Copyright by Joshua Dale Bryant 2017 The Dissertation Committee for Joshua Dale Bryant Certifies that this is the approved version of the following dissertation: Characterization of MTHFD2L Expression and Alternative Splicing and Loss of MTHFD1L Activity in Murine Embryos and Adults Committee: Dean R. Appling, Supervisor Michael Drew Richard Finnell David Hoffman Edward Mills Stefano Tiziani Characterization of MTHFD2L Expression and Alternative Splicing and Loss of MTHFD1L Activity in Murine Embryos and Adults by Joshua Dale Bryant, B.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin May 2017 Dedication I would like to dedicate this work to my parents, who have always been incredibly supportive of me and are the best people that I know. Acknowledgements I would first like to thank my advisor, Dr. Dean Appling, for his support and patience with me and for helping me learn how to think about science. I would also like to thank my committee members Drs. Drew, Finnell, Hoffman, Mills, and Tiziani. I would especially like to thank Dr. Drew for teaching me how to do the behavioral experiments and Dr. Tiziani for his help with the metabolomics experiments. I would also like to thank two Tiziani lab members: Dr. Enrique Sentandreu for acquiring the LC-MS data and Shannon Sweeney for data processing and answering an inordinate number of questions from me. I thank Dr. Hélène Ipas for advice and assistance with the Seahorse XFp. I thank Appling lab members Dr. Minhye Shin and Dr. Jessica Momb for their assistance with embryo dissections and their discussion and advice about my experiments. Finally, I would like to thank my parents. They have always been there for me beyond anything that I deserve, and I could have never made it this far without them. v Characterization of MTHFD2L Expression and Alternative Splicing and Loss of MTHFD1L Activity in Murine Embryos and Adults Joshua Dale Bryant, Ph.D. The University of Texas at Austin, 2017 Supervisor: Dean R. Appling In Eukaryotes, folate-dependent one-carbon (1C) metabolism is a highly compartmentalized process in which mitochondria play a central role. Defects in folate metabolism are associated with diseases such as cancer, Alzheimer's disease, and neural tube defects (NTDs). 1C units are attached to tetrahydrofolate (THF) and carried in various oxidation states between folate-dependent enzymes. There is an exchange of 1C units across the mitochondrial membrane, with 1C donors such as serine and glycine being oxidized to formate in the mitochondria, which is then released into the cytoplasm. 1C units in the cytoplasm can be used for the synthesis of purines, thymidylate, and methionine for the methyl cycle. The core of the pathway in both compartments is catalyzed by the methylene-tetrahydrofolate (MTHFD) gene family. These enzymes + catalyze the reversible interconversion between CH2-THF, CH -THF, CHO-THF, and formate. The cytoplasmic protein MTHFD1 is trifunctional and carries the CH2-THF dehydrogenase, CH+-THF cyclohydrolase, and 10-CHO-THF synthetase activities necessary to carry out these interconversions. In the mitochondria, two bifunctional isozymes, MTHFD2 and MTHFD2L, carry the dehydrogenase/cyclohydrolase (D/C) activities. The monofunctional enzyme MTHFD1L is responsible for the synthetase vi activity. MTHFD2 is only expressed in embryos and transformed cells, and the enzyme responsible for the D/C activity in adults was unknown until the recent discovery of MTHFD2L. In this work, characterization of the expression of MTHFD2L in mouse embryos and adults is described. Expression of MTHFD2L in embryos was found to be switched on between embryonic days 8.5-10.5, and remains high throughout development. MTHFD2L is also widely expressed in adults, with highest expression in brain and lung. A splice variant of MTHFD2L lacking exon 8 was found to be abundant in embryos but was not catalytically active in vitro or in vivo. MTHFD1L is an essential protein, and SNPs in MTHFD1L are associated with increased risk for Alzheimer's disease and NTDs in humans. Loss of MTHFD1L activity in adult mice with and without a folate deficient diet was investigated. Indications of sex-dependent behavioral anomalies were found, with evidence for genotype-dependent hyperactivity in male mice and diet-dependent anxiety in female mice, but further investigation of these findings is warranted. Finally, metabolic defects associated with NTDs and growth restriction in MTHFD1L-null (Mthfd1lz/z) embryos were identified. Glycolysis, the TCA cycle, and the metabolism of methionine, purines, and multiple amino acids were found to be disrupted in Mthfd1lz/z embryos. These altered metabolic pathways suggest potential future therapies for preventing NTDs in humans. vii Table of Contents List of Tables ........................................................................................................ xii List of Figures ...................................................................................................... xiii Chapter 1: Introduction ............................................................................................1 1.1 Folate-Dependent One-Carbon Metabolism .............................................1 1.1.1 Cellular Folates .............................................................................1 1.1.1.1 Folate Structure .................................................................1 1.1.1.2 Folate transport .................................................................3 1.1.2 Compartmentalization of One-Carbon Metabolism ......................5 1.1.2.1 Overview of Folate-dependent One-Carbon Cycle ...........5 1.1.2.2 Mitochondrial One-Carbon Metabolism ...........................8 1.1.2.3 Cytoplasmic One-Carbon Metabolism ...........................11 1.1.2.4 Nuclear One-Carbon Metabolism ...................................13 1.2 Folate Metabolism in Embryogenesis and Neurulation ..........................14 1.2.1 Mammalian Neural Tube Closure ...............................................14 1.2.2 History of Folic Acid Fortification in the United States .............16 1.2.3 Mouse Models of Folate Metabolism and Neural Tube Defects 17 1.2.3.1 NTD Mutants Responsive to Folate ................................17 1.2.3.2 NTD Mutants not Responsive to Folate..........................18 Chapter 2: Characterization of expression of MTHFD gene family and alternative splicing of MTHFD2L ..................................................................................20 2.1 Introduction .............................................................................................20 2.2 Methods and Materials ............................................................................22 2.2.1 Materials .....................................................................................22 2.2.2 Mouse Work................................................................................22 2.2.3 RNA Isolation and cDNA Synthesis...........................................22 2.2.4 Real Time PCR ...........................................................................23 2.2.5 In situ hybridization of whole mouse embryos ...........................25 2.2.6 5,10-Methylene-THF Dehydrogenase Assay ..............................25 viii 2.2.7 In Vivo Yeast Complementation Assay ......................................26 2.2.8 Submitochondrial localization of MTHFD2L lacking exon 8 ....27 2.3 Results .....................................................................................................28 2.3.1 Expression of MTHFD Genes During Mouse Embryonic Development ...............................................................................28 2.3.2 Expression of MTHFD Genes in Male and Female Adult Mice 30 2.3.3 MTHFD2L Alternative Splicing .................................................30 2.4 Discussion ...............................................................................................42 Chapter 3: Characterization of loss of function of MTHFD1L in adult mice .......47 3.1 Introduction .............................................................................................47 3.2 Materials and Methods ............................................................................49 3.2.1 Mouse Work................................................................................49 3.2.2 MTHFD1L conditional knock out ..............................................50 3.2.3 Behavioral experiments ..............................................................51 3.2.3.1 Open Field Test ...............................................................51 3.2.3.2 Elevated Plus ...................................................................51 3.2.3.3 Delay Fear Conditioning .................................................52 3.2.3.4 Morris Water Maze .........................................................52 3.2.4 Immunoblotting...........................................................................53 3.2.5 Statistics ......................................................................................54 3.3 Results .....................................................................................................55 3.3.1 Preliminary Study .......................................................................55 3.3.2 Main Study ..................................................................................62