THE EFFECT OF MISOPROSTOL ON NEURONAL STEM CELL MIGRATION AND DIFFERENTIATION DENIS ADIGAMOV A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOLOGY YORK UNIVERSITY TORONTO, ONTARIO February 2020 © Denis Adigamov 2020 ii Abstract MisoprostoL is a drug commonLy used for medicaL termination of pregnancy, and improper use has been Linked to neurodeveLopmentaL disorders such as Moebius syndrome and Autism Spectrum Disorder (ASD). It is an analog of prostagLandin type E signaLLing Lipids, Like PGE1 and PGE2, and binds the same E-Prostanoid receptors, which reguLate mechanisms of neurodeveLopment. Previous research from the Crawford lab found that PGE2 affected migration, differentiation, and expression of genes associated with ASD in NE-4C neuroectodermaL stem ceLLs, and neurite eLongation in Neuro-2A neurobLastoma ceLLs. The objective of this study was to determine if misoprostoL alters the same ceLLuLar behaviors in NE-4C and Neuro-2A ceLLs. Time-Lapse microscopy was used to observe migration, neurosphere morphoLogy, and neurite extension in the ceLL modeLs. MisoprostoL treatment decreased migration (speed and distance traveLLed) of NE-4C stem ceLLs in a dose-dependent manner. Expression of stem ceLL marker Oct4 and neuronal marker MapT demonstrated a misoprostoL induced deLay in NE-4C differentiation. This was associated with changes in neurosphere morphoLogy and expression of adhesion moLecuLe genes, Cdh2 and NCAM. In differentiating Neuro-2A ceLLs, chronic misoprostoL exposure eLongated primary neurites and branches, but decreased branch density. These resuLts demonstrate the infLuence of misoprostoL on ceLLuLar mechanisms of migration, differentiation, and neurite eLongation, suggesting that prenataL exposure may aLter these processes, resuLting in abnormaL neurodeveLopment. iii Dedication I wouLd Like to dedicate this thesis to my famiLy. To my parents Rustam and ELLa Adigamov, for doing everything you couLd to support me in my goaLs. You have both sacrificed so much so that I wouLd have everything I needed. If you had not immigrated to Canada those 20 odd years ago, I wouLd never have had the opportunities that I do now. Most of aLL, I want to dedicate this work to my brother, ALexander Adigamov, you have aLways been my number one supporter. You are aLways the happiest to hear about my progress, and aLways there for me if I need something. You inspire me to work hard every day and remind me that I shouLd never squander the opportunities that Life may throw at me. To those mentioned above, thank you dearLy. I wiLL aLways be gratefuL for your undying faith in me, and work hard to make you proud. As such, I dedicate this work to you, in honour of your never-ending support. iv Acknowledgements I wouLd Like to acknowLedge some of the wonderfuL peopLe that have been so important in shaping my graduate experience. Thank you, Ashby KissoondoyaL, for introducing me to dungeons and dragons and mentoring me in ceLL cuLture procedures and western bLot anaLysis. You have been a friend to me from the start and reaLLy heLped guide and integrate me into the Lab when I joined. Thank you, Christine Wong, for teaching me protein and RNA/DNA isoLation, and being a roLe modeL of what I consider to be the ideaL scientist. Thank you, Ravneet Rai-BhogaL, for demonstrating fierce work ethic and integrity. Thank you Eizaaz Ahmad for passing on the torch to me when I joined the Lab. Thank you, IsabeL Bestard-Lorigados, for teaching me quantitative PCR anaLyses, and hoarding aLL the dice. Thank you, Sarah WheeLer, for participating in inteLLectuaL discussions, and for being a compassionate friend when I needed it. Thank you to Stephen Anthony Rose, HarshiLL PateL, and Arshad Imrit for heLping me Learn Python and further my understanding of statistics. Thank you, Keenan SterLing and Sasha Udhesister, for your support in my finaL semesters. Thank you, LiLy SoLomon- Harris, for your Love, care, and support in my finaL moments Leading up to the defense. Thank you, Kyle BeLozerov, for being a great friend and mentor throughout my studies since BIOL-4061. Thank you, Dorota Crawford, for guiding my growth as a scientist and aduLt throughout the program. Your beLief and encouragement heLped to push me when I needed it most. And thank you to anyone eLse that I may have missed for taking part in this journey. I have Learned a Lot and grown as an academic and scientific thinker. This experience wiLL aLways be a part of me. v Table of Contents Abstract ------------------------------------------------------------------------------------------------------------------- ii Dedication ---------------------------------------------------------------------------------------------------------------- iii