Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2016 Evidence That Myo-Inositol Plus Ethanolamine Elevates Plasmalogen Levels And Lends Protection Against Oxidative Stress In Neuro-2A Cells Isaie Sibomana Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Biomedical Engineering and Bioengineering Commons Repository Citation Sibomana, Isaie, "Evidence That Myo-Inositol Plus Ethanolamine Elevates Plasmalogen Levels And Lends Protection Against Oxidative Stress In Neuro-2A Cells" (2016). Browse all Theses and Dissertations. 1674. https://corescholar.libraries.wright.edu/etd_all/1674 This Dissertation is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. EVIDENCE THAT MYO-INOSITOL PLUS ETHANOLAMINE ELEVATES PLASMALOGEN LEVELS AND LENDS PROTECTION AGAINST OXIDATIVE STRESS IN NEURO-2A CELLS A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy By ISAIE SIBOMANA B.S., National University of Rwanda, 1990 Agricultural Engineering, Polytechnic University of Bobo-Dioulaso (Burkina Faso), 1999 M.Phil., Entomology, University of Ghana, 2002 M.S., Wright State University, 2011 ____________________________ 2016 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL December 16, 2016 I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY SUPERVISION BY Isaie Sibomana ENTITLED Evidence that Myo-Inositol plus Ethanolamine Elevates Plasmalogen Levels and Lends Protection Against Oxidative Stress in Neuro-2A Cells BE ACCEPTED IN PARTIAL FULFILLMENT OF THEREQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy ___________________ Nicholas V. Reo, Ph.D. Dissertation Director ______________________ Mill W. Miller, Ph.D. Director, Biomedical Sciences Ph.D. Program ______________________ Robert E.W. Fyffe, Ph.D. Vice President for Research and Dean of the Graduate School Committee on Final Examination ____________________ Nicholas J. DelRaso, Ph.D. _____________________ Michael L. Raymer, Ph.D. _____________________ James E. Olson, Ph.D. _____________________ Jeffery M. Gearhart, Ph.D. Abstract Sibomana Isaie, Ph.D., Biomedical Sciences Ph.D. Program, Wright State University, 2016. Evidence that myo-inositol plus ethanolamine elevates plasmalogen levels and lends protection against oxidative stress in Neuro-2A cells Plasmalogens are glycerophospholipids abundant in brain and heart tissues. Evidence suggests that they have antioxidant properties. Studies from our laboratory showed that rats treated with myo-inositol plus ethanolamine (ME) have elevated ethanolamine plasmalogens (PE-Pls) in brain and are protected against phosphine- induced oxidative stress. We hypothesized that ME elevates PE-Pls levels and protects against oxidative stress through oxidation of its vinyl ether bond. We tested this hypothesis in Neuro-2A cell culture and assessed the effects of treatments with myo- inositol (M), ethanolamine (Etn), or a combination (ME) on the: (1) effects on phospholipid (PL) classes, especially Etn PLs; (2) effects on cell viability in response to H2O2-induced oxidative stress; and (3) molecular species of Etn PLs preferentially affected by ME and H2O2 treatments, especially PE-Pls and their degradation byproducts – lyso-phosphatidylethanolamines (LPE). 31P NMR data show that treating the cells with equimolar amounts (500 M) of M or Etn for 24 h did not influence PL levels, but ME yielded a 3-fold increase in both PE-Pls and PE (p<0.001). Cells exposed to 650 M H2O2 for 24 h decreased cell viability to 53% ± 1.7. While pretreatment with M or ME significantly increased cell survival to 62% ± 1.2 or 80% ± 0.6, respectively (p<0.05), Etn alone had no effect. Mass spectrometry showed that ME preferentially elevated the iii levels of PE-Pls species containing saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) by 60%, while PE-Pls containing polyunsaturated fatty acids (PUFA) increased by only 10%. H2O2 caused a significant decrease in PE-Pls (27%), producing a 39% increase in LPE and a 4-fold increase in glycerophosphoethanolamine (GPE), but had no impact on PE levels, suggesting that LPE and GPE were primarily byproducts of PE-Pls degradation. Surprisingly, all these effects were blocked by pre-treating cells with ME prior to H2O2 exposure. Taken together, these data suggest that a preferential increase in PE-Pls species containing SFA+MUFA in response to ME may protect cells from H2O2-induced oxidative stress. The mechanism for this effect is unclear, but further investigations to understand these processes may help to develop neuroprotective approaches to alleviate the progression of neurodegenerative diseases/disorders. iv TABLE OF CONTENTS CHAPTER 1: INTRODUCTION AND SPECIFIC AIMS ........................................... 