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Stem-Like Cells and Glial Progenitors in the Adult Mouse Suprachiasmatic Nucleus

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Stem-Like Cells and Glial Progenitors in the Adult Mouse Suprachiasmatic Nucleus STEM-LIKE CELLS AND GLIAL PROGENITORS IN THE ADULT MOUSE SUPRACHIASMATIC NUCLEUS Dilshan H. Beligala A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2019 Committee: Michael E. Geusz, Advisor Pascal Bizarro Graduate Faculty Representative George S. Bullerjahn HowarD C. Cromwell Paul F. Morris © 2019 Dilshan H. Beligala All Rights Reserved iii ABSTRACT Michael E. Geusz, Advisor Reports have described cells with stem-like protein expression in the hypothalamic suprachiasmatic nucleus (SCN), which contains the principal circadian pacemaker of the body. Additionally, there are oligodendrocyte progenitor cells (OPCs) scattered throughout the SCN and other brain areas with reported abilities to differentiate into neurons and glia. The SCN is a particularly good structure for studying adult neurogenesis because its cellular manipulation has known quantifiable effects on specific parameters of circadian rhythms. The objectives of this study were to characterize stem and progenitor cells in the SCN and to study neurogenesis from SCN OPCs in vitro. We first performed a meta-analysis to identify the expression of stem cell- related genes in the SCN and then used defined serum-free media for inducing stem and progenitor cell proliferation in SCN explant cultures, identified by immunocytochemistry and confocal microscopy. In the meta-analysis, we analyzed 25 genes associated with stem cell maintenance and increased motility, out of which over 90% were expressed at higher levels in the SCN than in other brain areas. In explant cultures maintained in stem and progenitor cell medium (SPM), cells expressed stem cell proteins: SOX2, nestin, MSI2 and OCT4. Explant cultures had ongoing mitotic activity and extensive cell loss. Despite neuronal loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging. The circadian rhythm in SCN gene expression persisted in brain slice cultures in SPM. SCN explants maintained in NeuralX medium supporting OPC proliferation, formed a cell monolayer and a suspended cell culture that included 87% OPCs. These cells were then induced to differentiate into neurons, which were identified by immunocytochemistry and electrical impulses recorded with microelectrode arrays. In differentiating cultures, a subset of OPCs formed iv oligodendrocytes that myelinated nascent neurons. These results provide evidence that the mature SCN has cells with regenerative properties providing plasticity that may enable circadian rhythms to adjust to changing environmental timing cues, seasonal behavioral cycles or aging. These immature cells can be used to prepare an SCN cell line that may provide a consistent source of rhythmic cells that would enable simpler genetic manipulation of key mammalian clock genes. v I would like to dedicate this work to my family and my mentors for being great pillars of support vi ACKNOWLEDGMENTS On the very outset of this dissertation, I would like to extend my sincere and heartfelt gratitude to all the personages who have helped me in this endeavor. Without their guidance, help and motivation, this would not have been a success. First and foremost, I would like to pay my gratitude to my advisor, Associate Professor Michael E. Geusz, for his time and effort spent on continuously leading, advising and encouraging me throughout this research. His passion for challenges has given me inspiration, his vast knowledge has given me guidance and his enthusiasm in research has given me motivation. I truly feel lucky to work under the supervision of such a talented advisor. I am extremely thankful for my committee members, professors George S. Bullerjahn, Paul F. Morris, Howard C. Cromwell. and Pascal Bizarro, whose stimulating motivation and valuable ideas were extremely helpful in completing my research successfully. They have been a constant source of knowledge and support throughout the years. Further, I am indebted to my collaborators Dr. Rae Silver (Columbia University), Dr. Joseph LeSauter (Columbia University), Dr. Astha Malik (Cincinnati Children’s Hospital), Dr. Peter Lu (BGSU) and Ms. Lorena Alvarez (BGSU) for their immense contribution towards my research experiments and publications. I would also like to acknowledge the undergraduate students who helped me with data analysis throughout the years: Mr. Hugh J. McQuillen, Ms. Hayley Ruff, Ms. Erin Tepe, Mr. Ifeanyichukwu Amujiogu, and Ms. Amanda Fairbairn. In addition, I gratefully acknowledge the support of all the academic and non-academic staff members in the Department of Biological Science, Bowling Green State University, especially Ms. Dorothy Laforce, Ms. Susan