The Role of DEPTOR in Intrauterine Growth Restriction A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI’I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN Developmental and Reproductive Biology May 2021 by Lance Gregory Nunes Thesis Committee: Johann Urschitz, Chairperson Peter Hoffmann Jesse Owens Keywords: Placenta, DEPTOR, mTOR, sonoporation, Acknowledgements I would like to take the opportunity to thank the many people who have helped me along the way and made this thesis research come to fruition. I would like to express my deepest appreciation to my thesis committee chairperson, mentor, and friend, Dr. Johann Urschitz, for his thoughtful insight, guidance, patience, and support throughout this Master’s degree. Words can’t express how thankful I am for everything you have helped me with over the last four and a half years. Thank you to my thesis committee members, Dr. Peter Hoffmann and Dr. Jesse Owens, for their time, patience, expertise, and support throughout this research. Both of you have helped me in too many ways to detail here, but please know that I am very grateful for it all. Thank you to all of my colleagues and classmates at the Mānoa Institute of Biogenesis Research and at the John A. Burns School of Medicine, who have provided their kindest assistance when I needed it most. To my past and current lab mates, thank you for all the great memories and invaluable support in many aspects of this thesis research. I would like to acknowledge my family – my mom, dad, and sister – whose continued love and support in countless ways have made all my life accomplishments a possibility. I cannot thank you three enough for everything you do and continue to do for me. Last, but most certainly not least, I would like to thank my loving fiancée, Sara, who has been there for me through it all. Your unending love and support continues to help push me to be the best version of myself and for that I am eternally thankful. 2 Abstract Maternal obesity or insufficient nutrient supply during pregnancy can result in fetal overgrowth and fetal growth restriction, respectively, and cause serious health problems for both the mother and the fetus. Dysregulated placental nutrient transport is believed to be a mediator for both conditions. The mammalian target of rapamycin (mTOR) is thought to be the nutrient sensor of the placenta. mTOR is comprised of two complexes, mTORC1 and mTORC2. Both mTORC1 and mTORC2 have downstream targets that regulate cell growth, proliferation, ion transport, cytoskeletal remodeling, and more. Intrauterine Growth Restriction (IUGR) is associated with decreased nutrient transport and restricted nutrient availability for the fetus. It has been shown that mTOR is downregulated in placentas of pregnancies complicated by IUGR. The DEP-domain containing mTOR interacting protein (DEPTOR) has recently been shown to be an endogenous inhibitor of mTOR. We hypothesized that placenta-specific DEPTOR knockdown (KD) results in restoration of mTOTRC1/2 signaling, normalizing placental amino acid transport capacity and preventing IUGR. In the first part of this thesis, we generated a transgenic mouse model that expressed a placenta-specific DEPTOR KD cassette to study the in vivo effects of placental DEPTOR KD. Results showed that trophoblastic DEPTOR inhibition leads to increased expression of mTORC1/2 signaling, increased activity of the two main placental amino acid transport systems, and a marked increase in birth weight. In the second part of this thesis, we aimed to optimize parameters for placental sonoporation, a minimally invasive in vivo gene delivery technique that utilizes ultrasound and DNA conjugated lipid microbubbles. We observed successful placental transgene expression after sonoporation following intravenous injection of DNA conjugated lipid microbubbles. Taken together, this thesis has elucidated the role of 3 placental DEPTOR in the regulation of mTORC1/2 signaling and fetal nutrient transport, providing insight into the molecular mechanisms that govern fetal growth and, ultimately, health. Furthermore, the successful sonoporation trials illustrate the possibility for this technique to be used as an alternative to transgenics for studying in vivo gene modulation, as well as a potential adaptable technique for the treatment of complications due to placental dysregulation. 4 Table of Contents CHAPTER 1 – LITERATURE REVIEW .......................................................................................... 9 1.1 MATERNAL NUTRITION AND FETAL PROGRAMMING ....................................................................... 9 1.2 THE PLACENTA - STRUCTURE ..................................................................................................... 10 1.3 THE PLACENTA - FUNCTION ...................................................................................................... 12 1.4 COMPARISON OF HUMAN AND MOUSE PLACENTA ........................................................................ 15 1.5 INTRAUTERINE GROWTH RESTRICTION ........................................................................................ 17 1.6 MECHANISTIC TARGET OF RAPAMYCIN (MTOR) IN INTRAUTERINE GROWTH RESTRICTION ................... 19 1.7 MTOR AND THE DEP-DOMAIN CONTAINING MTOR INTERACTING PROTEIN (DEPTOR) ..................... 21 CHAPTER 2 IN VIVO PLACENTAL DEPTOR KD – TRANSGENIC MOUSE MODEL ......................... 22 2.1 INTRODUCTION ...................................................................................................................... 22 2.2 BACKGROUND INFORMATION ................................................................................................... 23 2.2.1 Transgenesis and Transposases .................................................................................. 23 2.2.2 RNA Interference ......................................................................................................... 26 2.2.3 Plasmid Cloning ........................................................................................................... 28 2.3 METHODS ............................................................................................................................. 31 2.3.1 Construction of plasmid DNA ...................................................................................... 31 2.3.2 Animals ....................................................................................................................... 32 2.3.3 Oocyte Collection ........................................................................................................ 32 2.3.4 Sperm Collection ......................................................................................................... 33 2.3.5 Transposase-EnHanced Pronuclear Injection .............................................................. 33 2.3.6 Embryo culture and embryo transfer .......................................................................... 34 2.3.7 Genotyping .................................................................................................................. 34 2.3.8 Germline Transgenic Assay ......................................................................................... 34 2.3.9 Transgene copy number assays .................................................................................. 35 2.3.10 Breeding .................................................................................................................... 35 2.3.11 Fetus and Placenta Collection ................................................................................... 36 2.3.12 TropHoblast Plasma Membrane (TPM) Isolation ...................................................... 36 2.3.13 Western Blot mTOR signaling ................................................................................... 37 2.3.14 Amino Acid System Transporter Uptake Assay ......................................................... 37 2.4 RESULTS ................................................................................................................................ 39 2.4.1 Clone Validation in Cells .............................................................................................. 39 2.4.2 Detection of Transgene in Founding Transgenic Mice ................................................ 40 2.4.3 Validation of Germline Transgenesis in DEPTOR KD Mouse Line ................................ 40 2.4.3 Generation of Mice for Experimental Analysis ............................................................ 42 2.4.4 Evaluation of Fetal and Placental WeigHt ................................................................... 42 2.4.5 Validation of DEPTOR KD in Transgenic Mouse Line ................................................... 43 2.4.6 Evaluation of the Effect of DEPTOR Knockdown on mTOR signaling in the TPM ........ 44 2.4.7 Evaluation of the Effect of DEPTOR Knockdown on Nutrient Transporters in TPM .... 46 CHAPTER 3 – PLACENTAL GENE DELIVERY - SONOPORATION ................................................. 49 5 3.3.1 INTRODUCTION ................................................................................................................... 49 3.2 BACKGROUND INFORMATION ................................................................................................... 50 3.2.2 Ultrasound Gene Delivery - Sonoporation ................................................................... 50 3.2.3 Ultrasound Transducers .............................................................................................