TLR4-activated microglia have divergent effects on oligodendrocyte lineage cells DISSERTATION Presented in partial fulfillment of the requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University Evan Zachary Goldstein, BA Neuroscience Graduate Program The Ohio State University 2016 Dissertation Committee: Dr. Dana McTigue, Advisor Dr. Phillip Popovich Dr. Jonathan Godbout Dr. Courtney DeVries Copyright by Evan Zachary Goldstein 2016 i Abstract Myelin accelerates action potential conduction velocity and provides essential metabolic support for axons. Unfortunately, myelin and myelinating cells are often vulnerable to injury or disease, resulting in myelin damage, which in turn can lead to axon dysfunction, overt pathology and neurological impairment. Inflammation is a common component of CNS trauma and disease, and therefore an active inflammatory response is often considered deleterious to myelin health. While inflammation can certainly damage myelin, inflammatory processes also benefit oligodendrocyte (OL) lineage progression and myelin repair. Consistent with the divergent nature of inflammation, intraspinal toll-like receptor 4 (TLR4) activation, an innate immune pathway, kills OL lineage cells, but also initiates oligodendrogenesis. Soluble factors produced by TLR4-activated microglia can reproduce these effects in vitro, however the exact factors are unknown. To determine what microglial factors might contribute to TLR4-induced OL loss and oligodendrogenesis, mRNA of factors known to affect OL lineage cells was quantified in TLR4-activated microglia and spinal cords (chapter 2). Results indicate that TLR4-activated microglia transcribe numerous factors that induce OL loss, OL progenitor cell (OPC) proliferation and OPC differentiation. However, some factors upregulated after intraspinal TLR4 activation were not ii upregulated by microglia, suggesting that other cell types contribute to transcriptional changes in vivo. Many factors produced by TLR4-activated microglia have no known effects on OL lineage cells. In chapter 3, colony stimulating factor 3 (CSF3) and interleukin 7 (IL-7) were identified as factors produced by TLR4-activated microglia that might contribute to OPC proliferation or differentiation. Indeed, CSF3 injection into the uninjured spinal cord promoted OPC proliferation. Conversely, IL-7 injection into the uninjured spinal cord promoted OPC differentiation. Although it is unclear if these factors act directly on OPCs or if they initiate paracrine responses, CSF3 and IL-7 represent novel oligodendrogenic factors produced by TLR4-activated microglia. In chapter 4, an innate TLR4-induced iron chelation response was harnessed to sequester iron in a model of intraspinal iron accumulation. Because excess iron induces progressive neuronal and OL death, we hypothesized that intraspinal TLR4 activation (with lipopolysaccharide; LPS) would enhance iron sequestration by CNS macrophages and reduce subsequent cytotoxicity. LPS co-injected with iron did promote in vivo iron storage as detected by increased ferritin-expressing microglia and intraspinal iron protein mRNA expression characteristic of iron sequestration. Nevertheless, this approach was not neuroprotective against progressive neuronal or glial loss. In fact, Iron+LPS injection reduced OL replacement from OPCs. iii Collectively, this work demonstrates that activation of a single inflammatory receptor on microglia (TLR4) is capable of divergent effects on every stage of OL lineage progression. Thus, modulation of specific factors during demyelinating insults is a more advantageous therapeutic approach than general anti- inflammatory agents. iv Dedication To my family and friends: past, present, and future v Acknowledgments First and foremost, I must acknowledge and thank my advisor Dr. Dana McTigue. You have been a wonderful advisor and mentor. When I joined your lab, I was a naïve college graduate with an interest in oligodendrocytes. Over the years, you have taught me so many things, from how to be a scholar, to how to always be prepared with food and extra batteries in case of a blizzard! You pushed me to do the best work possible, and always had good advice when I knocked on your office door. Thank you for everything. I would also like to thank the rest of my committee members. Dr. Phillip Popovich, thank you for all your constructive criticism. You have certainly helped shape me as a scientist. Dr. Jonathan Godbout, thank you for teaching me neuroimmunology my first year in graduate school, and for all the subsequent advice on my dissertation work. Dr. Courney Devries, thank you for your words of encouragement and honest opinions on my work. All of the work that I have completed throughout my graduate career would not have been