1 1.1 Plasmalogens: Structure and Cellular Role ............................................................... 1 1.1 SPECIFIC AIMS ....................................................................................................... 2 CHAPTER 2: BACKGROUND ...................................................................................... 4 2.1 Phospholipids in Mammalian Cells .......................................................................... 4 2.2 Plasmalogens in Neural Membranes ......................................................................... 6 2.3 Biosynthesis of Ethanolamine Plasmalogens in Brain .............................................. 8 2.4 Phospholipid Homeostasis in Neural Membranes .................................................. 12 2.5 Role of Plasmalogens in Neurological Diseases/Disorders .................................... 13 2.6 Antioxidant Properties of Plasmalogens ................................................................. 16 2.7 Goal of the Present Study ........................................................................................ 19 2.8 Hypothesis and Strategy .......................................................................................... 20 CHAPTER 3: METHODS ............................................................................................. 22 3.1 Nuclear Magnetic Resonance (NMR) Analyses ..................................................... 22 3.1.1 Cell line and culture conditions ............................................................................... 22 3.1.2 Neuro-2A cell treatment with myo-inositol +/- ethanolamine ............................. 22 3.1.3 Neuro-2A cell treatment with hydrogen peroxide ................................................. 23 3.1.4 Evaluation of cell viability ....................................................................................... 23 v 3.1.5 Evaluation of apoptosis ............................................................................................. 24 3.1.6 Lipid and aqueous phase extractions ...................................................................... 26 3.1.7 Protein assays ............................................................................................................. 28 3.1.8 Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy of lipid extracts .. 29 3.1.9 Proton NMR of aqueous extracts ............................................................................ 30 3.1.10 Phosphorus-31 NMR of aqueous extracts ............................................................ 30 3.1.11 NMR data processing .............................................................................................. 30 3.2 Ultra Performance Liquid Chromatography (UPLC)-Mass Spectrometry (LC- MS/MS) Analyses ......................................................................................................... 31 3.2.1 Chemicals ................................................................................................................... 31 3.2.2 Methods ...................................................................................................................... 32 3.3 Statistical analyses ........................................................................................................ 39 CHAPTER 4: RESULTS AND DISCUSSION FOR NMR DATA ............................ 40 4.1 RESULTS................................................................................................................ 40 4.1.1 Phospholipid profiles in Neuro-2A cells ................................................................ 40 4.1.2 Myo-inositol plus ethanolamine increases myo-inositol, phosphoethanolamine, phospholipid pool and ethanolamine phospholipids in N2A cells ..................... 43 4.1.3 Cells treated with myo-inosiol plus ethanolamine show elevated ethanolamine plasmalogens and are protected from H2O2-induced oxidative stress ................ 51 vi 4.1.4 Myo-inositol plus ethanolamine blocks the H2O2-mediated decrease in ethanolamine plasmalogens in N2A cells .............................................................. 54 4.1.5 Myo-inositol plus ethanolamine blocks the H2O2-mediated increase in lyso- ethanolamine lipids and glycerophosphoethanolamine in N2A cells ................. 59 4.2 DISCUSSION ......................................................................................................... 62 4.2.1 Phospholipid profiles in