University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School March 2020 Mass Spectrometry Discovery-Based Proteomics to Examine Anti- Aging Effects of the Nutraceutical NT-020 in Rat Serum Samantha M. Portis University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the Bioinformatics Commons, and the Neurosciences Commons Scholar Commons Citation Portis, Samantha M., "Mass Spectrometry Discovery-Based Proteomics to Examine Anti-Aging Effects of the Nutraceutical NT-020 in Rat Serum" (2020). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/8279 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 Discovery-Based Proteomics to Examine Anti-Aging Effects of the Nutraceutical NT-020 in Rat Serum by Samantha M. Portis A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Medical Sciences with a concentration in Neuroscience Department of Molecular Pharmacology and Physiology College of Medicine University of South Florida Major Professor: Paul R. Sanberg, Ph.D., D.Sc Co-Major Professor: Paula C. Bickford, Ph.D. Kevin Nash, Ph.D. Dominic D’Agostino, Ph.D. Brent Small, Ph.D. Date of Approval: March 28, 2020 Keywords: aging, proteome, serum, inflammation, bioinformatics Copyright © 2020, Samantha M. Portis Dedication I dedicate this work to my family, Alan, Candace, Carolyn, and Jimmy, my brother, Patrick, and my partner, Will. Without your unfailing support and encouragement, this would not have been possible. Thank you for your love and support throughout this process. Acknowledgements This work was made possible by the guidance and support of my mentors, Dr. Paula C. Bickford and Dr. Paul R. Sanberg. I would also like to thank Dr. Dominic D’Agostino, Dr. Brent Small, and Dr. Kevin Nash for your advice, recommendations, and feedback during the development of this project. I also want to extend my sincere gratitude to Dr. Dale Chaput from the Proteomics Core Facility at the College of Arts and Sciences, who helped me immensely on this project, and my good friend Dr. Jared Ehrhart who was an invaluable source of support during my first few years of graduate school. I would also like to thank Dr. Edwin Weeber, who has moved on to another institution, but who nonetheless served as a valuable member of my committee while he was here and was one of the finest professors with whom I have studied. Thank you all for your advice and encouragement throughout my burgeoning academic career. Table of Contents List of Tables ................................................................................................................................. ii List of Figures .............................................................................................................................. iv Abstract ........................................................................................................................................ vi Chapter 1: Introduction ................................................................................................................ 1 Aging ................................................................................................................................ 1 Cell Autonomous and Cell Non-Autonomous Changes: Effects on the Stem Cell Niche ................................................................................................................... 4 Parabiosis Studies ................................................................................................ 5 Sex Differences .................................................................................................... 9 Graft Studies ...................................................................................................... 11 Nutraceuticals for Aging and Age-related Degenerative Diseases ................................ 12 Polyphenolic Compounds and Diet .................................................................... 14 NT-020 ............................................................................................................... 17 Summary of Approaches and Project Aims ................................................................... 28 Chapter 2: Proteomics ............................................................................................................... 30 Mass Spectrometry and Proteomics .............................................................................. 30 Tissue-Specific Proteomics Using Serum ...................................................................... 33 Sample Preparation and Albumin Depletion....................................................... 34 LC-MS/MS .......................................................................................................... 38 Analysis .............................................................................................................. 38 Chapter 3: Effects of Nutraceutical Intervention on Serum Proteins in Aged Rats .................... 40 Abstract .......................................................................................................................... 40 Introduction .................................................................................................................... 41 Materials and Methods ................................................................................................... 44 Animals ............................................................................................................... 44 Sample Preparation ............................................................................................ 45 LC-MS/MS .......................................................................................................... 46 Analysis .............................................................................................................. 46 Results ........................................................................................................................... 47 Change of Protein Expression as a Function of Age .......................................... 47 NT-020 Counteracts Upregulated Immune Response in Aged Rat Serum ........ 47 Autophagy Pathway Protein Cathepsin A is Upregulated with NT-020 .............. 48 Macrophage Stimulating 1 Decreases with NT-020 ........................................... 49 Complement System Proteins were Altered by NT-020 ..................................... 50 Discussion ...................................................................................................................... 51 Chapter 4: Conclusions and Future Directions .......................................................................... 71 Conclusions .................................................................................................................... 71 Overview ............................................................................................................ 71 i Aging and NT-020 .............................................................................................. 72 Serum Data ........................................................................................................ 73 Autophagy and Aging ......................................................................................... 75 Macrophage Stimulating 1 and Complement Proteins ....................................... 76 Apolipoproteins ................................................................................................... 77 Future Directions ............................................................................................................ 78 Study Limitations ................................................................................................ 78 Future Studies .................................................................................................... 79 References ................................................................................................................................. 81 Appendix A: The Role of Glycogen Synthase Kinase-3 Signaling in Neruodevelopment and Fragile X Syndrome .............................................................................................. 91 Appendix B: GFAP Expression and Social Deficits in Transgenic Mice Overexpressing Human sAPPα .................................................................................................... 100 Appendix C: Potential Autoepitope within the Extracellular Region of Contactin-Associated Protein-like 2 in Mice .......................................................................................... 114 ii List of Tables Table 1. Expression of proteins in old rat serum compared to young ........................................ 56 Table 2. Expression of proteins in old NT-020 serum versus old control ................................... 56 Table 3. Full list of proteins detected ......................................................................................... 56 iii List of Figures Figure 1. Prevalence of disease by age ........................................................................................ 1 Figure 2. NT-020 promotes CD34+ cell proliferation .................................................................