Characterization of the DJ-1 Knockout Rat Model of Parkinson’s Disease A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in the Neuroscience Graduate Program of the College of Medicine 2019 By Tara Leigh Kyser B.S., Ursinus College Advisor: Kim B. Seroogy, Ph.D. Committee Chair: Mark Baccei, Ph.D. Christina Gross, Ph.D. Michael Williams, Ph.D. David Yurek, Ph.D. Abstract Parkinson’s disease (PD) is a complex neurodegenerative disorder with a plethora of symptoms categorized as motor and non-motor. Historically, research on PD has focused mainly on degradation of the nigrostriatal pathway. However, degeneration of additional brainstem regions, including the locus coeruleus (LC) and dorsal raphe nucleus (DRN), and dysfunction of their associated noradrenergic and serotonergic neurotransmitter systems, respectively, also contribute to disease pathology. Nevertheless, the etiology of the vast majority of PD cases remains unknown. The overall goal of our work was to examine the relatively novel DJ-1 knockout (KO) rat model of PD in an effort to expand our knowledge of the manifestation and progression of PD. Complete loss of the protein DJ-1 leads to an autosomal recessive form of PD. The DJ-1 KO rat was created with the objective of generating an animal model with a more robust PD phenotype than seen previously in DJ-1 KO mice. Although a few studies on these mutant rats have noted some promising PD-like features, they have not always been in agreement. Here, we conducted a more thorough analysis of the DJ-1-deficient rat model, hypothesizing that we would uncover aberrant motor and non-motor behaviors, altered neurotransmitter levels and reduced neuronal survival in PD-relevant brain regions. In Aim 1, we performed a battery of motor and non-motor tasks at various ages to expand the behavioral characterization of the DJ-1 KO rat. Consistent with a PD phenotype, the DJ-1 KO rats demonstrated a reduction in rears, stride length, and grooming time, compared to wild-type (WT) control rats. However, the DJ-1-deficient rats took more steps than WT controls in several motor tasks, inconsistent with PD-like behavior. The non-motor findings were also mixed, in that DJ-1 KO rats exhibited deficits in short-term memory, but also better olfactory detection and increased sucrose intake during the sucrose preference task. In Aim 2, we examined monoamine levels using neurochemistry to determine if DJ-1 KO rats showed altered i levels of monoamines in brain regions associated with PD. We used immunohistochemistry and unbiased stereology to examine the substantia nigra pars compacta, LC, and DRN for monoaminergic neuronal degeneration. Depending on age, we detected increases in the dopamine metabolite 3,4 dihydroxyphenylacetic acid in the striatum and of the serotonin metabolite 5-hydroxyindoleucetic acid in the ventral midbrain and hippocampus of DJ-1-deficient rats. Norepinephrine (NE) levels in the hippocampus of DJ-1 KO rats were lower than controls. DJ-1 KO rats demonstrated reduced numbers of noradrenergic LC neurons than WT rats and a loss of serotonergic neurons in the DRN, regardless of age. In Aim 3, because of the association of neuroinflammation with PD, we evaluated activated microglia in several PD- related brain regions in DJ-1 KO rats. Only the LC displayed activated microglia when compared to WT rats, suggesting limited involvement of detrimental microglial mechanisms in the DJ-1- deficient brain. Overall, DJ-1-deficient rats demonstrate a PD-like phenotype in several behavioral, neurochemical and morphological aspects of the disease. Moreover, the data suggest that DJ-1 KO rats will be particularly useful in investigating prodromal stages of PD. ii iii Acknowledgements Over the course of my dissertation I have had people who have given me guidance and support, and I would like to thank these individuals. First, I would like to thank my advisor Dr. Kim Seroogy. Thank you for allowing me to join the lab. I appreciate all the guidance you have given me and for allowing me to follow my interests in the lab. Thank you for all the advice you have given me and for making me a better scientist. I wish to thank my committee members Mark Baccei, Michael Williams, Nina Gross, and David Yurek for all of their helpful suggestions and individual guidance. I want to thank Mark Baccei for being the head of my committee and helping me through the graduation process. I appreciate at his advice and support as both a committee member and director of the Neuroscience Graduate Program. I would like to thank Michael Williams for his help with the behavioral testing as well as statistical analysis. Thanks