Bioenergetic abnormalities in schizophrenia 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 in the Graduate Program in Neuroscience of the College of Medicine by Courtney René Sullivan B.S. University of Pittsburgh, 2013 Dissertation Committee: Mark Baccei, Ph.D. (chair) Robert McCullumsmith, M.D., Ph.D. (advisor) Michael Lieberman, Ph.D. Temugin Berta, Ph.D. Robert McNamara, Ph.D. ABSTRACT Schizophrenia is a devastating illness that affects over 2 million people in the U.S. and displays a wide range of psychotic symptoms, as well as cognitive deficits and profound negative symptoms that are often treatment resistant. Cognition is intimately related to synaptic function, which relies on the ability of cells to obtain adequate amounts of energy. Studies have shown that disrupting bioenergetic pathways affects working memory and other cognitive behaviors. Thus, investigating bioenergetic function in schizophrenia could provide important insights into treatments or prevention of cognitive disorders. There is accumulating evidence of bioenergetic dysfunction in chronic schizophrenia, including deficits in energy storage and usage in the brain. However, it is unknown if glycolytic pathways are disrupted in this illness. This dissertation employs a novel reverse translational approach to explore glycolytic pathways in schizophrenia, effectively combining human postmortem studies with bioinformatic analyses to identify possible treatment strategies, which we then examine in an animal model. To begin, we characterized a major pathway supplying energy to neurons (the lactate shuttle) in the dorsolateral prefrontal cortex (DLPFC) in chronic schizophrenia. We found a significant decrease in the activity of two key glycolytic enzymes in schizophrenia (hexokinase, HXK and phosphofructokinase, PFK), suggesting a decrease in the capacity to generate bioenergetic intermediates through glycolysis in this illness. Notably, we did not detect protein changes in enzymes or transporters in this pathway in the DLPFC. This suggests the bioenergetic interplay of astrocytes and neurons in schizophrenia is highly complex and may not be fully appreciated at the region-level. Thus, we utilized a cell-level approach (laser capture microdissection) to generate populations of cells enriched for astrocytes or neurons cut from layers 3 and 5 of the DLPFC of schizophrenia and control ii subjects. We found significant mRNA changes in glycolytic enzymes (HXK1, PFKM, PFKL, and glucose-6-phosphate isomerase, GPI), lactate transporters (monocarboxylate transporter 1, MCT1), and glucose transporters (glucose transporter 1, GLUT1 and GLUT3) in pyramidal neurons in schizophrenia. Interestingly, we did not find any changes in astrocytes. This suggests a neuron-specific deficit in glycolytic pathways in the DLPFC in schizophrenia, which could contribute to pathophysiology of this illness. To build on these findings, we performed bioinformatic analyses to examine the implications of an altered bioenergetic profile in schizophrenia. We first sought to replicate our findings in additional cohorts of schizophrenia and control subjects. We probed 2 independent transcriptomic datasets (Stanley Medical Research Institute Online Genomics Database and Mount Sinai Microarray Dataset) for our metabolic targets. Supporting our hypothesis, we found several glycolytic enzymes and transporters to also be dysregulated in schizophrenia in these databases. Next, we utilized the Library of Integrated Network- Based Cellular Signatures (LINCS) database to generate transcriptional signatures containing differentially expressed genes associated with bioenergetic abnormalities in schizophrenia. Using these signatures, we performed enrichment analyses with Enrichr to probe for connected pathways and biological significance and found hits for cell metabolism, proliferation, immunity, and inflammation pathways. Furthermore, we compared our disease signatures to a library of “drug activity transcriptional signatures” to identify possible perturbagens with the ability to “reverse” the disease signature and inform future preclinical experiments. Top perturbagens included peroxisome proliferator-activated receptor (PPAR) agonists, capable of bolstering metabolic pathways and possibly reversing cognitive deficits. iii To further elucidate the role of bioenergetics in cognitive dysfunction, we examined metabolic pathways in the GluN1 knockdown (KD) model of schizophrenia. This genetic model has 10% of normal functioning n-methyl-D-aspartate receptor (NMDA) receptors levels and exhibits several endophenotypes of schizophrenia including impaired social interaction, increased stereotypic behaviors, and decreased performance in spatial and working memory tasks. Using mass spectrometry and pathway analyses, we found abnormal metabolic pathways in GluN1 KD mice, as well as decreases in lactate and glucose transporter transcripts. With the goal of reversing these deficits, we selected