of luxol fast blue, MBP immunohistochemistry, and electron microscopy. Remyelination is evident with thin myelin sheaths after the forth week post lesion in a small 1.5µl lesion (Jeffery and Blakemore, 1995). Macrophage depletion, as demonstrated with other demyelination models, impairs subsequent remyelination (Kotter et al., 2001).

White matter lesions induced by LPC in general have reduced MBP transcription and decrease in neurofilament immunohistochemistry associative with axonal aberrations (Jean et al., 2002).

Replacement of normal rodent chow with a diet containing 0.2% cuprizone

(bis-cyclohexanone oxaldihydrazone) treated chow for 4 to 6 weeks in young

C57Bl/6 mice creates a demyelination globally throughout the white matter regions in the CNS, mostly detectable in the corpus callosum (Zatta et al., 2005).

Cuprizone is a copper chelator that produces a copper deficiency and demyelination (Zatta et al., 2005). Demyelination becomes apparent at 3 weeks post cuprizone chow feeding and is complete by 5 weeks (Cammer and Zhang,

1993; Arnett et al., 2001). Immunohistochemical detection of demyelination includes reduction in MBP staining and subsequent axonal pathology indicated by neurofilament staining (Blakemore and Franklin, 2008). Quantitative protein blots of MBP protein have also been used to indicate myelin damage (Zatta et al., 2005). Assessment of myelin in the corpus callosum may also be achieved through luxol fast blue staining, toluidine blue and subsequent electron

34 microscopy to indicate aberrations in myelin. Mature oligodendrocytes are ablated and thought to follow activation of macrophages and microglia (Hiremath et al., 1998). A minute amount of inflammation is present; however, this occurs in the absence of T lymphocytes because of the presence of an intact blood brain barrier (Arnett et al., 2002; McMahon et al., 2002).

Experimental autoimmune encephalomyelitis (EAE) in mice is produced by active immunization of mice using whole brain or spinal cord homogenates or purified myelin proteins or by adoptive transfer using activated myelin specific T cells (Cross et al., 1991). It is the prototypic model where paralytic disease is caused by immune response to CNS antigens. EAE is primarily produced by generating T cell mediated immunity to CNS antigens (Yan et al., 2003). Active subcutaneous immunization using MOG35-55 peptide or MOG1-125 peptide emulsified in complete Freud’s adjuvant (CFA) supplemented with Myobacterium tuberculosis with two injections of pertussis toxin 2 days apart induces chronic

EAE in C57Bl/6 mouse strains. The disease is evident 10-12 days post immunization, peaking by the end of the second week. Inflammatory demyelination, oligodendrocyte and neuronal cell death, and axonal loss occur within the first couple weeks of disease (Williams et al., 1994). This model allows for the assessment of potential therapies for MS. Immunization with PLP1-139 peptide in a similar manner as above induces a relapsing-remitting disease similar to MS. Pathology of the first attack is mostly inflammatory (Williams et al.,

1994). Subsequent attacks include inflammation, demyelination, and axonal loss(Halachmi et al., 1992). Additionally, T cells activation, dendritic cell

35 involvement, and macrophage and astrocyte activation are involved in both disease models of EAE (Bauer et al., 1995). Disease severity in EAE seems to be correlative to the recruitment of macrophages that mediate axonal injury and myelin loss (Benveniste, 1997; Bjartmar et al., 2003). The key steps to modulation of EAE include activation of reactive CD4+ T cells in the periphery, proinflammatory T cells (Ponomarev et al., 2005) and monocyte transmigration into the blood brain barrier, local inflammation and activation of antigen presenting cells such as microglia and macrophages, and finally destruction of oligodendrocytes, myelin sheaths and subsequent axonal damage (Williams et al., 1994; Bauer et al., 1995; Yan et al., 2003). Disease advancement traditionally transpires in a progressive fashion beginning with loss of tail tone and hind limb paralysis, hind and forelimb paralysis, and death (Bai et al., 2009).

Remyelination success and failure in MS

Remyelination is dependent on the population of immature cells that still have the capacity of proliferation, migration and differentiation to become myelinating oligodendrocytes. These progenitors are known to persist into adulthood and are marked by expression of NG2 and platelet derived growth factor alpha (PDGFRα), similar to those found during development (Hinks and

Franklin, 1999). These cells are known to proliferate and migrate to areas of demyelination. Spontaneous remyelination generally occurs in the response to demyelination in a number of experimental models by the generation of new oligodendrocytes (Blakemore et al., 2000). Mature oligodendrocytes themselves

36 do not divide normally; on the other hand, precursor cells can give rise to them in optimal conditions. However, this differentiation may be incomplete.

Demyelinated axons leave the neurons vunerable to attack and accounts for the cognitive decline in patients with MS (Bjartmar et al., 2003). Additional factors that may contribute to axonal damage include cytokines, proteases, superoxides, activated CD8+ T cells, nitric oxides, and glutamate toxicity (Blackmore and

Letourneau, 2006). Supplementary, activated microglia and astrocytes could play a role in the pathology as well.

Repair of demyelinated lesions are influenced by aging, sex, size of lesions and extent of oligodendrocyte precursor cell affliction (Sim et al., 2002).

The lesion occupies a dynamic environment of inhibitory molecules, lack of growth factors, and scar formation due to astrocytes. To understand remyelination, a large amount of studies on what is occurring in experimental models of demyelination have been extensively studied but to no avail.

Oligodendrocyte precursor cells expressing NG2 are able to survive demyelination process; however, they fail to remyelinate or are waiting for a signal to remyelinate (Franklin et al., 1997). Later differentiated oligodendrocytes, marked by galactrocerebroside (O1), transplanted into areas of demyelination fail to remeylinate although they express and can form myelin sheaths (Rosano et al., 1999). Conversely, oligodendrocyte progenitor cells isolated expressing A2B5 and then transplanted are able to remyelinate successfully (McTigue et al., 2006). This has been attributed to the cells responsiveness to environmental cues or migration from subventricular zones in

37 the brain and spinal cord (outside of lesions) (Franklin and Ffrench-Constant,

2008). Nonetheless, this is speculation and remains to be proven.

Primary hypothesis of this dissertation

Chemokines and cytokines play a role in a number of cell types in the

CNS. These secreted and signaling molecules play important roles in migration, proliferation, and differentiation of cells within and outside the CNS, including immune cells. Positioning of cells during development is thought to be partially mediated by chemokines and cytokines and their receptors. These expression patterns may determine the migratory and proliferative capacity of each cell type to some extent. The continued expression of chemokines in the adult CNS remains to be understood completely. However, the expression of chemokines has been determined to be upregulated in response injury, thus modulating proliferation, migration and differentiation of glia. The work presented in the dissertation effort was based upon this idea. The role of chemokines, specifically

CXCL1/CXCR2 signaling, in the injured CNS is the focus of this work. CXCL1 signaling is known to halt migration of oligodendrocyte precursor cell migration and induces proliferation. CXCL1 is secreted by subsets of astrocytes and is upregulated around MS lesions in patients, thereby potentially modulating secondary pathology and astrogliosis. CXCR2 is expressed by astrocytes and oligodendrocytes in addition to neutrophils. Modulation of CXCR2/CXCL1 signaling by inhibitors may increase the number of oligodendrocyte precursor cell migration into areas of lesion in experimental models of MS. Inhibition of CXCR2

38 signaling may decrease the pathological response of immune cells in the CNS, and astrocyte reactivity. Altering the CXCR2/CXCL1 signaling has the potential for being a therapeutic for the treatment of demyelination disorders, in so much as, transforming signaling could change inflammatory reaction in addition to enhancing capacity of oligodendrocytes to remyelinate (Fig 1.6).

39

Figure 1.1. Cell lineage of astrocytes and oligodendendrocyte in the central nervous system.

Oligodendrocyte precursor cells, expressing A2B5, NG2, and PDGFa, are initially specified from neural stem cells, as are type I astrocyte and neurons. OPCs have the capacity in vitro to constitutively differentiate into mAb identifiable O4+ pre-oligodendrocyte or type II astrocytes if exposed to bone morphogenetic proteins. Subsequently, O4 expressing pre-oligodendrocytes begin to express galactocerebroside are termed immature oligodendrocytes and label with mAb O1. Immature oligodendrocytes can either progress to myelinating cells under specific cues and factors to survive or undergo apoptosis. Cells that do not undergo programmed cell death mature further and usually do not respond to the mitogen PDGF. Myelinating cells, the most mature oligodendrocytes, express myelin proteins including myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG).

40

Figure 1.2. Saltatory conduction associated with myelination and clustering of sodium channels promoting efficient axonal transduction.

Myelin the fatty insulation surrounding axons of the neuron promote clustering of sodium channels and aids in the rapid conduction of action potentials in the CNS. Myelination of axons promotes the jumping of axonal signal from one node to the next in the area occupied by a high concentration of sodium channels to regenerate axon potentials (A). Depolarization becomes discontinous, called saltatory conduction, thus making conduction faster than non-myelinated axons. A continuous wave of depolarization spread sequentially along bare axonal membranes in unmyelinated axons (B).

41

Figure 1.3. Oligodendrocyte specification occurs in the ventral spinal cord and cell proliferation is conducted by locally derived signals from astrocytes.

Oligodendrocyte precursor cells are directed to white matter regions by cues that are attractive and repulsive. In white matter regions, OPCs encounter the chemokine CXCL1, secreted locally by astrocytes during development. CXCL1 signaling through the receptor CXCR2 expressed on OPCs halts cell migration and allows cells to become more responsive to mitogen PDGF. Cell proliferation is enhanced and allows oligodendrocytes to myelinate the developing axons.

42

Figure 1.4. Canonical CXCL1/CXCR2 signaling resulting in modulation of proliferation, differentiation, and migration in a number of cell types.

The chemokine receptor,CXCR2, is a member of G protein coupled receptor (GPCR) family. CXCR2 binds CXCL1, a soluble chemoattractive cytokine of the ELR+ family of CXC chemokines. Interaction of CXCR2/CXCL1 activates intracellular processes that modulate proliferation, differentiation, and migration. In general GPCR, 7 transmembrane receptors, bind heterotrimeric G protein complexes, composed fo three subunits (α,β,γ). Activation of G protein complexes (in the case of CXCR2, Gi proteins), activates further intracellular processes including phospholipase C (PLC) leading to phosphatydylinositol (PI) induction and calcium increase, leading to PI3 kinase. PI3 kinase activation products can reorganize actin cytoskeleton componenents leading to migration and chemotaxis. Modulation of receptor signaling can be mediated by arrestins that bind to the cytoplasmic end of GPCRs preventing G protein interaction, modulating receptor internalization and recycling.

43

Figure 1.5. Disability progression in different types of Multiple Sclerosis.

Multiple Sclerosis, a major demyelination disorder is classifed into different types based on the remissions and disability. In relapsing remitting forms of MS (RR) (50% of total cases of MS) recovery may not be complete after relapse and disability progresses over time. Secondary progressive MS (SP) (30%) frequently begins as RR however, over time there is a decrease in the number of remissions occurring and the progression of disability is rapid. Primary progressive (10%) presents with no clear remission of lesions or symptoms and results in continuous accumulation of disability. (Compston and Coles, 2002; Benedict and Bobholz, 2007)

44

Figure 1.6. Model of CXCL1/CXCR2 modulation in reducing immune mediated pathology and enhancement of migration and differentiation of oligodendrocyte progenitor cells.

Stimulation of CXCR2 following binding of CXCL1 enhances the production of proinflammatory cytokines and upregulates astrogliosis promoting disability of repair in demyelination injury. However, inhibition of this pathway decreases T cell entry into the CNS and therefore prevents further damage by proinflammatory cytokines secreted by T cells and also the activation of astrocytes. Additionally, decreasing the CXCL1 induced inhibition of migration of OPC, allows OPCs to occupy areas of demyelination. Further, inhibition of CXCR2 signaling promotes differentiation of OPCs that have entered lesion areas and therefore promotes remyelination in areas of demyelination. Therefore by inhibiting CXCR2, the detrimental effects of blocking migration of OPCs, reducing attacks by astrocytes and immune cells, and promoting differentiation, results in enhanced repair from demyelination injury.

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CHAPTER 2: INHIBITION OF CXCR2 SIGNALING PROMOTES RECOVERY IN MODELS OF MULTIPLE SCLEROSIS

ABSTRACT

Multiple sclerosis (MS) is a neurodegenerative disease characterized by demyelination/remyelination episodes that ultimately fail. Chemokines and their receptors have been implicated in myelination and remyelination failure.

Chemokines regulate migration, proliferation and differentiation of immune and neural cells during development and pathology. Previous studies have demonstrated that the absence of the chemokine receptor CXCR2 results in both disruption of early oligodendrocyte development and long term structural alterations in myelination. Histological studies suggest CXCL1, the primary ligand for CXCR2, is upregulated around the peripheral areas of demyelination suggesting this receptor/ligand combination modulates responses to injury. Here we show that in focal LPC induced demyelinating lesions, localized inhibition of

CXCR2 signaling reduced lesion size and enhanced remyelination while systemic treatments were relatively less effective. Treatment of spinal cord cultures with a CXCR2 antagonist reduced CXCL1 induced A2B5+ cell proliferation and increased differentiation of myelin producing cells. More critically, treatment of myelin oligodendrocyte glycoprotein peptide 35-55 peptide- induced EAE mice, an animal model of multiple sclerosis, with a small molecule antagonist against CXCR2 results in increased functionality, decreased lesion load, and enhanced remyelination. Our findings demonstrate the importance of antagonizing CXCR2 in enhancing myelin repair by reducing lesion load and

46 increasing functionality in models of multiple sclerosis and thus provide a therapeutic target for demyelinating diseases.

INTRODUCTION

Multiple sclerosis (MS) is a devastating heterogeneous inflammatory demyelinating disorder of the central nervous system (CNS) affecting young adults (Davis, 1970; Antel, 1999; Compston and Coles, 2002; Benedict and

Bobholz, 2007). Hallmarks of the disease include recurrent demyelinating episodes that result in the progression of neurological deficits due to the slowing and ultimately failure of axonal conduction (Davis, 1970; Waxman, 1977;

Blakemore et al., 2000; Baumann and Pham-Dinh, 2001; Nashmi and Fehlings,

2001; Bjartmar et al., 2003). Remyelination frequently occurs during early stages of disease, however this fails during later disease stages and the number of persistent demyelinating lesions increases (Chari, 2007).

The majority of current therapies for MS target the inflammatory response that is distinctive of the disease (Miller et al., 1998; Hohol et al., 1999; Miller et al., 1999; McFarland and Martin, 2007; Trapp and Nave, 2008). Although active inflammation predominates in the initial stages of the disease, the neurological disabilities associated with the chronic disease accumulates independent of inflammatory mediators (McFarland and Martin, 2007; Frohman et al., 2008) and long-term recovery requires myelin repair. Models of MS include the experimental autoimmune encephalomyelitis (EAE) (Cross et al., 1991), where widespread demyelination is largely a consequence of immunological attack on resident CNS myelin following immunization by specific myelin peptides. Focal

47 demyelinated lesions can be induced by direct injection of lysolecithin (LPC)

(Ousman and David, 2000; Bambakidis et al., 2003; Mi et al., 2005) where demyelination is largely a consequence of chemical dissolution of myelin sheaths. Finally, cuprizone intoxication (Zatta et al., 2005) results in demyelination largely as a consequence of metabolic perturbations. In the present study, we utilize lysolecithin lesions and MOG35-55 peptide induced EAE to assess the efficacy of inhibiting the CXCR2 chemokine receptor in mediating demyelination and remyelination.

Chemokines, or chemoattractant cytokines, comprise a family of inducible secreted molecules of small molecular weight (8-10kDa) (Hesselgesser and

Horuk, 1999; Fernandez and Lolis, 2002; Laing and Secombes, 2004), that function as activators of leukocytes, wound healing, modulators of angiogenesis, and tumorigenesis (Robinson et al., 2001; Smith et al., 2005; Charo and

Ransohoff, 2006). The functions of chemokines have been primarily implicated in modulation of immune coordination and inflammation (Fernandez and Lolis,

2002; Flynn et al., 2003; Laing and Secombes, 2004; Charo and Ransohoff,

2006) where they contribute to the localization of specific cells with fine spatio- temporal precision (Charo and Ransohoff, 2006). Chemokine receptors are G- protein coupled receptors (GPCRs) usually linked to pertussis toxin sensitive Gi proteins (Bajetto et al., 2001). The receptor CXCR2 is the primary mediator of signaling by the chemokine CXCL1 (Katancik et al., 2000), a soluble secreted chemoattractive molecule of the glutamine-leucine-arginine (ELR) family of CXC chemokines. Their interaction initiates intracellular processes that modulate a

48 number of cellular events, including migration, proliferation, and differentiation

(Bajetto et al., 2001; Miller, 2002; Tsai et al., 2002). Several studies have characterized the expression and function of chemokine receptors on neural cells in the vertebrate CNS. For example, the CXCR4 receptor and its cognate ligand

SDF1 have been implicated in the control of neuronal precursors in the hippocampus and cortex (Imitola et al., 2004). During oligodendrocyte development, signaling through CXCR2 in combination with platelet derived growth factor AA promotes oligodendrocyte precursor proliferation (Robinson et al., 1998; Robinson et al., 2001; Woodruff et al., 2004). Furthermore, the CXCR2 signaling pathway contributes to the correct localization of oligodendrocytes in white matter tracts of the spinal cord (Miller, 2002; Tsai et al., 2002; Padovani-

Claudio et al., 2006).

Elevated levels of CXC chemokine expression are associated with a range of pathological CNS insults including infection, tumors, ischemia and demyelination (Liu et al., 1993; Spanaus et al., 1997; Filipovic et al., 2003; Keane et al., 2004; Sue et al., 2004; Omari et al., 2006; Valles et al., 2006), and this may reflect ingress of immune cells in response to local expression. For example, overexpression of CXCL1 in oligodendrocytes induces neutrophil invasion and astrogliosis (Tani et al., 1996b; Tanabe et al., 1997).

Overexpression of CXCL1 in astrocytes resulted in a milder course of EAE disease pathology (Omari et al., 2009) and upregulation of CXCL1 has been reported at the periphery of MS lesions coincident with the accumulation of OPCs

(Omari et al., 2005). Furthermore, proliferation of OPCs has been determined in

49 organotypic human fetal slice cultures to be dependent on CXCL1/CXCR2 signaling (Filipovic and Zecevic, 2008). By contrast, CXCR2 null animals have been reported to have increased NG2+ cells that may facilitate recovery of demyelination (Padovani-Claudio et al., 2006). Here we demonstrate that in a chemical demyelination model (LPC), and an immune mediated demyelination model (MOG35-55 peptide induced EAE), blocking CXCR2 enhances recovery and remyelination in spinal cords of affected animals suggesting inhibition of CXCR2 may represent a potential target for myelin repair.

MATERIALS AND METHODS

Animals and Induction of EAE

All animal procedures were conducted according to approved guidelines established by the National Institutes of Health Animal Protection Guidelines and were approved by Institutional Animal Care and Utilization Committee of

Case Western Reserve University School of Medicine

The chronic EAE model was induced in C57BL/6 mice using synthetic myelin oligodendrocyte glycoprotein peptide (MOG35-55, 200ul at 200µg/animal).

Mice were injected subcutaneously with an emulsion of MOG35-55 peptide mixed with complete Freud’s adjuvant with 500µg Mycobacterium tuberculosis followed by two intraperitoneal (IP) injections of 500µg pertussis toxin, one immediately after immunization and a second 24hrs later (Bai et al., 2009). Clinical scores were obtained on a 5 point scoring system in which a score of 0 equates to no clinical symptoms ; 1, limp tail; 2, paralysis of one limb; 3, paralysis of two hind

50 limbs; 4, paralysis of front limbs; 5, death as previously described (Bai et al.,

2009). Treatment of animals with either CXCR2 antagonist (Tocris; 8µg/kg)(Fig

2.2) or vehicle was begun when the animals showed the initial signs of disease.

In general, this occured 10-14 days post immunization on a daily basis for two- four weeks post disease induction.

At the conclusion of the study, animals were sacrificed and tissue from the spinal cord and splenocytes were collected and cultured for antigen reexposure.

Tissue was reexposed to MOG35-55 peptide (20µg) for 24 hours. Culture media was collected and analysized for cytokine secretion using Liqui-Chip system implemented by the Stephen Miller lab at Northwestern University in collaboration with the Myelin Repair Foundation. Cells from this study were additionally fixed and analyzed after a 24hour BrdU pulse to assess cell proliferation in response to peptide exposure. Immunohistochemistry was assessed using BrdU antibody after 2N HCl permeabilization (20minutes at room temperature) and double staining was conducted using CD3 antibody (1:200 in

0.03% PBST). Incubation in primary antibody was conducted at room temperature for 30 minutes followed by Alexa Fluor 488 (CD3) and Alexa Fluor

594 (BrdU), both antibodies at 1:500 dilution in 0.03% PBST. Coverslips were inverted and placed on slides mounted with Vectashield with DAPI. Total cells were counted per coverslip in addition to those marked by CD3 positive markers and BrdU to assess proliferation.