to Nina Gross for being on my committee and all her thoughtful questions that made my dissertation better. Lastly, I would like thank David Yurek for traveling the long distance to the University of Cincinnati for the committee meetings and for sharing your vast knowledge of Parkinson’s disease I would like to thank the Neuroscience Graduate Program for accepting me into the program. I want to thank the past (Deb Cummins and Sharon Weber) and present (Ana Madani) program coordinators and manager for their help with a lot of the paperwork and for making sure I turned everything in on time. Thank you to the past (Jim Herman and Kim Seroogy) and present (Mark Baccei) program directors for allowing me the opportunity to conduct research at UC. Also, thanks to my fellow students for shared knowledge, encouragement, and the good times we shared. iv To members of the Seroogy Lab, past and present, thank you for all of your help in lab and without you I would not have gotten this far. Kerstin Lundgren, thank you for being the anchor in lab and teaching me a lot of valuable laboratory techniques such as in situ hybridization. To Ann and Sarah, thank you for all the laughs and being very supportive lab mates. I would like to thank all of the undergraduates who helped me over the years, and most especially Adam Dourson for helping me greatly in the last couple years of my dissertation work. Importantly, I wish to acknowledge the financial support of NIH and the James J. and Joan A. Gardner Family Center for Parkinson's Disease and Movement Disorders. Last, but not least, I would like to thank my family, friends, and dog Ollie. Thanks to my mom and dad for having faith in me and being supportive of my pursuits. To my Aunt Colleen and Grandmom, thank you for driving all the way out to Cincinnati to support me during my defense. And to the rest of my family who could not make it out, thank you for your love and encouragement. To my dog, Ollie, thank you for all the cuddles and being the best dog a person could ask for. Thanks to Becky Bailey, for going through this graduate student process with me and listening to me when I needed it the most. To Kenton Woodard, thank you for picking me up when I was down and for always being there for me no matter how far we are from each other. Last, but definitely not least, thanks to Amanda Stover. I really could not have gotten through this whole process without you. You are there for me through thick and thin, making me laugh and letting me know that you have faith in me when I had lost faith in myself. I look forward to being there for you while you go through your own dissertation process. v Table of Contents Abstract i Acknowledgements iv List of Tables and Figures 3 Abbreviations 5 Chapter 1: Introduction Parkinson’s disease 10 Risk factors for Parkinson’s disease 16 Animal models 22 The substantia nigra pars compacta and Parkinson’s disease 35 The dorsal raphe nucleus and Parkinson’s disease 38 The locus coeruleus and Parkinson’s disease 41 Olfaction and Parkinson’s disease 43 Depression and Parkinson’s disease 44 Anxiety and Parkinson’s disease 45 Cognitive Impairment and Parkinson’s disease 46 DJ-1 and Parkinson’s disease 47 Objectives 50 Specific Aims 50 References 51 Chapter 2: Characterization of Motor and Non-Motor Behavioral Alterations in the Dj-1 (PARK7) Knockout Rat Abstract 132 Introduction 133 Methods 136 Results 143 Discussion 156 References 164 1 Chapter 3: Altered Monoamine Levels and Cell Degeneration of Monoaminergic Nuclei Abstract 178 Introduction 179 Methods 182 Results 186 Discussion 193 References 201 Chapter 4: Evaluation of Activated Microglia in Aged DJ-1 Knockout Rats Abstract 211 Introduction 212 Methods 216 Results 219 Discussion 224 References 227 Chapter 5: General Discussion Discussion 239 Limitations and Caveats 244 Future Studies/Conclusions 246 References 247 2 List of Tables and Figures Chapter 1 Table 1. List of known non-motor symptoms in Parkinson’s disease 13 Table 2. Braak staging of Lewy body pathology 14 Table 3. Environmental risk factors associated with Parkinson’s disease 20 Table 4. Genetic risk factors of Parkinson’s disease 21 Chapter 2 Figure 1. Evaluation of spontaneous activity in DJ-1 KO and WT rats 145 at 4, 7, and 13 months of age. Figure 2. Assessment of sensorimotor changes during the adhesive 146 removal task at 4,7, and 13 months of age for WT and DJ-1 KO rats Figure 3. Evaluation of postural instability in the adjusting step task at 4, 147 7, and 13 months of age for WT and DJ-1 KO rats Figure 4. Analysis of gait changes in WT and DJ-1 KO rats at 2 and 13 148 months of age Figure 5.