SINGLE-MOLECULE MICROSCOPY STUDIES OF INTERACTIONS BETWEEN ALZHEIMER’S AMYLOID-β (1-40) AND AMYLOID-β (1-42) ON THE MEMBRANE by Chun-Chieh Chang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biophysics) in the University of Michigan 2014 Doctoral Committee: Professor Ari Gafni, Co-Chair Professor Duncan G. Steel, Co-Chair Assistant Professor Michael M. A. Sutton Assistant Professor Sarah Veatch Professor Kristen J. Verhey © Chun-Chieh Chang 2014 DEDICATION This work is dedicated to my parents, Chien-Chen Chang and Bi-Chu Kuan, who raised a little boy and support him flying oversea to pursue his dream. ii ACKNOWLEDGMENTS I would like to express my sincere appreciation and thanks to my gracious and respectful advisors, Professor Ari Gafni and Duncan Steel. Their training in critical thinking, supports in my future career, and mentoring in the attitude of becoming a better scientist have nourished me during the past five years. Their sense of humor also makes me enjoy my work and be creative. I would also like to thank all my thesis committee, Professors Kristen Verhey, Michael Sutton, and Sarah Veatch for serving as the scientific council and providing valuable ideas. Brilliant, fun and thoughtful co-workers also fill up the joy in Chem 4070. I would like to thank the current graduate student Kian Kamgar-Parsi for discussions in experimental design, programming, and fun YouTube clips as well. Thanks to undergraduate Kaylee Van Dommelen for the help of culturing SH-SY5Y cell lines. I would like to express my deep and grateful appreciations to previous Gafni/Steel lab members who are also lifelong friends; Kathleen Wisser and Joseph Schauerte, Ph.D., were great co-workers and mentors who taught me everything that I needed to learn to become an independent researcher. They also served as a family who took care of my life outside the research. I would like to thank Dr. Robin Johnson for valuable scientific discussions and establishing the first single-molecule live-cell imaging protocol in the lab. Dr. Johnson’s research attitude is what I always look up to. Thanks to Dr. Pam Wong and Pavithra Aravamudhan for bringing the fun to every Gafni/Steel lab’s social event. Without all these beloved members, my research life in Chem 4070 could not be accomplished with blessed. Collaborators are an essential part for great research. I would like to thank John Christian Althaus and Cynthia J. L. Carruthers from Professor Sutton’s lab for preparing rat’s primary neuronal cells with excellent quality and consistency. I would also like to thank Dr. Elin Edwald for thorough guidance for the particle tracking algorithm and data iii interpretation even during the preparation period of her dissertation defense. I appreciate Dr. Krishnan Raghunathan for the generous help of the dissertation preparation, and I thank to people in Veatch’s lab for sharing the cell culture room. I also would like to thank David Rowland for the help of simulating particle diffusion movies which validated the particle tracking algorithm and analysis. Being part of the Biophysics and a student at the University of Michigan is proud. I have to thank all the Biophysics 2009 fellows, Teppei Shirakura, Leela Ruckthong, Josh Jasensky, Veronica Taylor, Stephen Norris, and Anthony Mustoe for fighting for the homework and sharing research and fun. I would like to thank the Biophysics staff, Sara Grosky and Ann Titus, for taking care of my student life. As a teaching assistant for five semesters, I have to thank to all the biophysics faculties and students for giving me the opportunity to learn and grow. I would especially thank all the friends from Michigan Taiwanese Student Association, Optical Society at the University of Michigan, and Taiwan Biomedical Journal Club. These five years in Michigan cannot be more wonderful without those memorable funs and supports. Finally, family is everything. I would like to give my sincere thanks to my parents for their unconditional love and support for almost three decades. I would also like to thank my caring, loving, and supportive wife, Tzu-Hui Wang. Your sweet laughter and encouragements are the driving force toward the completion of my PhD and our better future. iv TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGMENTS ................................................................................................. iii LIST OF FIGURES .......................................................................................................... vii ABSTRACT ....................................................................................................................... ix CHAPTER 1 - INTRODUCTION ...................................................................................... 1 1-1 Alzheimer’s Disease at a Glance .............................................................................. 1 1-2 Amyloid-β Production .............................................................................................. 2 1-3 Structural Differences between Amyloid-β Isoforms ............................................... 3 1-4 Membranes Accelerate Aβ Aggregation ................................................................... 5 1-5 Amyloid Hypothesis and Aβ Toxicity ...................................................................... 6 1-6 Aβ42:Aβ40 Ratio, Interaction, and Toxicity ............................................................ 9 1-7 Thesis Summary ...................................................................................................... 11 CHAPTER 2- METHODS ................................................................................................ 13 2-1 Rationale for the Selection of Single-Molecule Microscopy .................................. 13 2-2 Model Membrane System ....................................................................................... 