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Lysolecithin induced demyelination and delivery of antagonists

Twelve week old female Sprague-Dawley rats (220-240 grams) were anesthetized. Following a laminectomy at thoracic vertebrae level 10, three microliters of 1 percent LPC (L-α- lysophosphatidyl-choline, lysolecithin) (Sigma,

St. Louis, MO) in 0.9 % sodium chloride solution were microinjected using a pulled glass pipette into the dorsal column of the spinal cord. For double injection of either CXCR2 neutralizing antibody (R&D systems, 100µg/ml) or

CXCR2 antagonist (Tocris, 100µg/ml) (Fig 2.2) or appropriate vehicle controls, animals were anesthetized 2 days post lesion and injected with either 3µl CXCR2 antibody or 3µl CXCR2 antagonist, using the same paradigm as above.

Systemic delivery of CXCR2 antagonist (Tocris, 8µg/kg) was performed IP on the day of surgery and everyday thereafter. Animals were then allowed to recover for

10 days prior to sacrifice (Fig 2.1).

Primary spinal cord cultures

Mixed cell cultures were prepared from postnatal day 3 rat spinal cords and plated on poly-L-lysine (PLL) coated coverslips. The media was changed the following day and cells allowed to grow for 3 days. Cells were grown in media consisting of DMEM, 10ng/ml platelet derived growth factor AA

(PDGFAA), 10ng/ml fibroblast growth factor (bFGF), 1% FBS, and N2 supplement. Cells were treated with small molecule inhibitor against CXCR2 at various concentrations (40ng/ml, 80ng/ml, 160ng/ml) and/or the ligand CXCL1

(0.5 ng/ml) overnight and the effect on OPC development assessed.

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Immunocytochemistry of primary cell cultures

Cells were fixed in 4% paraformaldehyde and incubated in primary antibody for 30 minutes in PBST (0.03% triton) (MBP: SMI99, Sternberger

Monoclonals, 1:500) followed by corresponding secondary antibodies and mounted using Vectashield with DAPI (Vector Laboratories Burlingame, CA).

Labeling with O4, A2B5 and O1 was performed on live cells. Cells were post- fixed using 5% acetic acid in methanol. To analyze proliferation of cells in S phase, bromodeoxyuridine (BrdU) (10µM) was added to the media at least 18 hours prior to fixation. Images were collected using a Leica DM5000B microscope and Leica Applications Suite Software.

Immunohistochemistry and immunofluorescence

Animals were perfused with 4% PFA in saline. Transverse frozen sections of spinal cord were dried on slides and stored at -80oC. Sections were rinsed blocked in 0.03% PBST and 5% NGS and incubated in primary antibody overnight (GFAP: Dako 1:500; Iba1: Wako 1:250; MBP: SMI99, Sternberger

Monoclonals 1:500, Iba1: Dako, 1:100, ED1: Santa Cruz, 1:100). Sections were rinsed and incubated in anti-rabbit IgG or anti-mouse IgG fluorescently conjugated secondary antibodies (Sigma, 1:500). The sections were briefly rinsed and mounted using Citifluor mounting media (Ted Pella, Redding PA).

Images were collected using a Leica DM5000B microscope and Leica

Applications Suite software. The number of Iba1 positive cells was counted in

53 defined regions of spinal cord grey and white matter to determine relative amount of microglia and macrophages in response to CXCR2 treatment in EAE animals.

The mean cell number was determined from at least 4 different sections taken from 2 different animals. Data was pooled and presented as mean+/- standard deviation.

Electron Microscopy

Animals were anesthetized and perfused initially with PBS followed by 3% formaldehyde/3% gluteraldehyde in 0.1M sodium cacodylate buffer, pH 7.4

(Electron Microscopy Sciences, Hatfield, PA). Tissue was dissected and 300µm thick vibratome sections post fixed in 1% OsO4. Samples from EAE animals were taken from lumbar segments of the spinal cord. Sections from lysolecithin

(LPC) animals were selected from midlesion as indicated by luxol fast blue staining as previously described (Mi et al., 2005). Sections were dehydrated through a series of ethanol dilutions, stained using uranyl acetate and embedded in a Poly/Bed812 resin (Polysciences Inc., Warrington, PA). Thin (1µm) sections were stained with toluidine blue. Ultrathin (0.1µm) from matching areas of experimental and control tissue blocks were cut and visualized using an electron microscope (JEOL1200CX) at 80kV. G ratios were calculated by dividing the axonal area and myelin thickness for randomly selected myelinated axons (Fig

2.4). Three separate measurements of radial thickness of myelin were taken for each axon and averaged to provide mean myelin thickness.

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Statistical analyses

Data was analyzed using t-test having shown equal variances in Microsoft

Excel software. Statistical significance was set to p<0.05. Values are described in the text as mean +/- standard deviation followed by the p-value, and plotted as mean +/- two standard errors of the mean.

RESULTS

Local delivery of CXCR2 blocking reagents reduce the size of LPC-induced lesions

To determine whether CXCR2 antagonists alter the rate of recovery in models of demyelination and remyelination, the effect of local injection of neutralizing anti-CXCR2 antibody on lesion load was assayed in a lysolecithin model (Bambakidis et al., 2003; Mi et al., 2005). Lysolecithin injection induces rapid local demyelination that is mostly developed by 2 days post injection.

Direct injection of a CXCR2 neutralizing antibody into a region adjacent to the lesion 2 days after LPC injection had a profound effect on lesion volume(Fig 2.3;

2.5). Animals that received control antibodies had a lesion volume of 5 +/-

2.8mm3 after a survival of 10 days and the lesions appeared to contain relatively little myelin (Fig 2.5 A,C, E, and G). By contrast, in animals that received neutralizing CXCR2 antibodies, the lesion size was reduced to 1+/- 1.3mm3 after

10 days and only the central core was devoid of myelin (Fig 2.5 B, D, F, and G).

Three dimensional reconstruction of the area of the lesion occupying the spinal cord revealed a reduction in rostral caudal and lateral extent of the lesion in anti-

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CXCR2 treated animals although the lesions still spanned the depth of the dorsal column (Fig 2.5 F and G) (n=5). To confirm the specificity of the anti-

CXCR2 antibody effects, the studies were repeated using a single injection of a small molecule CXCR2 inhibitor. Local injection of the Tocris CXCR2 antagonist

(Busch-Petersen, 2006) resulted in smaller LPC lesions and increased number of myelinated axons at 10 days post insult (Fig 2.6). For example, the proportion of myelinated fibers was less than 6 +/- 4% in the middle lesion of animals that received vehicle only (Fig 2.6 A, C and E), and there were substantial cellular infiltrates throughout the lesion while similar areas of CXCR2 antagonist treated animals (Fig 2.6 B, D and E) contained a significant proportion (54 +/- 7%) of thinly myelinated axons indicative of remyelinated axons scattered throughout the lesion and fewer infiltrated cells. Ultrastructural studies confirmed the presence of relatively thinly myelinated axons (arrows) scattered throughout the lesion (Fig 2.6D). The lesions that develop in response to LPC injections are primarily a result of direct chemical demyelination rather than immune cell infiltration (Ousman and David, 2000), and these studies suggest that treatment with CXCR2 antagonists significantly accelerates myelin repair in a non- inflammatory setting through a mechanism that likely includes a direct effect on oligodendrocytes and their precursors (Robinson et al., 1998; Tsai et al., 2002).

Systemic delivery of CXCR2 antagonist does not significantly enhance recovery of LPC induced demyelination

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To determine whether CXCR2 inhibition in the LPC lesions was primarily local or systemic, the CXCR2 antagonist was delivered IP directly after lysolecithin lesions (Fig 2.7). Injection of CXCR2 antagonist daily for 10 days after induction of a dorsal spinal cord LPC lesion had only a small effect on lesion volume (2.6mm in experimental versus 3mm in controls). Consistent with this limited effect of systemic blockade of CXCR2 on lesion size, morphological studies demonstrated only a marginal although significant effect on repair in the

LPC model. In middle areas of lesions in control animals (Fig 2.7C) 94 +/- 2% of the axons were demyelinated (asterisks) and 6+/-3% were myelinated, and in animals that received CXCR2 antagonists (Fig 2.7D) 76+/-4% axons were demyelinated while 24 +/- 6% were remyelinated (arrows) (p=0.004) (Fig 2.7E).

In all cases the total number of axons and the proportion that were affected by

LPC injection were not different suggesting, there is not a protective effect on lesion formation following systemic inhibition of CXCR2. Further analyses suggested no significant difference in MBP expression in the lesion or cellularity of the lesion with systemic CXCR2 inhibition (data not shown) suggesting that the primary benefit derived from direct injections reflected local inhibition of CXCR2 signaling on CNS cells.

CXCR2 antagonists decrease CXCL1 induced OPC proliferation and increases OPC differentiation in vitro

Previous studies suggest that OPCs express the CXCR2 receptor and respond directly to CXCL1 stimulation (Robinson et al., 1998; Tsai et al., 2002).

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To assess whether CXCR2 antagonists had direct effects on neural cells, the antagonist was added to spinal cord cultures to determine the effect on oligodendrocyte differentiation (Fig 2.8). Spinal cord cultures from P3 animals were grown for 3 days and treated for one day with CXCL1 (Fig 2.8 B,E, and H), the CXCR2 antagonist (Fig 2.8 C, F, and I) or control medium (Fig 2.8 A, D, and

G). Oligodendrocyte precursors were identified through binding of mAb A2B5. In the presence of CXCL1 (0.5ng/ml), the proportion of A2B5+ cells increased due to proliferation (46% p=0.002) (Fig 2.8B). This increase was abolished by treatment with CXCR2 antagonist (26%), and the residual cells were weakly

A2B5+ and appeared to have a larger cell body and more differentiated phenotype suggesting treatment with CXCR2 antagonist promoted OPC differentiation (Fig 2.8C). Cultures exposed to CXCR2 antagonist contained increased O1+cells (20 +/- 4% versus 34 +/- 3%) and 50% more MBP positive cells than those in control conditions (18 +/- 1% versus 52 +/- 5% p=0.007) (Fig

2.8 D-I). Not only were there increased number of differentiated oligodendrocytes but their morphology was also substantially different. More of the MBP+ cells contained sheet like processes in the presence of CXCR2 antagonist than in control conditions (Fig 2.8I). Consistent with previous studies,

(Tsai et al., 2002) the presence of CXCR2 antagonists reduced the proliferation of A2B5+ cells (42 +/- 4% BrdU+/A2B5+ cells) in the presence or absence of

CXCL1 (data not shown). These data indicate that the CXCR2 antagonist blocks the mitogenic effects of CXCL1 and facilitates OPC differentiation.

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Functional improvement in EAE mice treated with CXCR2 antagonist

While analyses of recovery from lysolecithin (LPC) lesions has many advantages for the study of myelin repair, the immunological aspects of demyelinating disease such as MS are effectively modeled by experimental allergic encephalitis (EAE). Previous studies have implicated CXCR2 in the progression of EAE (Carlson et al., 2008), and to investigate whether systemic treatment with CXCR2 antagonists reduced disease burden, animals immunized with the MOG35-55 peptide (Bai et al., 2009) were treated daily with 8µg/kg

CXCR2 antagonist (Tocris) systemically while control animals received vehicle only (Fig 2.9). Treatments were initiated when immunized animals began to show functional deficits. On average this occurred during the second week following immunization. Animals that received vehicle rapidly developed increasing functional deficits (Fig 2.9A). EAE animals initially presented with flaccid tail and hindlimb weakness that progressed over a three-week period to forelimb paralysis. Some animals were severely compromised by day 24 and were subsequently sacrificed. By contrast, animals that received CXCR2 antagonists initially demonstrated functional deficits including hindlimb weakness but, with continued treatment, all animals (n=12) exhibited some functional recovery and no animals progressed to forelimb paralysis. Indeed, a subset

(9/12) of animals had total functional recovery after two weeks of treatment (Fig

2.9A). These data suggest that even after the onset of disease antagonizing

CXCR2 signaling promotes functional recovery in a chronic model of EAE.

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Enhanced myelination and reduced lesion load in EAE mice treated with

CXCR2 inhibitor

Histological analyses of CXCR2 antagonist and vehicle treated EAE animals supported the functional assessment. Animals treated with the CXCR2 antagonist had a reduced lesion load in the spinal cord. Luxol fast blue staining showed smaller demyelinated lesions after 14 days of treatment with CXCR2 antagonist (Fig 2.9C) compared to vehicle controls (Fig 2.9B). Although CXCR2 treated animals were not entirely free of demyelinated lesions, quantitation of relative lesion load by 3D reconstruction of the area occupied by lesions demonstrated a reduction of 39 +/- 2 % (3.3mm3 vehicle versus 1.3mm3 CXCR2 antagonist treated animals) in the extent of spinal cord demyelination between the EAE control and CXCR2 antagonist treated animals (Fig 2.9F; p=0.001).

Ultrastructural analyses confirmed that following 14 days of treatment with

CXCR2 antagonist, lesion areas from treated animals contained larger numbers of remyelinated axons (Fig 2.10D and E) than non-treated EAE controls (Fig

2.10C and E). For example, while greater than 90% of the axons were demyelinated (asterisks) in lesion areas from non-treated EAE animals, less than

25% of axons were demyelinated in antagonist treated animals (Fig 2.10; p<0.0001). Many of the myelinated axons in treated animals had altered G ratios consistent with remyelination (Fig 2.10F). Vehicle only treated EAE animals had a ratio close to 1 suggesting the myelinated axons were not remyelinated. By contrast, CXCR2 antagonist treated animals had a mean ratio closer to 3 indicative of thin myelin sheaths which indicates remyelination (Figure 2.10F;

60 p=0.02). To confirm the effects of blocking CXCR2 on demyelination/remyelination, sections from control and treated EAE animals were labeled with antibodies to the major myelin protein MBP (Fig 2.11). Inhibition of

CXCR2 increased the intensity of MBP labeling by 63% over control EAE animals (Fig 2.11A-D and reduced the size of the demyelinated areas) suggesting that oligodendrocytes are being recruited and/or protected in lesion areas by reducing CXCR2 signaling.

Infiltration and activation of macrophage/microglial cells are reduced in

MOG35-55 induced EAE animals treated with CXCR2 inhibitors

The reduction in demyelinated areas in CXCR2 antagonist treated EAE animals correlated with a reduction in inflammatory cell infiltration. For example,

H&E stained sections from vehicle EAE animals revealed substantial cell infiltrates associated with lesion areas (Fig 2.9D) characteristic of lesion pathology and the recruitment of immune cells. By contrast, in animals treated with the CXCR2 inhibitor only rare mononuclear cells were seen in presumptive lesion areas (Fig 2.9E).

The preservation of cytoarchitecture and reduction in vascular cuffing was most clearly seen in toluidine blue stained 1µm sections (Fig 2.10A and B) and confirmed at the ultrastuctural level. Vehicle treated EAE animals (Fig 2.10A) had significant cuffing and infiltration while CXCR2 antagonist treated EAE animals

(Fig 2.10B) exhibited fewer cells around blood vessels and infiltration into the parenchyma of the spinal cord. Microglia were present in all spinal cords but

61 their reactivity, defined by a more stellate phenotype and shorter process, was reduced in animals treated with CXCR2 antagonists (Fig 2.11E-H). Vehicle- treated EAE animals contained large numbers of Iba1+ cells within lesions (Fig

2.11E) as well as in adjacent grey and white matter areas of the spinal cord.

Animals treated with CXCR2 antagonists, however, demonstrated microglial activation associated with smaller lesions and a 50+/- 5% decrease of Iba1 cells in adjacent grey and white matter regions (Fig 2.11F and H). EAE animals that were removed from treatment after 1 week exhibited a return of demyelination in the absence of inflammation (Fig 2.12B). These animals also exhibited a return in functional impairment associated with demyelination (data not shown). In contrast, EAE animals that were treated with CXCR2 antagonists for a longer period of time (2 weeks), demonstrated an enhancement in remyelination in toluidine blue sections (Fig 2.12A and C) although they retained some inflammatory cell infiltration into the parenchyma of the spinal cord (Fig 2.12A).

Taken together, our studies suggest that antagonism of CXCR2 signaling promotes functional recovery and facilitates the establishment of normal cytoarchitecture in the spinal cord in animal models of CNS demyelination.

DISCUSSION

Current therapies for demyelinating diseases such as MS are largely focused on regulating the immunological attack on neural tissue. Successful long term functional recovery in MS is likely to require substantial myelin repair. Here we demonstrate that blocking signaling through the chemokine receptor CXCR2

62 promotes functional recovery in two demyelinating models, and OPC differentiation is enhanced. Focal injections of CXCR2 antibody, or a CXCR2 antagonist, enhanced myelin repair in LPC lesions and promoted the differentiation of OPCs in culture. Systemic injection of CXCR2 antagonist resulted in limited enhancement of repair likely reflective of the limited involvement of immune cells in this model. Consistent with this hypothesis, systemic treatment with CXCR2 antagonist resulted in significant functional recovery in a model of chronic EAE. This functional recovery was correlated with a reduced disease burden. For example, compared to vehicle treated EAE animals, those that received daily IP injections of CXCR2 antagonist had smaller demyelinated lesions and higher levels of myelin basic protein expression in the spinal cord that was correlated with higher numbers of remyelinated axons.

Treatment with CXCR2 antagonist also reduced the extent of microglial activation and the degree of inflammatory cell infiltration.

The enhanced recovery from CNS demyelinating lesions mediated by antagonists of CXCR2 likely reflects influences on both CNS and immune cells.

Within the CNS, CXCR2 has been reported to be expressed on a range of cells including OPCs and other glia (Luo et al., 2000; Nguyen and Stangel, 2001).

Within the spinal cord oligodendrocyte lineage, stimulation of CXCR2 on A2B5+

OPCs results in enhancement of PDGF driven proliferation and inhibition of cell migration (Tsai et al., 2002; Kadi et al., 2006). The accelerated repair in LPC lesions following local inhibition of CXCR2 signaling may reflect the ability of

OPCs to ignore the migrational blockade from locally expressed CXCL1 and

63 effectively repopulate the lesion. Alternatively, in order to progress to myelination, OPCs must complete their differentiation and mature. The control of differentiation has been shown to be inversely correlated with proliferation. If

OPCs are refractory to the mitogenic stimulation of CXCL1 through CXCR2 blockade, they are more likely to differentiate and myelinate. Consistent with this hypothesis, inhibition of CXCR2 in spinal cord cultures resulted in enhanced numbers of mature MBP+ oligodendrocytes. Other CNS cells have also been reported to express CXCR2 receptors including astrocytes. Astrocytes are thought to be a potential source of the ligand, raising the possibility of an autocrine loop that also contributes to the regulation of remyelination either through release of CXCL1 or other cytokines.

The robust functional recovery seen in EAE models after systemic delivery of CXCR2 antagonists may reflect CXCR2 signaling responses in immune tissues. Recent studies demonstrated that transfer of encephalitogenic TH17 cells into naive host animals induces both CXCL1 and CXCR2 expression

(Carlson et al., 2008). Furthermore, in relapsing remitting EAE, blockade of

CXCR2 resulted in functional improvement and a reduction in leukocyte infiltration. Likewise, CXCR2-/- animals were relatively refractory to disease induction. In this model, the functional deficits are largely a result of immune signaling since transplantation of CXCR2+ leukocytes into CXCR2-/- animals resulted in disease induction. Taken together with the current data from a chronic model of EAE, these data strongly suggest that CXCR2 signaling is a major contributor to immune mediated demyelination.

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The roles of the chemokine CXCL1 and its receptor CXCR2 in demyelination and remyelination are complex. In a model of demyelination induced by cuprizone intoxication, no apparent differences were seen in

CXCR2+/+ and CXCR2 null animals (Lindner et al., 2008). The basis for this discrepancy is unclear and may represent either differential mechanisms of demyelination, repair, or the quantitation of recovery. In a more complex transgenic animal model, recent studies suggest that overexpression of CXCL1 driven by the GFAP promoter in astrocytes reduces lesion load and enhances repair in models of relapsing and remitting EAE (Omari et al., 2009). By contrast, in the current studies, blocking CXCR2 signaling enhances recovery in chronic models of EAE and in LPC induced demyelination. Several factors may account for the apparent contradiction in these results. First, in vitro studies demonstrate that the levels of chemokine stimulation are critical in promoting OPC proliferation and inhibiting migration. Dose response analyses demonstrate that at high concentrations of CXCL1, OPC responses are negated, presumably, through receptor desensitization, a common characteristic of chemokine receptors (Mueller et al., 1997; Richmond et al., 1997). Thus, in the setting of

EAE, astrocytes are highly responsive and, presumably, the levels of CXCL1 are highly elevated. This might be expected to lead to a transient stimulation of OPC proliferation while more persistent high level ligand expression leads to desensitization of the receptor facilitating functional recovery in relapsing EAE

(Richmond et al., 1997). Elevated levels of CXCL1 may stimulate receptors other than CXCR2. For example, while CXCR2 is the major receptor for CXCL1,

65 there is substantial promiscuity in ligand receptor interactions, and other receptors such as CXCR1 are known to bind the ligand. The functional role of these alternate receptors is currently unknown. Finally, although overexpression of CXCL1 by oligodendrocytes in a transgenic model leads to leukocyte accumulation in the CNS (Smith et al., 1983; Omari et al., 2009), overexpression in astrocytes did not appear to do so. Thus, the effect of the chemokine in this model may be restricted to specific regions of the CNS parenchyma that harbor differentially responsive cells.

