Oncogenic Parallels in Alzheimer Disease
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ONCOGENIC PARALLELS IN ALZHEIMER DISEASE by ARUN K. RAINA Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisors Dr. Mark A. Smith & Dr. Xiongwei Zhu Department of Pathology Case Western Reserve University Janurary 2005 Copyright © 2005 by Arun K. Raina All rights reserved ii CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ARUN K. RAINA candidate for the Ph.D. degree *. (signed)___George Perry___________________________ (chair of the committee) ____Mark A. Smith__________________________ ____Xiongwei Zhu___________________________ ____Robert B. Petersen__________________________ ____David Boothman__________________________ (date) ____________________ *We also certify that written approval has been obtained for any proprietary material contained therein. iii Dedication To Bhairavah Uma, Autar, Arvind and P iv Table of Contents List of Tables 7 List of Figures 8 Acknowledgements 11 Abbreviations 12 Abstract 16 Chapter 1 Introduction 18 1.1 Historical 18 1.2 Definition of Dementias 19 1.3 Epidemiology of AD 19 1.4 Pathology of AD 19 a. Neurofibrillary Tangles 20 b. Senile Plaques 22 1.5 Clinical Features of AD 22 1.6 Etiopathogenesis of AD 23 a. Age 23 b. Genetics 24 i. Amyloid β Protien Precursor (AβPP) 24 ii. Presenilins 1 and 2 25 iii. ApoE 25 c. Oxidative Stress 27 d. Cell Cycle and AD 28 1 Chapter 2 Oxidative Stress in AD 30 2.1 Introduction 30 a. Oxidative Stress in AD 30 b. Oxidative Events and Apoptosis in AD 32 c. Oxidative and Cell Cycle Events in AD 33 d. Oxidative Stress - An Adaptation in AD? 34 e. Oxidative Relevance to Lesions 35 f. Neuroprotection Within Vulnerable Neurons in AD 37 2.2 Experimental Hypothesis 37 2.3 Methods and Materials 40 a. Tissue Section Preparation 40 b. Antibodies/Protein Preparations 40 c. Immunocytochemistry 41 2.4 Results 42 a. NQO1 42 b. Bleomycin Hydrolase 42 2.5 Discussion 43 a. NQO1 43 b. Bleomycin Hydrolase 45 2.6 Conclusions 46 2.7 Relevant Publications 53 2 Chapter 3 Cell Cycle Dysfunction in AD 57 3.1 Introduction 57 a. The Cell Cycle and Control Mechanisms 57 b. Reappearance of Cell Cycle Markers in AD 59 c. Association of the Mitotic Phenotype with AD Neuropathology 60 3.2 Experimental Hypothesis 63 3.3 Methods and Materials 63 a. Tissue section preparation 63 b. Immunocytochemistry 64 c. Immunoblots 65 3.4 Results 65 a. Mrg 15 65 b. CARB 66 c. P27 67 3.5 Discussion 69 a. Mrg 15 69 b. CARB 70 c. P27 73 3.6 Conclusions 75 3.7 Relevant Publications 92 3 Chapter 4 Apoptotic Avoidance as a Feature in AD 94 4.1 Introduction 94 a. Programmed Cell Death and Apoptosis - Basic Concepts 94 b. Apoptotic Avoidance in AD 96 4.2 Experimental Hypothesis 98 4.3 Methods and Materials 99 a. Tissue Section Preparation 99 b. Immunocytochemistry 100 c. Antibodies 100 d. Adsorption Experiments 101 e. Immunoblotting and Immunodotting 101 f. Electron Microscopy 103 g. Cells 103 h. Determination of Cell Death 104 i. Transfection and Selection 104 j. RT-PCR 105 4.4 Results 105 a. Upstream Caspases 105 b. Downstream Caspases 106 c. Bcl-w 106 4.5 Discussion 110 a. Caspases 110 b. Bcl-w 113 4 4.6 Conclusions 116 4.7 Relevant Publications 125 Chapter 5 Oncogenic Parallels in AD and Conclusions 127 5.1 Introduction 127 a. Oxidative