14 2-2-1 Preparation of a Supported Lipid Bilayer ........................................................ 15 2-2-2 Glass Cleaning ................................................................................................. 16 2-3 Total Internal Reflection Fluorescence (TIRF) Microscopy .................................. 16 2-3-1 TIRF Data Acquisition ..................................................................................... 18 2-3-2 Fluorescence Recovery after Photobleaching (FRAP) .................................... 18 2-3-3 Single Particle Tracking and Lateral Diffusion Analysis ................................ 20 2-3-4 Oligomer-Size Calibration using Photobleaching or Fluorescence Intensity .. 21 2-4 Confocal Microscopy .............................................................................................. 23 2-5 Förster Resonance Energy Transfer (FRET) .......................................................... 29 2-6 Peptide Preparation ................................................................................................. 35 v 2-7 Primary Rat Hippocampal Cell Culture .................................................................. 35 CHAPTER 3 - STUDIES OF Aβ40 AND Aβ42 INTERACTIONS ON A PLANAR LIPID BILAYER .............................................................................................................. 37 3-1 Motivation for Model Membrane Studies ............................................................... 37 3-2 Aβ40, Aβ42, and the Aβ40:Aβ42 mixture primarily exist as monomers in solution at nanomolar concentrations and do not exhibit additional oligomerization over 120 hours .............................................................................................................................. 39 3-3 Membrane-bound Aβ monomers and some dimers are mobile and tightly associate with the membrane ........................................................................................................ 40 3-4 Membranes immobilize some dimers and all higher-order oligomers ................... 49 3-5 Free Aβ40 is more readily incorporated into existing immobile oligomers than free Aβ42, whereas oligomers of the Aβ40:Aβ42 mixture remain unaltered ...................... 51 CHAPTER 4 - STUDIES OF Aβ40 AND Aβ42 INTERACTIONS ON THE PRIMARY NEURON NEURITES...................................................................................................... 56 4-1 Motivation for study of Stoichiometry of Aβ40 and Aβ42 on the primary neuron neurites .......................................................................................................................... 56 4-2 FRET Confirms Aβ40 and Aβ42 Form Heterogeneous Species on Neurites ......... 57 4-3 Aβ40 and Aβ42 Form Mainly Dimers on Neurites and Show Little Growth upon Incubation ...................................................................................................................... 59 4-4 Number of Heterogeneous Species (i.e., oligomers comprised of both Aβ40 and 42) Increases Over Time due to Continuous Binding of Aβ42 to Heterogeneous Oligomers on the Neurites .............................................................................................................. 61 4-5 Heterogeneous Oligomers are Larger than Homogeneous Oligomers ................... 64 4-6 Determining the Relative Fractions of 40 and 42 in Heterogeneous Oligomers .... 66 CHAPTER 5 -DISCUSSION AND CONCLUSIONS ..................................................... 69 5-1 Introduction ............................................................................................................. 69 5-2 Aβ Oligomerization on the model membrane ......................................................... 69 5-3 Aβ Oligomerization on the neuronal cells .............................................................. 78 5-4 Conclusion from the model membrane to the cell membrane ................................ 84 5-5 Future Directions .................................................................................................... 87 REFERENCES ................................................................................................................. 88 vi LIST OF FIGURES Figure 1-1. Aβ production pathway .................................................................................... 3 Figure 1-2. Sequence of Aβ40 and Aβ42 ........................................................................... 5 Figure 1-3. Schematic diagram depicting three possible mechanisms of Aβ-induced membrane damage: carpeting, pore formation and the detergent effect. ............................ 9 Figure 2-1. Formation of lipid bilayer on a cover glass. ................................................... 16 Figure 2-3. Lipid bilayer remains uniform and diffusible after incubating with Aβ for 150 hours .................................................................................................................................. 20 Figure 2-4. Two photobleaching steps indicate that the oligomer is a dimer. .................. 22 Figure 2-6. Fluorescence lifetime imaging microscopy and lifetime fitting .................... 26 Figure 2-7. Sample with Aβ40-HL555 shows shorter fluorescence lifetime spots than the control sample ................................................................................................................... 28 Figure 2-8. FRET is only detected when Aβ40 is mixed with Aβ42 ................................ 31 Figure 2-9. Distance of a hetero-dimer (N-terminus to N-terminus) on neurite and the glass surface ...................................................................................................................... 33 Figure 2-10. Aβ oligomer size is determined by its fluorescence intensity ...................... 