We propose several characteristics that position CXCR2 as an attractive target for therapeutic development in demyelinating diseases such as multiple sclerosis. The receptor influences response in OPCs, the primary target cells mediating repair, as well as in peripheral leukocytes, the primary effector cells in immune mediated demyelination. In addition, CXCR2 antagonists have been utilized as potential therapeutics in other inflammatory based diseases (Keane et al., 2004; Gorio et al., 2007). For example, clinical trials are currently ongoing to evaluate CXCR2 antagonists for the treatment of chronic obstructive pulmonary disease, asthma, and cystic fibrosis based on the modulation of neutrophil function (Barnes, 2001; Traves et al., 2004; Govindaraju et al., 2006). Taken together, current data suggests that CXCR2 antagonists may enhance myelin repair in demyelinating diseases such as multiple sclerosis.

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3 l 1% LPC 2D

3 l antibody (100 g/mL) 7D

Figure 2.1. Paradigm for local injection of antibody or small molecule inhibitor after lysolecithin lesions.

Female Sprague-Dawley (200-224g) rats were anesthetized by intramuscular injection of a rodent cocktail. Vertebral column exposure was done using a scalpel and scissors. Animals were stabilized using forceps in order to arch their backs for spinal cord exposure. Borosilicate pulled glass capillary micropipettes were used to deliver a 1% lysolecithin (lysophosphatidyl choline or LPC, Sigma, St. Louis,MO) attached to a micro4 microsyringe pump (World Precision Instruments) at T10/11 region of the spinal cord. The micropipette was inserted through the dura into the dorsal spinal cord and retracted slightly before 3µl of 1% LPC was injected into the dorsal column at a rate of 0.25µl per minute. Animals were allowed to recover for at least 1 day prior to second injection following the same procedure however with either 3µl of 100ng/ml anti-CXCR2 antibody or 100ng/ml IgG control or 160ng/ml CXCR2 small molecule inhibitor or PBS. After the second injection, animals were allowed to recover 10 days post- lysolecithin injection.

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Figure 2.2 Structor of small molecule inhibitor against CXCR2. First developed by Glaxo Smith Kline and displays 150 fold selectivity for

CXCR2 compared to CXCR1.

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Figure 2.3. Lesion volume quantification in lysolecithin lesions.

By utilizing a 3 dimensional reconstruction program, lesion volume in EAE and lysolecithin lesions were able to be calculated. Histological staining using the myelin stain, Luxol fast blue, was done through the entire lesion containing region of the spinal cord. On each section, the entire area of the spinal cord section, the central canal, and the area occupied by lesion was measured. Additionally on each section, 6 points along the grey matter were also selected to ensure proper alignment of the spinal cord sections. Quantification of areas occupied by lesion in respect to the entire spinal cord area was calculated.

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y A x

z

Figure 2.4. Myelin thickness/axonal diameter measurements in EAE and lysolecithin lesions.

This image illustrates the axon in cross section captured using electron microscopy with tracings of the axonal perimeter in red and three separate measurements of myelin thickness in blue (x,y,z). An average of the myelin thickness was calculated and divided by the axonal perimeter to determine their ratio in all models of demyelination. Axon Perimeter= A; Myelin thickness= (x+y=z)/3; Myelin to axon ratio= [(x+y+z)/3]/A.

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Figure 2.5. Local delivery of anti-CXCR2 antibodies reduces the size of

LPC induced demyelinated lesions.

Vibratome sections labeled with Luxol fast blue (A-D) of LPC lesions 10 days after induction treated with isotype control IgG (A and C) or neutralizing antibodies against CXCR2 (R&D Systems; 100µg/ml) (B and D) 2 days after lesion induction. Longitudinal sections (A and B) and cross sections (C and D) from mid-lesion areas demonstrate a reduction in lesion volume in anti-CXCR2 treated animals (B and D) compared to isotype controls (A and C). Three dimension reconstruction of LPC lesions (yellow) from isotype controls (E) and anti- CXCR2 treated animals (F). Comparison of the average LPC lesion volume in IgG control and anti-CXCR2 treated animals. n = 8 p= 0.005 (G). Bar = 100µm

71 in A-D (G). In E and F, blue = central canal, yellow = lesion, and red = outline of the spinal cord.

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Figure 2.6. Local delivery of CXCR2 antagonists enhances remyelination in

LPC lesions.

Labeling of 1 µm sections through the middle LPC lesion area with Toluidine blue (A and B) demonstrates an increase in the number of myelinated axonal profiles in animals that received a single injection of a CXCR2 antagonist 2 days after LPC injection. Ultrastructural analyses confirmed the presence of unmyelinated (asterisks) and thinly myelinated (arrows) axons in experimental animals (D). Lesions from control animals contain largely unmyelinated (asterisks) axons and a higher number of cell bodies (C). Quantification of the proportion of remyelinated versus demyelinated axons in lesions from vehicle and small molecule antagonist treated animals demonstrated a 20-fold increase (p=0.002) in remyelinated axons versus control with direct delivery into LPC lesions and a 9-fold increase in remyelinated axons (p=0.004) with systemic delivery (E). Bar = 20µm in A and B and 2µm in C and D.

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Figure 2.7. Systemic delivery of CXCR2 antagonists has limited effect on repair of LPC lesions.

Labeling of 1µm sections with toluidine blue (A and B) demonstrated no significant change in overall lesion size in animals treated daily with IP injections of CXCR2 antagonist, although experimental animals demonstrated an apparent reduction in the cellularity of lesions (B and D). Electron microscopy confirmed the limited effect of systemic inhibition of CXCR2 in promoting LPC lesion repair (C and D). Occasional thinly myelinated axons (arrows) were present scattered throughout the lesion in CXCR2 antagonist treated animals (D) that were absent in controls (C) which had a higher number of unmyelinated axons (asterisks). The myelin surrounding remyelinating axons was frequently uneven, possibly reflecting active remyelination. Bar in A and B = 20 µm and C and D = 2µm.

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Figure 2.8. Inhibition of CXCR2 promotes the differentiation of spinal cord

OPCs in vitro.

Spinal cord mixed cell cultures (P3) grown for 5 days in vitro contain 35% A2B5+ cells (A) and approx 19% O1+ (D) and 17% MBP+ cells (G). Exposure to 0.05ng/ml CXCL1 for 24hrs results in an increase in A2B5+ cells (46%) (B) but a decrease in O1+ (12%) (E) and MBP+ (12%) cells (H). By contrast, exposure to CXCR2 antagonist (40ng/ml) for 24hrs results in a decrease in A2B5+ cells (26%) (C) but an increase in O1+ (31%) (p=0.05) (F) and MBP+ cells (50%) (p=0.007) (I) to the proportion of immunopostive cells compared to DAPI+ cell numbers (J). Data represents mean +/- standard deviation taken from 2 coverslips from at least 3 separate experiments. Bar = 20µm in A-F and Bar = 40µm in G-I.

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Figure 2.9. Systemic inhibition of CXCR2 results in functional improvement in MOG35-55 peptide induced EAE animals.

Injection of MOG35-55 peptide induces a robust functional deficit after 7-10 days that plateaus in the 2nd week (circles) (A). Systemic daily delivery of CXCR2 inhibitor (8µg/kg) results in a slowing of disease progression and functional recovery that is maintained with continuous treatment (squares)(A). Data represent mean clinical scores for 12 animals from 3 separate studies. Luxol fast blue staining on transverse spinal cord sections showed areas of demyelination in control EAE animals (B) that were significantly reduced in animals treated with CXCR2 antagonists (C). Control EAE animals showed significant cellular infiltrates in areas of demyelination (D) that were reduced in animals treated with CXCR2 antagonists (E). Quantitation of the relative lesion load in the spinal cord of control EAE animals and CXCR2 antagonist treated animals at day 28

76 after immunization (F). Data represents the mean +/- standard deviation taken from 20 Luxol stained sections from each of 4 animals in each group. p= 0.0001. Bar = 100µm in B and C and 50µm in D and E.

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Figure 2.10. Systemic inhibition of CXCR2 results in decreased cell infiltration and increased remyelination in MOG35-55 induced EAE.

In control EAE animals, 28 days after disease induction, lesion areas in the spinal cord were characterized by demyelination and cellular infiltrates (A). By contrast, in EAE animals treated daily with CXCR2 antagonists the extent of demyelination and cellular infiltration is substantially reduced (B). Ultrastructural studies confirmed the presence of extensive demyelinated axons (asterisks) in lesion areas associated with substantial cell infiltration (C). In animals treated with CXCR2 antagonist (D) the number of demyelinated axons was reduced and the number of thinly myelinated (arrows) axons increased consistent with widespread remyelination. Quantification of the relative numbers of

78 demyelinated (asterisks), remyelinated (arrows), and unaffected axons in lesion areas of control CXCR2 antagonist treated EAE animals (E). The number and proportion of demyelinated axons is significantly decreased in CXCR2 antagonist treated animals compared to controls (90% vs 25%) while the number and proportion of remyelinated axons was significantly increased (10% vs 65%) (p= 0.000002) (E). The total number of axons was not significantly different in treated and untreated animals and the proportion of unaffected axons was below 10% in both groups (E). Remyelinated axons were characterized as having thinner myelin sheaths and quantification of 20 axons per lesion from 5 lesion areas in each of 3 animals demonstrated a significant reduction (p= 0.02) in myelin thickness/axon diameter ratio in treated animals (F). Control EAE animals had a ratio close to 1 suggesting little remyelination while CXCR2 antagonist treated animals had a mean ratio around 3, indicative of substantially thinner myelin sheaths (F). Bar = 75µm in A and B and 2 µm in C and D.

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Figure 2.11. Systemic treatment with CXCR2 antagonists results in increased MBP and decreased Iba1 expression in EAE animals.

Control EAE animals demonstrated reduced MBP labeling (A and C) and high levels of Iba1+ cells in lesion areas (arrowheads) (E and G. By contrast, animals treated with CXCR2 antagonist demonstrated higher levels of MBP expression (B and D) and fewer Iba1+ cells (F and H)in lesion areas (arrowheads). The number of Iba1+ cells in CXCR2 antagonist treated EAE animals was decreased by 50 +/- 5% in grey and white matter regions compared to vehicle treated animals (G and H). Bar = 500µm in A, B, E and F and 200µm in C, D, G and H.

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Figure 2.12. Long-term but not short-term treatment with CXCR2 antagonist results in sustained remyelination.

EAE animals treated with CXCR2 antagonist systemically for a period of 2 weeks and then removed from treatment demonstrated persistent remyelination 1 week later (A, C). By contrast, EAE animals removed from CXCR2 antagonist treatment after 1 week of treatment demonstrated a lack of remyelination, although there was an apparent reduction in inflammatory cell infiltration (B, D). Bar = 50µm.

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CHAPTER 3: REGULATION OF ASTROGLIOSIS BY THE CHEMOKINE

CXCL1

ABSTRACT

Multiple sclerosis (MS) is a neurodegenerative disease characterized by a series of demyelination/remyelination episodes. Chemokines and their receptors have been implicated in the process of remyelination failure (Filipovic et al.,

2003; Omari et al., 2005). It seems likely the actions of chemokines are complex and involve multiple cell types. Functional chemokine receptors have been demonstrated in CNS neurons, astrocytes, oligodendrocytes, and microglia under physiological conditions and activated chemokines regulate migration, proliferation and differentiation of immune and CNS cells during development and pathology (Glabinski et al., 1995; Eng et al., 1996; Glabinski et al., 1998;

Bona et al., 1999; Hesselgesser and Horuk, 1999; Huang et al., 2000; Omari et al., 2005; Valles et al., 2006; Szczucinski and Losy, 2007). In secondary progressive MS, chemokine expression by astrocytes is thought to play a role in microglia/macrophage activation and neurodegeneration. The activation of astrocytes themelves may contribute to pathology after neural insults. Astrocyte activation following injury has been demonstrated to cause upregulation of a number of inhibitory molecules such as chondroitin sulfate proteoglycans, creating a nonpermissive glia scar that blocks axonal regeneration. Previous studies in our laboratory demonstrated a disruption of early oligodendrocyte development and long term structural alterations in myelination and glia in the absence of chemokine receptor CXCR2 suggesting a role of oligodendrocytes

82 and astrocytes in the signaling of CXCR2. CXCR2 knockout mice were resistant to cuprizone induced demyelination in the corpus callosum suggesting an enhancement of myelination by enhancing migration and differentiation and/or due to a decrease in the astrogliotic response promoting repair. OPC proliferation is regulated by CXCL1 secreted by subsets of astrocytes (Robinson et al., 1998). Additionally, CXCL1 decreases OPC migration into presumptive white matter areas during development (Tsai et al., 2002; Padovani-Claudio et al., 2006). CXCL1 is known to be expressed in the periphery of MS lesions, and, therefore, the secretion by astrocytes may modulate astrogliosis and prevent oligodendrocytes from entering a lesion and remyelinating (Filipovic et al., 2003;

Omari et al., 2006). Determining the downstream signaling events may allow for modulation of this pathway by small molecule inhibitors to promote repair by decreasing astrogliosis. Here I show that rodent astrocytes express CXCL1 and

CXCR2. The expression of CXCR2 is developmentally expressed and persists into adulthood. NG2+ and GFAP+ cells respond to CXCL1 by increasing expression and secretion of CSPGs. Treatment of purified astrocyte cultures with CXCL1 for 24 hours resulted in upregulatation of GFAP protein and increased GFAP+ cell numbers. Treatment of purified astrocyte cultures with

CXCL1 upregulates a number of cytokines implicated in modulation of EAE disease severity, suggesting a role for CXCL1 in mediating pathology in models of demyelination. CXCL1 was additionally upregulated around the periphery of lysolecithin induced demyelinating lesions. This suggests a role for CXCL1 in

83 regulating astrogliosis, and the responses of astrocytes to chemokine stimulation may contribute to the presistance of damage.

INTRODUCTION

Many CNS insults are characterized by axonal damage, demyelination, and presence of glial scarring. This glial scarring is a consequence of astrocyte and glial precursor responses and is manifested by upregulation of a number of intermediate filament proteins including glial fibrillary acidic protein (GFAP), vimentin, and NG2 (Fawcett and Asher, 1999; Jones et al., 2002; Fitch and

Silver, 2008). In addition, glial scarring is typically associated with upregulation of a number of extracellular matrix proteins known as chondroitin sulfate proteoglycans (CSPGs), which create a nonpermissive environment for axonal regeneration (Silver and Miller, 2004). The glial scar is thought to create a chemical and physical barrier preventing axonal repair and possibly remyelination in injured tissue. The precise role of astrogliosis and the formation of glial scars is controversial. It remains uncertain as to whether astrogliosis is detrimental or benefical to repair. Ablation of reactive astrocytes in transgenic mice has demonstrated that these cells are important for the spatiotemporal regulation of inflammation after CNS injury (Bush et al., 1999; Faulkner et al.,

2004). An understanding of the glial scar complex environment, its purpose, and its influences on cell migration, proliferation, and differentiation will be beneficial

84 to designing techniques to modulate ongoing damage, improve remyelination and improve axonal function after injury.

As glial cells progress through the stages of differentiation towards a mature astrocytic phenotype, they upregulate GFAP, which is the most prominent intermediate filament in mature astrocytes (Kalman and Ajtai, 2001). After injury, these cells also upregulate their GFAP expression that contributes to the glial scar (Fitch and Silver, 2008). In time points after injury, NG2+/GFAP- cells respond to mitogenic cues to upregulate their CSPG expression (Fuller et al.,

2007). Thus, it is possible that GFAP+ and NG2+ cells respond to certain cues present at areas of damage and lesions to cause production of a non-permissive or permissive environment, respectively, for remyelination to occur.

Chemokines, or chemoattractant cytokines, comprise a family of inducible secreted molecules of small molecular weight (8-10kDa) (Hesselgesser and

Horuk, 1999), which function as activators of leukocytes, wound healing, modulation of angiogenesis, and tumorigenesis. Most of the knowledge related to chemokines has been derived from the immune system due to traditional implications in modulation of immune coordination and inflammation (Fernandez and Lolis, 2002; Laing and Secombes, 2004). Chemokines and their receptors localize cells with fine spatio-temporal precision (Charo and Ransohoff, 2006).

Chemokines are classified into four subfamilies based on the number of conserved cysteines at their amino terminus (Hesselgesser and Horuk, 1999).

CXC chemokines, whose expression is restricted to higher vertebrates, are further classified according to the presence or absence of a glutamate-lysine-

85 arginine (ELR) motif on their amino terminus adjacent to the first cysteine residue(Wang, 2003). CXCL1, previously known as Gro , is a member of the

ELR containing CXC chemokines who preferentiall binds to CXCR2 (Sai et al.,

2004).

Chemokine receptors are G-protein coupled receptors (GPCRs) usually linked to pertussis toxin sensitive Gi proteins (Bajetto et al., 2001). The chemokine receptor,CXCR2 binds CXCL1 and their interaction stimulates intracellular processes that modulate a number of cellular events, including migration, proliferation, and differentiation (Bajetto et al., 2001; Robinson and

Franic, 2001; Miller, 2002; Tsai et al., 2002). During early postnatal development in mice, CXCL1 can enhance PDGF induced proliferation and halt migration of OPCs (Tsai et al., 2002). CXCR2 signaling in response to CXCL1 had been found to help position OPCs into white matter (Tsai et al., 2002). It also locally modulates responses to PDGF, enhancing proliferative response of

OPCs (Robinson et al., 1998; Robinson and Franic, 2001).

Elevated levels CXC chemokine expression in the CNS is found in infection, tumors, ischemia and demyelination (Liu et al., 1993; Spanaus et al.,

1997). Overexpression of CXCL1 in oligodendrocytes induces neutrophil invasion and astrogliosis (Tani et al., 1996b; Tani et al., 1996a). The absence of

CXCR2 leads to changes in the oligodendrocyte lineage during development

(Padovani-Claudio et al., 2006). CXCR2 activation inhibits OPC migration and

OPC accumulation at the periphery of MS lesions where there is upregulation of

CXCL1 (Omari et al., 2005). NG2+ cells increase in the CXCR2 knockout mice

86 developmentally may facilitate recovery of the disease enhancing migration and proliferation of OPCs (Padovani-Claudio et al., 2006). An understanding of the mechanism by which CXCR2 mediates glial cell proliferation and morphology would be advantageous in defining possible mechanisms for repair of demyelinating lesions. Therefore, its role in demyelinating lesions would be valuable in providing a potential therapeutic.

Given that CXCL1 and CXCR2 are known to modulate glial cell proliferation and migration, we investigated the expression of CXCR2 and

CXCL1 on GFAP+ and NG2+ cells. This lead us to analyze the secretion of

CSPG in CXCL1 activated astrocytes, in order to model what occurs after injury.

These data demonstrates a role for CXCL1 signaling via CXCR2 in producing an astrogliotic response as judged by the expression and secretion of CSPG.

MATERIALS AND METHODS

Animals and Reagents

All animal procedures were conducted in according to approved guidelines set forth by the National Institutes of Health Animal Protection Guidelines and were approved by Institutional Animal Care and Utilization Committee of Case

Western Reserve University School of Medicine.

Primary spinal cord cultures and purification of astrocytes, OPCs, and mixed cell populations

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Purified astrocyte cultures were prepared from postnatal day 3 rat or mouse spinal cords. Spinal cords were removed and the meninges were detached. Chopped tissue was dissociated by incubation in 0.25% trypsin and

EDTA in calcium/magnesium free DMEM for 25 minutes in a 37o water bath.

Trypsin reaction was stopped by addition of 10% fetal bovine serum (FBS) in

DMEM and cells were triturated using a firepolished pipette and DNase. Cells were then filtered through a 40 m nitex filter and plated onto poly-L-lysine (PLL) coated flask. After 24 hours at 37o, cells were shaken at 200rpms at 37oCelsius overnight to remove cells that were not adherent and therefore not astrocytes, and media was changed. A week later, cells were treated with complement lysis to remove any remaining oligodendrocyte lineage cells. Cells were incubated in

A2B5, O4, and O1 antibodies for 30 minutes at 37o Celsius. Guinea pig complement sera (Sigma) was added to cells and incubated at 30o Celsius for 30 minutes causing lysis of cells that was bound to the specific antibodies. The cells were then removed by rinsing the flasks with 10%FBS in DMEM. Media was then changed, and cells were allowed to recover for 5-7 days. For analysis, cells were plated onto PLL coated coverslips and allowed to recover for 2-3 days prior to serum removal and treatment of CXCL1 (0.5ng/ml and 10ng/ml). Cells were assessed for purity by immunocytochemistry on sister coverslips. Resulting cell populations were of >85% astrocyte lineage.

