Chemical and Metabolomic Analyses of Cuprizone-Induced
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CHEMICAL AND METABOLOMIC ANALYSES OF CUPRIZONE-INDUCED DEMYELINATION AND REMYELINATION A Thesis Presented to The Graduate Faculty of the University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Chemistry Alexandra Taraboletti May 2017 CHEMICAL AND METABOLOMIC ANALYSES OF CUPRIZONE-INDUCED DEMYELINATION AND REMYELINATION Alexandra Taraboletti Dissertation Approved: Accepted: _________________________________ _________________________________ Advisor Department Chair Dr. Leah Shriver Dr. Christopher Ziegler _________________________________ _________________________________ Committee Member Dean of College Dr. Richard Londraville Dr. John Green _________________________________ _________________________________ Committee Member Dean of Graduate School Dr. Sailaja Paruchuri Dr. Chand Midha _________________________________ _________________________________ Committee Member Date Dr. Chrys Wesdemiotis _________________________________ Committee Member Dr. Christopher Ziegler ii ABSTRACT The cuprizone intoxication model of demyelination and remyelination has long been used to test myelin regenerative therapies for neurodegenerative diseases such as multiple sclerosis. Mice develop reversible and region-specific oligodendrocytosis and demyelination when fed this small molecule copper chelator. While the histopathology of the model has been well documented, to date, there is no consensus on cuprizone’s cellular mechanism of action, and whether it involves the chelation of copper in vivo. Additionally, a variant model has been employed that combines rapamycin with cuprizone, causing more robust demyelination. In this model, rapamycin is hypothesized to suspend oligodendrocyte differentiation via the disruption of mTOR. In this work, the spatial and temporal effects of cuprizone were extensively studied in the in vivo model, and compared to a newly developed in vitro model. Global metabolomic and lipidomic profiling were utilized as analytic tools to determine biochemical pathways altered by cuprizone, rapamycin, or cuprizone and rapamycin treatments. Furthermore, oligodendrocyte differentiation in relation to mTOR was explored with transcriptomics. Together this work utilizes OMICS technology paired with analytical techniques to investigate both the chemical nature of cuprizone and the biological function of the oligodendrocyte, gaining insight into the molecular interplay that occurs during the processes of demyelination and remyelination. iii DEDICATION “Isn’t nice to know a lot? And a little bit not.” -Stephen Sondheim (I Know Things Now, Into the Woods) iv ACKNOWLEDGMENTS The whole of this research has taken me on a long journey, starting in a seemingly empty lab as a one initial graduate student, and culminating in a group that has almost tripled. The lab has so much support and is filled with exciting energy, all in pursuit of many different scientific hypotheses. I was honored to start my academic path alongside my advisor and mentor Dr. Leah Shriver, who has instilled in me an extensive and diverse amount of knowledge. One day I too hope to have as detailed a grasp on human metabolism as her. I also have been shaped by my wonderful fellow students and lab- mates He Huang, Celina Cahalane, Hannah Baumann, and Rashmi Binjawadaji that I have had the honor to work alongside in our lab. I thank you especially for your endless support, and all of the coffee and snacks we have shared. I also want to thank my immediate family, Eva, Sabrina, and Matthew, as well as all of my extended family for their encouragement throughout my academic career; I hope I’ve made you proud. Thank you as well to all the new and wonderful friends I have made in my move to Ohio. I am so delighted to have a met a brilliant team of adventurers and fellow nerds in Mena Klittich, Jake Hill, Katherine and Erik Willett, and Carolyn and Jake Scherger. Thanks, Shadowmere’s Avengers for all the late nights, great food, and laughs. I was also very fortunate to befriend an excellent group of fellow graduate students, including Megan Klufas, Shaun Christe, Louis Ray, Paul Mallory, Dan Morris, Joel Caporoso, Roger Shi, v Farai Gombedza and many others who have all been so supportive during this expedition; my growth as a person, both emotionally and mentally, has been directly tied to these friendships. Finally, the work on my thesis would not have been completed without collaboration with the knowledgeable members of Renovo Neural, gracious funding from the Choose Ohio First Bioinformatics scholarship, and support from our NIH 1R15 GM119074-01 grant. vi TABLE OF CONTENTS Page LIST OF FIGURES ................................................................................................... xii LIST OF TABLES ................................................................................................... xiv LIST OF SCHEMES ..................................................................................................xv ABBREVIATIONS .................................................................................................. xvi CHAPTER I .................................................................................................................1 1.1 Multiple Sclerosis ................................................................................................... 1 1.1.1 Neuropathological Characteristics of Multiple Sclerosis ................................. 3 1.1.2 Remyelination ................................................................................................... 7 1.1.3 Animal models of Multiple Sclerosis ............................................................. 10 1.2 The cuprizone animal model ................................................................................. 13 1.2.1 Pathology and clinical symptoms ................................................................... 14 1.2.2 Proposed cuprizone mechanisms .................................................................... 19 1.3 Global metabolomics ............................................................................................ 21 1.3.1 Mass spectrometry .......................................................................................... 23 1.3.1.1 Separation .............................................................................................. 23 1.3.1.2 Ionization ............................................................................................... 25 1.3.1.3 Mass analyzer......................................................................................... 27 vii 1.3.2 Applications .................................................................................................... 29 1.3.2.1 Targeted metabolomics .......................................................................... 29 1.3.2.2 Untargeted metabolomics ...................................................................... 30 1.3.2.2.1 Bioinformatics.................................................................................. 32 1.3.2.3 Lipidomics ............................................................................................. 37 CHAPTER II ..............................................................................................................40 2.1 Introduction ........................................................................................................... 40 2.2 Methods................................................................................................................. 43 2.2.1 Absorbance Spectroscopy ............................................................................... 43 2.2.2 NMR Spectroscopy ......................................................................................... 44 2.2.3 Mass Spectrometry.......................................................................................... 44 2.3 Results and Discussion ......................................................................................... 45 2.3.1 Absorbance spectroscopy of CPZ, CuCPZ, and active site mimics ............... 45 2.3.2 NMR Spectroscopy of CPZ, and active site mimics....................................... 49 2.3.3 Mass Spectrometry of CPZ, CuCPZ, and active site mimics ......................... 52 2.4 Conclusions ........................................................................................................... 56 CHAPTER III .............................................................................................................57 3.1 Introduction ........................................................................................................... 57 3.2 Methods................................................................................................................. 61 viii 3.2.1 Chemicals ........................................................................................................ 61 3.2.2 Cell Culture ..................................................................................................... 62 3.2.3 Preparation of CPZ Solution ........................................................................... 62 3.2.4 Live/Dead Cell Viability Assay and MTT assay ............................................ 62 3.2.5 Immunofluorescence ....................................................................................... 63 3.2.6 Cuprizone and CuCPZ Absorbance Assay ..................................................... 64 3.2.7 SRM-MS Analysis of CPZ Uptake in Cells ..................................................