Exploring Novel Therapies for Motor and Non-Motor Symptoms in a Mouse Model of Multiple Sclerosis. by Curtis Alexander Benson A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Centre for Neuroscience University of Alberta © Curtis Alexander Benson, 2016 Abstract Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by neurodegeneration, inflammation and demyelination. Symptoms of multiple sclerosis not only include motor deficits, but also secondary symptoms of pain, depression and anxiety. The purpose of this thesis was firstly to understand the underlying mechanism of these symptoms and secondly to investigate possible treatments. The first half of this thesis investigates the role of several key neurotransmitter systems in the progression and symptomatology of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. The second half of this thesis examines whether exercise can modify the non-motor symptoms associated with EAE. Experiments in chapter 2 examined the effect of phenelzine (PLZ) in EAE. Daily PLZ treatment, starting seven days after disease induction, delayed EAE onset, reduced disease severity in the chronic phase and was associated with improvements in exploratory behaviour and a novel measure of sickness behaviour. EAE mice treated with daily PLZ showed a recovery in the levels of serotonin (5-HT) and norepinephrine (NE). In particular, EAE mice treated with PLZ showed a normalization of 5-HT levels in the ventral horn of the spinal cord. However, no differences were observed in the amount of T cell infiltration, microglia/macrophage reactivity, demyelination or axonal injury in PLZ-treated spinal cords. In chapter 3, we conducted experiments in which PLZ treatment only began when mice with EAE exhibited the first clinical signs of the disease, to determine whether PLZ could have beneficial effects in an already established disease state. Using this more clinically relevant treatment approach, we found that PLZ treatment reduced the severity of clinical signs and improved exploratory behaviours for the duration of the experiment in mice with EAE. These ii improvements were associated with higher levels of γ-aminobutyric acid (GABA), 5-HT, dopamine (DA) and NE in the brain and spinal cord. In chapter 4, work was undertaken to assess whether exercise could improve both the motor deficits and secondary symptoms of EAE. Allowing EAE mice 1 hour every day of voluntary running led to a significant delay in the onset of disease symptoms. The development of mechanical allodynia was assessed using Von Frey hairs and indicated that wheel running had a modest positive effect on the pain hypersensitivity associated with EAE. These behavioural changes were associated with decreased CNS inflammation, reduced oxidative stress and less mitochondrial protein expression. Unlike the case in chapters 2 and 3, voluntary wheel running led to only minor changes in neurotransmitter levels. The work done in chapter 5 further investigates the mitochondrial changes that occur during early EAE by examining the proteins involved in mitochondrial fission, fusion and biogenesis. EAE animals had increased levels of both the outer mitochondrial membrane protein Tom20 and the complex IV protein (COX IV) within the dorsal horn of the spinal cord compared to control mice. This increase was associated with an increase in the mitochondrial fission mediator, Drp1, a decrease in phosphorylated Drp1 at ser616, a decrease the fusion protein mitofusin 2 (MFN2), and a decrease in the expression of peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1α). The work in this thesis demonstrated the therapeutic potential of the monoamine oxidase (MAO) inhibitor PLZ and of daily exercise in MS. This study led to the identification of the role of several key neurotransmitters and also highlights mitochondrial dysfunction in the secondary symptoms of the disease. Further research into these mechanisms may lead to the development of novel therapies for MS. iii Preface This thesis is the original work of C. Benson. The projects and protocols used in this thesis have been approved by the University of Alberta animal care and use committee. Chapter 1 of thesis is an expanded version of the paper published by Benson C and Kerr BJ (2014) Pain and cognition in multiple sclerosis. Curr Top Behav Neurosci 20:201-15. C. Benson was involved in concept formation and manuscript composition and editing. B.J. Kerr was involved in manuscript editing and was the supervisory author. Chapter 2 of this thesis has been published as Musgrave T1, Benson C1, Wong G, Browne I, Tenorio G, Rauw G, Baker GB, Kerr BJ., (2011). The MAO inhibitor phenelzine improves functional outcomes in mice with experimental autoimmune encephalomyelitis (EAE). Brain