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Translational Profiling Reveals the Transcriptome of Leptin Receptor Neurons and Its Regulation by Leptin

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Translational Profiling Reveals the Transcriptome of Leptin Receptor Neurons and Its Regulation by Leptin TRANSLATIONAL PROFILING REVEALS THE TRANSCRIPTOME OF LEPTIN RECEPTOR NEURONS AND ITS REGULATION BY LEPTIN by Margaret B. Allison A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Integrative Physiology) In the University of Michigan 2015 Doctoral Committee: Professor Martin G. Myers Jr., Chair Associate Professor Carol F. Elias Professor Malcolm J. Low Professor Suzanne Moenter Professor Audrey Seasholtz Before you leave these portals To meet less fortunate mortals There's just one final message I would give to you: You all have learned reliance On the sacred teachings of science So I hope, through life, you never will decline In spite of philistine defiance To do what all good scientists do: Experiment! -- Cole Porter There is no cure for curiosity. -- unknown © Margaret Brewster Allison 2015 ACKNOWLEDGEMENTS If it takes a village to raise a child, it takes a research university to raise a graduate student. There are many people who have supported me over the past six years at Michigan, and it is hard to imagine pursuing my PhD without them. First and foremost among all the people I need to thank is my mentor, Martin. Nothing I might say here would ever suffice to cover the depth and breadth of my gratitude to him. Without his patience, his insight, and his at times insufferably positive outlook, I don’t know where I would be today. Martin supported my intellectual curiosity, honed my scientific inquiry, and allowed me to do some really fun research in his lab. It was a privilege and a pleasure to work for him and with him. I also have to thank the many members of the Myers lab over the years. Research is sometimes a solitary endeavor, but I was lucky to pursue it in very good company. Particular thanks go out to Megan Greenwald-Yarnell, Christa Patterson Polidori, Amy Sutton, and Paula Goforth in this regard. In addition to being a challenging and thoughtful group with whom to pursue scientific investigation, they made coming into lab each day a pleasure. A special acknowledgement has to be extended to Dave Olson. Scientifically, without his eGFP-L10a mice, none of this dissertation would have been possible. More ii valuable to me by far was his mentorship and friendship. He was an irreplaceable source of insight for many of my projects, and his thoughtful approach to science is one I hope to emulate in the future. He also deserves a thank you for putting up with me for so long. I know it was painful, and he may be scarred for life. A host of people remained to be thanked, and I will try to get them all. Thank you: to my dissertation committee, for their thoughtful and valuable questions, advice, and support over the years; to the MSTP program, including Ron, Ellen, Hilkka, and Laurie, for welcoming me to the University, and for their ability to solve almost any problem that a graduate or medical student might face; to MIP, including Michele Boggs, Scott Pletcher, Sue Moenter, and Ormond Macdougald, for their assistance in navigating the sometimes treacherous waters of graduate school; to Matthew Brady, for getting me hooked on research, and for introducing me to Martin; to my fellow MIP students for their good company at Pub nights; to AARC for giving me a reason to wake up in the morning; and to Alex, Steve, and Andrew, for sushi nights that gave me something to look forward to every week. I should note that some of this work has been published previously. The introduction is derived from a review article that appeared in the Journal of Endocrinology in October 2014. The second chapter of this dissertation is currently in press at Molecular Metabolism (2015). The material in chapters 3 and 4 has not been published. I have to thank Christa Patterson in particular for her help in generating this data. Without her expert hypothalamic and brainstem dissections, I would not have been able to perform many, if not all, of the TRAP-Seq experiments described. iii Additionally, the efforts of the University of Michigan DNA Sequencing and Bioinformatics cores were instrumental in the success of this project. Finally, to my family: I love you very much. Thank you for supporting me in all of my endeavors, for investing so much in my education, and for reminding me that there are few problems that a strong cocktail, a long run, or a good night’s sleep can’t solve. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………………………………………..ii LIST OF FIGURES…………………………………………………………………………………………………..vi LIST OF TABLES…………………………………………………………………………………………………….vii LIST OF APPENDICES…………………………………………………………………………………………….viii ABSTRACT…………………………………………………………………………………………………………...ix CHAPTER 1. INTRODUCTION…………………………………………………………………………………1 2. TRAP-SEQ DEFINES MARKERS FOR NOVEL POPULATIONS OF HYPOTHALAMIC AND BRAINSTEM LEPRB NEURONS……………………………………………………………25 3. TRANSCRIPTIONAL AND TRANSLATIONAL PROGRAMS INDUCED BY LEPTIN IN LEPRB NEURONS………………………………………………………………………………………….59 4. REGULATION OF THE LEPRB TRANSCRIPTOME BY LEPTIN AND LEPRB-STAT3 SIGNALING…………………………………………………………………………………………87 5. CONCLUSIONS AND FUTURE DIRECTIONS…………………………………………139 APPENDICES……………………………………………………………………………………………………….150 v LIST OF FIGURES Figure 1.1 Hypothalamic leptin action………………………………………………………………………….....18 1.2 Leptin signaling and biological functions………………………………………………………….19 2.1 LepRbeGFP-L10a mice allow for TRAP-Seq in LepRb neurons………………………………..44 2.2 Transcripts enriched in hypothalamic and brainstem LepRb neurons………………46 2.3 Secreted proteins enriched or de-enriched in hypothalamic and brainstem LepRb neurons……………………………………………………………………………………………………………………….47 2.4 Pdyn expression in hypothalamic LepRb neurons…………………………………………….48 2.5 Distribution of Tac1-positive LepRb neurons……………………………………………………50 2.6 Colocalization of LepRb and CRH in the lateral hypothalamus………………………...51 2.7 Colocalization of LepRb and VIP in the brainstem…………………………………………...52 2.8 Metabolic phenotype of LepRPdynKO mice………………………………………………….......53 3.1 ATF3 is induced in hypothalamic LepRb neurons……………………………………………..79 3.2 ATF3 induction by leptin in POMC and NPY neurons…………………..…………………..81 3.3 ATF3 is induced by fasting in AgRP neurons…………………………………………………….82 4.1 SERPINA3N colocalization with LepRb, POMC, and AgRP neurons………………...119 4.2 Fold change in diet induced obese vs ob/ob mice………………………………………….123 4.3 Conditional ablation of STAT3 from LepRb neurons……………………………………….125 4.4 Fold change in STAT3LepRKO vs ob/ob mice……………………………………….…………..127 vi LIST OF TABLES Table 3.1 Acute leptin treatment………………………………………………………………………………...77 3.2 QPCR for TRAP-isolated vs whole hypothalamic RNA..................….……………….79 4.1 Fold change in gene expression in ob/ob and leptin treated mice ……………….117 4.2 Fold change in neuropeptide expression in ob/ob and leptin treated mice….120 4.3 Fold change in gene expression in diet-induced obese mice..…………..………….121 4.4 Fold change in gene expression in STAT3LepRKO mice……….………………………….128 4.5 Fold change in gene expression in STAT3LepRKO mice (cont)…………………………130 4.6 QPCR analysis of gene expression in STAT3LepRKO and Leprs/s mice………………133 vii LIST OF APPENDICES Appendix 1. Transcripts enriched in hypothalamic LepRb neurons……………………………...............150 2. Transcripts enriched in brainstem LepRb neurons..............….……………………………...181 3. Fold change in gene expression in ob/ob, DIO, and leptin treated mice ……………..205 viii ABSTRACT Two thirds of American adults are overweight and at risk for complications such as Type 2 diabetes, heart disease, stroke, and fertility problems. The adipose hormone, leptin, signals via the long isoform of its receptor (LepRb) in the central nervous system to regulate diverse determinants of energy balance, including food intake, energy expenditure, and neuroendocrine output. Previous studies have demonstrated that the lack of leptin or its receptor promotes hyperphagic obesity among other phenotypes. Importantly, the identity of many LepRb subpopulations, as well as the transcriptional effects of leptin in these populations, remain almost entirely unknown. Recently, the optimization of Translating Ribosome Affinity Purification (TRAP) technology has allowed for the isolation of mRNA from specific neuronal populations via the cre-dependent induction of affinity-tagged ribosomes. We first examined the transcriptome of LepRb neurons to identify markers for LepRb subpopulations. We isolated mRNA from mouse hypothalamic and brainstem LepRb cells by TRAP and analyzed it by RNA-seq (TRAP-Seq). TRAP-Seq defined the LepRb neuron transcriptome and revealed novel markers for previously unrecognized subpopulations of LepRb neurons. LepRb mRNA was enriched for markers of peptidergic neurons, including Pdyn. Pdyncre-mediated ablation of Leprflox in Pdyn neurons (LepRbPdynKO mice) blunted energy expenditure to promote obesity during high-fat feeding. To determine the regulation of the LepRb transcriptome by leptin, we employed LepRb- specific TRAP-seq on mRNA isolated from the hypothalami of mice treated with exogenous leptin, genetically leptin-deficient (ob/ob) mice, mice exposed to diet induced obesity (DIO), and mice in which STAT3 had been specifically ablated from LepRb neurons. Exogenous leptin treatment induced a number of transcription factors and intracellular proteins but did not affect neuropeptide transcription/translation. In contrast, states of extreme leptin deprivation ix or repletion, such as in untreated ob/ob mice or in DIO mice, induced changes in multiple neuropeptide species, many
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