A2B5+ cell populations were prepared by immunopanning from postnatal day 2 rat spinal cords as described previously (Robinson, 1998). Resulting cell

88 population were >90% oligodendrocyte lineage and grown in DMEM with Hong’s

N2 supplement and 10ng/ml recombinant PDGF.

Mixed cell cultures were derived from postnatal day 2 rat spinal cords and dissociated via standard techniques. Cells were grown in media consisting of

DMEM, 10ng/ml PDGF, 10ng/ml fibroblast growth factor (FGF), 1% FBS, and

Hong’s N2 supplement. Cells were maintained for a maximum of 3 days for experiment.

Immunocytochemistry and immunofluorscence of primary cell cultures and assessment of purity

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and then rinsed three times in PBS. Cells were then incubated in primary antibody for 30 minutes in blocking buffer, which varied for each antibody. Cells were washed in PBS and incubated in corresponding secondary antibodies for 30 minutes in blocking buffer, washed in PBS thoroughly, and mounted onto Superfrost slides using Vectashield with DAPI (Vector Laboratories

Burlingame, CA). Images were collected using a Leica DM5000B microscope and Leica Applications Suite Software.

Bromodeoxyuridine incorporation

To analyze proliferation of cells in S phase, bromodeoxyuridine (BrdU)

(10 M) was added to the media at least 18 hours prior to fixation in 4% paraformaldehyde diluted in PBS for 30 minutes. After washing and prior to cell

89 marker staining, the nuclear envelope was permeabilized with 2N hydrochloric acid (HCl) for 45 minutes at room temperature. Cells were then gently washed with 1XPBS 3 times 15 minutes each. Cells were then incubated in primary antibody (mouse anti-bromodeoxyuridine; Roche 1:50) diluted in 10%NGS/0.3%

Triton in 1X PBS for 30 minutes at room temperature. Cells were washed by dipping coverslips in 1XPBS for 10 seconds 5 times. Cells were incubated with goat anti-mouse IgG conjugated with an Alexa Fluor (1:200) secondary antibody.

Coverslips were then washed in 1XPBS well and finally in distilled water and mounted.

Analysis of chondroitin sulfate proteoglycan secretion and expression and cytokine profiling

Media was collected after various treatments and stored at -20 degrees

Celsius from astrocyte purified cell populations for analysis of CSPG (CS-56) secretion. Dot blots were performed using the BioRad Vacu-Dot 96 well apparatus, according to the manufacturer’s instructions (BioRad Laboratories

Hercules, CA) to determine relative amounts of CS-56 protein that was secreted into media of astrocyte cultures. Nitrocellulose was soaked in tris-buffered saline

(TBS) and placed in the BioRad Vacu-Dot. After rinsing the wells with TBS, 50 l or 100 l of conditioned media from dose response and time course culture assays were loaded into the wells and allowed to pass through the nitrocellulose by passive filtration. After rinsing 2 times with TBS, the membrane was removed from the BioRad Vacu-Dot and blocked for several hours in 2% milk dissolved in

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TBS/0.2% Tween20 (TBST) at room temperature. The blot was incubated in mouse IgM anti-CS56 (1:2000, Sigma) in blocking buffer overnight at 4 degrees

Celsius. After rinsing in TBS, blots were incubated with goat anti-mouse IgM-

HRP (1:5000, MP Biomedicals, Aurora, OH) in block for 2 hours at room temperature. Blots were rinsed and developed with Super Signal West Pico

Chemiluminescent detection reagents (Pierce, Rockford IL) and exposed to X-ray film. Protein was quantified using Adobe Photoshop CS2 software.

The cytokine profiles were conducted using an R&D systems Profiler rat cytokine profile assay A according to manufacturer’s protocol. The same media from dot blot studies were also assayed using cytokine profile. Media from astrocytes with 1% FBS was used as a control, and treatments of CXCL1 were conducted for 1 day at 10ng/ml or 0.5ng/ml in 1% FBS in DMEM. However, it was determined that either concentration of CXCL1 mediated the same effects.

Therefore, for the conclusion and quantification of the study 10ng/ml CXCL1 treatments for 1 day were used throughout.

Quantification of cytokine profile and dot blot assays were conducted using Image J software analysis of pixel intensity. By repeating each study 4 times, using different astrocyte cultures, we were able to confidently quantify the results using Student paired t-test in Microsoft Excel.

RNA isolation and real time PCR of CXCR2, CXCL1, and chondroitin sulfate proteoglycans

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RNA isolation was from purified cultures or whole brain or spinal cord lysates. Cells or tissue were homogenized in Trizol and chloroform was added.

The cells or tissue was spun and the organic layer was removed and placed in isopropanol alcohol. This was incubated for 10 minutes at room temperature.

The resulting mixture was spun and a pellet containing RNA was washed in cold ethanol. The pellet was spun and dried at room temperature until reconstitution in DEPC water. Random primers were used to reverse transcribe cDNA from total RNA samples. Real time PCR was performed using the Gene Expression and Genotyping of the Case Comprehensive Cancer Center (P30CA43703).

Lysolecithin induced demyelination

Twelve week old female Sprague-Dawley rats weighing approximately

220-229 grams were anesthetized with an intramuscular (i.m.) injection of ketamine hydrochloride, xylazine hydrochloride, and acepromazine. Midline incisions were performed and a laminectomy of the T10 thoracic vertebrae performed to expose the spinal cord. Three microliters of 1 percent LPC (L-α- lysophosphatidyl-choline, lysolecithin) (Sigma, St. Louis, MO) in 0.9 % sodium chloride solution was microinjected using a pulled glass pipette into the dorsal column of the spinal cord at a rate of 0.25 microliters per minute to generate a reproducible localized demyelinating lesion. Incisions were surgically closed.

Post-operatively, animals received a subcutaneous injection of 5 milliliters of saline to promote hydration. Animals were allowed to recover and sacrificed at various days post-lesion.

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Immunohistochemistry using CXCL1 antibodies

For CXCL1 staining, animals were anesthetized as above and perfused with PBS. The spinal cords were then dissected out and placed in Bouin’s fixative. The sections were cut on a vibrating microtome at 35 m and allowed to dry at room temperature overnight then stored at -20 degrees Celsius until immunostaining could be completed.

Immunohistochemistry and immunofluorescence

Animals were perfused and fixed in 4% PFA as demonstrated above.

Spinal cord were cross sectioned using cryostat and allowed to dry and placed at

-80oC until staining could be performed. Sections were rinsed of excess O.C.T.

The sections were blocked in 0.03% PBST and 10% NGS. Primary antibody was diluted in blocking solution and incubated overnight at 4oC (GFAP; Dako 1:500;

CSPG 1:500 (no triton) Sigma). Sections were washed 3 times10 minutes each at room temperature with 1XPBS. Sections were incubated in anti-rabbit IgG or anti-mouse IgM fluorescently conjugated secondary antibodies (1:500) for 1 hour at room temperature. Slides were then washed in 1X PBS 3 times 10 minutes and incubated in DAPI (1:1000; Invitrogen) to label nuclei for 10 minutes. The sections were briefly rinsed with 1XPBS then distilled water and mounted using

Citifluor mounting media (Ted Pella, Redding PA). Images were collected using a Leica DM5000B microscope and Leica Applications Suite software.

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RESULTS

Distinct subsets of astrocytes express the chemokine CXCL1 and its receptor CXCR2.

Although CXCL1 has been suggested to be present in demyelinating injury and expressed by astrocytes, the expression of CXCL1 and its receptor in the developing CNS has yet to be elucidated. CXCL1 and its receptor expression may dictate the precise spatial and temporal regulation of oligodendrocyte precursors and astrocyte differentiation. Previous studies, using frontal lobe mixed cultures, suggest CXCL1 is secreted by a subset of astrocytes

(Robinson, 1999). This expression may control proliferation of OPCs or create a potential for autocrine signaling back onto astrocytes. Therefore, we assessed the expression of CXCL1, CXCR2, and CXCR1 in subsets of purified cell cultures

(A2B5+, GFAP+, mixed cells) derived from the postnatal spinal cords of rats.

Messenger RNA and staining assessed the expression of CXCR2, CXCL1 and other chemokine receptors and their dependence on time in culture, varying from

2 weeks to 4 weeks in vitro. CXCR2 was expressed in astrocytes (Fig 3.1) and oligodendrocyte progenitor cell populations (data not shown) cultured from postnatal rat spinal cords. Consistent with previous studies, CXCL1 mRNA was expressed in purified postnatally derived astrocyte cultures (Fig. 3.1). However, expression of the receptor was dependent on the time in vitro. During earlier culture periods of 2 weeks there were lower expression of CXCR2 as compared to 4 weeks in culture (data not shown). Furthermore, morphology of the GFAP expressing astrocytes changes with time in culture. The cells exibit a more

94 flattened morphology and seem to be less stellate as their younger counterparts.

Therefore, the expression of CXCR2 is induced in a precise culture time frame suggesting that astrocyte maturation or the generation of a reactive astrocyte population is enhanced over time in culture.

Glial precursors express CXCR2

Astrocyte expression of CXCR2 and CXCL1 suggests a role for

CXCL1/CXCR2 signaling in modulating astrocytes response. Short term cultures of astrocytes derived from early postnatal spinal cord presented with GFAP+ cell expression. However, there were additional cells that did not label with GFAP antibodies that were labeled with NG2+, potentially a glial precursor (Fig 3.3).

Cells were immuno-labeled for a variety of cell type specific markers to determine if these cells could be identified as precursors of the astrocyte lineage. These turned out to be NG2+ cells, and they did not coexpress GFAP markers.

Recently, it has been shown that NG2+ cells responding to a cortical knife wound can differentiate into astrocytes, supporting the hypothesis that NG2+ cells act as astrocyte precursors in certain situations (Penton-Rol et al., 2008). Additionally, there were cells present that expressed varying levels of GFAP protein, suggesting GFAP protein expression varies in culture conditions (Fig 3.3) (Soula et al., 1990). Additionally, there may be more differentiated astrocytes (low

GFAP expressing astrocytes) and those more activated in response to culture conditions (high GFAP expressing astrocytes). Therefore, it is possible these

NG2+ cells then differentiate into GFAP-expressing astrocytes which have

95 differing levels of GFAP expression. However, with addition of CXCL1, we observed increased proliferation via BrdU incorporation. This increase in proliferation was present in NG2+ GFAP- and GFAP+ NG2- cells (data not shown). Therefore, both cell types respond to ligand addition with increased proliferation.

Astrocyte cultures maintained for long periods of time (4 weeks) in 10%

FBS were assessed to determine what cell types were persistant in culture.

These cells continued to express GFAP however they did not coexpress NG2 proteins. The GFAP+ cells expressed protein at differing levels and demonstrated a flattened morphology (Fig 3.3). Under normal conditions (i.e., without addition of chemokine, CXCL1), these 4 week cultures of cells increased

CSPG expression (Fig 3.2). Additionally, at 1 day post treatment with 0.5ng/ml

CXCL1 and 10ng/ml CXCL1, there was induction of secretion of CSPG greater than that of control cultures (Fig 3.2). The expression of CSPG on the GFAP+ cells were located on the end of the processes when treated with CXCL1; however, in control cultures the CSPG expression remained more around the cytoplasm surrounding the nucleus (Fig 3.2).

CXCL1 stimulates astrogliosis in mature astrocytes

Upon addition of CXCL1 into these purified astrocyte cultures, there was an increase in expression and secretion of chondroitin sulfate proteoglycans

(CSPGs) and proinflammatory cytokines in addition to a migratory signal,

CXCL3. This suggests a role for CXCL1 in regulating the establishment of a

96 chemical barrier for astrogliosis. Cytokine profiling was conducted to determine potential downstream effects of CXCL1 stimulation. Astrocytes are known to secrete a number of cytokines. Therefore, conditioned media from purified astrocyte cultures in addition to cellular extracts were collected to analyze cytokine protein expression in response to 1 day of CXCL1 stimulation. Dot blots were analyzed using a chemiluminescent kit and relative pixel intensity was calculated using positive and negative controls. A number of proinflammatory cytokines such as LIX, TIMP1 and sICAM were altered by CXCL1 treatment (Fig

3.4). TIMP1 was decreased by 64% (p<0.02); sICAM increased by 162%

(p<0.02) than control; LIX was upregulated 98365% (p<0.02) than control cultures. TIMP1, LIX and sICAM have been found to upregulated in CSF in reponse to an acute MS lesion in patients. In addition migratory signals such as

CXCL3 were also induced with CXCL1 treatment although to a lesser degree

(p=0.07). An internal control was also present with CINC expressing (also known as CXCL1) only in astrocyte cultures treated with CXCL1 (Fig 3.4).

Traumatic injuries to the CNS result in acute infiltration of the lesion core with ED1+ microglia and NG2+ glial precursors (Levine, 1994; Penton-Rol et al.,

2008). Previously in our laboratory, it was demonstrated that LPC-induced demyelinating lesions into the dorsal column resulted in activation of GFAP+ cells

(Fuller et al., 2007). CXCL1 has been shown in rodents to act synergistically with platelet derived growth factor (PDGFAA) to induce proliferation of OPCs in culture (Robinson and Franic, 2001). Studies utilizing the jimpy mutant mouse demonstrate elevated levels of CXCL1 that correlate with increased NG2+ cells

97 proliferation in vivo (Wu et al., 2000b). Previous studies additionally demonstrated OPC migration being arrested with CXCL1 presence in vitro (Tsai et al., 2002). Therefore, modulation of OPC migration via NG2+ and dampening

GFAP cell populations may be mediated by CXCL1 via CXCR2. CXCR2 protein was demonstrated to be upregulated surrounding lesions as indicated by histological staining (Fig 3.5). However, the cell type responsible for this was unable to be determined due to antibody avaliability. In this sense, CXCR2 is affected by injury to the dorsal column; however, it is unclear as to what cell type co-expresses. Presumably, CXCR2 could be expressed on leukocytes or blood cells in addition to OPCs and GFAP expressing cells.

CXCL1 is upregulated in demyelinating lesions

To determine whether upregulation of CXCL1 is present in demyelinating lesions in the rat model, we immunostained sections derived from lysolecithin induced demyelination of the dorsal columns. There was indeed upregulation of

DAB+ CXCL1 + staining in LPC lesions 3 days post lesion (Fig 3.6 top right). The

CXCL1+ cells were of stellate morphology however, because of antibody constraints the determination of cell phenotype could not be determined.

Further in adjacent sections, there was increased CXCR2 staining in cells being recruited to the lesion (Fig 3.6 lower right). These cells seemed to be of stellate morphology and were distributed throughout the white matter. It has been suggested that NG2 expressing cells are OPC and are oligodendrocyte type 2 cells, which can differentiate into oligodendrocytes or type 2 astrocytes (Raff et

98 al., 2001); however, in vitro NG2 expressing glia can be driven to differentiate into astrocytes or oligodendrocytes (Zhu et al., 2008). The signals modulating the mechanism of pathway selection have yet to be determined.

To determine in vivo effects of CXCL1, direct injection of CXCL1 into dorsal columns of spinal cord of adult rats was performed without injury. Animals were sacrified at 6hrs, 12hrs and 3 days post injection. There was an absence of an induction of GFAP protein expression at either time point as indicated by immunohistochemistry (data not shown). Additionally, CS-56 protein immunohistochemistry also ceased to be upregulated in response to CXCL1 injection.

CXCL1 delivery intrathecally induces GFAP expression

Upregulation of a number of astrogliotic responses in culture suggests a role for CXCL1 in modulating astrogliosis. To determine whether elevation of

CXCL1 would promote astrogliosis in vivo, we implanted an Alzet osmotic pumps to administer into the cisterna magna to infuse CXCL1 (10µg/ml) or control saline at a rate of 1µl/ hour for 3 days. Perfusion of animals occurred on various days post-implantation (1 day, 2 days and 3 days); however, this made no difference in the results (data not shown). GFAP immunostaining was conducted on brain and spinal cord sections from control and CXCL1 infused animals (Fig 3.7).

There was a definitive upregulation of GFAP in the spinal cords of animals infused with CXCL1. On the other hand, the brain from control and CXCL1 infused animals seemed to be indistinguishable from each othe (Fig 3.7).

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Therefore, without injury to the CNS, prolonged exposure to CXCL1 alone can produce an astrogliotic response in the spinal cord.

DISCUSSION

Data presented in this study has provided new insights into the actions of chemokines in modulating astrogliosis in areas of injury. These data demonstrate expression of CXCR2 and CXCL1 in astrocytes and raises the possibility that an autocrine loop exists in astrocytes to produce a response in astrocytes. In response to CXCL1 addition, astrocytes upregulate GFAP+ cell numbers and levels of protein expression. The production of inhibitory molecules, such as chondroitin sulfate proteoglycans is a hallmark of gliosis. In this study, CXCL1 addition to astrocytes promoted secretion into media and expression on GFAP+ cells of CSPGs. Further, the secretion of a number of cytokines was intitated with treatement of astrocytes with CXCL1 which include

TIMP-1, LIX, and sICAM are modulators of injury models. TIMP-1 knockout animals have exacerbations of disease in chronic models of Multiple Sclerosis

(Crocker et al., 2006; Thorne et al., 2009). LIX is a secreted molecule found to be upregulated in astrocytes of early postnatal rodent brains who have been treated with lipopolysacchride (LPS), an endotoxin that elicits a strong immune response (Rovai et al., 1998). Soluble ICAM has been found to be induced in the cerebrospinal fluid extracted from patients with clinical MS and those with idiopathic optic neuritis during an inflammatory attack and not found in serum from patients (Petersen et al., 1998; Acar et al., 2005).

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CXCL1 infusion into intrathecal cerebrospinal fluid chronically produces an astrogliotic response with induction of GFAP protein in the spinal cord.

CXCL1/CXCR2 signaling blockade results in a decrease in GFAP upregulation in areas of demyelination.

Inflammation in the CNS is a source of cytokines and can lead to upregulation of inhibitory molecules that further hinders repair in pathology or injury. The injury or pathology inhibits neurite outgrowth and potentially inhibits myelination. Injured neurons in the adult CNS have a limited capacity to regenerate. In demyelination injury, the axons become stripped of their myelin sheaths and are susceptible to downstream attacks potential by inflammatory cells or cytokines or inhibitory molecules. This non-permissive environment is either set up by chemokines and cytokines or is a downstream effect of the injury or pathology.

Chemokines may regulate the release of proinflammatory cytokines and also inhibitory molecules in response to injury. A number of chemokines and cytokine are upregulated in response to demyelination injury, stroke, neuritic plaques, and in experimental closed head injury.

Chemokines are known modulators in the development of the nervous system as are extracellular matrix molecules, such as CSPGs. CXCL1 will induce proliferation yet halts migration of OPCs in vitro. CXCR2 knockout animals have a reduction in the amount of GFAP protein in addition to MBP protein. This suggests a role for CXCL1/CXCR2 signaling in regulating oligodendrogenesis and astrogenesis in the developing brain and spinal cord.

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Subsets of astrocytes are known to secrete CXCL1, whether this is in response to injury is unknown. However, CXCL1 is known to be upregulated around demyelinating lesions in MS patients and in experimental models of MS. By correlation, it is possible CXCL1 secretion by astrocytes is enhanced in an injury setting. Further, astrocytes proliferate and upregulate GFAP in response to injury. Whether this is a result of CXCL1 secretion or upstream of CXCL1 is the focus of this work. Astrocytes proliferate and upregulate GFAP with exposure to

CXCL1 after serum withdrawl. Addtionally, inhibitory cytokines and extracellular matrices are upregulated in response to CXCL1 treatment in vitro. Additionally,

CXCL1 perfusion into CSF also produces an astrogliotic response in areas of increased exposure to CXCL1. It is possible, in these studies; the brain never became exposed to CXCL1 due to the small protein size (10kDa) and short half life and the rostral caudal flow of CSF. Additionally, the small induction of GFAP protein seen in the spinal cord and absence in the brain could be due to desentization of the receptor and therefore, dampening the GFAP/CSPG protein response. This would account for the lack of difference in animals perfused with

CXCL1 versus saline.

CXCL1 in astrocytes is an inducer of astrogliosis with upregulation of

GFAP and secretion of CSPGs and regulation of secretion of other detrimental cytokines. A recent paper suggests overexpression of CXCL1 in astrocytes promotes neuroprotection and remyelination (Omari et al., 2009). However, this may mimic results seen in CXCR2 knockout animals (Padovani-Claudio et al.,

2006), in that there was an increase in OPCs but reduction in MBP. Additionally,

102 the transgenic animals overexpressing CXCL1 used in Omari et al., 2009, the potentially a reduction in the number of OPCs migrating into lesion areas due to

CXCL1/PDGF migratory blockade (Tsai et al., 2002). Therefore, the researchers may have had cells entering and proliferating but never had functional remyelination. Additionally, astrocyte overexpressing CXCL1 may have been secreting other factors such as CSPGs and cytokines that further impeded repair, however this was not assessed. Short term exposure to CSPGs and cytokines may be beneficial to repair however, long term secretion of CXCL1 thereby secretion of cytokines and CSPGs may imped repair. The signaling between astrocytes and oligodendrocytes via CXCL1/CXCR2 needs to be studied more extensively to determine the downstream effects.

