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Mechanism of U18666a-Mediated Cell Death in Cultured Neurons

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Mechanism of U18666a-Mediated Cell Death in Cultured Neurons MECHANISM OF U18666A-MEDIATED CELL DEATH IN CULTURED NEURONS KOH CHOR HUI VIVIEN (B.Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements I would like to thank: My supervisor, Assistant Professor Steve Cheung Nam Sang, for the opportunity to work in his lab and his guidance in this research project. The Biomedical Research Council (BMRC) of the Agency for Science, Technology and Research (A*STAR) of Singapore for the Research Assistant position and financial support (R-183-000-082-305). This work was also financially supported by the National Medical Research Council (NMRC) of Singapore (R-183-000-075-213) and the Academic Research Fund of the National University of Singapore (R-183- 000-060-214). Associate Professor Matthew Whiteman from the Department of Biochemistry for his guidance in oxidative stress studies. Dr. Ou Keli and members of his lab from Agenica Research Pte. Ltd., and Dr. Zhang Dao Hai from the Department of Laboratory Medicine of the National University Hospital for their expertise in proteomics and the use of their facilities. Dr. Li Qiao-Xin from the Department of Pathology of the University of Melbourne for her assistance in Aβ studies; Dr. Qu Dianbo from the Institute of Molecular and Cell Biology for his assistance in radioactive work; Ms Chan Yee Gek from the Department of Anatomy for her assistance in electron microscopy; Jayapal Manikandan from the Department of Physiology for his assistance in GeneSpring™. Ms Seet Sze Jee, Ms Huang Shan Hong and Ms Siau Jialing from the Department of Biochemistry for their technical assistance. I am especially grateful to: Dr. Peng Zhao Feng for his immense patience in teaching and demonstrating all proteomics techniques, as well as the innumerable encouragements and tremendous help given in more ways than one. Choy Meng Shyan for his wise and sensible advices, innovative suggestions, quick and perceptive remarks, and selfless guidance in many things throughout these years. My parents and friends for their understanding and support. This thesis is dedicated to my pet chicken, Jeffrey (deceased), with love. i Table of Contents Acknowledgements i Table of Contents ii Summary ix List of Tables xii List of Figures xiii List of Abbreviations xvi List of Publications xix Chapter 1 Introduction 1 1.1 General Introduction 2 1.2 Literature Review 7 1.2.1 Overview of the central nervous system 7 1.2.2 Neuronal Cell Death 7 1.2.2.1 Cell death in neurodegeneration 8 1.2.3 Niemann-Pick disease type C (NPC) 9 1.2.3.1 Overview of Niemann-Pick diseases 9 1.2.3.2 Characteristics of NPC 9 1.2.3.3 NPC and inhibition of intracellular cholesterol 10 transport 1.2.3.4 Niemann-Pick C proteins 11 1.2.4 Cholesterol 12 1.2.4.1 Cholesterol biosynthesis 13 1.2.4.2 Cholesterol homeostasis in the brain 13 1.2.5 Alzheimer’s disease (AD) 15 1.2.5.1 Molecular pathology of AD 15 1.2.5.2 Cleavage of APP into Aβ 16 1.2.5.3 Cholesterol and AD 16 1.2.6 Bridging NPC and AD 18 ii 1.2.7 U18666A: The widely-studied inhibitor of cholesterol 18 transport 1.2.7.1 Overview of hydrophobic amines and steroids 18 1.2.7.2 Class-2 amphiphiles 19 1.2.7.3 Studied effects of U18666A 19 Chapter 2 Materials and Methods 23 2.1 Materials 24 2.1.1 Buffers and solutions 24 2.1.2 Consumables 24 2.1.3 Drugs 25 2.1.4 Primary antibodies 25 2.1.5 Secondary antibodies 26 2.1.6 Protein assay kits 27 2.2 Methods 28 2.2.1 Preparation of mouse primary cortical neurons 28 2.2.2 Culture and maintenance of adherent cell lines 29 2.2.3 MTT assay 30 2.2.4 Release of lactate dehydrogenase (LDH) 31 2.2.5 LIVE/DEAD® viability and cytotoxicity assay 31 2.2.6 Annexin V staining 32 2.2.7 Hoechst staining 32 2.2.8 Transmission electron microscopy 33 2.2.9 Preparation of protein lysates for Western blotting 33 2.2.10 Sodium dodecyl sulfate polyacrylamide gel electrophoresis 34 (SDS-PAGE) 2.2.11 Western blotting 35 iii 2.2.12 Filipin staining 35 2.2.13 Cholesterol measurement 36 2.2.14 Fluorometric caspase activity measurement 37 2.2.15 Measurement of intracellular ATP and glutathione (GSH) 38 2.2.16 Measurement of proteasome activity 39 2.2.17 Measurement of mitochondrial membrane potential 39 2.2.18 Determination of intracellular oxidative stress by 2’,7’- 40 dichlorofluorescin diacetate (DCFH-DA) 2.2.19 Measurement of lipid peroxidation 41 2.2.20 Assessment of oxidized proteins 42 2.2.21 Analysis of DNA base modifications by gas chromatography- 43 mass spectrometry (GC-MS) 2.2.22 Measurement of β-amyloid (Aβ) 45 2.2.23 Determination of amyloid precursor protein (APP) derivatives 46 2.2.24 Immunocytochemistry 46 2.2.25 Measurement of cyclin-dependent kinase 5 (cdk5) activity 47 2.2.26 