University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 11-19-2015 Mass Spectrometry-Based Investigation of APP- Dependent Mechanisms in Neurodegeneration Dale Chaput University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Molecular Biology Commons Scholar Commons Citation Chaput, Dale, "Mass Spectrometry-Based Investigation of APP-Dependent Mechanisms in Neurodegeneration" (2015). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/5921 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Mass Spectrometry-Based Investigation of APP-Dependent Mechanisms in Neurodegeneration by Dale Chaput A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cell Biology, Microbiology, and Molecular Biology College of Arts and Sciences University of South Florida Co-Major Professor: Stanley M. Stevens, Jr, Ph.D. Co-Major Professor: Jaya Padmanabhan, Ph.D. Kristina Schmidt, Ph.D. Patrick Bradshaw, Ph.D. Date of Approval: November 2nd, 2015 Keywords: proteomics, phosphoproteomics, stable-isotope labeling, Alzheimer’s Disease Copyright © 2015, Dale Chaput Dedication I dedicate this to my parents, Tim and Lori, my brother, Noel, and my grandma, Sheila. Without your persistent support along the way, this would not have been possible. Thank you for always encouraging me and pushing me to challenge myself. Acknowledgements This was made possible by the guidance and support of both my mentors Dr. Stanley M Stevens, Jr., and Dr. Jaya Padmanabhan. I also want to thank Dr. Kristina Schmidt and Dr. Patrick Bradshaw for your advice and feedback during the development of this project. I also need to extend a special thank you to Dr. Kent Seeley and Dr. Harris Bell-Temin who helped me immensely through my first few years of graduate school. Finally, I’d like to thank Dr. Anne Messer for taking a chance and hiring an inexperienced undergrad and giving me the confidence to pursue my dreams. Thank you all for your advice and encouragement, teaching me to solve problems and think critically as I have grown into a young scientist. Table of Contents List of Tables ................................................................................................................................................ iv List of Figures ................................................................................................................................................ v Abstract ........................................................................................................................................................ vii Chapter 1 – Introduction ............................................................................................................................... 1 Alzheimer’s Disease ......................................................................................................................... 1 Amyloid Precursor Protein ............................................................................................................... 3 Proteolytic Processing ......................................................................................................... 4 Function .............................................................................................................................. 7 Hypotheses of Alzheimer’s Disease Pathogenesis ........................................................................... 8 Amyloid Cascade Hypothesis .............................................................................................. 9 Aberrant Cell Cycle Re-entry ................................................................................ 10 Proteomics & Alzheimer’s Disease ................................................................................................ 11 Mass Spectrometry and Proteomics ................................................................................. 11 Proteomic Studies Using Cell Models ............................................................................... 14 Proteomic Studies Using Animal Models .......................................................................... 17 C.Elegans Models ................................................................................................. 17 Mouse Models ..................................................................................................... 17 Rat Models ........................................................................................................... 20 Proteomic Studies Using Human Tissue ........................................................................... 20 Significance of Protein Phosphorylation in Alzheimer’s Disease ................................................... 24 Phosphoproteomics .......................................................................................................... 26 Phosphoproteomic Studies ............................................................................................... 27 Cell Models .......................................................................................................... 27 Mouse Models ..................................................................................................... 27 Human Tissue ....................................................................................................... 28 Summary of Approaches and Project Aims ................................................................................... 30 Chapter 2: Global Proteomic Analysis of a Cell Model of Alzheimer’s Disease .......................................... 32 Summary ........................................................................................................................................ 32 Introduction ................................................................................................................................... 33 B103 and B103-695 cells ................................................................................................... 33 Cell-Cycle Mediated Protein Expression Changes ............................................................ 33 Inflammation-Mediated Protein Expression Changes ...................................................... 34 Materials and Methods .................................................................................................................. 35 Protein Kinase A (PKA) Knockdown .................................................................................. 35 SILAC Labeling and Roscovitine, Taxol, and LPS Treatments ............................................ 35 LC-MS/MS and Data Analysis ............................................................................... 36 Cell Synchronization and Treatments ............................................................................... 36 Results and Discussion ................................................................................................................... 37 i Global Proteomic Analysis of B103 and B103-695: Additional Experiments .................... 37 Cell-Cycle Mediated Protein Expression Changes ............................................................ 39 Cell Synchronization and Preliminary Phosphorylation Analysis ......................... 41 Inflammation Mediated Protein Expression Changes ...................................................... 43 Chapter 3: Phosphoproteomic Analysis of a Cell Model of Alzheimer’s Disease and Validation Using Cultured Primary Neurons and Human Brain Tissue .................................................................. 46 Abstract .......................................................................................................................................... 46 Introduction ................................................................................................................................... 47 Materials and Methods .................................................................................................................. 49 B103 and B103-695 Phosphoproteomic Analysis ............................................................. 49 Cell Culture and SILAC Labeling ........................................................................... 49 Sample Preparation and Phosphopeptide Enrichment ....................................... 50 LC-MS/MS ............................................................................................................ 50 Database Searching and Consensus Motif Analysis ............................................. 50 Transgenic Mouse Tissue .................................................................................................. 51 Oligomeric Aβ42 Preparation ........................................................................................... 52 B103 and B103-695 Cell Culture and Aβ Treatment ......................................................... 52 Primary Neuron Culture and Aβ Treatment ..................................................................... 52 Nuclear Fractionation ....................................................................................................... 53 Human Brain Tissue .........................................................................................................