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Msc Thesis Prenatal Alcohol Exposure Programs Neural Gene Expression

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Msc Thesis Prenatal Alcohol Exposure Programs Neural Gene Expression PRENATAL ALCOHOL EXPOSURE PROGRAMS STEADY-STATE GENE EXPRESSION AND THE GENE EXPRESSION RESPONSE TO INFLAMMATION IN THE ADULT RAT BRAIN by Katarzyna Anna Stepien B.Sc., The University of Guelph, 2009 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2013 © Katarzyna Anna Stepien, 2013 Abstract Prenatal alcohol exposure results in alterations in numerous physiological systems, including neuroendocrine and neuroimmune systems. The purpose of this study was to determine whether prenatal ethanol exposure results in long-term alteration of neural gene expression, particularly in genes related to neuroendocrine and neuroimmune function. Utilizing a well-established animal model of prenatal ethanol exposure, ethanol was administered to pregnant Sprague-Dawley dams throughout gestation in a liquid diet fed ad libitum (36% calories derived from ethanol). Maltose-dextrin was isocalorically substituted for ethanol in a liquid control diet for a pair-fed group, and a control group received a pelleted control diet ad libitum. In young adulthood, an adjuvant-induced arthritis paradigm was utilized, where female offspring were injected with either saline or complete Freund’s adjuvant, to induce an inflammatory response and elucidate dysregulated neuroimmune pathways. Gene expression was analyzed in the prefrontal cortex and hippocampus at both the peak and resolution of arthritis using whole genome gene expression microarrays. Within saline-injected animals, prenatal alcohol exposure alone resulted in significant changes in gene expression in both the prefrontal cortex and hippocampus. Included were multiple genes related to, cell death, transcriptional regulation, neuronal signaling and neurodevelopment. Among the genes involved in neurodevelopment, Acs13 has also been shown to be variably methylated in humans according to in utero exposure to environmental factors. Prenatal alcohol exposure also altered the gene expression response to adjuvant-induced arthritis. Many genes showed a significantly different pattern of expression in ethanol-exposed animals compared to both pair-fed and control, in both prefrontal cortex and hippocampus. These genes were either differentially up- or downregulated in ethanol-exposed compared to control animals or failed to show the adjuvant-induced change in regulation shown by controls. As well, several of these genes were mediators of the response to immune or stress challenge, such as Lcn2 and Bhlhe40. Genes found to be differentially expressed in this study are potential mediators contributing to the long-term alterations in neuroendocrine and neuroimmune function observed in prenatal alcohol exposure. ii Preface The animal work described in this thesis (breeding, handling, and termination) was conducted primarily by Xinqi Zhang, with assistance from other members of the Weinberg lab. The tissues used for gene expression analysis were dissected by Tamara Bodnar, Linda Ellis, and Kasia Stepien. Subsequent lab work, analyses, and writing were conducted primarily by Kasia Stepien, with some help from Sarah Neumann in optimization of the RNA extraction and amplification protocols. All animal work approved by the UBC Animal Care Committee and conducted under the Animal Care Certificate number A05-1187. iii Table of Contents Abstract .............................................................................................................................................................. ii Preface ............................................................................................................................................................... iii Table of Contents .............................................................................................................................................. iv List of Tables ..................................................................................................................................................... vi List of Figures .................................................................................................................................................. vii List of Abbreviations ..................................................................................................................................... viii Acknowledgements ........................................................................................................................................... ix Dedication ........................................................................................................................................................... x Chapter 1: Introduction ................................................................................................................................... 1 1.1 Prenatal alcohol exposure and Fetal Alcohol Spectrum Disorders ..................................................... 1 1.1.1 Clinical significance of prenatal alcohol exposure ......................................................................... 1 1.1.2 Animal models of prenatal alcohol exposure ................................................................................. 2 1.2 Fetal programming by prenatal alcohol exposure .............................................................................. 3 1.2.1 Fetal programming: the developmental origins of health and disease ........................................... 3 1.2.2 Long term effects of PAE on the stress response ........................................................................... 4 1.2.3 Long term effects of PAE on the immune response ....................................................................... 6 1.3 Alterations in neural gene expression: a potential mechanism of fetal programming by prenatal alcohol exposure .............................................................................................................................................. 7 1.4 Rationale and thesis objectives ........................................................................................................... 9 Chapter 2: Materials and Methods ............................................................................................................... 10 2.1 Breeding and prenatal alcohol exposure ........................................................................................... 10 2.2 Induction of adjuvant-induced arthritis ............................................................................................ 10 2.3 Termination of animals ..................................................................................................................... 10 2.4 Tissue dissection and RNA extraction .............................................................................................. 11 2.5 Microarray assaying of whole genome gene expression .................................................................. 11 2.6 Data pre-processing, quality control, and exploratory data analysis ................................................ 11 2.7 Differential expression analysis ........................................................................................................ 12 2.8 Gene ontology and pathway analysis ............................................................................................... 12 2.9 Validation of microarray results ....................................................................................................... 13 Chapter 3: Results ........................................................................................................................................... 15 3.1 Overview of analyses and main findings .......................................................................................... 15 3.2 Exploratory data analysis.................................................................................................................. 16 3.3 Prenatal alcohol exposure alters steady-state gene expression in PFC and HPC ............................. 26 3.3.1 Genes altered by PAE at a steady-state level of gene expression ................................................ 26 iv 3.3.2 GO categories altered by PAE at a steady-state level of gene expression ................................... 27 3.3.3 Validation of gene expression changes by RT-qPCR .................................................................. 27 3.3.4 Genes showing common, graded, or differential effects of PAE and pair-feeding ...................... 28 3.4 Prenatal alcohol exposure alters the gene expression response to an inflammatory challenge in PFC and HPC ......................................................................................................................................................... 44 3.4.1 Incidence and severity of adjuvant-induced arthritis ................................................................... 44 3.4.2 Genes differentially altered in PAE compared to control animals in response to adjuvant exposure .................................................................................................................................................... 44 3.4.3 GO categories differentially altered in PAE compared to control animals in response to adjuvant exposure .................................................................................................................................................... 45 Chapter 4:
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