Stress, AD and Oncogenesis 127 b. Dysmitotic Mechansisms, AD and Oncogenesis 128 c. Loss of Apoptosis, AD and Oncogenesis 129 5.2 Experimental Hypothesis 131 5.3 Methods and Materials 133 a. Tissue Section Preparation 133 b. Immunocytochemistry 133 c. Immunoblots 135 5.4 Results 135 a. ADAM 135 b. BRCA-1 137 c. Phospho-retinoblastoma (pRb) 138 5.5 Discussion 138 a. ADAM 138 b. BRCA-1 141 c. Phospho-retinoblastoma (pRb) 144 5.6 Overall Conclusions 146 5.7 Future Directions 148 5 a. Apoptotic Avoidance 148 b. Functional Consequences of BRCA-1 Misexpression in Post- Mitotic Neurons 149 5.8 Relevant Publications 163 Bibliography 164 6 List of Tables Table 2.1 48 Quantitative comparison of NQO1 immunoreactivity in AD and control cases. Table 3.1 77 Cell cycle markers that are seen in AD Table 3.2 78 The AD phenotype resembles a mitotic phenotype. Table 5.1 150 BRCA-1 (Ab-1) immunoreactive AD cases and respective predispositional factors. Table 5.2 151 Antisera to Rb phosphorylational sites that immunoreacts with AD neuropathology. 7 List of Figures Figure 2.1 49 NQO1 in AD. Figure 2.2 50 Immunocytochemical localization of BH. Figure 2.3 51 Immunoreactivity of BH-absorption analysis. Figure 2.4 52 NQO1 and its role in AD. Figure 3.1 79 Cell cycle control overview. Figure 3.2A 80 The G1-S checkpoint control. Figure 3.2B 81 Cell-cycle related proteins: overlapping functions. Figure 3.3 82 Mrg 15 – Role in cell cycle. Figure 3.4 83 Mrg 15 immunocytochemistry. Figure 3.5. 84 Western analysis of Mrg 15. Figure 3.6 85 Immunocytochemical localization of CARB. Figure 3.7 86 CARB immunocytochemistry: Absorption studies. Figure 3.8 87 CARB immunoblot analysis. Figure 3.9 88 p27 immunocytochemistry. 8 Figure 3.10 89 p27/τ overlap. Figure 3.11 90 p27 immunocytochemistry: absorption studies. Figure 3.12 91 p27 quantification. Figure 4.1 117 An overview of the apoptotic process. Figure 4.2 118 Caspase 8 in AD. Figure 4.3 119 Caspase 3 in AD. Figure 4.4 120 Bcl-w immunocytochemistry. Figure 4.5 121 Bcl-w electron microscopy. Figure 4.6 122 Bcl-w immunoblots. Figure 4.7 123 Effect of Aβ on Bcl-w. Figure 4.8 124 Bcl-w transfection. Figure 5.1 152 Parallels between AD and oncogenesis. Figure 5.2 153 Immunocytochemistry of ADAMs 1 and 2 in AD Figure 5.3 154 Immunocytochemistry of ADAMs 1 and 2 in controls Figure 5.4 155 Immunocytochemistry of ADAMs 1 and 2: Adsorption studies 9 Figure 5.5 156 Immunoblot analysis of ADAMs 1 and 2 Figure 5.6 157 BRCA-1 immunocytochemistry Figure 5.7 158 BRCA-1 immunoblot analysis Figure 5.8 159 BRCA-1 is absent from control NFT Figure 5.9 160 Phospho-retinoblastoma immunocytochemistry Figure 5.10 161 Effects of BRCA-1 overexpression – Assays Figure 5.11 162 Effects of BRCA-1 overexpression – JNK/ERK studies 10 Acknowledgments This work would not be possible if it were not for close support from many people on a daily basis. I would like to offer my heartfelt appreciation to all of them for taking the time and giving the effort to make all of this possible. I would like to thank Mark A. Smith, my mentor, for opening the window of science for me and allowing me to have fun in science. I would also like to thank Xiongwei Zhu and George Perry for the constant support in both scientific and nonscientific matters. I have truly enjoyed the teaching