Behav Immun 25:1677-1688. T. Musgrave and C. Benson contributed equally to this work. T Musgrave was responsible for data collection and analysis of Figures 2.1 and 2.2 with assistance from C. Benson. C. Benson was responsible for data collection, analysis and preparation of figures 2.3-2.7 with assistance from G. Wong on figures 2.3 and 2.6. G. Rauw, G. Tenorio and I. Browne provided technical assistance. C. Benson, T. Musgrave and B.J. Kerr were involved in concept formation and manuscript composition and editing. G.B. Baker and B.J. Kerr were the supervisory authors. Chapter 3 of this thesis has been published as Benson C, Wong G, Tenorio G, Baker GB, Kerr BJ. (2013) The MAO inhibitor phenelzine can improve functional outcomes in mice with established clinical signs in experimental autoimmune encephalomyelitis (EAE). Behav Brain Res 252:302-311. C. Benson was responsible for data collection and analysis of figures 3.1, 3.5, 3.6 and 3.7. G. Wong, with direction and assistance from C. Benson, collected data for figures iv 3.2, 3.3 and 3.4. G. G. Tenorio provided technical assistance. C. Benson and B.J. Kerr were involved in concept formation and manuscript composition and editing. G.B. Baker and B.J. Kerr were the supervisory authors. Chapter 4 of this thesis has been published as Benson C1, Paylor JW1, Tenorio G, Winship I, Baker G, Kerr BJ. (2015). Voluntary wheel running delays disease onset and reduces pain hypersensitivity in early experimental autoimmune encephalomyelitis (EAE). Exp Neurol 271:279-290. C. Benson C and J.W. Paylor contributed equally to this project. C. Benson was responsible for data collection, analysis and preparation of figures 4.1, 4.2, 4.8 and 4.9. C. Benson performed immunohistochemistry and J.W. Paylor conducted image analysis and quantification for figures 4.3, 4.4, 4.5, 4.6, and 4.7. G. Tenorio provided technical assistance. I. Winship supervised the work of J.W. Paylor. C. Benson and B.J. Kerr were involved in concept formation and manuscript composition and editing. G.B. Baker and B.J. Kerr were the supervisory authors. Chapter 5 of this thesis is unpublished work. C. Benson was responsible for data collection and analysis for figures 5.1, 5.2, 5.3, 5.4, 5.5 and 5.6. S. Yousuf, M. Hubert and G. Tenorio provided technical assistance for figures 5.5 and 5.6. C. Benson and B.J. Kerr were involved in concept formation and manuscript composition and editing. B.J. Kerr were the supervisory authors. v “Every day is a new day. It is better to be lucky. But I would rather be exact. Then when luck comes you are ready.” ― Ernest Hemingway, The Old Man and the Sea vi Acknowledgements I would first like to thank my supervisor Dr. Bradley Kerr for all his support and allowing me to find my passion for scientific research. All the past and present members of the Kerr Lab deserve thanks for creating a supportive, collaborative and fun place to do my PhD. Especially, Camille, Travis, Liam, Katherine, Kevin and Saad. Thank you to everyone who provided me with technical advice and support. In particular, Gustavo Tenorio for keeping me sane by doing the Von Frey hair’s in many of my experiments and Gail Rauw for helping me to avoid Murphy’s Law when running the HPLC. Grace Wong and Wes Paylor deserve special credit for their diligent and impeccable cell staining and counting in chapters 3 and 4 of this thesis. I would like to thanks the department of Anesthesiology and Pain Medicine, the department of Pharmacology, Neuroscience and Mental Health Institute and the Multiple Sclerosis Society of Canada for their funding and support throughout my PhD work. I want to thank all my friends for their loyalty, motivation and the many games of darts. Thank you to my Mom, Dad and Haley for their unending support and for all the times they had to answer “what is Curtis doing again?” Finally, to Megan, thank you for listening, understanding and attempting to learn the areas of the brain. You push me to learn, explore and follow my dreams. I will always be grateful for your encouragement, kindness and love. vii Table of Contents CHAPTER 1 ................................................................................................................................... 1 General Introduction ....................................................................................................................... 1 1.1 MULTIPLE SCLEROSIS .................................................................................................... 2 1.2 BRIEF EPIDEMIOLOGY OF MS ...................................................................................... 2 1.2.1 MS Risk Factors.............................................................................................................