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INVESTIGATION of Mrna OXIDATION in ALZHEIMER's

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INVESTIGATION of Mrna OXIDATION in ALZHEIMER's INVESTIGATION OF mRNA OXIDATION IN ALZHEIMER’S DISEASE DISSERTATION Presented in partial fulfillment of the requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xiu Shan, Bachelor in Medicine *********** The Ohio State University 2005 Dissertation Committee: Approved by Dr. Chien-liang Glenn Lin, Advisor Dr. Jeff Kuret ------------------------------------------- Dr. James Van Brocklyn Advisor Dr. Mike Zhu Neuroscience Graduate Studies Program ABSTRACT Oxidative modifications to vital cellular components have been described in association with Alzheimer’s disease (AD). Cytoplasmic RNA oxidation is a prominent feature of vulnerable neurons in AD affected regions. However, the role of RNA oxidation in the pathogenesis of AD is unknown. This dissertation examined the magnitude of oxidized mRNAs in AD, identified oxidized mRNA species, and characterized the consequence of mRNA oxidation. An immunoprecipitation method was developed to isolate, characterize and quantify oxidized mRNAs in AD brain. The data showed that 30-70% of mRNAs are oxidized in frontal cortices of AD as compared to age-matched normal control. Identification of oxidized mRNA species revealed that mRNA oxidation in AD is not random but selective. Many identified oxidized mRNA species have been implicated in the pathogenesis of AD. Quantitative analysis revealed that some mRNA species are more susceptible to oxidative damage for which the relative amounts of oxidized transcripts reach 50-70%. To understand whether oxidation of mRNAs is involved in the pathogenesis of AD, the consequence of mRNA oxidation was studied using in vitro translation system and cell lines. mRNA oxidation may have deleterious effects by interfering ii with normal translational process or by a toxic gain-of-function. Examination of the protein expression from oxidized mRNAs revealed that mRNA oxidation causes a decrease of protein level and associated activity, which may result from the abnormal association of polyribosomes with oxidized mRNAs during the process of translation. Furthermore, we observed that microinjection of oxidized mRNAs into neuronal cells caused cell death, suggesting that increased mRNA oxidation could be lethal to cells. Studies in primary neuronal cultures revealed that RNA oxidation is an early event far preceding cell death induced by insults associated with AD. Neurons are specifically susceptible to RNA oxidation. Some mRNA species highly oxidized in AD brain are also present in oxidized mRNA pool of oxidative insult-treated cultures. A decrease of protein level was observed to oxidized mRNA species in cultures. Taken together, these studies indicate the possibility of oxidized mRNAs to interfere with physiological functions of cells and also implicate the potential contribution of RNA oxidation in the pathogenesis of AD. iii Dedicated to my parents and sisters iv ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor, Dr. Chien-liang Glenn Lin for providing me with an opportunity to work in and learn from his research group. I will always appreciate his supervision on my graduate study and research. I would also like to thank the members of my dissertation committee – Dr. Jeff Kuret, Dr. James Van Brocklyn, and Dr. Mike Xi Zhu – for providing me with many helpful inputs during the course of my thesis research. The following individuals are acknowledged for providing me access to their equipments or their inputs related to said equipments: Dr. Georgia A. Bishop (Department of Neuroscience) and Dr. James S. King (Department of Neuroscience) for access to the Zeiss inverted microscope, Dr. David Suffen (Department of Pharmacology) and Ju Young Kim (Department of Pharmacology) for access to the SW60 ultracentrifuge rotor, Dr. Richard W. Burry (Department of Neuroscience) and Kathy Wolken (Campus Microscope and Imaging Facility) and Brian Kemmenoe (Campus Microscope and Imaging Facility) for access to the Zeiss laser-scanning confocal microscope. v I thank Johns Hopkins Alzheimer’s Disease Research Center Brain Bank and ALS Brain Bank, Harvard Brain Tissue Resource Center and University of Maryland Brain and Tissue Banks for Developmental Disorders for providing AD and normal control brain tissues. I would like to thank the current and past members of the Lin research group, Dr. Hirofumi Tashiro, Dr. Matthew E. R. Butchbach, Dr. Hong Guo, Ms. Liching Lai, Ms. Yueming Chang, Ms. Guilian Tian and Mr. Michael P. Stockinger, for their support during the course of my thesis research. Especially, I would like to thank Dr. Hirofumi Tashiro for sharing the project with me and Dr. Matthew E. R. Butchbach for providing me many helpful insights for my experiment