that I was offered. Additionally, I would like to thank Peggy Harris for her showing me how to do my first experiment and for her constant support over the years. I would also like to thank Sandi Siedlak for her help throughout these years. Of course without Beth Kumar and her efficient help in so many areas I would be quite handicapped. I have truly enjoyed their help, support, and friendship. I have great pleasure in knowing and working with my colleagues, Hyoung-gon Lee, Mike Marlatt, Gemma Casadesus, Kate Webber, and others that have worked in the lab. I am also indebted to my committee members, David Boothman and Robert B. Petersen. Finally, I would especially like to thank Catherine A. Rottkamp for her immense help throughout my studies and for being a consummate friend. 11 Abbreviations 8OHG 8-hydroxyguanosine AD Alzheimer disease ADRDA Alzheimer disease and Related Disorders Association AGE Advanced glycation end products ApoE Apolipoprotein E ATP Adenosine triphosphate Aβ Amyloid beta AβPP Amyloid beta protein precursor bFGF Basic fibroblast growth factor BH Bleomycin hydrolase BRCA1 Breast cancer gene 1 CAK CDK activating kinase CARB CIP 1 regulator of cyclin B CDK Cyclin dependent kinase CERAD Consortium to Establish a Registry for Alzheimer's Disease CKI CDK inhibitor CNS Central Nervous System CSF Cerebrospinal fluid DAB Diaminobenzadine DCS Donor Calf Serum DHFR Dihydrofolate reductase DSB Double strand breaks 12 ECM Extracellular matrix EDTA Ethylenediaminetetraacetic Acid EEG Electroencephalography EGFR Epidermal growth factor receptors G6PD Glucose 6 phosphate dehydrogenase GSH Glutathione GSHPx Glutathione peroxidase GSSG-R Reduced Glutathione GST-NQO1 Glutathione S transferase NQO1 HAT Histone acetyltransferase HNE Hydroxynonenal HO-1 Heme Oxygenase-1 HPG Hypothalamic-pituitary-gonadal IAP Inhibitor of apoptosis protein JNK Jun N-terminal kinase LDH Lactate dehydrogenase LDL Low Density Lipopprotein MCI Mild Cognitive Impairment MEKK Mitogen-activated protein kinase kinase kinase MMP Matrix metalloproteinases MORF Mortality factor 4 MRG MORF4-Related Gene on chromosome 15 MRI Magnetic Resonance Imaging 13 NAD Nicotinamide Adenine Dinucleotide NADP Nicotinamide adenine dinucleotide phosphate NADPH Nicotinamide adenine dinucleotide phosphate reduced NGF Nerve Growth Factor NGS Nerve growth serum NIA National Institute of Aging NINCDS National Institute of Neurological & Communicative Disorders & Stroke NO Nitrous oxide NQO1 NADPH Quinone Oxidoreductase 1 NSAIDS Nonsteroidal anti-inflammatory drugs PARP Poly (ADP-ribose) polymerase PBS Phosphate buffered Saline PCNA Proliferating cell nuclear Antigen PCR Polymerase Chain Reaction PET Positron Emission Tomography PGEX-KG Bacterial Expression vector PHF Parahelical filaments PLK Polo like Kinase PMSF Phenylmethanesulfonyl fluoride PS1/2 Presenilin 1/2 PUFA Polyunsaturated fatty acid RAGE Receptor for advanced glycation end products ROS Reactive oxygen species 14 SAPK Stress-activated protein kinase SDS Sodium dodecyl sulfate SEK SAPK kinase SOD Superoxide dismutase SPECT Single Photon Emission Computed Tomography SSB Single strand breaks TAK1 TGF-β activating kinase 1 TBS Tris Buffered Saline TGFβ Transforming growth factor-β 15 ONCOGENIC PARALLELS IN ALZHEIMER DISEASE Abstract by ARUN K.