Isolation of total RNA 48 2.2.27 Quantification of total RNA and determination of RNA 48 integrity 2.2.28 Agarose gel electrophoresis 49 2.2.29 One-cycle complementary DNA (cDNA) synthesis 49 2.2.30 Cleanup of double-stranded cDNA 50 2.2.31 Synthesis of biotin-labeled complementary RNA (cRNA) 50 2.2.32 Cleanup of biotin-labeled cRNA 50 2.2.33 Fragmentation of cRNA for target preparation 51 iv 2.2.34 Microarray analysis 52 2.2.35 Eukaryotic target hybridization 52 2.2.36 Washing, staining and scanning of probe arrays 53 2.2.37 Microarray data analysis 54 2.2.38 Sample preparation for proteomics analysis 55 2.2.39 Cleanup and quantification of protein for proteomics analysis 55 2.2.40 Immobilized pH gradient (IPG) strip rehydration 56 2.2.41 First-dimension isoelectric focusing (IEF) 57 2.2.42 Equilibration of IPG strips 58 2.2.43 Second-dimension SDS-PAGE 58 2.2.44 Silver staining 59 2.2.45 Analysis of 2D gels 60 2.2.46 Enzymatic digestion of protein spots 61 2.2.47 Mass spectrometry 61 2.2.48 Database searching and identification of proteins 62 2.2.49 Statistical analyses 63 Chapter 3 Cellular Signaling of U18666A-Mediated Cell Death 64 Part I Apoptotic cell death and accumulation of intracellular free 65 cholesterol 3.1 Introduction 66 3.2 Results 71 3.2.1 U18666A induces significant cell death only in primary 71 cortical neurons 3.2.2 U18666A-mediated cell death in primary cortical neurons is 73 apoptotic v 3.2.3 Caspase-3 activation is correlated with U18666A-mediated 74 neuronal apoptosis 3.2.4 Cholesterol accumulates in primary cortical neurons treated 75 with U18666A 3.2.5 Depletion of intracellular cholesterol attenuates cell death in 76 U18666A-treated cortical neurons 3.3 Discussion 91 Part II Intracellular free radical production and β-amyloid 96 accumulation 3.4 Introduction 97 3.5 Results 101 3.5.1 U18666A treatment leads to loss of intracellular ATP and 101 glutathione (GSH), decrease in proteasome activity, and mitochondrial depolarization 3.5.2 U18666A leads to increased intracellular reactive oxygen 102 species (ROS) and lipid peroxidation in primary cortical neurons 3.5.3 U18666A induces protein oxidation and DNA damage in 103 primary cortical neurons 3.5.4 Co-treatment of U18666A with vitamin E attenuates cell death 104 during U18666A-induced oxidative stress in primary cortical neurons 3.5.5 U18666A leads to cleavage of APP and accumulation of Aβ in 105 primary cortical neurons 3.6 Discussion 117 Part III Role of caspases, calpains, kinases and cell cycle 125 regulatory proteins 3.7 Introduction 126 3.8 Results 130 vi 3.8.1 U18666A induces caspase and calpain activation in primary 130 cortical neurons 3.8.2 U18666A induces hyperphosphorylation of tau in primary 132 cortical neurons 3.8.3 U18666A decreases the kinase activity of Cdk5 and the 133 protein level of p35 in primary cortical neurons 3.8.4 U18666A decreases the protein levels of phosphorylated 133 GSK3, p44/42 MAPK and SAPK/JNK in primary cortical neurons 3.8.5 U18666A increases the protein levels of phospho-p53 and 134 activates cell cycle machinery in primary cortical neurons 3.9 Discussion 147 Chapter 4 Global Gene Expression Profile of U18666A-Mediated 156 Neuronal Apoptosis 4.1 Introduction 157 4.2 Results 162 4.2.1 Determination of RNA integrity and cRNA fragmentation 162 before target hybridization onto GeneChip® probe arrays 4.2.2 Differential gene expression after U18666A treatment in 163 primary cortical neurons 4.2.3 Cluster analysis of genes differentially expressed after 165 U18666A treatment in primary cortical neurons 4.2.4 Validation of differential gene expression through Western 170 blot analysis 4.3 Discussion 192 Chapter 5 A Proteomics Approach to the Study of U18666A- 207 Mediated Neuronal Apoptosis 5.1 Introduction 208 5.2 Results 214 vii 5.2.1 Optimization of experimental conditions for 2D gel 214 electrophoresis 5.2.2 Global protein expression profiles after U18666A treatment in 215 primary cortical neurons 5.2.3 Differentially-expressed proteins after U18666A treatment in 216 primary cortical neurons 5.2.4 Correlation of proteomics changes to corresponding gene 217 alterations in U18666A-treated cortical neurons 5.3 Discussion 233 Chapter 6 General Discussion and Future Work 242 6.1 General Discussion 243 6.2 Future Work 249 References 251 Appendices 289 Appendix I Media for preparation of mouse primary cortical neurons 290 Appendix II Buffers and reagents for SDS-PAGE and Western blotting 291 Appendix III Master mixes and buffers for microarray analysis 293 Appendix IV Gel formulations and solutions for proteomics analysis 295 viii Summary U18666A (3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one) is a well-known cholesterol transport-inhibiting drug widely used to mimic the cellular effects of Niemann-Pick disease type C (NPC).
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