INVESTIGATION OF CYTOTOXICITY AND ION FLUX INDUCED BY VARIOUS AGGREGATION STATES OF AMYLOID-BETA PEPTIDES by Panchika Prangkio A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biomedical Engineering) in The University of Michigan 2011 Doctoral Committee: Associate Professor Michael Mayer, Chair Associate Professor David Samuel Sept Assistant Professor Mohamed E.H. El-Sayed Assistant Professor Sarah Veatch ฝงชนกู ําเนิดคลาย้ คลึงกนั ใหญ่ย่อมเพศผิวพรรณ แผกบาง้ ความรู้อาจเรียนกนทั นั กนหมดั เวนแต้ ่ชวดั่ ีกระดาง้ ห่อนแก้ ฤาไหว - King Rama V Born men are we all and one, Brown black by the sun cultured. Knowledge can be won alike, Only the heart differs from man to man. -Translated by M.R. Seni Pramoj © Panchika Prangkio All rights reserved 2011 DEDICATION To my parents for your love, great support, and encouragement ii ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Michael Mayer, for providing me an opportunity to work with such an important research topic. I have learned a great deal of knowledge while being in his laboratory. I really appreciate his patience, guidance for my research, and his advices for my future career. I also would like to show my gratitude to the rest of my dissertation committees: Dr. David Sept, for his time and valuable suggestions on statistical analyses. Dr. Mohamed El-Sayed, for providing access to equipments in his laboratory, as well as great suggestions on my works. Dr. Sarah Veatch, for her insights and suggestions on my works. The work of this thesis would not have been possible without helps and supports from my colleagues, family and friends. I would like to thank these people whom I will address in the following: Dr. Ricardo Capone, for his mentoring at the beginning of my works, and providing a lot of great advices. Dr. Jerry Yang, for providing a number of molecules for the studies and also his valuable suggestions on my research. iii Dr. Raymond Scott Turner, and Dr. Indu Saluja, for their suggestions regarding clinical perspectives Dr. Divya Rao, for her guidance and valuable discussions in biochemistry perspectives Erik Yusko, for his helps with TEM imaging, and his suggestions on my thesis Brandon Bruhn, for his feedbacks on my thesis. Felipe Garcia Quiroz, Anna Sauer, Kevin Lance, Axel Fanget, for their contributions in data collection and helpful discussions on my research. The other current and former lab members, Kim Horger, Dr. Haiyan Liu, Yazan Billeh, Marian Adamson, Dr. Sheereen Majd, Dr. Jeff Uram, Dr. Daniel Estes. I really appreciate their helpful suggestions, especially feedbacks during group seminars, and their friendship over the years. I am really grateful to know all of you. My parents and family, for your unconditional love, supports, encouragement, and for shaping me the way I am today. Nirand Pisutha-arnond, for being supportive, encouraging, and also being a great help with programming. My friends, especially Niravun Pavenayotin, Thitiporn Sukaew, Phapanin Charoenpol, Rhatarporn Kessom, Sirarat Sarntivijai, and Tul Suriyaluck, for their friendship, supports and sharing wonderful moments in Michigan Lastly, I would like to thank the Thai government for a prestigious scholarship, which allows me to study abroad and experience the world. iv TABLE OF CONTENTS DEDICATION……………………………………………………………………….…………....ii ACKNOWLEDGEMENTS……………………………………………………………………...iii LIST OF FIGURES……………………………………………………………………………..xii LIST OF TABLES……………………………………………………………………............xvii LIST OF ABBREVIATIONS…………………………………………………………….…..xviii ABSTRACT…………………………………………………………………………….............xx CHAPTER 1. Introduction to Alzheimer’s Disease and Amyloid beta (Aβ) peptides..................................................................................................…..........1 1.1 General overview of Alzheimer’s disease (AD)……………..………….............1 1.2 Amyloid-β peptides and their roles in AD pathology…..…….........................2 1.2.1 Amyloid cascade hypothesis………....................................................2 1.2.2 Molecular properties of amyloid β.......................................................4 1.3 Studies of ion channel hypothesis.................................................................11 1.3.1 Ion channel hypothesis.......................................................................11 v 1.3.2 Membrane model systems and techniques to study the Aβ-lipid membrane Interaction........................................................................15 1.4 Summary of thesis.........................................................................................20 References…………………………………………………………..………………....22 2. Amyloid beta (Aβ) Ion Channels in Artificial Lipid Bilayers and Neuronal Cells…………………………………………………………...