In these studies, we have demonstrated CXCL1 secretion by astrocytes produces an autocrine signaling event by CXCR2. This cascade promotes secretion of factors detrimental to repair, for example CSPGs and inflammatory cytokines. CXCL1/CXCR2 signaling in astrocytes promotes upregulation of

GFAP+ cell numbers and GFAP protein levels in vitro and in vivo. By decreasing the astrogliosis elicited by CXCL1, this will promote repair in demyelination and other injuries of the CNS.

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Figure 3.1. CXCR2 and CXCL1 mRNA is expressed by purified astrocytes in vitro.

Astrocytes were grown in vitro for 2 weeks in 10%FBS and then serum starved for 2 days. DNA was extracted from astrocytes, reverse transcribed and probed for CXCR2 and CXCL1 using standard PCR techniques. Primers were used from R&D systems and also confirmed using previously published primer sequences. GAPDH was used as a control housekeeping gene. CXCR2 and CXCL1 was expressed by the purified astrocytes in vitro

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Figure 3.2. CSPG protein is secreted and expressed by astrocytes in response to CXCL1 treatment.

Astrocyte purified cultures were grown for 2 weeks and plated at the same density and then serum starved prior to treatment for 1 day. Media was extracted and cells were stained. Media was run on a dot blot to determine secretion of CSPG protein into media. Cells were double stained for CS-56 and GFAP. CS-56 was found to be expressed throughout the cell however, on those treated with CXCL1 for 24 hours there was increased CSPG immunostaining on the “processes” of GFAP expressing cells.

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Figure 3.3. CXCL1 treatment increases the number and protein levels of

GFAP in astrocyte cultures.

Purified astrocytes were grown and plated at the same density and then serum starved prior to treatment. CXCL1 was added to culture media for 24 hours. Cells were fixed and stained or processed using standard western blot techniques. Immunohistochemistry indicated a higher amount of GFAP expressing astrocytes. Further, protein extraction and western blotting confirmed the presence of more GFAP protein expression in cells treated with CXCL1 for 24 hours.

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Figure 3.4. Cytokine profile in astrocyte conditioned media treated with

CXCL1 demonstrate in upregulation of inflammatory mediators and migratory signals.

Using R&D systems rat cytokine profiler array A to distinguish whether other cytokines are controlled by CXCL1 in culture conditions. Astrocyte purified cultures were treated for 24 hours with CXCL1 (10ng/ml) or control media after being serum starved for 24 hours. Conditioned media was extracted and used for array using manufacturer’s protocol. Dots were visualized using chemiluminscent standard techniques. 1: positive control; 2: CINC-1; 3:

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CX3CL1; 4: sICAM-1; 5: TIMP-1 control; 6: positive control; 7: LIX; 8: negative control.

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Figure 3.5. CXCR2 protein is expressed in the periphery 3 days post LPC demyelination.

LPC lesions were induced into the dorsal column of the spinal cord. Three days later animals were perfused and CXCR2 protein expression was assessed using DAB immunostaining. This confirmed CXCR2 to be expressed in the periphery surrounding lesions at 3 days post demyelination. Potential cell types expressing the protein include neutrophils, astrocytes and OPCs.

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Figure 3.6. The chemokine CXCL1 is upregulated around LPC lesion 3 days post demyelination and CXCR2 is expressed by GFAP expressing cells within the lesion.

LPC lesions were induced in the dorsal column of the spinal cord. Animals were processed 3 days later for CXCL1 using DAB indirect immunohistochemistry and CXCR2/GFAP immunohistochemistry. CXCL1 expression was induced as a result of demyelination (top right). CXCR2 protein was expressed by GFAP expressing cells in addition to other cell types, possibly neutrophils and oligodendrocytes (bottom right). CXCR2 is expressed in nonlesioned tissue in but not by GFAP expressing cells (bottom left). The chemokine CXCL1 and its congnate receptor CXCR2 are expressed following LPC induced demyelination.

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Figure 3.7. Constant intrathecal delivery of CXCL1 enhances GFAP immunohistochemistry suggesting an astrogliotic response in the spinal cord.

Catheter application was done through the cisterna magna and delivery of either PBS or various concentrations of CXCL1 ligand were given at 1ul/hr increments for 3 days. At this time animals were perfused and processed for IHC. GFAP immunoreactivity was elevated in animals receiving CXCL1.

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CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS

The work presented in this thesis was initiated to determine the role of chemokines, in particular CXCL1/CXCR2 signaling, in the injured CNS and assess whether by blocking CXCR2 there is enhancement of myelination after lesion formation. Two models of demyelination were utilized. One, EAE, consists of an immune mediated event that is followed by a number of other events including astrogliosis and demyelination and the second, local injection of

LPC, is primarily a local dissolution of myelin but without the primary involvement of the immune system. CXCR2 inhibition in EAE produced a recovery of function with systemic delivery of a small molecule inhibitor. In the LPC model, systemic delivery of CXCR2 inhibitor promoted a small amount of remyelination however; direct delivery into the local LPC lesion of inhibitors of CXCR2 signaling produced a reduction of lesion volumes and the promotion of remyelination.

Therefore, by utilizing two models with two different modalities of disease this thesis was able to test inhibitors against CXCR2 and determine the components that regulate remyelination in response to lack of chemokine signaling.

Modulation of CXCR2 signaling in LPC and EAE has produced consistent results that point to CXCR2 inhibitors as having the potential to be a therapeutic for the treatment of MS.

Current therapeutic treatments for MS remain focused on immunmodulation and have yet to focus on the pathology associated with demyelination. The ideal therapy for the treatment of MS would focus on three

112 main arms of disease: induction of pathology, immunomodulation, and inhibition of repair. Induction of pathology remains to be understood fully. The etiology of the disease remains uncertain and may reflect a combination of genetic, environmental, virus induction, previous injury history, and nutrition. However, the most effective therapies would target both immune and CNS cells. CXCR2 is a potential candidate for modulating two distinct aspects of MS. For example,

CXCR2 inhibition depresses the immune cell attacks possibly by decreasing leukocyte infiltration into the CNS and mediation of further pathology in addition to astrogliosis and by decreasing halt of OPC migration mediated by

CXCL/PDGF signaling. In addition, CXCR2 inhibition also promotes the differentiation of OPCs which enhances remyelination in areas of demyelination as a result of pathology.

Expression of a number of chemokines has been determined to be upregulated in response to injury thus regulating migration of cells into and out of injured areas in the CNS and other areas of the animal and human. Chemokines have been found to be upregulated around neuritic plaques in AD brains, closed head brain injury, around MS lesions, and also around lesions in the skin.

Chemokines are known modulators of immune cells outside the CNS and are expressed highly by lymphocytes. However, it is unclear whether lymphocyte entry into the CNS due to disease is modulated via chemokines and their receptors.

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Astrogliosis may confer reason for recovery in animal models of demyelination

In these studies, astrocytes responded to CXCL1 through CXCR2 and upregulated their GFAP+ cell numbers and protein levels and CSPG secretion.

Several cell types, including astrocytes, are known to express receptors for chemokines and cytokines including CXCR2, and this has been confirmed in this thesis. The activity of CXCR2 in astrocytes may have multiple down stream effects. For example, my data suggests that stimulation of CXCR2 modulates the astrogliotic responses which could be regulated by other immune molecules and in turn modulate secretion of other cytokines and chemokines. For example, in a recent study, it was shown CXCL1 secretion by human astrocytes was dependent on IL-6 (Filipovic and Zecevic, 2008). It is possible that CXCL1 secretion by astrocytes modulates autocrine signaling via CXCR2 to cause secretion of inflammatory cytokines, such as TNFα and IFNγ, or secretion of known inhibitors of repair, chondroitin sulfate proteoglycans (CSPGs).

Astroctyes are known to produce IL-1, IL-6, IL10, IFNα, IFNβ, TNFα, TGFβ,

RANTES, IL-8, MCP-1, IP-10 (Miljkovic et al., 2007). Additionally, astrocytes are known mitogen and antigen induced producers of IL-17 and IFNγ in leukocytes

(Miljkovic et al., 2007).

Recent work in other laboratories has utilized an in vitro model system for astrogliosis (Hou et al., 1995). This model system involved wounding a confluent purified culture by scratching the astrocytes creating an in vitro scar. By utilizing this model system, we may be able to tease out in vitro the role of chemokines in

114 modulating astrogliotic response. This would allow for testing of inhibitors of specific chemokines, such as CXCL1, in modulation of astrogliosis in a culture model. Additionally, it would be possible to analyze the expression of chemokine receptors and chemokine secretion in response to injury of astrocytes in confluent astrocyte culture.

Neuroprotection elicited by inhibition of CXCR2 in demyelinating lesions

I have proposed that inhibition of CXCR2 enhances remyelination in different models of demyelination and that this remyelination is a major factor in the functional recovery seen in EAE. It is possible, however that an additional consequence of inhibiting CXCR2 is that there is less neural cells loss because blocking CXCR2 signaling results in neuroprotection and it is this neuroprotection that results in functional recovery.

The role of neuroprotection in lysolecithin- induced demyelination is more complex. Since the timing of lesion induction is directly correlated with LPC injection and there was a delay of 48hrs before delivery of the CXCR2 antagonist e the window for neuroprotection may have passed. However, lysolecithin lesions do not reach maximum until 7 days post lesion and inhibiting CXCR2 may promote recovery by protecting the development of pathology after day two. The mechanisms by which CXCR2 inhibition enhances neuroprotection iis not well understaood. It may be that since chemokine signaling promotes the infiltration of monocytes into lesioned CNS, inhibiting that infiltration results in reduced cellular damage by monocytes and thus protection of axons and glial cells in a lesion.

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In EAE animals, the amount of demyelinated axons was decreased in animals treated with CXCR2 antagonists. The presence of higher numbers of relatively thick myelin sheaths around intact axons raises the possibility that some xxons may have been protected by inhibition of CXCR2 and never underwent demyelination. The existence of thinly myelinated axons, however supports the hypothesis that remyelination had occurred, the realitve contribution of the two effects is currently unclear.Additionally, if animals were treated with

CXCR2 antagonists later in the disease course, when most axons are demyelinated, the animals failed to undergo functional recovery consistent with the notion that the axons were too damaged and were unable to be remyelinated. However, an alternative hypothesis may be the OPCs were depleted by immunological and astrogliotic insults and therefore, unable to migrate and differentiate into myelinating oligodendrocytes and engage demyelinated axons. Furthermore, in EAEshort term treatment with CXCR2 antagonists resulted in initial recovery however after cessation of treatment animals regressed functionally and histologically. However, EAE animals treated for longer periods of time remained functionally improved and remyelination was evident. By inhibiting the pathological insults, recovery was able to be elicited with CXCR2 inhibition. Ceasing treatment however, caused regression in disease; therefore, the axons were not protected to the extent of promoting continued remyelination.

In future work, experiments need to assess the value of neuroprotection in both models of demyelination. This may be accomplished by varying the time

116 from demyelination initiation to treatment with CXCR2 antagonists and assessment of the proportion of protected axons. If neuroprotection is a major factor then shortterm treated animals should have greater protection than those in which the insult to treatment interval is longer.

Chemokine receptor/ligand promiscuity of binding

One of the characteristics of the chemokine system is the significant promiscuity that exists between the different ligands and receptors. For example, although CXCR2 is the primary receptor for CXCL1, there is evidence that other receptors such as CXCR1 may bind CXCL1 with lower affinity. The binding of small molecule inhibitors against CXCR2 could have prevented any CXCL1 signaling thereby affecting not only the high affinity binding of CXCL1 to CXCR2 but also to CXCR1. There is 80% sequence homology between CXCR2 and

CXCR1. CXCR1 signaling could be effected if the inhibitors aren’t selective to

CXCR2; thereby, not only affecting the CXCR2 downstream signaling events.

CXCR1 has also been found to be upregulated in MS lesions as with CXCR2, however, mice and rats have not been found to have CXCR1 expressed and only expressed the functional CXCR2 homologue (Stillie et al., 2009). The rat ortholog of CXCR1 does not signal with any of the presumptive chemokines tested (Dunstan et al., 1996). There has been a receptor homologue/ortholog, called mCXCR1-like, found in mice to be expressed in spleen, kidneys, liver, and

CD4+ T cells, although no known chemokine induced G protein activation therefore, it is unlikely to be analogous to human CXCR1 (Moepps et al., 2006).

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These data suggest that the effects reported in this study are likely mediated through the CXCR2 receptor.

The contribution of the immune system to models of demyelination and potential targets for therapeutics

Current MS therapies predominately target the immune response in patients. This allows for some recovery of attacks initiated through proinflammatory cytokines and potentially results in decreasing the amount of inflammation within the CNS. Chemokines are known modulators of immune responses and previous work has implicated chemokines and their receptors in modulation of infiltration of leukocytes into CNS parenchyma. They are also found to be expressed highly in chronic demyelination models. In this thesis work, decreasing CXCL1/CXCR2 signaling enhances repair in a chronic model and acute focal model of demyelination. The chronic EAE model initiated through injection of myelin peptides elicits a strong immune component that may have been modulated by inhibition of CXCR2. However, the acute model also demonstrated repair with inhibition of CXCR2, and this model has only a weak immune response. Although, inhibition of CXCR2 may have been diminishing the immune response by decreasing leukocyte entry through the blood brain barrier, this is unlikely to be its only mode of action. CXCR2 inhibition in focal lesions, which have a weak immune response, also promoted remyelination in a relatively short time frame. Therefore, while in EAE, inhibition of CXCR2 may

118 have been decreasing the proinflammatory cytokines, it also was directly affecting remyelination in CNS tissue.

The functional recovery seen in EAE animals treated with CXCR2 antagonists is likely to reflect multiple cellular events. EAE animals recovered relatively quickly from the hindlimb paralysis associated with EAE. This recovery could reflect a reduction in inflammation and proinflammatory cytokines. The animals treated with CXCR2 inhibitor did have reduction in the number of lymphoid cells entering into the spinal cord parenchyma from the blood vessels, so this hypothesis is possible. An inflammatory condition in the CNS, such as what occurs in MS and EAE, promotes lymphocytes and macrophages crossing the BBB which leads to edema, inflammation and demyelination. Interactions of the endothelium of the barrier and leukocytes are essential to disease induction

(Engelhardt, 2006, , 2008). Affecting lymphocyte rolling, arrest, and migration thereby decreasing edema and the response of the immune system could be the sole foundation for recovery (Brown, 2002). Although, data was also generated to point to recovery in a largely non-immune mediated demyelination event, LPC.

The chemokine, CXCL1, found to be upregulated around MS lesions has also been sited as being a regulator of sodium currents and neuronal excitability in small diameter sensory neurons (Wang et al., 2008). The activation of these currents could lead to hyperexcitability and have been known to promote nociception in nerve injury and inflammatory models of pain. Therefore, by decreasing CXCL1/CXCR2 signaling it is possible reduction in excitability was

119 occurring allowing axons to survive and promote neuroprotection and subsequent remyelination.

Not all effects of the hyperstimulated immune system in the CNS may be detrimental. Growth cones of axons will collapse when they contact oligodendrocytes/CNS myelin in vitro (Caroni et al., 1988). Treatment with antibody against axonal growth inhibitor NogoA, promotes sprouting (Buffo et al.,

2000). Binding partners for NogoA include MAG and OMgp, mature oligodendrocyte proteins (Wang et al., 2002; Sandvig et al., 2004).

Demyelination results in myelin proteins needed to be cleared for remyelination and possibly recovery. In animal models in which macrophages were depleted, there was a worsening in pathology in models of demyelination (Triarhou and

Herndon, 1985; Kotter et al., 2001) further implicating the need for macrophage and immune cell activation for remyelination to occur. Preliminary experiments, using the lyslecithin model in nude rats, however, pointed to a fact that the immune system does play a small role in the original demyelination events. It seems there must be a clearance of myelin debris to trigger remyelination. If there is myelin proteins still present, there may not be a recovery of remyelination in the LPC model. Lesions in these animals appeared smaller; however, they were only assessed using Luxol fast blue staining and could have marked myelin proteins that were still present in the lesion (Fig 4.1). This is a potential new project and branch that came out of this thesis work that should be explored in the future.

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Modulation of blood brain barrier and inflammation

Regulatory mechanisms modulating the opening and closure of the blood brain barrier could contribute to recovery in models of demyelination following inhibition of CXCR2 signaling. Blood brain barrier breach is a common hallmark of many neurodegenerative diseases, including MS. This leakage allows for the entry of T-cells, cytokines, chemokines and, inflammatory mediators in addition to the potential of infectious agents. These can all trigger secondary inflammation present in the brain which may be mediated in part by chemokines.

The downstream consequences of chemokine receptor stimulation are wide ranging. Intracellular cascades by activation of G-protein coupled receptors, to which CXCR2 belongs, involves activation of matrix metalloproteinases (MMPs) to release tethered ligands (Shah and Catt, 2004).

Induction and activation of MMPs is known to be induced by CXCR2 (Li et al.,

2003; Chakrabarti and Patel, 2005) and stimulation of other chemokine receptors. Matrix metalloproteinases (MMPs) key role is in degradation of extracellular matrix proteins for remodeling and cellular migration. MMPs have been implicated in degradation of the blood brain barrier seen in many neurological diseases (Duchossoy et al., 2001). MMP-2, MMP-3, MMP-7 and

MMP-9 are expressed by peripheral macrophages and reported to be within active MS lesions (Leppert et al., 1998; Chakrabarti and Patel, 2005; Larsen et al., 2006; Ulrich et al., 2006). Further, they are known to be expressed by perivascular leukocytes (Li et al., 2003). This activation could be controlled by a number of chemokines, CXCL9, CXCL10, CCL2, CCL3, CCL5, in addition to

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CXCL1 (Sellebjerg and Sorensen, 2003). Therefore, blocking MMP signaling events via inhibitors may ameliorate an upregulation of other detrimental factors other than pro-inflammatory cytokines.

A distinctive feature of MS is the appearance of cuffs of lymphocytes around small blood vessels. It is thought CD4+ T cells infiltrate early and CD8+

T cells follow later in disease course. The T-cells then go on to secrete proinflammatory and anti-inflammatory cytokines. Lymphocytes migrating across the blood brain barrier upregulate a number of inflammatory mediators which may contribute to pathology of MS. Neutophils are one of the first set of cells to migrate in the region of injury in response to local expression of CXCL1, because neutrophils express CXCR2 on their cell surface. Neutrophils potentiate the inflammatory response and allow lymphocytes to enter the brain. I propose based on the data I presented in this thesis that the inhibition of CXCR2 may prevent neutrophils from crossing the blood brain barrier and prevent subsequent pathology. Consistent with this hypothesis, I demonstrate that in animals with

EAE that are treated with inhibitors of CXCR2, there is a substantial decrease in the number of infiltrating lymphoid cells into lesion areas.

Expression of CXCL1/CXCR2 and signaling in vivo

The spectrum of neural cells that express CXCL1 and CXCR2 in vivo is still unclear. Due to the lack of quality of the antibodies, double labeling in vivo was unsuccessful. However, single labeling with antibodies to CXCR2 was found to be around lysolecithin lesions. In addition, CXCL1 has also been

122 demonstrated to be around lysolecithin lesions, so the potential exists for local signaling. In previous studies in this laboratory, CXCL1 was shown to be secreted by subsets of astrocytes (Robinson et al., 1998). Here I have extended these studies to show CXCR2 and CXCL1 was expressed around lysolecithin lesions but this could have been expressed by infiltrating leukocytes or blood cells. It seems likely that CXCL1 is expressed by astrocytes around demyelinating lesions and thus is poised to contribute to demyelination/remyelination. Consistent with this idea, cultured astrocytes express CXCR2 mRNA, and it may be that expression of the receptor is upregulated in an injury setting. It is possible that once CXCL1 gets secreted, among other cytokines, in response to injury; this may recruit the cognate receptor to be expressed on the surface of each particular cell. On the other hand, too much secretion could cause desentization of receptors and other downstream effects (Mueller et al., 1997; Richmond et al., 1997).