35 Figure 3-1. Oligomer size distributions for 4 nM Aβ40, Aβ42, and a 1:1 mixture of Aβ40:Aβ42 in solution ...................................................................................................... 39 Figure 3-2. Identifying mobile particles. .......................................................................... 41 Figure 3-3. The trajectories of mobile species .................................................................. 42 Figure 3-4. MSD curve analysis. ...................................................................................... 44 Figure 3-5. Diffusion coefficient and particle motion. .................................................... 45 Figure 3-6-1. Aβ40’s MSD-Tau curves ............................................................................ 46 Figure 3-6-2. Aβ42’s MSD-Tau curves ............................................................................ 47 vii Figure 3-6-3. Mixed species’ MSD-Tau curves ............................................................... 48 Figure 3-7. A comparison of the size distribution between Aβ in solution and membrane- bound immobile Aβ indicates that the membrane selectively incorporates dimers and higher-order oligomers rather than the monomer ............................................................. 50 Figure 3-8. Extensive wash of the lipid bilayer does not affect the population of immobile oligomers........................................................................................................................... 52 Figure 3-9. Size distribution and density of membrane-bound immobile Aβ .................. 54 Figure 4-1. Mixed Aβ40-HL555 and Aβ42-HL647 are incubated with neurons and show FRET ................................................................................................................................. 58 Figure 4-2. Aβ40 or Aβ42 oligomers form mainly dimers and show little growth on neuritis............................................................................................................................... 60 Figure 4-3. Heterogeneous species increases over time due to continuous binding of Aβ42 to the neurites .......................................................................................................... 62 Figure 4-4. Diagram of the number of Aβ40 and Aβ42 oligomers on the neurites .......... 63 Figure 4-5. Heterogeneous oligomers are larger than homogeneous oligomers .............. 65 Figure 4-6. Aβ42 fraction in the heterogeneous oligomers increases dramatically over time but not Aβ40 ............................................................................................................. 67 Figure 5-1. SEM image of pre-cleaned cover glass shows no detectable defect .............. 71 Figure 5-2. Low-lying structures in terms of potential energy are shown for the Aβ42 monomer ........................................................................................................................... 72 Figure 5-3. Dimer structures in the membrane ................................................................. 73 Figure 5-4. Primary inserted conformations of the Aβ peptide ........................................ 74 Figure 5-5. Models that summarize the structural properties of monomer, dimer, and different Aβ isoforms on the membrane. .......................................................................... 78 Figure 5-6. Summary of synergistic interactions between Aβ40 and Aβ42 on the neurons ........................................................................................................................................... 83 Figure 5-7. Hypothesis that explains how Aβ40 and Aβ42 interact on the cell membrane ........................................................................................................................................... 86 viii ABSTRACT Two amyloid-β peptides (Aβ40 and Aβ42) feature prominently in the extracellular brain deposits associated with Alzheimer’s disease. While Aβ40 is the prevalent form in the cerebrospinal fluid, the fraction of Aβ42 increases in the amyloid deposits over the course of disease development. The low in vivo concentration (pM-nM) and metastable nature of Aβ oligomers have made identification of their size, composition, cellular binding sites and mechanism of action challenging and elusive. Furthermore, recent studies have suggested that synergistic effects between Aβ40 and Aβ42 alter both the formation and stability of various peptide oligomers and as well as their cytotoxicity. These studies often utilized Aβ oligomers that were prepared in solution and at μM peptide concentrations. Here we utilized various single-molecule microscopies to follow peptide binding and association on the model membrane as well as the primary cultured neurons under physiological Aβ concentrations. At these concentrations monomers constitute the dominant Aβ species in solution. These monomers tightly associate with the model membrane and are highly mobile, whereas trimers and higher-order oligomers are largely immobilized. The Aβ dimer appears to exist in a metastable state that can be either mobile or immobile. Additionally, oligomer growth on the model membrane occurs more rapidly for Aβ40 than for Aβ42 while oligomer growth is largely inhibited for a 1:1 Aβ40:Aβ42 mixture. Interestingly, when the neuronal cells were exposed to a 1:1 mixture of nM Aβ40:Aβ42, significantly larger membrane-bound oligomers developed compared to those formed from either peptide alone. Fluorescence resonance energy transfer experiments at the single molecule level reveal that these larger oligomers contained both Aβ40 and Aβ42, but that the growth of these oligomers was predominantly by addition of Aβ42. Both pure peptides form very few oligomers larger than dimers, but either cell membrane bound Aβ40/42 complex, or ix