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Phosphorylation and Sequence Dependency of Neurofilament Protein Oxidative PHOSPHORYLATION AND SEQUENCE DEPENDENCY OF NEUROFILAMENT PROTEIN OXIDATIVE MODIFICATION IN ALZHEIMER DISEASE By QUAN LIU Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. George Perry Department of Pathology CASE WESTERN RESERVE UNIVERSITY January, 2005 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. I grant to Case Western Reserve University the right to use this work, irrespective of any copyright, for the University’s own purpose without cost to the University or to its students, agents and employees. I further agree that the University may reproduce and provide single copies of the work, in any format other than in or from microforms, to the public for cost of reproduction. Quan Liu (sign) iii DEDICATION To my parents, my lovely fiancée, and family members Be proud of me for having double “D”s and partially it is yours. iv TABLE OF CONTENTS Title Page i Signature Sheet ii Copyright wavier. iii Dedication. iv Table of Contents. 1 List of Tables 5 List of Figures 6 Acknowledgements 9 List of Abbreviations 11 Abstract 15 Chapter 1 General Introduction 16 1.1 Introduction of Alzheimer Disease 17 1.1.1 Clinical 17 1.1.2 Pathology 17 1.1.3 Etiology. 18 1.1.4 Pathogenesis and Hypothesis models. 19 1.1.5 Research Relevance: Oxidative Stress and AD Pathogenesis. 24 1.2 Introduction of Neurofilament Proteins 27 1.2.1 General Information 27 1.2.2 History 28 1.2.3 Structure 28 1.2.4 Expression. 30 1 1.2.5 Assembly 32 1.2.6 Transport 34 1.2.7 Posttranslational Modifications 35 1.2.8 Degradation. 37 1.2.9 Functions. 38 1.2.10 Animal Models.. 38 1.2.11 Neurofilaments and Neurodegenerative Diseases. 45 1.2.12 Summary. 52 1.3 Introduction of Tau Protein 54 1.3.1 General Information 54 1.3.2 History. 54 1.3.3 Tau gene and Tau Expression. 55 1.3.4 Tau Protein Structure 56 1.3.5 Posttranslational Modifications of Tau. 56 1.3.6 Tau Degradation 58 1.3.7 Tau Functions. 59 1.3.8 Tau Polymerization. 60 1.3.9 Animal Models 61 1.3.10 Tau and Neurodegenerative Diseases (Tauopathies) 64 1.3.11 Summary. 69 1.4 Introduction of Oxidative Modification. 70 1.4.1 Free radical Theory of Aging 70 1.4.2 Free Radicals. 70 2 1.4.3 The Primary Sources of Free Radicals 71 1.4.4 Physiological Damages Caused by Free Radicals 73 1.4.5 Antioxidant Systems. 73 1.4.6 Oxidative Stress in The Nervous System 76 1.4.7 HNE and HNE Modification in AD 80 1.4.8 Summary. 80 1.5 Research Relevance 83 Chapter 2 Oxidative Modification of Neurofilament Proteins 84 2.1 Introduction 85 2.2 Materials and Methods. 94 2.3 Results 99 2.4 Discussion 111 Chapter 3 Cellular Protective Function of Neurofilament Heavy Subunit 121 3.1 Introduction 122 3.2 Materials and Methods. 128 3.3 Results 132 3.4 Discussion 140 Chapter 4 Oxidative Modification of Tau Protein Contribute to NFT Formation. 144 4.1 Introduction 145 4.2 Materials and Methods. 155 4.3 Results 159 4.4 Discussion 165 3 Chapter 5 Conclusion and Future Work. 169 References. 176 Appendix Publications, Abstracts and Conferences 235 4 List of Tables Table 1.1 Comparison of the structures of neurofilaments and other type IV filaments 29 Table 1.2 The summary and comparison of NF subunits knock-out and overexpression mouse models 42 Table 2.1 Comparison of all the proteins and peptides in these studies. 116 Table 2.2 Protein database search for KSP repeats. 117 Table 2.3 All the control peptides used in these studies did not show high level of HNE adduct. 118 5 List of Figures Figure 1.1 Oxidative stress is a prominent central event in AD pathogenesis. 25 Figure 1.2 Comparison of the structures of neurofilam- ents and other type IV filaments. 31 Figure 1.3 Schematic model of neurofilament assembly 33 Figure 1.4 Regulatory functions of NF phosphorylation 36 Figure 1.5 Hypothetical relationships among neurofil- ament proteins, oxidative stress, cell injury and tissue damage 39 Figure 1.6 The structure of HNE and its adducts with amino acids. 81 Figure 2.1 Phosphorylation state of NFH regulates level of HNE-NFH adduct 100 Figure 2.2 Tau protein did not show the similar effect. 101 Figure 2.3 C-terminus of NFH is very reactive with HNE. 102 Figure 2.4 Nonphospho-20 AA KSP peptides are not able to be intensively modified by HNE. 104 Figure 2.5 Phospho-AKSPV peptide is modified by HNE. 105 Figure 2.6 Synthetic K-S-P peptides with and without phosphation show different reactivity with HNE 106 Figure 2.7 Synthetic K-S peptides with and without phosphorylation did not show significant 6 difference in the formation of HNE adduct. 