The Pennsylvania State University The Graduate School Intercollege Program of Neuroscience THE INTERACTION OF INFLAMMATION AND GENDER/SEX IN BIPOLAR DISORDER A Dissertation in Neuroscience by Caitlin E. Millett Ó 2018 Caitlin E. Millett Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2018 The dissertation of Caitlin E. Millett was reviewed and approved* by the following: Erika FH Saunders Chair, Department of Psychiatry Director, Mood Disorders Program Dissertation Advisor Chair of Committee Patricia (Sue) Grigson Professor, Neural and Behavioral Science James Connor Distinguished Professor of Neurosurgery, Neural and Behavioral Sciences and Pediatrics Vice Chair of Neurosurgery Research Director, Center for Aging and Neurodegenerative Diseases Julio Fernandez-Mendoza Associate Professor, Department of Psychiatry Colin Barnstable Professor and Chair, Neural & Behavioral Sciences Co-Director of the Graduate Program in Neuroscience *Signatures are on file in the Graduate School iii ABSTRACT In recent decades, inflammatory/immune activation has emerged as an important factor in the onset and progression of mood disorders. Increased pro-inflammatory markers have been observed in the peripheral blood, cerebrospinal fluid, and postmortem brain tissue of individuals with Bipolar Disorder (BD) and Major Depressive Disorder (MDD). Further, pro- inflammatory cytokine concentrations may decline during periods of normal mood (euthymia). There is evidence for a causal relationship between peripheral concentrations of the cytokine IL-6 in childhood, which can predict the onset of depressive symptoms in adulthood. Here we have proposed that gender and/or sex moderates the relationship between inflammation and mood in BD. Women and men are known to have different symptomatology and comorbidities associated with a diagnosis of BD; women have higher rates of post-traumatic stress disorder (PTSD) and eating disorders, whereas men have higher rates of alcohol and cannabis abuse and conduct disorder. Further, women with BD are known to experience depressive symptoms at the onset of the illness, whereas men are more likely to experience mania at onset. Moreover, there are known sex-based differences in inflammatory/immune activation. In general females have more robust cell- mediated and humoral immune responses to antigenic challenges compared with males. Women had higher self-reported feelings of depression and social isolation after endotoxin injection compared to men in a recent report. In contrast, it is known that the female sex hormone 17-β estradiol (E2) is broadly anti-inflammatory and confers protection against sepsis – a life-threatening complication of infection. iv Our research aims to address gaps in knowledge related to inflammation and gender/sex in BD. In our first study (Chapter 2) we examined peripheral biomarkers from human participants with symptomatic BD. The biomarkers of interest were (1) neopterin – a marker of the acute phase immune response that is released from activated macrophages, and (2) zinc – an essential biometal and antioxidant in the human body. We postulated that neopterin would be increased in symptomatic BD and zinc would be decreased. Further, we hypothesized that gender/sex would moderate the relationship between biomarker concentration and mania and depression severity. Our results indicated that peripheral zinc concentrations were in fact reduced in symptomatic BD compared to a healthy control population. Further, we found that zinc concentrations were associated with severity of depressive symptoms in women with BD. In contrast, neopterin concentrations were associated with mania severity in men with BD. In this way, our findings underscore the importance of analyzing gender differences in biomarker studies. This research will help to advance the field of personalized medicine for BD. In our second study (Chapter 3), we postulated that male and female mice would respond differently to an injection of the endotoxin lipopolysaccharide (LPS) – a component of the cell wall of gram-negative bacteria. Further, we expected that there would be a sex- based dichotomy in regard to the levels of oxidative stress and apoptosis in the brain after LPS injection. We found that males had higher levels of depressive-like behavior after LPS, compared to saline treated males. Further, we found increased protein levels of superoxide dismutase in the male hippocampus. 28 days after the LPS injection, males showed signs of increased apoptosis in the hippocampus – higher cleaved caspase 3 protein levels and increased TUNEL staining. Females overall appeared more resilient against LPS administration – they did not show signs of apoptosis or oxidative stress in the v hippocampus and did not increase depressive-like behavior. However, females appeared more ‘sick’ than males, indicated by decreased mobility in the open field test. Overall our study indicated that (1) inflammation may underlie hippocampal atrophy in some cases in males, (2) females appear to have a different behavioral response to inflammation compared to males, (3) females are more resilient against deleterious effects on LPS on the hippocampus compared to males. vi TABLE OF CONTENTS LIST OF FIGURES ................................................................................................... viii LIST OF TABLES ..................................................................................................... xi ACKNOWLEDGEMENTS......................................................................................... xii Chapter 1 Introduction .............................................................................................. 1 1.1 The History of Melancholy ........................................................................... 1 1.1.1 Hysteria ............................................................................................... 2 1.2 Mood Disorders Defined .............................................................................. 4 1.3 Global Impact .............................................................................................. 7 1.4 Burden......................................................................................................... 9 1.5 Gender/sex Influences Psychopathology and Prevalence ........................... 11 1.6 Inflammation in Mood Disorders .................................................................. 13 1.6.1 Inflammation Mechanisms to Depressive Symptoms ........................... 16 1.6.2 Inflammation and Gender/Sex.............................................................. 22 1.7 Animal Models ............................................................................................ 24 1.8 The Antioxidant Zinc .................................................................................... 25 1.9 Summary and Specific Aims ........................................................................ 30 1.10 References……………………………………………………………………….34 Chapter 2 Peripheral zinc and neopterin concentrations are associated with mood severity in bipolar disorder in a gender-specific manner .................................... 43 2.1 Abstract ....................................................................................................... 43 2.2 Introduction ................................................................................................. 44 2.3 Methods ...................................................................................................... 46 2.3.1 Participants .......................................................................................... 46 2.3.2 Rating Scales ....................................................................................... 46 2.3.3 Sample Collection and Biomarker Analysis .......................................... 47 2.3.4 Statistical Analysis ............................................................................... 48 2.4 Results ........................................................................................................ 48 2.4.1 Demographics and Clinical Data .......................................................... 48 2.4.2 Peripheral Biomarkerd in BD and HC ................................................... 50 2.4.3 Differential Association of Zinc, Neopterin, and Mood by Gender ......... 53 2.4.4 Follow-up Zinc and Mood State Analysis ............................................. 56 2.5 Discussion ................................................................................................... 57 2.6 References .................................................................................................. 63 Chapter 3 The sex-specific effects of LPS on depressive-like behavior and oxidative stress in the hippocampus of the mouse............................................. 68 vii 3.1 Abstract ....................................................................................................... 68 3.2 Introduction ................................................................................................. 70 3.3 Methods ...................................................................................................... 72 3.3.1 Animals ................................................................................................ 72 3.3.2 Experimental Design ............................................................................ 72 3.3.3 Behavioral Analysis.............................................................................