Downstream signaling components of CXCR2

The downstream signaling components of CXCR2 activation are currently unclear. It has been reported, like other GPCRs they mediate their signaling components via ERK, JAK/STAT, JNK and PI-3 kinase. In a recent paper, it was demonstrated that CXCL1 is expressed by subsets of neurons in Alzheimer’s disease brains of mice. Further, CXCL1 additon to mixed cortical cell cultures triggered hyperphosphorylation of tau proteins (hallmark sign of AD), and activation of Erk1/2 and PI-3 kinase (Xia and Hyman, 1999; Xia and Hyman,

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2002). In human fetal oligodendrocyte progenitor cells, CXCL1 addition triggered

OPC proliferation and activation of Erk1/2 and release of IL-6 (Filipovic and

Zecevic, 2008). In models of hypoxia/ischemia CXCR2 blockade results in protection from JNK mediated apoptosis (Wang et al., 2006). The JNK pathway is also involved in T cell differentiation so by blocking this pathway by antagonizing CXCR2, there may be an inhibition of apoptosis in addition to downregulation of inflammation. JAK/STAT pathway mediates a number of cytokine events intracellularly; therefore CXCL1 signaling may mediate secondary effects due to a potentially complex singaling cascade. Since chemokines are important mediators of inflammatory reaction and pathogenesis of many neurodegenerative diseases has been linked to inflammation, this has raised questions as to what signaling pathway is elicited in response to chemokine signaling. It is possible in the setting of pathology in the brain and spinal cord, there is upregulation of secretion of a number of chemokines, including CXCL1, potentially by astrocytes and microglia, which initiates downstream signaling events mediating proinflammatory and anti-inflammatory cytokines to protect and/or mediate repair.

Stability of antagonists

A major concern with the utilization of small molecule inhibitors to regulate biological properties is tracking of the molecule into target areas. While biologics and antibodies are longer lasting and more stable, they have their own issues including passage through the blood brain barrier and the generation of an immune response.

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Although, the demyelination models utilized in this thesis work were associated with blood brain barrier breach and it seems highly likely that the antagonist entered the brain to have effects on oligodendrocyte maturation and migration while the efficacy of the treatment indicates a significant difference between those treated with antagonist versus vehicle only control and a number of unknowns remain. For example, the intrathecal concentration of the agonist and its presistance and distribution are unclear. It would be interesting to tag the antagonist with a fluorescent tag in order to trace its diffusion and localization in particular models of pathology. On the other hand, this modification may alter the structure and thereby alter its actions or not allow for it to pass through the blood brain barrier. Perhaps more critically, since several cell types (OPCs, astrocytes, and neutrophils) are known to express the receptors, the cell that is critical for enhanced repair is important to determine. Target cell identification could be determined by utilizing genetic modifications using cell specific promoters. This would allow for knockout of CXCR2 in GFAP+ (astrocytes) ,

MBP (oligodendrocytes), or Iba1 (microglia) expressing cells. Additionally, by utilizing transgenic mice with loxP sites on either side of CXCR2 crossed with transgenic mice with Cre under control of cell specific promoters to excise

CXCR2 with tamoxifen. This would reveal the role of CXCR2 in specific cell types and be under temporal control so that development of OPCs would not be affected as in previous studies (Padovani-Claudio et al., 2006).

Analysis of serum and cerebrospinal fluid (CSF) levels of antagonist over time would also allow for correct dosage to be tailored to the half maximal

125 inhibitory concentration (IC50). A dose-response curve would need to be constructed to examine the effect of different concentrations of antagonist on reversing agonist activity. The quantitative measurement will indicate the effectiveness of the inhibitor to inhibit a given biological process; in this case, this could be conducted in vitro using migration or proliferation assay. The current assumption is that these antagonists do not stay in the system for very long and are rapidly lost. In these studies, the antagonist was delivered on a daily basis.

Dose-response curve construction would potentially allow for delivery of a more concentrated antagonist having quicker repair and smaller lesion recovery than those seen with daily delivery. Serum and CSF level analysis would allow for concentration of dosage to be more clearly defined. Construction of dose- response curve would determine the toxicity of the antagonist and the correct dosage to promote repair in models of demyelination.

Alternate chemokine targets alone or in combination with CXCR2 inhibitors

Studies have linked a number of chemokines to demyelinating disorders.

For example, CXCR12/SDF1 signaling has been implicated in a number of neurological disorders, including demyelinating diseases (Calderon et al., 2006).

In addition, CCL chemokines are known modulators of leukocyte trafficking, and therefore, could modulate demyelination/remyelination epidsodes similar to

CXCL1 (McMahon et al., 2001; Ousman and David, 2001; Babcock et al., 2003).

Activated astrocytes express CCL2, CCL3, CCL5, and CCL10 (Sun et al., 1997;

Takami et al., 1997; Carter et al., 2007). Consistent with the role for additional chemokines, numerous chemokine expression patterns coincide with the

126 peripheral areas of demyelinating lesions (Sun et al., 1997; McManus et al.,

1998; Simpson et al., 1998; Szczucinski and Losy, 2007). Inhibitors to chemokines are commercially available and by utilizing in vivo models of demyelination and other inflammatory mediated diseases, these may be potential candidate for therapy. It is possible that in combination, these inhibitors may be a powerful tool in combating inflammation and triggering remyelination, thus repair, in MS. Alternatively, it may be that many of the chemokines expressed in the areas of inflammation and demyelination are a consequence of the disease and would provide poor therapeutic targets for MS. The efficacy of putative therapeutic targets is going to depend largely on how many and how important the downstream effects are.

Ex vivo applications of brain lesions may be helpful in understanding role of T cells and other cells outside the CNS

In the models of MS utilized in this thesis work, the blood brain barrier was compromised. Compromization of the blood brain barrier, whether in disease or injury, allows for the influx of lymphocytes making the disease prognosis worse.

To eliminate the functional influences of systemic cells from bone marrow, central nervous ex vivo system would allow for cells to be maintained in a precise environment without the contribution of macrophages, neutrophils, and lymphocytes. Such a system is provided for by slice cultures. The slice preparation entails vibratome cutting a postnatal day 3 rat brain coronally and maintaining the slice for 28 days on a membrane allowing for diffusion of media

127 with growth factors on one side and oxygen/CO2 on the opposing side. By utilizing slice cultures, it is possible to modulate remyelination or developmental myelination in a dish. Additionally, factors, such as CXCR2 inhibitors or other chemokine inhibitors, can be added into the culture media with ease. This method would allow for study of developmental myelination such an ex vivo system of brain slices could also be used for future work. Demyelinating the corpus callosum of adult rats using LPC stereotactically prior to slice preparation would allow for demyelination to be assessed in an injury model. Although, macrophages, lymphocytes, neutrophils in addition to astrocytes become activated very quickly after injury, an ex vivo culture system would provide a downstream model system for the effects of various inhibitors or modulators of myelination (growth factors, MMPs, ect). In the current work, inhibitor was not delivered until 2 days post LPC in the spinal cord. Therefore, 24-48 hours would be a suitable window for extracting the brains and following through with the ex vivo slice culture system. Application of inhibitors or growth factors would be given in the culture media as well. Moreover, the developmental and ex vivo method would accommodate a high thoroughput screening of a number of molecules or concentrations. A cavet to these studies, is the acquiring of images. Since the slices are thicker than the normal cryostat sections, imaging becomes an issue. Nonetheless, imaging of remyelination events can be done using confocal microscopy or electron microscopy.

Other demyelination disease models and the efficacy for CXCR2 inhibitors

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A number of demyelination models are utilized in a number of laboratories to examine the molecules controlling demyelination/remyelination events. There are advantages and disadvantages to every model of demyelination studied.

However, combination of the information from a number of models may enhance the discovery of potential therapeutics, though, the expense and time associated with each model needs to be taken into account. However, other models suggest other evidence for therapeutic advances for inhibitors of CXCR2. PLP induced EAE in SJL strain of mice produces a relapsing remitting type of diease typical of MS patients early in their disease course. This model also induces a number of phases or expression of cytokines and chemokines which eventually spread. The importance of using this model to investigate the efficacy of the

CXCR2 inhibitor is that it would allow for delivery of inhibitor(s) at specific stages of disease to determine where CXCR2 inhibition is operative. Essentially, PLP- induced EAE studies should be conducted at a stage in which the disease was allowed to go through one phase of relapse and recovery and then treat to determine if inhibition of CXCR2 modulates the frequency of relapse and/or disease severity at relapse. Additionally, it would be important to test the role of

CXCR2 in later stages of disease. The hypothesis of whether inhibition of

CXCR2 is acting on resident OPCs or on stem cells outside the CNS migrating in to remyelinate could be tested because OPCs would presumably be extensively depleted at the later stages of disease.

Other models of inflammation or pathology may be mediated via CXCR2.

Inflammatory diseases such as acute inflammation in lungs, keratinitis, and

129 rheumatoid arthritis may be initiated by leukocyte entry and destruction mediated by these cells entering the parenchyma of the affected tissue. CXCR2 inhibition may benefit patients affected with inflammatory and other autoimmune diseases by inhibiting the inflammation and thereby allowing for repair of affected tissue.

Conclusions and potential of CXCR2 inhibitors for the treatment of demyelination disorders

The studies presented in this thesis demonstrate a role for CXCR2 in regulating oligodendrocyte development, astrogliosis and the progression of demyelinating diseases. The inhibition of CXCR2 decreases lesion load and induced remyelination in two models of MS. Antagonizing CXCR2 enhances functional recovery in addition to decreasing immune cell infiltration in EAE.

CXCL1/CXCR2 signaling promotes astrogliosis in vitro and in vivo with upregulation of GFAP. Further, CXCL1 addition to astrocyte culture results in upregulation of GFAP protein and CSPG secretion. Antagonizing CXCR2 reduces astrogliosis and immune cell infiltration, and promotes remyelination.

However, remyelination must be initiated by migration of OPCs into areas of demyelination. CXCR2 inhibition decreases CXCL1 induced inhibition of OPCs migration and therefore, allows OPCs to migrate into areas of injury. Further,

CXCR2 inhibition promotes OPCs to differentiate into myelin producing MBP+ cells. As a result of the reduction of immune and non-immune attacks combined with enhanced migration and differentiation of OPCs, remyelination is able to be

130 successful in areas of demyelination. Hence, CXCR2 inhibitors could be a beneficial therapeutic for the treatment of demyelinating disorders, such as

Multiple Sclerosis.

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Figure 4.1. Lysolecithin lesions in nude rats are decreased compared to control animals as indicated by histology.

Lysolecithin lesions initiated into the dorsal columns of T10/11 control Sprague- Dawley rat or RNU nude rats from Charles River. Animals were processed for histology 3 days post lesion. The smaller lesion size is attributed to the reduction in the clearance of myelin debris as a result of lack of T cells, and thus diminished macrophage activation.

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BIBLIOGRAPHY

Aarli JA (1983) The immune system and the nervous system. J Neurol 229:137-154. Abbott NJ (2002) Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat 200:629-638. Abbott NJ, Revest PA, Romero IA (1992) Astrocyte-endothelial interaction: physiology and pathology. Neuropathol Appl Neurobiol 18:424-433. Acar G, Idiman F, Kirkali G, Ozakbas S, Oktay G, Cakmakci H, Idiman E (2005) Intrathecal sICAM-1 production in multiple sclerosis--correlation with triple dose Gd-DTPA MRI enhancement and IgG index. J Neurol 252:146-150. Aloisi F (2001) Immune function of microglia. Glia 36:165-179. Antel J (1999) Multiple sclerosis--emerging concepts of disease pathogenesis. J Neuroimmunol 98:45-48. Araque A, Sanzgiri RP, Parpura V, Haydon PG (1999) Astrocyte-induced modulation of synaptic transmission. Can J Physiol Pharmacol 77:699-706. Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK, Ting JP (2001) TNF alpha promotes proliferation of oligodendrocyte progenitors and remyelination. Nat Neurosci 4:1116-1122. Arnett HA, Hellendall RP, Matsushima GK, Suzuki K, Laubach VE, Sherman P, Ting JP (2002) The protective role of nitric oxide in a neurotoxicant-induced demyelinating model. J Immunol 168:427-433. Azari MF, Profyris C, Karnezis T, Bernard CC, Small DH, Cheema SS, Ozturk E, Hatzinisiriou I, Petratos S (2006) Leukemia inhibitory factor arrests oligodendrocyte death and demyelination in spinal cord injury. J Neuropathol Exp Neurol 65:914-929. Babcock AA, Kuziel WA, Rivest S, Owens T (2003) Chemokine expression by glial cells directs leukocytes to sites of axonal injury in the CNS. J Neurosci 23:7922-7930. Bacci A, Verderio C, Pravettoni E, Matteoli M (1999) The role of glial cells in synaptic function. Philos Trans R Soc Lond B Biol Sci 354:403-409. Bachmann R, Eugster HP, Frei K, Fontana A, Lassmann H (1999) Impairment of TNF-receptor-1 signaling but not fas signaling diminishes T-cell apoptosis in myelin oligodendrocyte glycoprotein peptide-induced chronic demyelinating autoimmune encephalomyelitis in mice. Am J Pathol 154:1417-1422. Bai L, Lennon DP, Eaton V, Maier K, Caplan AI, Miller SD, Miller RH (2009) Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune response and promote endogenous repair in animal models of multiple sclerosis. Glia. Bajetto A, Bonavia R, Barbero S, Schettini G (2002) Characterization of chemokines and their receptors in the central nervous system: physiopathological implications. J Neurochem 82:1311-1329. Bajetto A, Bonavia R, Barbero S, Florio T, Schettini G (2001) Chemokines and their receptors in the central nervous system. Front Neuroendocrinol 22:147-184. Bambakidis NC, Wang RZ, Franic L, Miller RH (2003) Sonic hedgehog-induced neural precursor proliferation after adult rodent spinal cord injury. J Neurosurg 99:70-75.

133

Baracskay KL, Kidd GJ, Miller RH, Trapp BD (2007) NG2-positive cells generate A2B5-positive oligodendrocyte precursor cells. Glia 55:1001-1010. Barnes DA, Huston M, Perez HD (1998) TNF-alpha and IL-1beta cross- desensitization of astrocytes and astrocytoma cell lines. J Neuroimmunol 87:17-26. Barnes PJ (2001) Cytokine modulators as novel therapies for airway disease. Eur Respir J Suppl 34:67s-77s. Barres BA, Raff MC (1993) Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons. Nature 361:258-260. Barres BA, Raff MC (1999) Axonal control of oligodendrocyte development. J Cell Biol 147:1123-1128. Barres BA, Jacobson MD, Schmid R, Sendtner M, Raff MC (1993) Does oligodendrocyte survival depend on axons? Curr Biol 3:489-497. Barres BA, Burne JF, Holtmann B, Thoenen H, Sendtner M, Raff MC (1996) Ciliary neurotrophic factor enhances the rate of oligodendrocyte generation. Mol Cell Neurosci 8:146-156. Bartholdi D, Schwab ME (1997) Expression of pro-inflammatory cytokine and chemokine mRNA upon experimental spinal cord injury in mouse: an in situ hybridization study. Eur J Neurosci 9:1422-1438. Bauer J, Huitinga I, Zhao W, Lassmann H, Hickey WF, Dijkstra CD (1995) The role of macrophages, perivascular cells, and microglial cells in the pathogenesis of experimental autoimmune encephalomyelitis. Glia 15:437- 446. Baumann N, Pham-Dinh D (2001) Biology of oligodendrocyte and myelin in the mammalian central nervous system. Physiol Rev 81:871-927. Becher B, Bechmann I, Greter M (2006) Antigen presentation in autoimmunity and CNS inflammation: how T lymphocytes recognize the brain. J Mol Med 84:532-543. Ben-Hur T, Ben-Yosef Y, Mizrachi-Kol R, Ben-Menachem O, Miller A (2006) Cytokine-mediated modulation of MMPs and TIMPs in multipotential neural precursor cells. J Neuroimmunol 175:12-18. Benarroch EE (2005) Neuron-astrocyte interactions: partnership for normal function and disease in the central nervous system. Mayo Clin Proc 80:1326- 1338. Benedict RH, Bobholz JH (2007) Multiple sclerosis. Semin Neurol 27:78-85. Benveniste EN (1997) Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis. J Mol Med 75:165-173. Berry M, Ibrahim M, Carlile J, Ruge F, Duncan A, Butt AM (1995) Axon-glial relationships in the anterior medullary velum of the adult rat. J Neurocytol 24:965-983. Bjartmar C, Wujek JR, Trapp BD (2003) Axonal loss in the pathology of MS: consequences for understanding the progressive phase of the disease. J Neurol Sci 206:165-171. Blackmore M, Letourneau PC (2006) Changes within maturing neurons limit axonal regeneration in the developing spinal cord. J Neurobiol 66:348-360.

134

Blakemore WF, Franklin RJ (2008) Remyelination in experimental models of toxin- induced demyelination. Curr Top Microbiol Immunol 318:193-212. Blakemore WF, Smith PM, Franklin RJ (2000) Remyelinating the demyelinated CNS. Novartis Found Symp 231:289-298; discussion 298-306. Blomstrand F, Aberg ND, Eriksson PS, Hansson E, Ronnback L (1999) Extent of intercellular calcium wave propagation is related to gap junction permeability and level of connexin-43 expression in astrocytes in primary cultures from four brain regions. Neuroscience 92:255-265. Bona E, Andersson AL, Blomgren K, Gilland E, Puka-Sundvall M, Gustafson K, Hagberg H (1999) Chemokine and inflammatory cell response to hypoxia- ischemia in immature rats. Pediatr Res 45:500-509. Bonavia R, Bajetto A, Barbero S, Pirani P, Florio T, Schettini G (2003) Chemokines and their receptors in the CNS: expression of CXCL12/SDF-1 and CXCR4 and their role in astrocyte proliferation. Toxicol Lett 139:181-189. Brown KA (2002) Lymphocyte trafficking, survival and proliferation. Trends Immunol 23:275-277. Buffo A, Zagrebelsky M, Huber AB, Skerra A, Schwab ME, Strata P, Rossi F (2000) Application of neutralizing antibodies against NI-35/250 myelin-associated neurite growth inhibitory proteins to the adult rat cerebellum induces sprouting of uninjured purkinje cell axons. J Neurosci 20:2275-2286. Bugga L, Gadient RA, Kwan K, Stewart CL, Patterson PH (1998) Analysis of neuronal and glial phenotypes in brains of mice deficient in leukemia inhibitory factor. J Neurobiol 36:509-524. Bullon MM, Alvarez-Gago T, Fernandez-Ruiz B, Aguirre C (1984) Glial fibrillary acidic protein (GFAP) in spinal cord of postnatal rat. An immunoperoxidase study in semithin sections. Brain Res 316:129-133. Busch-Petersen J (2006) Small molecule antagonists of the CXCR2 and CXCR1 chemokine receptors as therapeutic agents for the treatment of inflammatory diseases. Curr Top Med Chem 6:1345-1352. Bush TG, Puvanachandra N, Horner CH, Polito A, Ostenfeld T, Svendsen CN, Mucke L, Johnson MH, Sofroniew MV (1999) Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice. Neuron 23:297-308. Butt AM, Ransom BR (1989) Visualization of oligodendrocytes and astrocytes in the intact rat optic nerve by intracellular injection of lucifer yellow and horseradish peroxidase. Glia 2:470-475. Butt AM, Duncan A, Berry M (1994) Astrocyte associations with nodes of Ranvier: ultrastructural analysis of HRP-filled astrocytes in the mouse optic nerve. J Neurocytol 23:486-499. Butzkueven H, Emery B, Cipriani T, Marriott MP, Kilpatrick TJ (2006) Endogenous leukemia inhibitory factor production limits autoimmune demyelination and oligodendrocyte loss. Glia 53:696-703. Butzkueven H, Zhang JG, Soilu-Hanninen M, Hochrein H, Chionh F, Shipham KA, Emery B, Turnley AM, Petratos S, Ernst M, Bartlett PF, Kilpatrick TJ (2002) LIF receptor signaling limits immune-mediated demyelination by enhancing oligodendrocyte survival. Nat Med 8:613-619.

