TRANSGENERATIONAL EPIGENETIC INHERITANCE OF THE STRESS RESPONSE IN RATS OLENA BABENKO Bachelor of Science, Biology, Precarpathian National University, Ukraine, 2007 Master of Science, Biology, Precarpathian National University, Ukraine, 2008 Master of Science, Biology, University of Lethbridge, Canada, 2010 A Thesis Submitted to the School of Graduate Studies of the University of Lethbridge in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY Department of Neuroscience University of Lethbridge LETHBRIDGE, ALBERTA, CANADA © Olena Babenko, 2015 TRANSGENERATIONAL EPIGENETIC INHERITANCE OF THE STRESS RESPONSE IN RATS OLENA BABENKO Date of Defence: June 23, 2015 Dr. G. Metz Co-Supervisor Professor Ph.D. Dr. I. Kovalchuk Co-Supervisor Professor MD, Ph.D. Dr. U. Wieden-Kothe Thesis Examination Committee Member Associate Professor Ph.D. Dr. F. Eudes Thesis Examination Committee Member Ph.D. Dr. T. Russell Internal Examiner Assistant Professor Ph.D. Dr. K. Riabowol External Examiner, University of Calgary, Calgary, Alberta Professor Ph.D. Dr. R. Gibb Chair, Thesis Examination Committee Associate Professor Ph.D. Dedicated to my parents, Nataliya and Mykola, and to my grandparents, Nina and Mykola, Kateryna and Oleksandr. iii ABSTRACT Recent human and animal studies indicate that stressful experiences in utero may have long-term consequences on the future mental wellbeing of the offspring across several generations. Epigenetic mechanisms, such as microRNA expression and DNA methylation are prone to change in response to hostile environmental factors. The main goal of this study was to investigate transgenerational changes in gene and microRNA expression, as well as in DNA methylation patterns in the placenta and developing brain in response to stress in a rat model. Our results demonstrated that ancestral exposure to stress during gestation causes significant changes in gene expression as well as epigenetic changes in placenta and embryonic cortex in the F2 and F3 generations. In addition, our results showed that assessing changes in placenta can be used to predict pathological processes of the newborn brain, thus providing an opportunity for early diagnosis of neurological and psychiatric diseases. iv ACKNOWLEDGEMENTS First and foremost, I would like to thank my parents and family for their love, support and encouragement. I would like to express my sincere gratitude to my supervisors Dr. Gerlinde Metz and Dr. Igor Kovalchuk for providing me with an excellent opportunity to work in their laboratories and complete my PhD degree. Without their guidance and support this dissertation would not have been possible. I would like to thank my committee members, Dr. Ute Wieden-Kothe and Dr. Francois Eudes for their helpful advices during my PhD journey. In addition, I would like to thank all my colleagues from the Metz and Kovalchuk laboratories, especially Jerrah Robbins, Erin Falkenberg, Slava Ilnytskyy, Andrey Golubov, Ping Li, and Mieke Heyns for their help. I would also like to thank all my friends for their love and support. v TABLE OF CONTENTS THESIS EXAMINATION COMMITTEE MEMBERS ............................................... ii DEDICATION ............................................................................................................................... iii ABSTRACT ........................................................................................................................ iv ACKNOWLEDGEMENTS ................................................................................................. v TABLE OF CONTENTS .................................................................................................... vi LIST OF TABLES ............................................................................................................... x LIST OF FIGURES ........................................................................................................... xii LIST OF ABBREVIATIONS ............................................................................................ xiv Chapter 1: General Introduction ......................................................................................... 1 Stress-induced Perinatal and Transgenerational Epigenetic Programming of Brain Development and Mental Health .......................................................................................... 1 Introduction .............................................................................................................................. 2 Transgenerational epigenetic inheritance: significance and definitions............................... 2 Epigenetic reprogramming during mammalian embryonic development ............................ 5 Transgenerational epigenetic changes in mammals ............................................................. 6 Metastable epialleles in mice ............................................................................................... 7 “Paramutation-like” effects in mice and the role of sperm RNA in transgenerational epigenetic inheritance .......................................................................................................... 8 Implication of sperm RNA in transgenerational inheritance of stressful experiences ....... 10 Transgenerational non-genetic effects ............................................................................... 11 Transgenerational effects of endocrine disruptors ............................................................. 12 Transgenerational effects of stress ..................................................................................... 13 Transgenerational effects of diet in mice ........................................................................... 15 Transgenerational epigenetic inheritance in humans ......................................................... 16 The Dutch Famine Cohort Study ....................................................................................... 16 The Överkalix Cohort Study .............................................................................................. 17 Fetal programming ................................................................................................................ 18 MicroRNAs (miRNA) are important epigenetic regulators of brain development ............ 20 miRNAs in placenta ........................................................................................................... 21 DNA methylation in the embryonic brain .......................................................................... 21 DNA methylation in placenta ............................................................................................ 26 Partially methylated domains and their role in brain and placenta .................................... 26 Histone modifications and chromatin remodeling in the brain .......................................... 27 Stress and epigenetic regulation ............................................................................................ 28 Stress: general terms and definitions................................................................................. 28 Role of stress in brain development ................................................................................... 29 Experience-dependent programming of stress response and epigenetic marks ................. 31 Stress-induced epigenetic changes in placenta may be associated with epigenetic changes in brain ............................................................................................................................... 35 Stress-induced epigenetic signatures of disease ................................................................. 37 miRNAs as markers of stress and disease .......................................................................... 37 Changes in brain DNA methylation in response to stress and disease ............................... 38 Stress during pregnancy and risks of neurological disorders later in life .......................... 40 Prenatal stress and risk of developing schizophrenia later in life ...................................... 41 Prenatal stress and risk of anxiety- and depression-related disorders later in life ............. 44 Prenatal stress and predisposition to attention deficit hyperactivity disorder and autism .. 48 Conclusion ............................................................................................................................. 50 vi Objectives and hypothesis .................................................................................................. 51 Chapter 2: Transgenerational effects of gestational stress on mRNA expression ..... 52 Introduction ............................................................................................................................ 52 Materials and methods ........................................................................................................... 53 Animals .............................................................................................................................. 53 Experimental groups .......................................................................................................... 53 Time course ........................................................................................................................ 54 Stress paradigm .................................................................................................................. 55 Tissue dissection and RNA extraction