Acknowledgements ---------------------------------------------------------------------------------------------------- iv Table of Contents --------------------------------------------------------------------------------------------------------v List of Tables ------------------------------------------------------------------------------------------------------------- vi List of Figures ----------------------------------------------------------------------------------------------------------- vii List of abbreviations and definitions --------------------------------------------------------------------------- viii 1 - INTRODUCTION ----------------------------------------------------------------------------------------------------- 1 1.1 - Lipid Signalling in the brain ------------------------------------------------------------------------------------------ 1 1.2 - Prostaglandin E2 signalling ------------------------------------------------------------------------------------------ 4 1.3 - The Role of Prostaglandin E2 in the nervous system --------------------------------------------------------- 7 1.4 – Abnormalities in the PGE2 signalling pathway and brain pathology ------------------------------------- 9 1.5 - The role of the COX-2/PGE2 signalling pathway in Autism – Molecular and behavioural evidence ----------------------------------------------------------------------------------------------------------------------- 11 1.6 – Misoprostol ----------------------------------------------------------------------------------------------------------- 13 1.7 - Experimental Model Systems ------------------------------------------------------------------------------------- 16 1.8 - Objectives and Hypothesis ---------------------------------------------------------------------------------------- 20 Study 1: The role of misoprostol in the migration of undifferentiated neuroectodermal (NE-4C) stem cells ------------------------------------------------------------------------------------------------------------- 21 Study 2: Misoprostol increases the growth of NE-4C neurospheres and delays differentiation 22 Study 3: The effect of misoprostol on neurite length, CREB phosphorylation, and branch density in Neuro-2A cells ---------------------------------------------------------------------------------------------------- 23 v 2 - METHODS ----------------------------------------------------------------------------------------------------------- 24 2.1 - Cell Culture ----------------------------------------------------------------------------------------------------- 24 2.2 - RNA and Protein Isolation ---------------------------------------------------------------------------------- 24 2.3 - Western Blot analyses --------------------------------------------------------------------------------------- 25 2.4 - Reverse Transcription and Polymerase Chain Reaction ---------------------------------------------- 26 2.5 - Quantitative Real-Time PCR -------------------------------------------------------------------------------- 27 2.6 - NE-4C Migration Timelapses ------------------------------------------------------------------------------- 30 2.7 - Differentiation of NE-4C cells and neurosphere induction ------------------------------------------ 30 2.8 - Measuring neurospheres ----------------------------------------------------------------------------------- 31 2.9 - Differentiation of Neuro-2A cells and neurite analysis ----------------------------------------------- 31 2.10 - Statistical Analysis ------------------------------------------------------------------------------------------ 32 3 - RESULTS -------------------------------------------------------------------------------------------------------------- 34 3.1 - STUDY 1: The role of misoprostol in the migration of undifferentiated neuroectodermal (NE- 4C) stem cells----------------------------------------------------------------------------------------------------------------- 34 3.1.1 - Dose dependent effect of Misoprostol on the migration of undifferentiated NE-4C stem cells--------------------------------------------------------------------------------------------------------------------- 34 3.1.2 - Misoprostol decreased the expression of CREB protein in NE-4C stem cells ----------------- 37 3.2 - STUDY 2: Misoprostol increases the growth of NE-4C neurospheres and delays differentiation ----------------------------------------------------------------------------------------------------------------------------------- 43 3.2.1 - Misoprostol slows the rate of differentiation-------------------------------------------------------- 43 3.2.2 - Misoprostol’s effect on the size of neurospheres --------------------------------------------------