135

Calderon TM, Eugenin EA, Lopez L, Kumar SS, Hesselgesser J, Raine CS, Berman JW (2006) A role for CXCL12 (SDF-1alpha) in the pathogenesis of multiple sclerosis: regulation of CXCL12 expression in astrocytes by soluble myelin basic protein. J Neuroimmunol 177:27-39. Cammer W, Zhang H (1993) Atypical localization of the oligodendrocytic isoform (PI) of glutathione-S-transferase in astrocytes during cuprizone intoxication. J Neurosci Res 36:183-190. Canning DR, Hoke A, Malemud CJ, Silver J (1996) A potent inhibitor of neurite outgrowth that predominates in the extracellular matrix of reactive astrocytes. Int J Dev Neurosci 14:153-175. Canoll PD, Kraemer R, Teng KK, Marchionni MA, Salzer JL (1999) GGF/neuregulin induces a phenotypic reversion of oligodendrocytes. Mol Cell Neurosci 13:79-94. Carlson T, Kroenke M, Rao P, Lane TE, Segal B (2008) The Th17-ELR+ CXC chemokine pathway is essential for the development of central nervous system autoimmune disease. J Exp Med 205:811-823. Caroni P, Savio T, Schwab ME (1988) Central nervous system regeneration: oligodendrocytes and myelin as non-permissive substrates for neurite growth. Prog Brain Res 78:363-370. Carson MJ, Reilly CR, Sutcliffe JG, Lo D (1998) Mature microglia resemble immature antigen-presenting cells. Glia 22:72-85. Carter SL, Muller M, Manders PM, Campbell IL (2007) Induction of the genes for Cxcl9 and Cxcl10 is dependent on IFN-gamma but shows differential cellular expression in experimental autoimmune encephalomyelitis and by astrocytes and microglia in vitro. Glia 55:1728-1739. Cayrol R, Wosik K, Berard JL, Dodelet-Devillers A, Ifergan I, Kebir H, Haqqani AS, Kreymborg K, Krug S, Moumdjian R, Bouthillier A, Becher B, Arbour N, David S, Stanimirovic D, Prat A (2008) Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system. Nat Immunol 9:137-145. Chakrabarti S, Patel KD (2005) Regulation of matrix metalloproteinase-9 release from IL-8-stimulated human neutrophils. J Leukoc Biol 78:279-288. Chalasani SH, Sabol A, Xu H, Gyda MA, Rasband K, Granato M, Chien CB, Raper JA (2007) Stromal cell-derived factor-1 antagonizes slit/robo signaling in vivo. J Neurosci 27:973-980. Chari DM (2007) Remyelination in multiple sclerosis. Int Rev Neurobiol 79:589-620. Charles P, Hernandez MP, Stankoff B, Aigrot MS, Colin C, Rougon G, Zalc B, Lubetzki C (2000) Negative regulation of central nervous system myelination by polysialylated-neural cell adhesion molecule. Proc Natl Acad Sci U S A 97:7585-7590. Charo IF, Ransohoff RM (2006) The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med 354:610-621. Chen SC, Leach MW, Chen Y, Cai XY, Sullivan L, Wiekowski M, Dovey-Hartman BJ, Zlotnik A, Lira SA (2002) Central nervous system inflammation and neurological disease in transgenic mice expressing the CC chemokine CCL21 in oligodendrocytes. J Immunol 168:1009-1017.

136

Chiu FC, Norton WT, Fields KL (1981) The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acidic protein, and the fibroblast-type filament protein, vimentin. J Neurochem 37:147-155. Compston A, Coles A (2002) Multiple sclerosis. Lancet 359:1221-1231. Conrad S, Schluesener HJ, Adibzahdeh M, Schwab JM (2005) Spinal cord injury induction of lesional expression of profibrotic and angiogenic connective tissue growth factor confined to reactive astrocytes, invading fibroblasts and endothelial cells. J Neurosurg Spine 2:319-326. Crocker SJ, Whitmire JK, Frausto RF, Chertboonmuang P, Soloway PD, Whitton JL, Campbell IL (2006) Persistent macrophage/microglial activation and myelin disruption after experimental autoimmune encephalomyelitis in tissue inhibitor of metalloproteinase-1-deficient mice. Am J Pathol 169:2104-2116. Cross AH, Hashim GA, Raine CS (1991) Adoptive transfer of experimental allergic encephalomyelitis and localization of the encephalitogenic epitope in the SWR mouse. J Neuroimmunol 31:59-66. Cross AK, Woodroofe MN (1999) Chemokines induce migration and changes in actin polymerization in adult rat brain microglia and a human fetal microglial cell line in vitro. J Neurosci Res 55:17-23. Cudrici C, Niculescu T, Niculescu F, Shin ML, Rus H (2006) Oligodendrocyte cell death in pathogenesis of multiple sclerosis: Protection of oligodendrocytes from apoptosis by complement. J Rehabil Res Dev 43:123-132. Davis CN, Chen S, Boehme SA, Bacon KB, Harrison JK (2003) Chemokine receptor binding and signal transduction in native cells of the central nervous system. Methods 29:326-334. Davis FA (1970) Pathophysiology of multiple sclerosis and related clinical implications. Mod Treat 7:890-902. De Groot CJ, Woodroofe MN (2001) The role of chemokines and chemokine receptors in CNS inflammation. Prog Brain Res 132:533-544. Decker L, Avellana-Adalid V, Nait-Oumesmar B, Durbec P, Baron-Van Evercooren A (2000) Oligodendrocyte precursor migration and differentiation: combined effects of PSA residues, growth factors, and substrates. Mol Cell Neurosci 16:422-439. Dubois-Dalcq M, Murray K (2000) Why are growth factors important in oligodendrocyte physiology? Pathol Biol (Paris) 48:80-86. Duchossoy Y, Arnaud S, Feldblum S (2001) Matrix metalloproteinases: potential therapeutic target in spinal cord injury. Clin Chem Lab Med 39:362-367. Dunstan CA, Salafranca MN, Adhikari S, Xia Y, Feng L, Harrison JK (1996) Identification of two rat genes orthologous to the human interleukin-8 receptors. J Biol Chem 271:32770-32776. Dziembowska M, Tham TN, Lau P, Vitry S, Lazarini F, Dubois-Dalcq M (2005) A role for CXCR4 signaling in survival and migration of neural and oligodendrocyte precursors. Glia 50:258-269. Eglitis MA, Mezey E (1997) Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci U S A 94:4080- 4085.

137

Eng LF (1985) Glial fibrillary acidic protein (GFAP): the major protein of glial intermediate filaments in differentiated astrocytes. J Neuroimmunol 8:203- 214. Eng LF, Ghirnikar RS (1994) GFAP and astrogliosis. Brain Pathol 4:229-237. Eng LF, Ghirnikar RS, Lee YL (1996) Inflammation in EAE: role of chemokine/cytokine expression by resident and infiltrating cells. Neurochem Res 21:511-525. Engelhardt B (2006) Molecular mechanisms involved in T cell migration across the blood-brain barrier. J Neural Transm 113:477-485. Engelhardt B (2008) The blood-central nervous system barriers actively control immune cell entry into the central nervous system. Curr Pharm Des 14:1555- 1565. Faulkner JR, Herrmann JE, Woo MJ, Tansey KE, Doan NB, Sofroniew MV (2004) Reactive astrocytes protect tissue and preserve function after spinal cord injury. J Neurosci 24:2143-2155. Fawcett JW, Asher RA (1999) The glial scar and central nervous system repair. Brain Res Bull 49:377-391. Fernandez EJ, Lolis E (2002) Structure, function, and inhibition of chemokines. Annu Rev Pharmacol Toxicol 42:469-499. Ferrari CC, Depino AM, Prada F, Muraro N, Campbell S, Podhajcer O, Perry VH, Anthony DC, Pitossi FJ (2004) Reversible demyelination, blood-brain barrier breakdown, and pronounced neutrophil recruitment induced by chronic IL-1 expression in the brain. Am J Pathol 165:1827-1837. Filipovic R, Zecevic N (2008) The effect of CXCL1 on human fetal oligodendrocyte progenitor cells. Glia 56:1-15. Filipovic R, Jakovcevski I, Zecevic N (2003) GRO-alpha and CXCR2 in the human fetal brain and multiple sclerosis lesions. Dev Neurosci 25:279-290. Fitch MT, Silver J (2008) CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure. Exp Neurol 209:294-301. Flynn G, Maru S, Loughlin J, Romero IA, Male D (2003) Regulation of chemokine receptor expression in human microglia and astrocytes. J Neuroimmunol 136:84-93. Fok-Seang J, DiProspero NA, Meiners S, Muir E, Fawcett JW (1998) Cytokine- induced changes in the ability of astrocytes to support migration of oligodendrocyte precursors and axon growth. Eur J Neurosci 10:2400-2415. Franklin RJ (2002) Why does remyelination fail in multiple sclerosis? Nat Rev Neurosci 3:705-714. Franklin RJ, Ffrench-Constant C (2008) Remyelination in the CNS: from biology to therapy. Nat Rev Neurosci 9:839-855. Franklin RJ, Gilson JM, Blakemore WF (1997) Local recruitment of remyelinating cells in the repair of demyelination in the central nervous system. J Neurosci Res 50:337-344. Frohman EM, Eagar T, Monson N, Stuve O, Karandikar N (2008) Immunologic mechanisms of multiple sclerosis. Neuroimaging Clin N Am 18:577-588, ix.

138

Fuller ML, DeChant AK, Rothstein B, Caprariello A, Wang R, Hall AK, Miller RH (2007) Bone morphogenetic proteins promote gliosis in demyelinating spinal cord lesions. Ann Neurol 62:288-300. Gallo V, Chittajallu R (2001) Neuroscience. Unwrapping glial cells from the synapse: what lies inside? Science 292:872-873. Gehrmann J (1996) Microglia: a sensor to threats in the nervous system? Res Virol 147:79-88. Gehrmann J, Matsumoto Y, Kreutzberg GW (1995) Microglia: intrinsic immuneffector cell of the brain. Brain Res Brain Res Rev 20:269-287. Giess R, Maurer M, Linker R, Gold R, Warmuth-Metz M, Toyka KV, Sendtner M, Rieckmann P (2002) Association of a null mutation in the CNTF gene with early onset of multiple sclerosis. Arch Neurol 59:407-409. Glabinski AR, Tuohy VK, Ransohoff RM (1998) Expression of chemokines RANTES, MIP-1alpha and GRO-alpha correlates with inflammation in acute experimental autoimmune encephalomyelitis. Neuroimmunomodulation 5:166-171. Glabinski AR, Tani M, Tuohy VK, Tuthill RJ, Ransohoff RM (1995) Central nervous system chemokine mRNA accumulation follows initial leukocyte entry at the onset of acute murine experimental autoimmune encephalomyelitis. Brain Behav Immun 9:315-330. Gorio A, Madaschi L, Zadra G, Marfia G, Cavalieri B, Bertini R, Di Giulio AM (2007) Reparixin, an inhibitor of CXCR2 function, attenuates inflammatory responses and promotes recovery of function after traumatic lesion to the spinal cord. J Pharmacol Exp Ther 322:973-981. Gottenberg JE, Chiocchia G (2007) Dendritic cells and interferon-mediated autoimmunity. Biochimie 89:856-871. Govindaraju V, Michoud MC, Al-Chalabi M, Ferraro P, Powell WS, Martin JG (2006) Interleukin-8: novel roles in human airway smooth muscle cell contraction and migration. Am J Physiol Cell Physiol 291:C957-965. Guenard V, Frisch G, Wood PM (1996) Effects of axonal injury on astrocyte proliferation and morphology in vitro: implications for astrogliosis. Exp Neurol 137:175-190. Halachmi E, Pinkus R, Miron S, Ben-Nun A, Werkele H, Berke G (1992) Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). II. Characteristics of astrocyte detachment mediated by myelin basic protein (MBP) specific CD4+ T lymphocytes. J Autoimmun 5:427-441. Hellings N, Baree M, Verhoeven C, D'Hooghe M B, Medaer R, Bernard CC, Raus J, Stinissen P (2001) T-cell reactivity to multiple myelin antigens in multiple sclerosis patients and healthy controls. J Neurosci Res 63:290-302. Hesselgesser J, Horuk R (1999) Chemokine and chemokine receptor expression in the central nervous system. J Neurovirol 5:13-26. Hildebrand C, Remahl S, Persson H, Bjartmar C (1993) Myelinated nerve fibres in the CNS. Prog Neurobiol 40:319-384.

139

Hinks GL, Franklin RJ (1999) Distinctive patterns of PDGF-A, FGF-2, IGF-I, and TGF-beta1 gene expression during remyelination of experimentally-induced spinal cord demyelination. Mol Cell Neurosci 14:153-168. Hiremath MM, Saito Y, Knapp GW, Ting JP, Suzuki K, Matsushima GK (1998) Microglial/macrophage accumulation during cuprizone-induced demyelination in C57BL/6 mice. J Neuroimmunol 92:38-49. Hohol MJ, Olek MJ, Orav EJ, Stazzone L, Hafler DA, Khoury SJ, Dawson DM, Weiner HL (1999) Treatment of progressive multiple sclerosis with pulse cyclophosphamide/methylprednisolone: response to therapy is linked to the duration of progressive disease. Mult Scler 5:403-409. Horuk R, Martin AW, Wang Z, Schweitzer L, Gerassimides A, Guo H, Lu Z, Hesselgesser J, Perez HD, Kim J, Parker J, Hadley TJ, Peiper SC (1997) Expression of chemokine receptors by subsets of neurons in the central nervous system. J Immunol 158:2882-2890. Hou YJ, Yu AC, Garcia JM, Aotaki-Keen A, Lee YL, Eng LF, Hjelmeland LJ, Menon VK (1995) Astrogliosis in culture. IV. Effects of basic fibroblast growth factor. J Neurosci Res 40:359-370. Hu QD, Cui XY, Ng YK, Xiao ZC (2004) Axoglial interaction via the notch receptor in oligodendrocyte differentiation. Ann Acad Med Singapore 33:581-588. Huang D, Han Y, Rani MR, Glabinski A, Trebst C, Sorensen T, Tani M, Wang J, Chien P, O'Bryan S, Bielecki B, Zhou ZL, Majumder S, Ransohoff RM (2000) Chemokines and chemokine receptors in inflammation of the nervous system: manifold roles and exquisite regulation. Immunol Rev 177:52-67. Imitola J, Raddassi K, Park KI, Mueller FJ, Nieto M, Teng YD, Frenkel D, Li J, Sidman RL, Walsh CA, Snyder EY, Khoury SJ (2004) Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci U S A 101:18117-18122. Jean I, Allamargot C, Barthelaix-Pouplard A, Fressinaud C (2002) Axonal lesions and PDGF-enhanced remyelination in the rat corpus callosum after lysolecithin demyelination. Neuroreport 13:627-631. Jeffery ND, Blakemore WF (1995) Remyelination of mouse spinal cord axons demyelinated by local injection of lysolecithin. J Neurocytol 24:775-781. Jessen KR, Thorpe R, Mirsky R (1984) Molecular identity, distribution and heterogeneity of glial fibrillary acidic protein: an immunoblotting and immunohistochemical study of Schwann cells, satellite cells, enteric glia and astrocytes. J Neurocytol 13:187-200. Jiang Y, Salafranca MN, Adhikari S, Xia Y, Feng L, Sonntag MK, deFiebre CM, Pennell NA, Streit WJ, Harrison JK (1998) Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis. J Neuroimmunol 86:1-12. Joe EH, Angelides KJ (1993) Clustering and mobility of voltage-dependent sodium channels during myelination. J Neurosci 13:2993-3005. Johnstone M, Gearing AJ, Miller KM (1999) A central role for astrocytes in the inflammatory response to beta-amyloid; chemokines, cytokines and reactive oxygen species are produced. J Neuroimmunol 93:182-193.

140

Jones LL, Yamaguchi Y, Stallcup WB, Tuszynski MH (2002) NG2 is a major chondroitin sulfate proteoglycan produced after spinal cord injury and is expressed by macrophages and oligodendrocyte progenitors. J Neurosci 22:2792-2803. Juedes AE, Hjelmstrom P, Bergman CM, Neild AL, Ruddle NH (2000) Kinetics and cellular origin of cytokines in the central nervous system: insight into mechanisms of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. J Immunol 164:419-426. Kadi L, Selvaraju R, de Lys P, Proudfoot AE, Wells TN, Boschert U (2006) Differential effects of chemokines on oligodendrocyte precursor proliferation and myelin formation in vitro. J Neuroimmunol 174:133-146. Kadiu I, Glanzer JG, Kipnis J, Gendelman HE, Thomas MP (2005) Mononuclear phagocytes in the pathogenesis of neurodegenerative diseases. Neurotox Res 8:25-50. Kalman M, Ajtai BM (2001) A comparison of intermediate filament markers for presumptive astroglia in the developing rat neocortex: immunostaining against nestin reveals more detail, than GFAP or vimentin. Int J Dev Neurosci 19:101-108. Kang J, Jiang L, Goldman SA, Nedergaard M (1998) Astrocyte-mediated potentiation of inhibitory synaptic transmission. Nat Neurosci 1:683-692. Kaplan MR, Cho MH, Ullian EM, Isom LL, Levinson SR, Barres BA (2001) Differential control of clustering of the sodium channels Na(v)1.2 and Na(v)1.6 at developing CNS nodes of Ranvier. Neuron 30:105-119. Katancik JA, Sharma A, de Nardin E (2000) Interleukin 8, neutrophil-activating peptide-2 and GRO-alpha bind to and elicit cell activation via specific and different amino acid residues of CXCR2. Cytokine 12:1480-1488. Keane MP, Belperio JA, Xue YY, Burdick MD, Strieter RM (2004) Depletion of CXCR2 inhibits tumor growth and angiogenesis in a murine model of lung cancer. J Immunol 172:2853-2860. Klawiter EC, Cross AH (2007) B cells: no longer the nondominant arm of multiple sclerosis. Curr Neurol Neurosci Rep 7:231-238. Kossmann T, Stahel PF, Lenzlinger PM, Redl H, Dubs RW, Trentz O, Schlag G, Morganti-Kossmann MC (1997) Interleukin-8 released into the cerebrospinal fluid after brain injury is associated with blood-brain barrier dysfunction and nerve growth factor production. J Cereb Blood Flow Metab 17:280-289. Kotter MR, Zhao C, van Rooijen N, Franklin RJ (2005) Macrophage-depletion induced impairment of experimental CNS remyelination is associated with a reduced oligodendrocyte progenitor cell response and altered growth factor expression. Neurobiol Dis 18:166-175. Kotter MR, Setzu A, Sim FJ, Van Rooijen N, Franklin RJ (2001) Macrophage depletion impairs oligodendrocyte remyelination following lysolecithin- induced demyelination. Glia 35:204-212. Krumbholz M, Theil D, Cepok S, Hemmer B, Kivisakk P, Ransohoff RM, Hofbauer M, Farina C, Derfuss T, Hartle C, Newcombe J, Hohlfeld R, Meinl E (2006) Chemokines in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment. Brain 129:200-211.

141

Laing KJ, Secombes CJ (2004) Chemokines. Dev Comp Immunol 28:443-460. Larsen PH, DaSilva AG, Conant K, Yong VW (2006) Myelin formation during development of the CNS is delayed in matrix metalloproteinase-9 and -12 null mice. J Neurosci 26:2207-2214. Larsen PH, Wells JE, Stallcup WB, Opdenakker G, Yong VW (2003) Matrix metalloproteinase-9 facilitates remyelination in part by processing the inhibitory NG2 proteoglycan. J Neurosci 23:11127-11135. Lassmann H, Schmied M, Vass K, Hickey WF (1993) Bone marrow derived elements and resident microglia in brain inflammation. Glia 7:19-24. Lee MA, Smith S, Palace J, Matthews PM (1998) Defining multiple sclerosis disease activity using MRI T2-weighted difference imaging. Brain 121 ( Pt 11):2095- 2102. Lees JR, Golumbek PT, Sim J, Dorsey D, Russell JH (2008) Regional CNS responses to IFN-gamma determine lesion localization patterns during EAE pathogenesis. J Exp Med 205:2633-2642. Leppert D, Ford J, Stabler G, Grygar C, Lienert C, Huber S, Miller KM, Hauser SL, Kappos L (1998) Matrix metalloproteinase-9 (gelatinase B) is selectively elevated in CSF during relapses and stable phases of multiple sclerosis. Brain 121 ( Pt 12):2327-2334. Levine JM (1994) Increased expression of the NG2 chondroitin-sulfate proteoglycan after brain injury. J Neurosci 14:4716-4730. Li A, Dubey S, Varney ML, Dave BJ, Singh RK (2003) IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol 170:3369-3376. Lindner M, Trebst C, Heine S, Skripuletz T, Koutsoudaki PN, Stangel M (2008) The chemokine receptor CXCR2 is differentially regulated on glial cells in vivo but is not required for successful remyelination after cuprizone-induced demyelination. Glia 56:1104-1113. Little AR, O'Callagha JP (2001) Astrogliosis in the adult and developing CNS: is there a role for proinflammatory cytokines? Neurotoxicology 22:607-618. Liu T, Young PR, McDonnell PC, White RF, Barone FC, Feuerstein GZ (1993) Cytokine-induced neutrophil chemoattractant mRNA expressed in cerebral ischemia. Neurosci Lett 164:125-128. Luo Y, Fischer FR, Hancock WW, Dorf ME (2000) Macrophage inflammatory protein-2 and KC induce chemokine production by mouse astrocytes. J Immunol 165:4015-4023. Man S, Ubogu EE, Williams KA, Tucky B, Callahan MK, Ransohoff RM (2008) Human brain microvascular endothelial cells and umbilical vein endothelial cells differentially facilitate leukocyte recruitment and utilize chemokines for T cell migration. Clin Dev Immunol 2008:384982. Mason JL, Suzuki K, Chaplin DD, Matsushima GK (2001) Interleukin-1beta promotes repair of the CNS. J Neurosci 21:7046-7052. Mayer M, Bhakoo K, Noble M (1994) Ciliary neurotrophic factor and leukemia inhibitory factor promote the generation, maturation and survival of oligodendrocytes in vitro. Development 120:143-153.