………………………..28 2.1 Introduction………………………………………………………………………...29 2.2 Results and discussion………………………….…………………………….....30 2.3 Conclusion……………………………….……….………………………………..46 2.4 Materials and methods………………………..………………………………….48 2.4.1 Chemicals………………………………………..………………………..48 2.4.2 Preparation of solutions of Aβ by purging HFIP with nitrogen gas….48 2.4.3 Quantification of HFIP concentrations by GC-MS analysis..………..49 2.4.4 Formation of planar lipid bilayers…………………………….…………49 2.4.5 Incorporation of Aβ into lipid membranes…………………..…............50 2.4.6 Current recordings……………………….…………..………….............51 2.4.7 Preparation of primary neurons………….……………………..............53 2.4.8 Cytotoxicity assay of HFIP with SH-SY5Y cells…………..…………...54 Acknowledgements………………………………………………………….….………….54 References………………………………………………………………………………….55 3. The Abundance of Aβ Oligomer (tetramers-18mers) Correlates with Pore Formation and Cytotoxicity………………………………………………………...61 3.1 Introduction……………………………………………………………….............62 vi 3.2 Results and discussion………………………………………………….............66 3.2.1 Pretreatment of Aβ with HFIP combined with HFIP removal is critical for the reproducibility of experiments……………………………..........66 3.2.2 Pore formation is maximal after incubating Aβ samples for 2-3 days…………………………………………………………………..…….66 3.2.3 Cytotoxicity is maximal after pre-incubating Aβ1-40 for ten days, and Aβ1-42 for one to ten days………………………………………………...71 3.2.4 During aggregation, the relative abundance of small Aβ oligomers decreases, of intermediate sizes undergoes a maximum, and of large sizes increases..…………….....................................................74 3.2.5 Pore formation and cytotoxicity correlate with the relative abundance of oligomers of intermediate size …….………….……………………..78 3.2.6 Multiple linear regression models describe the relationship between multiple Aβ oligomer species and pore formation or cell death.......80 3.2.7 Characterization of Aggregation of Aβ by thioflavin T binding assay, circular dichroism, and transmission electron microscopy……......84 3.3 Conclusion……..…………………..………………………………………….…..87 3.4 Materials and methods…………………………………………………………...89 3.4.1 Chemicals………………………………………………………….……...88 3.4.2 Lipid preparation…………………………………………………….…....89 3.4.3 Formation of planar lipid bilayers….…………………..………............89 3.4.4 Current recordings across planar lipid bilayer………………..............90 3.4.5 Preparation of Aβ…………….............................................................90 3.4.6 Cytotoxicity assay…...........................................................................93 3.4.7 SDS-PAGE/Western Blotting….........................................................94 vii 3.4.8 SDS-PAGE/silver staining…………...................................................95 3.4.9 Quantification of the relative abundance of Aβ Species....................96 3.4.10 ThT binding to aggregates of Aβ........................................................97 3.4.11 CD spectrometry…….….....................................................................97 3.4.12 Transmission electron microscopy imaging......................................98 3.4.13 Statistical analysis……….…………...................................................98 Chapter 3 Appendix………………………….………………………………………..99 References………..……………………………….………………………………….108 4. Membrane Disruption in Unilamellar Liposomes Induced by Amyloid-beta (Aβ) Peptides at Different Aggregation States……………...………………....113 4.1 Introduction………..………………………………………………….……...…..114 4.2 Results and discussion…………………………………………………….……116 4.2.1 Characterization of Aβ………..…………………...……………………116 4.2.2 Calibration curves of relative fluorescence ratio (FS/SR) as a function of pH………………………….………………………………...117 4.2.3 Leakage of protons induced by Aβ at various aggregation states……………………………………………………………………..121 4.2.4 Influence of membrane curvature on membrane permeabilization………………………………….………..……………126 4.2.5 Influence of lipid composition on membrane permeabilization………………………………………………………...127 4.2.6 Dye quenching assay…………………………………….…………….130 4.2.7 Membrane permeabilization of Aβ in planar lipid bilayers and liposomes are driven by different mechanisms ………….....……....132 viii 4.3 Conclusion..…..…………………….……......................................................136 4.4 Materials and methods………..…………………….……..............................137 4.4.1 Preparation of Aβ…..…………………….………...............................137 4.4.2 Preparation of liposomes…..……………………...............................137 4.4.3 Fluorescence measurement…..…………….…….............................138 Chapter4 Appendix..…………………………………………………………………140 Acknowledgements……………………………………………………..……………146 References………..….…………………………………………………………….…146 5. Self-Assembled,