108 Figure 2.8 HPLC-ESI-MS/MS confirms the removal of HNE from the HNE adduct after depho- sphorylation of the sample 109 Figure 2.9 Hypothetical model for the phosphorylation- regulated HNE modification in KSP motif. 121 Figure 3.1 HNE treatment induced higher level of NFH in M17 cells 133 Figure 3.2 HNE treatment induced higher phosphoryl- ation level of NFH. 134 Figure 3.3 MAPK were activated in M17 cells after HNE treatment 135 Figure 3.4 M17 differentiated cells with higher level of NFH showed significant protection form HNE cytotoxicity with LDH cytotoxicity assay 137 Figure 3.5 M17 differentiated cells with higher level of NFH showed significant protection form HNE cytotoxicity with LDH cytotoxicity assay 138 Figure 3.6 N2A cells showed significant protection with overexpressing NFH using trypan blue staining. 139 Figure 4.1 Summary of the specificity of seven NFT antibodies. 161 Figure 4.2 Recognition of various τ forms with and without dephosphorylation by antibodies to NFT. 162 7 Figure 4.3 Enhancement (fold increase) of antibody recognition by HNE modification 163 Figure 4.4 Immunocytochemistry on adjacent serial sections with the antibodies to NFT. 164 8 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my dissertation advisor, Professor George Perry, for his never-ending guidance and support during my Ph.D. research. He has continuously provided me with enthusiasm, vision, and wisdom, and inspired me from the beginning to the end. He also created an environment that gave me the flexibility to explore new ideas, while helping me to make critical decisions whenever the project was at a crossroad. He is an outstanding researcher to have as a role model for a Ph.D. student. I also want to thank my committee chair Professor Mark A. Smith, who provided knowledge and skills for me anytime during my Ph.D. studies, and motivated me to improve my basic knowledge in all aspects. It is he that really made me to write scientific papers and accomplish all the things in my Ph.D. training process. I have also had the good fortune to work with Professor Lawrence M. Sayre, who has helped me in every aspect of the thesis, which is deeply appreciated. I admire him for his broad knowledge, creative thinking, and deep insight in experimental design and ability for solving problems. Discussions with Professor Sayre directly led to the results of the neurofilament studies in chapter 2. I also thank Professor Shu G. Chen, who really helped me with the equipment and in producing good data. He also directly helped me with the methods and experimental design, which put me on the right track so many times. He has always been supportive for my research work and contributed extensively to my Ph.D. research. 9 It has been a privilege to work together with these intelligent and friendly people in my laboratory and co-laboratories. They have given me all the support from time to time. Here I send my gratitude to Sandra L. Siedlak, Peggy L.R. Harris, Beth Kumar, Xiongwei Zhu, Kazuhiro Honda, Paula I. Moreira, Dandan Wang, De Lin, David R. Sell, and Dr. Ricardo B. Maccioni, Dr. Jesus Avila, Dr. Mervyn J. Monteiro, Dr. Don W. Cleveland, Dr. Robert Salomon, Dr. Vincent Monnier, Dr. Michael Kinter, Dr. Touradj Solouki, Dr. Michael Strong, and so many other people that have already contributed to my Ph. D. research work. I really had a good time with them and had been honored to have all these colleagues to work with. Last, I would like to thank my family. All the family members are so supportive for my studies, my personal life and my future plans, in spite of they are thousand miles away or nearby. They are the best family members I know in my life. Surely they are worthy of my most sincerely appreciation. This work has been supported by the NIH and Alzheimer Association. Their assistance is gratefully acknowledged. 10 List of Abbreviations Aβ amyloid-β protein AβPP amyloid-β precursor protein AD Alzheimer’s disease ALE advanced lipoxidation end-product ALS Amyotrophic Lateral Sclerosis AP alkaline phosphatase AR alkoxyl radical BME. β-mercaptoethanol CaM. calmodulin MARK kinases Cdk5. Cyclin-dependent kinase 5 CNBr cyanogen bromide CNS central nervous system CMT. Charcot-Marie-Tooth Disease CSF. cerebrospinal fluid CWRU Case Western Reserve University DNPH.
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