142

McCandless EE, Piccio L, Woerner BM, Schmidt RE, Rubin JB, Cross AH, Klein RS (2008) Pathological expression of CXCL12 at the blood-brain barrier correlates with severity of multiple sclerosis. Am J Pathol 172:799-808. McFarland HF, Martin R (2007) Multiple sclerosis: a complicated picture of autoimmunity. Nat Immunol 8:913-919. McMahon EJ, Suzuki K, Matsushima GK (2002) Peripheral macrophage recruitment in cuprizone-induced CNS demyelination despite an intact blood-brain barrier. J Neuroimmunol 130:32-45. McMahon EJ, Cook DN, Suzuki K, Matsushima GK (2001) Absence of macrophage-inflammatory protein-1alpha delays central nervous system demyelination in the presence of an intact blood-brain barrier. J Immunol 167:2964-2971. McManus C, Berman JW, Brett FM, Staunton H, Farrell M, Brosnan CF (1998) MCP-1, MCP-2 and MCP-3 expression in multiple sclerosis lesions: an immunohistochemical and in situ hybridization study. J Neuroimmunol 86:20-29. McTigue DM, Tripathi R, Wei P (2006) NG2 colocalizes with axons and is expressed by a mixed cell population in spinal cord lesions. J Neuropathol Exp Neurol 65:406-420. McTigue DM, Tani M, Krivacic K, Chernosky A, Kelner GS, Maciejewski D, Maki R, Ransohoff RM, Stokes BT (1998) Selective chemokine mRNA accumulation in the rat spinal cord after contusion injury. J Neurosci Res 53:368-376. Merrill JE, Benveniste EN (1996) Cytokines in inflammatory brain lesions: helpful and harmful. Trends Neurosci 19:331-338. Mi S, Miller RH, Lee X, Scott ML, Shulag-Morskaya S, Shao Z, Chang J, Thill G, Levesque M, Zhang M, Hession C, Sah D, Trapp B, He Z, Jung V, McCoy JM, Pepinsky RB (2005) LINGO-1 negatively regulates myelination by oligodendrocytes. Nat Neurosci 8:745-751. Miljkovic D, Momcilovic M, Stojanovic I, Stosic-Grujicic S, Ramic Z, Mostarica- Stojkovic M (2007) Astrocytes stimulate interleukin-17 and interferon- gamma production in vitro. J Neurosci Res 85:3598-3606. Miller A, Shapiro S, Gershtein R, Kinarty A, Rawashdeh H, Honigman S, Lahat N (1998) Treatment of multiple sclerosis with copolymer-1 (Copaxone): implicating mechanisms of Th1 to Th2/Th3 immune-deviation. J Neuroimmunol 92:113-121. Miller DH, Molyneux PD, Barker GJ, MacManus DG, Moseley IF, Wagner K (1999) Effect of interferon-beta1b on magnetic resonance imaging outcomes in secondary progressive multiple sclerosis: results of a European multicenter, randomized, double-blind, placebo-controlled trial. European Study Group on Interferon-beta1b in secondary progressive multiple sclerosis. Ann Neurol 46:850-859. Miller RH (2002) Regulation of oligodendrocyte development in the vertebrate CNS. Prog Neurobiol 67:451-467.

143

Moepps B, Nuesseler E, Braun M, Gierschik P (2006) A homolog of the human chemokine receptor CXCR1 is expressed in the mouse. Mol Immunol 43:897- 914. Mueller SG, White JR, Schraw WP, Lam V, Richmond A (1997) Ligand-induced desensitization of the human CXC chemokine receptor-2 is modulated by multiple serine residues in the carboxyl-terminal domain of the receptor. J Biol Chem 272:8207-8214. Murphy CA, Hoek RM, Wiekowski MT, Lira SA, Sedgwick JD (2002) Interactions between hemopoietically derived TNF and central nervous system-resident glial chemokines underlie initiation of autoimmune inflammation in the brain. J Immunol 169:7054-7062. Nakazawa E, Ishikawa H (1998) Ultrastructural observations of astrocyte end-feet in the rat central nervous system. J Neurocytol 27:431-440. Namaka M, Turcotte D, Leong C, Grossberndt A, Klassen D (2008) Multiple sclerosis: etiology and treatment strategies. Consult Pharm 23:886-896. Nashmi R, Fehlings MG (2001) Mechanisms of axonal dysfunction after spinal cord injury: with an emphasis on the role of voltage-gated potassium channels. Brain Res Brain Res Rev 38:165-191. Ness JK, Valentino M, McIver SR, Goldberg MP (2005) Identification of oligodendrocytes in experimental disease models. Glia 50:321-328. Neumann H (2001) Control of glial immune function by neurons. Glia 36:191-199. Nguyen D, Stangel M (2001) Expression of the chemokine receptors CXCR1 and CXCR2 in rat oligodendroglial cells. Brain Res Dev Brain Res 128:77-81. Ni HT, Hu S, Sheng WS, Olson JM, Cheeran MC, Chan AS, Lokensgard JR, Peterson PK (2004) High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells. Brain Res Dev Brain Res 152:159- 169. Nieto-Sampedro M, Saneto RP, de Vellis J, Cotman CW (1985) The control of glial populations in brain: changes in astrocyte mitogenic and morphogenic factors in response to injury. Brain Res 343:320-328. Omari KM, John GR, Sealfon SC, Raine CS (2005) CXC chemokine receptors on human oligodendrocytes: implications for multiple sclerosis. Brain 128:1003- 1015. Omari KM, John G, Lango R, Raine CS (2006) Role for CXCR2 and CXCL1 on glia in multiple sclerosis. Glia 53:24-31. Omari KM, Lutz SE, Santambrogio L, Lira SA, Raine CS (2009) Neuroprotection and remyelination after autoimmune demyelination in mice that inducibly overexpress CXCL1. Am J Pathol 174:164-176. Ono K, Takii T, Onozaki K, Ikawa M, Okabe M, Sawada M (1999) Migration of exogenous immature hematopoietic cells into adult mouse brain parenchyma under GFP-expressing bone marrow chimera. Biochem Biophys Res Commun 262:610-614. Otto VI, Stahel PF, Rancan M, Kariya K, Shohami E, Yatsiv I, Eugster HP, Kossmann T, Trentz O, Morganti-Kossmann MC (2001) Regulation of chemokines and chemokine receptors after experimental closed head injury. Neuroreport 12:2059-2064.

144

Ousman SS, David S (2000) Lysophosphatidylcholine induces rapid recruitment and activation of macrophages in the adult mouse spinal cord. Glia 30:92-104. Ousman SS, David S (2001) MIP-1alpha, MCP-1, GM-CSF, and TNF-alpha control the immune cell response that mediates rapid phagocytosis of myelin from the adult mouse spinal cord. J Neurosci 21:4649-4656. Padovani-Claudio DA, Liu L, Ransohoff RM, Miller RH (2006) Alterations in the oligodendrocyte lineage, myelin, and white matter in adult mice lacking the chemokine receptor CXCR2. Glia 54:471-483. Park SK, Solomon D, Vartanian T (2001) Growth factor control of CNS myelination. Dev Neurosci 23:327-337. Parri HR, Gould TM, Crunelli V (2001) Spontaneous astrocytic Ca2+ oscillations in situ drive NMDAR-mediated neuronal excitation. Nat Neurosci 4:803-812. Penton-Rol G, Cervantes-Llanos M, Cabrera-Gomez JA, Alonso-Ramirez R, Valenzuela-Silva C, Rodriguez-Lara R, Montero-Casimiro E, Bello-Rivero I, Lopez-Saura P (2008) Treatment with type I interferons induces a regulatory T cell subset in peripheral blood mononuclear cells from multiple sclerosis patients. Int Immunopharmacol 8:881-886. Peters A (2004) A fourth type of neuroglial cell in the adult central nervous system. J Neurocytol 33:345-357. Petersen AA, Sellebjerg F, Frederiksen J, Olesen J, Vejlsgaard GL (1998) Soluble ICAM-1, demyelination, and inflammation in multiple sclerosis and acute optic neuritis. J Neuroimmunol 88:120-127. Peterson PK, Hu S, Salak-Johnson J, Molitor TW, Chao CC (1997) Differential production of and migratory response to beta chemokines by human microglia and astrocytes. J Infect Dis 175:478-481. Ponomarev ED, Shriver LP, Maresz K, Dittel BN (2005) Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J Neurosci Res 81:374-389. Raff M, Apperly J, Kondo T, Tokumoto Y, Tang D (2001) Timing cell-cycle exit and differentiation in oligodendrocyte development. Novartis Found Symp 237:100-107; discussion 107-112, 158-163. Raine CS, Wu E (1993) Multiple sclerosis: remyelination in acute lesions. J Neuropathol Exp Neurol 52:199-204. Rasband MN (2006) Neuron-glia interactions at the node of Ranvier. Results Probl Cell Differ 43:129-149. Remahl S, Hildebrand C (1990) Relations between axons and oligodendroglial cells during initial myelination. II. The individual axon. J Neurocytol 19:883-898. Rezaie P, Male D (1999) Colonisation of the developing human brain and spinal cord by microglia: a review. Microsc Res Tech 45:359-382. Richmond A, Mueller S, White JR, Schraw W (1997) C-X-C chemokine receptor desensitization mediated through ligand-enhanced receptor phosphorylation on serine residues. Methods Enzymol 288:3-15. Rinner WA, Bauer J, Schmidts M, Lassmann H, Hickey WF (1995) Resident microglia and hematogenous macrophages as phagocytes in adoptively transferred experimental autoimmune encephalomyelitis: an investigation using rat radiation bone marrow chimeras. Glia 14:257-266.

145

Robinson S, Franic LA (2001) Chemokine GRO1 and the spatial and temporal regulation of oligodendrocyte precursor proliferation. Dev Neurosci 23:338- 345. Robinson S, Tani M, Strieter RM, Ransohoff RM, Miller RH (1998) The chemokine growth-regulated oncogene-alpha promotes spinal cord oligodendrocyte precursor proliferation. J Neurosci 18:10457-10463. Robinson S, Cohen M, Prayson R, Ransohoff RM, Tabrizi N, Miller RH (2001) Constitutive expression of growth-related oncogene and its receptor in oligodendrogliomas. Neurosurgery 48:864-873; discussion 873-864. Rogister B, Ben-Hur T, Dubois-Dalcq M (1999) From neural stem cells to myelinating oligodendrocytes. Mol Cell Neurosci 14:287-300. Rosano C, Felipe-Cuervo E, Wood PM (1999) Regenerative potential of adult O1+ oligodendrocytes. Glia 27:189-202. Rovai LE, Herschman HR, Smith JB (1998) The murine neutrophil- chemoattractant chemokines LIX, KC, and MIP-2 have distinct induction kinetics, tissue distributions, and tissue-specific sensitivities to glucocorticoid regulation in endotoxemia. J Leukoc Biol 64:494-502. Sai J, Fan GH, Wang D, Richmond A (2004) The C-terminal domain LLKIL motif of CXCR2 is required for ligand-mediated polarization of early signals during chemotaxis. J Cell Sci 117:5489-5496. Sandvig A, Berry M, Barrett LB, Butt A, Logan A (2004) Myelin-, reactive glia-, and scar-derived CNS axon growth inhibitors: expression, receptor signaling, and correlation with axon regeneration. Glia 46:225-251. Sellebjerg F, Sorensen TL (2003) Chemokines and matrix metalloproteinase-9 in leukocyte recruitment to the central nervous system. Brain Res Bull 61:347- 355. Shah BH, Catt KJ (2004) Matrix metalloproteinase-dependent EGF receptor activation in hypertension and left ventricular hypertrophy. Trends Endocrinol Metab 15:241-243. Sheikh AM, Nagai A, Ryu JK, McLarnon JG, Kim SU, Masuda J (2009) Lysophosphatidylcholine induces glial cell activation: role of rho kinase. Glia 57:898-907. Sherman DL, Brophy PJ (2005) Mechanisms of axon ensheathment and myelin growth. Nat Rev Neurosci 6:683-690. Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5:146-156. Sim FJ, Zhao C, Penderis J, Franklin RJ (2002) The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation. J Neurosci 22:2451-2459. Simpson JE, Newcombe J, Cuzner ML, Woodroofe MN (1998) Expression of monocyte chemoattractant protein-1 and other beta-chemokines by resident glia and inflammatory cells in multiple sclerosis lesions. J Neuroimmunol 84:238-249.

146

Smith DF, Galkina E, Ley K, Huo Y (2005) GRO family chemokines are specialized for monocyte arrest from flow. Am J Physiol Heart Circ Physiol 289:H1976- 1984. Smith ME, Somera FP, Eng LF (1983) Immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis. Brain Res 264:241-253. Soula C, Sagot Y, Cochard P, Duprat AM (1990) Astroglial differentiation from neuroepithelial precursor cells of amphibian embryos: an in vivo and in vitro analysis. Int J Dev Biol 34:351-364. Spanaus KS, Nadal D, Pfister HW, Seebach J, Widmer U, Frei K, Gloor S, Fontana A (1997) C-X-C and C-C chemokines are expressed in the cerebrospinal fluid in bacterial meningitis and mediate chemotactic activity on peripheral blood- derived polymorphonuclear and mononuclear cells in vitro. J Immunol 158:1956-1964. Steinman L, Zamvil SS (2005) Virtues and pitfalls of EAE for the development of therapies for multiple sclerosis. Trends Immunol 26:565-571. Steinman L, Zamvil SS (2006) How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis. Ann Neurol 60:12-21. Stillie R, Farooq SM, Gordon JR, Stadnyk AW (2009) The functional significance behind expressing two IL-8 receptor types on PMN. J Leukoc Biol 86:529- 543. Stoll G, Jander S (1999) The role of microglia and macrophages in the pathophysiology of the CNS. Prog Neurobiol 58:233-247. Sue RD, Belperio JA, Burdick MD, Murray LA, Xue YY, Dy MC, Kwon JJ, Keane MP, Strieter RM (2004) CXCR2 is critical to hyperoxia-induced lung injury. J Immunol 172:3860-3868. Sun D, Hu X, Liu X, Whitaker JN, Walker WS (1997) Expression of chemokine genes in rat glial cells: the effect of myelin basic protein-reactive encephalitogenic T cells. J Neurosci Res 48:192-200. Sykova E, Vargova L, Prokopova S, Simonova Z (1999) Glial swelling and astrogliosis produce diffusion barriers in the rat spinal cord. Glia 25:56-70. Szczucinski A, Losy J (2007) Chemokines and chemokine receptors in multiple sclerosis. Potential targets for new therapies. Acta Neurol Scand 115:137-146. Takami S, Nishikawa H, Minami M, Nishiyori A, Sato M, Akaike A, Satoh M (1997) Induction of macrophage inflammatory protein MIP-1alpha mRNA on glial cells after focal cerebral ischemia in the rat. Neurosci Lett 227:173-176. Tanabe S, Heesen M, Berman MA, Fischer MB, Yoshizawa I, Luo Y, Dorf ME (1997) Murine astrocytes express a functional chemokine receptor. J Neurosci 17:6522-6528. Tani M, Glabinski AR, Tuohy VK, Stoler MH, Estes ML, Ransohoff RM (1996a) In situ hybridization analysis of glial fibrillary acidic protein mRNA reveals evidence of biphasic astrocyte activation during acute experimental autoimmune encephalomyelitis. Am J Pathol 148:889-896. Tani M, Fuentes ME, Peterson JW, Trapp BD, Durham SK, Loy JK, Bravo R, Ransohoff RM, Lira SA (1996b) Neutrophil infiltration, glial reaction, and

147

neurological disease in transgenic mice expressing the chemokine N51/KC in oligodendrocytes. J Clin Invest 98:529-539. Thorne M, Moore CS, Robertson GS (2009) Lack of TIMP-1 increases severity of experimental autoimmune encephalomyelitis: Effects of darbepoetin alfa on TIMP-1 null and wild-type mice. J Neuroimmunol 211:92-100. Tran EH, Prince EN, Owens T (2000) IFN-gamma shapes immune invasion of the central nervous system via regulation of chemokines. J Immunol 164:2759- 2768. Trapp BD, Nave KA (2008) Multiple sclerosis: an immune or neurodegenerative disorder? Annu Rev Neurosci 31:247-269. Traves SL, Smith SJ, Barnes PJ, Donnelly LE (2004) Specific CXC but not CC chemokines cause elevated monocyte migration in COPD: a role for CXCR2. J Leukoc Biol 76:441-450. Triarhou LC, Herndon RM (1985) Effect of macrophage inactivation on the neuropathology of lysolecithin-induced demyelination. Br J Exp Pathol 66:293-301. Tsai HH, Frost E, To V, Robinson S, Ffrench-Constant C, Geertman R, Ransohoff RM, Miller RH (2002) The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration. Cell 110:373-383. Uhm JH, Dooley NP, Oh LY, Yong VW (1998) Oligodendrocytes utilize a matrix metalloproteinase, MMP-9, to extend processes along an astrocyte extracellular matrix. Glia 22:53-63. Ulrich R, Baumgartner W, Gerhauser I, Seeliger F, Haist V, Deschl U, Alldinger S (2006) MMP-12, MMP-3, and TIMP-1 are markedly upregulated in chronic demyelinating theiler murine encephalomyelitis. J Neuropathol Exp Neurol 65:783-793. Valles A, Grijpink-Ongering L, de Bree FM, Tuinstra T, Ronken E (2006) Differential regulation of the CXCR2 chemokine network in rat brain trauma: implications for neuroimmune interactions and neuronal survival. Neurobiol Dis 22:312-322. Vos JP, Gard AL, Pfeiffer SE (1996) Regulation of oligodendrocyte cell survival and differentiation by ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and interleukin-6. Perspect Dev Neurobiol 4:39-52. Wang JG, Strong JA, Xie W, Yang RH, Coyle DE, Wick DM, Dorsey ED, Zhang JM (2008) The chemokine CXCL1/growth related oncogene increases sodium currents and neuronal excitability in small diameter sensory neurons. Mol Pain 4:38. Wang KC, Koprivica V, Kim JA, Sivasankaran R, Guo Y, Neve RL, He Z (2002) Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 417:941-944. Wang Y, Luo W, Stricker R, Reiser G (2006) Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine GRO/CINC-1. J Neurochem 98:1046-1060. Waxman SG (1977) Conduction in myelinated, unmyelinated, and demyelinated fibers. Arch Neurol 34:585-589.

148

Weiner HL (2008) A shift from adaptive to innate immunity: a potential mechanism of disease progression in multiple sclerosis. J Neurol 255 Suppl 1:3-11. Werner SR, Dotzlaf JE, Smith RC (2008) MMP-28 as a regulator of myelination. BMC Neurosci 9:83. Williams KC, Ulvestad E, Hickey WF (1994) Immunology of multiple sclerosis. Clin Neurosci 2:229-245. Wirenfeldt M, Babcock AA, Ladeby R, Lambertsen KL, Dagnaes-Hansen F, Leslie RG, Owens T, Finsen B (2005) Reactive microgliosis engages distinct responses by microglial subpopulations after minor central nervous system injury. J Neurosci Res 82:507-514. Woodruff RH, Fruttiger M, Richardson WD, Franklin RJ (2004) Platelet-derived growth factor regulates oligodendrocyte progenitor numbers in adult CNS and their response following CNS demyelination. Mol Cell Neurosci 25:252- 262. Wu DT, Woodman SE, Weiss JM, McManus CM, D'Aversa TG, Hesselgesser J, Major EO, Nath A, Berman JW (2000a) Mechanisms of leukocyte trafficking into the CNS. J Neurovirol 6 Suppl 1:S82-85. Wu Q, Miller RH, Ransohoff RM, Robinson S, Bu J, Nishiyama A (2000b) Elevated levels of the chemokine GRO-1 correlate with elevated oligodendrocyte progenitor proliferation in the jimpy mutant. J Neurosci 20:2609-2617. Xia M, Hyman BT (2002) GROalpha/KC, a chemokine receptor CXCR2 ligand, can be a potent trigger for neuronal ERK1/2 and PI-3 kinase pathways and for tau hyperphosphorylation-a role in Alzheimer's disease? J Neuroimmunol 122:55-64. Xia MQ, Hyman BT (1999) Chemokines/chemokine receptors in the central nervous system and Alzheimer's disease. J Neurovirol 5:32-41. Xu J, Ling EA (1994) Studies of the ultrastructure and permeability of the blood- brain barrier in the developing corpus callosum in postnatal rat brain using electron dense tracers. J Anat 184 ( Pt 2):227-237. Yamamura T, Konola JT, Wekerle H, Lees MB (1991) Monoclonal antibodies against myelin proteolipid protein: identification and characterization of two major determinants. J Neurochem 57:1671-1680. Yan SS, Wu ZY, Zhang HP, Furtado G, Chen X, Yan SF, Schmidt AM, Brown C, Stern A, LaFaille J, Chess L, Stern DM, Jiang H (2003) Suppression of experimental autoimmune encephalomyelitis by selective blockade of encephalitogenic T-cell infiltration of the central nervous system. Nat Med 9:287-293. Zatta P, Raso M, Zambenedetti P, Wittkowski W, Messori L, Piccioli F, Mauri PL, Beltramini M (2005) Copper and zinc dismetabolism in the mouse brain upon chronic cuprizone treatment. Cell Mol Life Sci 62:1502-1513. Zhu X, Bergles DE, Nishiyama A (2008) NG2 cells generate both oligodendrocytes and gray matter astrocytes. Development 135:145-157.

149