Schooner, Ms. Chris Hess and Mr. Steve Queen for their kindness and support in all administration and technical issues. I would also like to thank Ms. Jenifer Baranski of the BGSU Animal Care Facility for all the help with animal care and Dr. Marilyn Cayer for her support in confocal imaging. vii Furthermore, I am grateful to all the current and past Geusz lab members: Mr. Arpan De, Mr. Tyler Birkholz, and Ms. Katarina Coulson. I feel lucky to have the opportunity to work with such friendly lab mates. Finally, my deepest gratitude goes to my loving parents (Mr. Samarasinghe Beligala & Ms. Renuka Ranaweera), my brother (Mr. Lakshan Beligala) and my wife (Ms. Gayathri Beligala) for their support in my studies and for being there with me through thick and thin. I am forever indebted to all that they have done for me. Without their endless support and unconditional love, I wouldn’t have achieved this much. Thank you for being around and for never ending motivations I’ve been getting all this while. viii TABLE OF CONTENTS Page CHAPTER I: INTRODUCTION ........................................................................................... 1 The Suprachiasmatic Nucleus (SCN) ........................................................................ 1 Adult Neural Stem Cells (NSCs) ............................................................................... 5 Adult Neurogenesis in the Brain ................................................................................ 7 Adult Neurogenesis in Brain Regions other than DG and SVZ ................................ 9 Evidence of Cells with Stem Cell or Developmental Properties in the Adult Rodent SCN ............................................................................................................................ 12 CHAPTER II: A META-ANALYSIS CHARACTERIZING STEM-LIKE GENE EXPRESSION IN THE SUPRACHIASMATIC NUCLEUS AND ITS CIRCADIAN CLOCK .................................................................................................................................. 14 Preface ........................................................................................................................ 14 Introduction ................................................................................................................ 14 Materials and Methods …………………………………………………………… .. 18 Databases Queried ......................................................................................... 18 Procedure for Selecting Candidate Stem Cell-Related Genes ....................... 19 Results and Discussion .............................................................................................. 20 FSS Analysis .................................................................................................. 20 Evaluating Transcription Factors Expressed at High Levels in the SCN ...... 25 Genes Expressed in the SCN That Serve in Stem Cell-Regulating Pathways 28 Stem Cell-Related Genes Regulating SCN Circadian Rhythm Output or Phase Shifts through Cell-Cell Coupling ................................................................. 33 Summary of Candidate Stem-Like Gene Activity in Adult Mouse SCN ...... 36 ix Conclusions ................................................................................................................ 39 CHAPTER III: MUSASHI-2 AND RELATED STEM CELL PROTEINS IN THE MOUSE SUPRACHIASMATIC NUCLEUS AND THEIR POTENTIAL ROLE IN CIRCADIAN RHYTHMS………………………. ....................................................................................... 40 Preface ........................................................................................................................ 40 Introduction ................................................................................................................ 40 Materials and Methods ............................................................................................... 43 Animals .......................................................................................................... 43 Microdissected SCN Explant Cultures and Immunocytochemistry (ICC) .... 43 Live/Dead Staining ........................................................................................ 52 Explant Bioluminescence Imaging ................................................................ 52 Bioinformatics Analysis ................................................................................. 53 Results ....................................................................................................................... 61 Culture in SPM Causes Morphological and Cellular Changes in SCN Explants .......................................................................................................... 61 SCN Cells Express Multiple Stem Cell-Related
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