a top perturbagen from our drug discovery bioinformatic analysis (PPAR agonists) with the hypothesis that this drug intervention may help restore schizophrenia endophenotypes in this model. Pioglitazone (pio) is a synthetic ligand for PPARγ, which can alter the transcription and expression of glucose transporters, leading to changes in glucose uptake. We investigated the effects of (pio) treatment in the GluN1 KD model and found that pioglitazone treatment helped to restore explicit memory. This suggests bolstering metabolic pathways via pioglitazone may improve specific subtypes of cognition. This work has important implications for the treatment of cognitive illnesses with bioenergetic deficits such as schizophrenia. iv v ACKNOWLEDGEMENTS I want to extend great thanks to the many people who helped me reach success in this journey. You have all made me a better thinker, teammate, and person. First, I would like to thank my mentor, Dr. Robert McCullumsmith. Over the years, I realized just how lucky I was to work with you, and I am confident you were exactly what I needed. You pushed me to be the best scientist I could be, always supporting but never coddling me. I immediately noticed your great passion for research, as evidenced (you hate that phrase) by your Saturday phone calls to talk about awesome ideas that came to you about my project. You made me excited about science. I have appreciated the way you cared about my well-being, both personally and professionally. I always valued your enthusiasm and belief in me, and I feel honored to have been taken under your wing. I want to thank all the current and former members of the McCullumsmith laboratory. We have always prided ourselves on having an excellent lab milieu, and my career working with all of you is a testament to that. This work could never have been completed without you, and it has been a pleasure to work with you all. A special thanks to Sinead O’Donovan and Adam Funk, who have been there the duration of my time in the lab and never called any of my questions stupid or got annoyed with me for my numerous lab supply orders. You truly are some of the most gifted scientists I have met, and a joy to be around. I wouldn’t want to go to conferences or Christmas parties with any other postdocs. Also to Jennifer McGuire, Erica Carey, Emily Devine, Rachael Koene, Jarek Meller, Eduard Bentea, and Rebekka Meeks for their great help on these projects. The hard work you have done has not gone unnoticed. I would also like to extend a special thanks to my friends up north in Toronto, Canada. Dr. Amy Ramsey was kind enough to host me in her lab and work with her animals to complete the last chapter of my thesis. Our collaboration together has been wonderful, vi and you are an inspiring female role model. Living in another country for a month is not easy. Thus, I would also like to send a special thanks to Catharine Mielnik, who made Canada feel like a home. I learned more under your training than I could have ever hoped. You truly have a talent for what you do and teaching others, and my gratitude for our time together is great. Not only did I become a more well-rounded scientist, I gained a lifelong friend. I would next like to thank the Neuroscience Graduate Program, who proved repeatedly why I came to Cincinnati. Never have I seen faculty care so much about each individual student, and for this I never felt lost or alone. I would especially like to thank my dissertation committee: Dr. Mark Baccei, Dr. Robert McNamara, Dr. Michael Lieberman, and Dr. Temugin Berta, who guided me throughout this journey. Their feedback and thoughtful comments helped shape my ideas into excellent projects. Coming to UC was one of my best decisions thanks to the outstanding science and outstanding people. Of course, words cannot describe how amazing my family has been. From high school, to college, to graduate school, to say you were my biggest cheerleaders is an understatement. It is probably accurate to say you thought my work was cooler than I did, and your excitement for my future was never lost on me. You always made me feel special, and the pride you had in me motivated me every day. To my parents, no one has ever believed in me like you. To my mom, I am the luckiest daughter in the world to have someone who loves me enough to actually want to talk to me on the phone every few days. You have coached me through every part of life, and made me into the woman I am today. To my dad, who taught me that hard work pays off, you instilled in me the work ethic and drive that is essential to survive graduate school. You did everything for me along this journey, from rent money in college to moving me from 3rd floor apartments multiple times- I vii hope to attain your level of selflessness one day. You retain the right to go car shopping with me when I get a big girl job, a day you’ve always dreamed about. A huge thanks to my Grandma, I had no idea being away from you would be so hard.