and vigorous discussion for my manuscripts and kind suggestion for my postdoctoral position searching. I would like to thank our collaborator, Dr. Chandan Sen and Dr. Soma Chaki for helping us with the cell microinjection experiment. Finally, I would like to thank my family for their constant support throughout my education vi VITA August 24, 1977…………………..Born – Hebei Province, P.R.China Sept 1995 - June 2000 Bachelor in Medicine Beijing Medical University, Beijing, P.R.China May 1999 - June 2000 Undergraduate Research Assistant, Neuroscience Research Institute, Beijing Medical University, P.R.China Sept 2000 – present Graduate Research Associate Neuroscience Graduate Studies Program The Ohio State University PUBLICATIONS 1. Shan X, Tashiro H, and Lin CG (2003) The Identification and Characterization of Oxidized RNA in Alzheimer’s Disease (J Neurosci. 23:4913-4921) 2. Guo H, Lai L, Butchbach MER, Stockinger MP, Shan X, Bishop GA and Lin CG (2003) Increased expression of the glial glutamate transporter EAAT2 modulates excitotoxicity and delays the onset but not the outcome of ALS in mice. (Human Mol Genet 19:2519-2532) FIELDS OF STUDY Major field: Neuroscience vii TABLE OF CONTENTS Page ABSTRACT…...…………………………..………………………………………. ii ACKNOWLEDGMENTS…..…………………………...……………………….. v VITA……………………...………………...…………………..…………………. vii LIST OF TABLES…………………..…………….………………………..…….. xi LIST OF FIGURES………………………..…………………………………….. xii LIST OF SYMBOLS AND ABBREVATIONS……………………..…………...xiv Chapter 1 INTRODUCTION…………………………………………………… 1 1.1 Alzheimer’s disease…………………………………………..……………. 1 1.2 Reactive oxygen species regulatory pathway………………………..…….. 7 1.3 Oxidative stress hypothesis in Alzheimer’s disease………..……………… 9 1.4 Markers for oxidative stress………………………………………….…….. 18 1.5 Lipid peroxidation in Alzheimer’s disease………………………………….19 1.6 Protein oxidation in Alzheimer’s disease…………………………..…..….. 22 1.7 DNA oxidation in Alzheimer’s disease………………………………..……25 1.8 RNA oxidation in Alzheimer’s disease…………………………..….……...29 1.9 Oxidative stress in other neurodegenerative diseases………………..…….. 30 Chapter 2 QUANTIFICATION OF OXIDIZED mRNAS IN ALZHEIMER’S DISEASE..…………………..…………………………………………………….. 36 2.1 Abstract…………………..……………………...………………..……….. 36 2.2 Introduction………………..……………………………………………… 37 2.3 Materials and Methods…………………..………………………………..38 2.4 Results…………………….…………………………………………...…... 45 2.4.1 The quality of RNA from postmortem tissues………………….……… 45 2.4.2 Detecting of oxidized mRNA in AD by Northern blotting………….….46 2.4.3 Immunoprecipitation of oxidized mRNAs….…………………………..47 2.4.4 Quantification of the level of oxidized mRNAs in diseased and normal tissues……………………………..………………………………….……..... 52 2.5 Discussion………………………..………………...……………………… 54 viii Chapter 3 CHARACTERIZATION OF OXIDIZED mRNA SPECIES IN ALZHEIMER’S DISEASE………………………………..……………………... 58 3.1 Abstract......................................................................................................... 58 3.2 Introduction……………………………………..………………………… 58 3.3 Materials and Method………………………………………..……………60 3.4 Results……………………………………………………………………... 64 3.4.1 Molecular cloning and sequence analysis of oxidized mRNAs from Alzheimer’s brains……………………………………..………...……………64 3.4.2 Filter array to examine the susceptibility of individual mRNA species to oxidation in AD.……………………….……………………………………...70 3.4.3 Quantification of the level of oxidation for individual mRNA species by filter array and semi-quantitative RT-PCR………………………….……….. 72 3.4.4 Characterization of the selectivity of oxidation to individual mRNA species………………………………………………………………………... 75 3.5 Discussion…………………..…………………………………………...… 75 Chapter 4 THE CONSEQUENCE OF mRNA OXIDATION…………..…….. 81 4.1 Abtract………………………………………………………..…………… 81 4.2 Introduction…………………………………………………..…………… 82 4.3 Materials and Methods………………………………………………..….. 84 4.4 Results........................................................................................................... 91 4.4.1 RNA oxidation causes decreases of expression level and activity of the protein in rabbit reticulocyte lysate………………………………..…………. 91 4.4.2 RNA oxidation causes decreases of expression level and activity of the protein in cell lines………………………………………..………………….. 93 4.4.3 RNA oxidation causes the abnormal increase of polyribosome association……………………………………………………………………. 98 4.4.4 No truncated protein was generated from oxidized mRNA examined by immunoprecipitaion and Western blotting ……………..…………………… 102 4.4.5 Investigation of protein mutation from oxidized mRNA by SELDI- TOF…………………………………..………………………………… 104 4.4.6 mRNA oxidation could be lethal to cells……………………..………... 105 4.5 Discussion………………………………………..………………………... 108 Chapter 5 INVESTIGATION OF RNA OXIDATION IN CELL MODEL….. 115 5.1 Abstract…………………………………………….……………………....115 5.2 Introduction………………………………………….…………………….
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