BETA-SECRETASE TRANSGENIC MICE: EFFECTS OF BACE1 AND BACE2 ON ALZHEIMER’S DISEASE PATHOGENESIS by MATTHEW J. CHIOCCO Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Bruce T. Lamb Department of Genetics CASE WESTERN RESERVE UNIVERSITY May, 2005 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents Chapter 1: Introduction and Research Aims .............................................................10 Clinical Presentation of AD.......................................................................................10 AD Epidemiology......................................................................................................16 APP Processing and AD Genetics..............................................................................22 Amyloid Cascade Hypothesis ....................................................................................27 APP Secretases..........................................................................................................32 BACE1: The primary β-secretase ..............................................................................35 Regulation of β-secretase...........................................................................................39 Research Aims...........................................................................................................46 Chapter 2: Altered Amyloid-β Metabolism and Deposition in Genomic-Based β- secretase Transgenic Mice...........................................................................................50 ABSTRACT..............................................................................................................51 INTRODUCTION.....................................................................................................52 MATERIALS AND METHODS...............................................................................54 RESULTS .................................................................................................................61 DISCUSSION ...........................................................................................................73 Chapter 3: Spatial and Temporal Control of Age-related APP Processing in Genomic-based β-secretase Transgenic Mice.............................................................79 ABSTRACT..............................................................................................................80 INTRODUCTION.....................................................................................................81 MATERIALS AND METHODS...............................................................................83 1 RESULTS .................................................................................................................87 DISCUSSION ...........................................................................................................97 Chapter 4: Characterization of APP Processing in Genomic-based BACE2 Transgenic Animals...................................................................................................103 INTRODUCTION...................................................................................................104 MATERIALS AND METHODS.............................................................................105 RESULTS ...............................................................................................................109 DISCUSSION .........................................................................................................116 Chapter 5: Conclusions and Future Directions........................................................119 Summary.................................................................................................................119 Conclusions.............................................................................................................121 Future Directions.....................................................................................................127 Appendix....................................................................................................................137 Bibliography ..............................................................................................................139 2 List of Tables Chapter 1: Table 1-1: List of AD-associated APP mutations and the proposed effect on APP cleavage 25 Chapter 3: Table 3-1: Summary of alterations in brain-regional BACE1 expression and APP processing 99 3 List of Figures Chapter 1: Figure 1-1: APP protein processing pathway 23 Figure 1-2: Amyloid cascade hypothesis 29 Chapter 2: Figure 2-1: Generation of genomic-based human BACE1 transgenic mice 63 Figure 2-2: Human BACE1 mRNA and protein expression and quantitation 65 Figure 2-3: Human BACE1 immunohistochemical localization 67 Figure 2-4: APP processing and Aβ metabolism in BACE1 transgenics 69 Figure 2-5: Analysis of Amyloid-β deposition in BACE1 transgenic animals 70 Figure 2-6: Amyloid-β production and BACE1 protein expression by brain region 72 Chapter 3: Figure 3-1: Human BACE1 expression and Aβ production in neonatal and adult BACE1 transgenic mice 89 Figure 3-2: Human BACE1 protein expression by brain region in neonatal and adult BACE1 transgenic mice 91 Figure 3-3: Amyloid-β metabolism by brain region in neonatal and adult BACE1 and APP double transgenic mice 93 4 Figure 3-4: Impact of BACE1 expression on Aβ production by brain region in adult animals 95 Figure 3-5: Amyloid-β deposition profile in aged BACE1xAPP animals 96 Chapter 4: Figure 4-1: Generation of genomic-based human BACE2 transgenic mice and expression of human BACE2 mRNA 111 Figure 4-2: APP processing in BACE2 transgenics 113 Figure 4-3: Analysis of Amyloid-β deposition in BACE2 transgenic animals 115 Appendix: Figure A1: Aβ production by brain region in hemizgyous BACE1 x homozygous APP transgenics 137 Figure A2: Profile of Aβ species in human BACE1 transgenics by immunoprecipitation/mass spectrometry 138 5 Acknowledgements: I would like to start by thanking my advisor, Bruce Lamb, for “showing me the ropes” and teaching me how to tackle a scientific project. From the very beginning of my rotation project in the lab, Bruce has exhibited unlimited patience through my developing years as a young scientist. He always kept an optimistic perspective when the project took some unfortunate turns as well as congratulated me whenever I made advances. He managed to keep me motivated with encouraging words that kept me focused on my goals as a student. I have been grateful for his willingness to share his life experiences as a student, providing an incredible example of how to achieve success in all my scientific endeavors. I would also like to acknowledge all members of the laboratory who have helped me throughout my graduate training and helped to make lab an enjoyable professional environment. First, Laura Kulnane has been a superb lab mate who manages the lab with extraordinary precision, dedication, and a willingness to help out wherever possible. I am grateful to Brian Hock and Emily Lehman, my first baymate, as the senior lab mates who helped form my scientific foundation as great teachers and friends early in my graduate career. I would also like to thank Nick Varvel for his assistance with critical immunohistochemical experiments and someone who helped ease the pressures towards the end of my graduate training through both insightful scientific and light-hearted non- scientific discussions. Finally, I am especially grateful to Karen Mann for not only her perceptive comments on my writing, but also her energy and positive attitude that have been a daily source of motivation and encouragement. 6 I would like to thank my thesis committee, Mitch Drumm, Joseph Nadeau, Sanjay Pimplikar, and Helen Salz for their advice and guidance throughout the course of my project. I am especially grateful to Helen Salz and Sanjay Pimplikar who motivated and challenged me to “tell the story” of my project. I am also grateful to those friends at CWRU who helped graduate school life more enjoyable. I would also like to thank my family, especially my parents, for their patience and guidance over the past six years. Through their love and support, I was constantly reminded to keep striving for my goals and to always “keep smiling.” Finally, my wife, Laura, has been the bastion of support throughout my graduate career. I am eternally grateful for her unwavering support, patience, and constant source of tranquility. She has taught me how to be strong, how to endure, and how to persevere through all the challenges over the years. For her love and for inspiring me to achieve my goals as a graduate student, I will always be thankful. 7 Beta-secretase Transgenic Mice: Effects of BACE1 and BACE2 on Alzheimer’s Disease Pathogenesis Abstract by Matthew J. Chiocco Numerous investigations have demonstrated that genetic