University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2017 Acetate Supplementation Reduces Disease Progression, Alters Cns And Myelin Lipid Content, And Influences Cns And Myelin Protein Content In Mice Subjected To Experimental Autoimmune Encephalomyelitis Amber C. Chevalier Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Chevalier, Amber C., "Acetate Supplementation Reduces Disease Progression, Alters Cns And Myelin Lipid Content, And Influences Cns And Myelin Protein Content In Mice Subjected To Experimental Autoimmune Encephalomyelitis" (2017). Theses and Dissertations. 2186. https://commons.und.edu/theses/2186 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. 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ACETATE SUPPLEMENTATION REDUCES DISEASE PROGRESSION, ALTERS CNS AND MYELIN LIPID CONTENT, AND INFLUENCES CNS AND MYELIN PROTEIN CONTENT IN MICE SUBJECTED TO EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS By Amber Christine Chevalier Bachelor of Arts, Concordia College, 2012 Dissertation Submitted to the Graduate Faculty of the University of North Dakota In partial fulfillment of the requirements For the degree of Doctor of Philosophy Grand Forks, North Dakota December 2017 Copyright 2017 Amber Chevalier ii PERMISSION Title: Acetate Supplementation Reduces Disease Progression, Alters CNS and Myelin Lipid Content, and Influences CNS and Myelin Protein Content in Mice Subjected to Experimental Autoimmune Encephalomyelitis Department: Pharmacology, Physiology, and Therapeutics Degree: Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purpose may be granted by the professor who supervised by dissertation work or, in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or to the use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation. Amber Christine Chevalier December, 2017 iv TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES ............................................................................................................. xi ACKNOWLEDGEMENTS .............................................................................................. xii ABSTRACT ..................................................................................................................... xiii CHAPTER I. INTRODUCTION .......................................................................................1 Multiple Sclerosis ............................................................................1 Animal Models Of Multiple Sclerosis .................................4 Current Available Therapies for Multiple Sclerosis ..........10 Gaps in Knowledge ............................................................15 Lipid Synthesis, Lipid Breakdown, and Myelin Lipid Composition .......................................................................17 Glyceryl Triacetate.............................................................23 Overview of Dissertation ...................................................26 II. METHODS ................................................................................................28 Materials ........................................................................................28 Induction of EAE and Treatment .......................................29 Myelin Staining ..................................................................30 Behavioral Testing .............................................................31 Total Spinal Cord and Brain Lipid Extraction ...................31 v Myelin Brain Lipid Extraction ...........................................32 Thin Layer Chromatography..............................................33 Quantifying Phospholipid Content ....................................34 Quantifying Ganglioside Content ......................................34 Quantifying Esterified Fatty Acid Content ........................35 Quantifying Cholesterol and Cholesteryl Ester Content ....35 Protein Isolation .................................................................36 Total Protein Determination ..............................................36 Western Blot Analysis .......................................................36 Statistical Analysis .............................................................38 III. STUDY I ....................................................................................................39 Increasing Acetyl-CoA Metabolism Attenuates Injury and Alters Spinal Cord Lipid Content in Mice Subjected to Experimental Autoimmune Encephalomyelitis ............................39 Acetate Supplementation Attenuates Clinical Symptoms in Mice Subjected to EAE ...............................39 EAE Results in a Reduction of Spinal Cord Phospholipid That Was Not Found in EAE Mice Treated With Glyceryl Triacetate ......................................41 Acetate Supplementation Modulates Spinal Cord Fatty Acid Content in Mice Subjected to EAE ..................43 Acetate Supplementation Prevents the Loss of Cholesterol in EAE Mice but Does Not Alter the EAE-Induced Decrease in Cholesteryl Esters ....................46 Acetate Supplementation Alters Spinal Cord Ganglioside Content in Mice Subjected to EAE................48 vi Acetate Supplementation Returns Cytosolic Phospholipase A2 (cPLA2) to Baseline but EAE and Treatment Did Not Alter Phospholipase C (PLC) Levels .................................50 Acetate Supplementation Slightly Increases FluoroMyelinTM Intensity in Mice Subjected to EAE .......52 IV. STUDY II...................................................................................................54 Increasing Acetyl-CoA Metabolism Alters Spinal Cord Phosphatidylinositol Palmitic and Oleic Acid Levels in Mice Subjected to Experimental Autoimmune Encephalomyelitis ..........................................................................54 Acetate Supplementation Alters Esterified Fatty Acid Content in Phosphatidylinositol of EAE Mice ..........54 V. STUDY III .................................................................................................56 Increasing Acetyl-CoA Metabolism Increases Total Brain and Brain Myelin Lipid Content in Mice Subjected to Experimental Autoimmune Encephalomyelitis ....................................................56 Acetate Supplementation Increases Total Brain Phospholipid Levels in Mice Subjected to EAE ................56 Acetate Supplementation Increases Brain Myelin Phosphatidylinositol in Mice Subjected to EAE ................58 Acetate Supplementation Had No Effect on Brain Cholesterol and Cholesteryl Ester Content in Mice Subjected to EAE ...............................................................60 Acetate Supplementation Increases Total Brain GD3 Ganglioside and Brain Myelin GM1 Ganglioside Content in Mice Subjected to EAE..................................................60 Acetate Supplementation Alters Spinal Cord Oligodendrocyte Myelin Glycoprotein and Phosphorylated Acetyl-CoA Carboxylase Protein Levels in Mice Subjected to EAE but Had No Effect on Alkylglycerone Phosphate Synthase and Acyl-CoA Synthetase ..................................64 VI. DISCUSSION ............................................................................................68 Overall Conclusion of Dissertation ................................................68 vii Multiple Sclerosis May Be a Metabolic Disorder..............69 An Inflammatory Stimulus May Be Necessary to Shift the Acetyl-CoA Pool into Producing Lipid .............................76 Therapeutic Window Where Glyceryl Triacetate May Benefit Demyelinating and Metabolic Disorders ..............81 Acetate’s Role in Oligodendrocyte Maturation, Differentiation, and Function .............................................82 The Significance of CNS Lipid Changes Due to EAE and Treatment Related to Myelin Structure, Function, and Stability .......................................................85 The Importance to Develop Therapies that Promote Lipid Synthesis and/or Reduce Lipid Breakdown to Treat Multiple Sclerosis ..............................................................88 Future Studies ....................................................................93 VII. SUMMARY .............................................................................................105 APPENDICES .................................................................................................................109 List of Abbreviations ...........................................................................................110 Copyright Clearance ............................................................................................115