possible without so many wonderful people in the lab that have generously donated their time to me. To all the past and present technicians in the lab, thank you for all the time you spent helping me get to this point. I realize how lucky I am to have such wonderful technical support, and I know whichever lab I go to next will not compare. To all of my fellow graduate students, thank you vi for all of your friendships, support, and encouragement. I will try to keep in touch with as many of you as possible. To all the post-docs that have come through the lab, thank you for setting such a good example and for many friendships as well. To my parents, you both have been the inspiration that has gotten me to this point. The work that you have accomplished throughout your careers motivates me to do the best science and make as large an impact as I can. However, the love, time and attention that you have always afforded me are truly baffling. No matter how busy you were, I never had to ask twice for help on math homework, edits on an essay, or feedback on a dissertation. To Alexa, thank you for being a great big sister, always looking out for me and helping me navigate life. And finally, to Christine, thank you for being so supportive and understanding. I love you and can’t wait to start the next chapter of our lives. vii Vita 2006………………...James S. Rickards High School, Tallahassee, FL 2010………………...B.A. Biology & Psychology Case Western Reserve University, Cleveland, OH 2010 – 2016….…….Graduate Research Fellow The Ohio State University, Columbus, OH Publications Schonberg DL, Goldstein EZ, Sahinkaya FR, Wei P, Popovich PG, McTigue DM. Ferritin stimulates oligodendrocyte genesis in the adult spinal cord and can be transferred from macrophages to NG2 cells in vivo. J Neurosci, 32(16): 5374- 5384; 2012. PMID: 22514302 Hesp Z, Goldstein EZ, Miranda C, Kaspar, B, McTigue DM. Chronic oligodendrogenesis and remyelination after spinal cord injury in mice and rats. J Neurosci, 35(3): 1274-90; 2015 PMID:25609641 Goldstein EZ, Church JD, Hesp ZC, McTigue DM. Silver lining of inflammation: The beneficial effects of inflammation on myelination. Exp Neurol. In Press; 2016 PMID: 27151600 Fields of Study Major Field: Neuroscience viii Table of Contents Abstract…………………………………………………………………………………...ii Dedication………………………………………………………………………………..v Acknowledgments…..…………………………………………………………………..vi Vita………………………………………………………………………………………viii List of Tables…………………………………………………………………………….x List of Figures…………………………………………………………………………...xi Chapter 1. Introduction…………..…………………………………………………….1 Chapter 2. TLR4-activated microglia express factors known to influence OL lineage survival and oligodendrogenesis……………………………………………32 Chapter 3. CSF3 & IL-7 are novel oligodendrogenic factors produced by TLR4- activated macrophages/microglia.………………………………………………...…58 Chapter 4. Intraspinal TLR4 activation promotes iron storage but does not protect neurons or oligodendrocytes from progressive iron-mediated damage….….......91 Chapter 5. General conclusions and discussion………………………………….133 References……………………………………………………………………………151 ix List of Tables Table 1. Iron metabolism proteins……………………………………………………22 Table 2. TLR4-activated BMDMs increased transcription of cytokines and growth factors………………………………………………….……………….……………….70 Table 3. TLR4-activated BMDMs decreased transcription of cytokines and growth factors...……………………………………………………………………….……..…71 x List of Figures Figure 1. The stages of OL lineage progression have distinct morphological characteristics and express different proteins……………….……..………………..5 Figure 2. Inflammatory mediators affect all stages of myelination……...……..…13 Figure 3. Macrophage iron import, storage and export…………………………....24 Figure 4. In vivo and in vitro TLR4 activation induced transcriptional changes in factors responsible for OL apoptosis………..………………………………….…...40 Figure 5. In vivo and in vitro TLR4 activation induced transcriptional changes in factors that influence OL survival……………………..……………………………..42 Figure 6. In vivo and in vitro TLR4 activation increased transcription of factors that initiate OPC proliferation……………………………………………………..….44 Figure 7. In vivo and in vitro TLR4 activation induced transcriptional changes IL-6 family of cytokines.…………………………………………..………………………...45 Figure 8. In vivo and in vitro TLR4 activation increased IL-1β mRNA transcription. …………………………………………………………………………………..……….46 Figure 9. In vivo TLR4 activation increased CXCL12 mRNA transcription…......47 Figure 10. In vivo TLR4 activation increased TGFβ mRNA transcription....….…48 xi Figure 11. In vivo TLR4 activation increased BMP2 mRNA transcription.…..…..49 Figure 12. Bone marrow derived macrophages increased CSF3 and IL-7 mRNA after TLR4 activation………….………………………………………………..……..72