Ceramide Metabolism Regulates A Neuronal Nadph Oxidase Influencing Neuron Survival During Inflammation Item Type Thesis Authors Barth, Brian M. Download date 07/10/2021 02:29:56 Link to Item http://hdl.handle.net/11122/8999 CERAMIDE METABOLISM REGULATES A NEURONAL NADPH OXIDASE INFLUENCING NEURON SURVIVAL DURING INFLAMMATION A THESIS Presented to the Faculty of the University of Alaska Fairbanks in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY By Brian M. Barth, B.S., M.S. Fairbanks, Alaska August 2009 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3386045 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI Dissertation Publishing UMI 3386045 Copyright 2009 by ProQuest LLC. All rights reserved. This edition of the work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CERAMIDE METABOLISM REGULATES A NEURONAL NADPH OXIDASE INFLUENCING NEURON SURVIVAL DURING INFLAMMATION By Brian M. Barth RECOMMENDED: , ... f / M Advisory Committee Chah Chai ^Department of Chemistry and Biochemistry /? & f ) ./ APPROVED: Dean, C olleg£^ Natural Science'and“Mathemati cs /<C £>dan of the Graduate School 3 / / - Date Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract Inflammation is a major component of acute and chronic pathologies of the central nervous system, including psychiatric disorders. Microglia respond to pathogens, injury, and toxins by secreting inflammatory mediators including pro-inflammatory cytokines in an event known as neuroinflammation. This thesis research investigated a link between neuroinflammation and oxidative stress, and ultimately neurodegeneration. The cytokine tumor necrosis factor alpha was shown to stimulate a neuronal NADPH oxidase (NOX), specifically by stimulating the production of ceramide and ceramide-1 -phosphate via Mg2+-neutral sphingomyelinase (Mg2+-nSMase) and ceramide kinase. Intriguingly, glucosylceramide blocked NOX activation, linking ceramide neutralization directly to a decline in oxidative stress. Most importantly, NOX activity interfered with actin and sphingosine kinase-1 via oxidation, demonstrating a positive and detrimental feedback mechanism that impedes neuronal survival pathways. Interestingly, crude extracts from 2_j_ wild Alaskan bog blueberries showed the ability to interfere with Mg -nSMase, demonstrating a specific neuroprotective property of the berry. Altogether, this thesis research defined a key neuronal pathway linking inflammation to oxidative stress via ceramide metabolism, potentially allowing for future therapeutic development to improve neuronal function and survival. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Page Signature Page.............................. ....i Title Page.................... ii Abstract ...... iii Table of Contents .................. iv List of Figures......................... viii List of Tables ..................................... xi List of Abbreviations ..................... xii Acknowledgements ............................................................. xv Chapter 1 Introduction ........................ 1 1.1 CNS Health..................................................................................................... 1 1.1.1 Neuroinflammation ......................................................................... 1 1.1.2 Nutrition ...........................................................................................2 1.2 NADPH Oxidase ............................................................................................3 1.2.1 NADPH oxidase in phagocytes and other cells .......................... 3 1.2.2 Structure and function .....................................................................5 1.3 Sphingolipids ................................................................................................11 1.3.1 Sphingolipid metabolism ............................................................. 11 1.3.2 Neutral Sphingomyelinase ...........................................................14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V Page 1.3.3 Sphingosine K inase .......................................................................16 1.3.4 Gangliosides and neuronal membranes ......................................18 1.3.5 Interconversion of ceramide and sphingosine-1 -phosphate: a potential biostat ...........................................................................20 1.3.6 Regulation of eicosanoid biosynthesis ........................................22 1.3.7 Regulation of multi-drug resistant and invasive cancer 24 1.4 Rationale and significance of thesis-research ..........................................25 1.5 Aims, hypotheses, and methods of individual chapters .........................27 1.6 References .....................................................................................................31 Chapter 2 TNFcx-induced neuronal NADPH oxidase activity mediates oxidative damage to the neuronal actin cytoskeleton.......................68 2.1 Abstract......................................................................................................... 68 2.2 Introduction ................................................................................................. 69 2.3 Experimental Procedures ............................................................................71 2.4 Results ...........................................................................................................76 2.5 Discussion .....................................................................................................86 2.6 Acknowledgements .....................................................................................90 2.7 Contributions ............................................................................................... 90 2.8 References .....................................................................................................90 Chapter 3 TNFa- and Aj3-induced neutral sphingomyelinase activity regulates the neuronal NADPH oxidase and subsequent oxidative damage to sphingosine kinase-1.......................................... 98 3.1 Abstract......................................................................................................... 98 3.2 Introduction ...................................................................................................99 3.3 Materials and M ethods ..............................................................................100 3.4 Results .........................................................................................................105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VI Page 3.5 Discussion ...................................................................................................119 3.6 Acknowledgements ...................................................................................125 3.7 Contributions ............................................................................................. 125 3.8 References .................................................................. 125 Chapter 4 Ceramide kinase regulates TNFct-induced NADPH oxidase activity and eicosanoid biosynthesis in neuronal cells........... 132 4.1 Abstract....................................................................................................... 132 4.2 Introduction .................................................................................................133 4.3 Materials and Methods .................................................................134 4.4 Results.........................................................................................................140 4.5 Discussion .............................................................................................. 152 4.6 Acknowledgements ...................................................................................156 4.7 Contributions ............................................................................................. 156 4.8 References ...................................................................................................157 Chapter 5 Inhibition of NADPH oxidase by glucosylceramide confers chemoresistance............................. 164 5.1 Introductory Paragraph ............................................................................. 164 5.2 Main Text ...................................................................................................165 5.3 M ethods ................................................................................ 178 5.4 Acknowledgements ...................................................................................183 5.5 Contributions ............................................................................................184 5.6 References