THE MECHANISM OF APOLIPOPROTEIN E IN THE PROTEOLYTIC DEGRADATION OF Aβ By CHUNG-YING DANIEL LEE Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: GARY E. LANDRETH Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY May, 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Chung-Ying Lee candidate for the Ph.D. degree *. (signed) Dr. Evan Deneris (chair of the committee) Dr. Gary Landreth Dr. Bruce Lamb Dr. Jonathan Smith (date) January 27, 2012 *We also certify that written approval has been obtained for any proprietary material contained therein. -1- TABLE OF CONTENTS List of figures ......................................................................................................................5 Acknowledgement ..............................................................................................................7 Abstract ...............................................................................................................................9 Chapter 1 Introduction ................................................................................................... 11 Epidemiology and genetics of AD ................................................................................ 13 Aβ production and the amyloid cascade hypothesis ..................................................... 15 Apolipoprotein E ........................................................................................................... 18 Microglia ....................................................................................................................... 27 Aβ clearance mechanisms ............................................................................................. 29 Soluble Aβ clearance ................................................................................................ 30 Proteolysis of soluble Aβ ...................................................................................... 31 The role of apoE in soluble Aβ clearance ............................................................. 33 Fibrillar Aβ clearance ................................................................................................ 34 Microglial Aβ receptor complex ........................................................................... 35 Role of inflammation in fibrillar Aβ clearance ..................................................... 37 Antibody- and complement-mediated clearance of fibrillar Aβ ........................... 39 Cholesterol and Alheimer’s Disease ............................................................................. 41 Cholesterol in the CNS ................................................................................................. 42 Cholesterol synthesis .................................................................................................... 43 Cholesterol uptake and intracellular trafficking ........................................................... 44 Cellular cholesterol homeostasis .................................................................................. 45 Cholesterol excretion from the CNS ............................................................................. 47 -2- Cholesterol and Aβ production ..................................................................................... 48 Cholesterol homeostasis-related genes and AD ............................................................ 50 ATP-Cassette Transporter A1 .................................................................................... 51 Liver X Receptors ..................................................................................................... 52 Niemann-Pick Type C Proteins ................................................................................. 54 Conclusion and research objectives .............................................................................. 55 Figures .......................................................................................................................... 58 Chapter 2 Apolipoprotein E Promotes β-amyloid Trafficking and Degradation by Modulating Microglial Cholesterol Levels ....................................................................63 Abstract ......................................................................................................................... 64 Introduction ................................................................................................................... 65 Materials and methods .................................................................................................. 68 Results ........................................................................................................................... 76 Facilitation of Aβ degradation is a common feature of HDL apolipoproteins .......... 76 Facilitation of Aβ degradation relies on the cholesterol efflux function of apolipoproteins .......................................................................................................... 77 Degradation of Aβ is influenced by the cellular cholesterol levels .......................... 79 The transcription of Aβ degrading enzymes is not regulated by cholesterol levels.. 80 Cholesterol levels regulate Aβ transport ................................................................... 81 Cholesterol regulates the trafficking of Aβ-containing endosomes via the recruitment of Rab7 ...................................................................................................................... 82 Discussion ..................................................................................................................... 84 Figures .......................................................................................................................... 91 -3- CHAPTER 3 ApoE Acts as a chaperone to promote IDE-mediated Aβ degradation105 Abstract ....................................................................................................................... 106 Introduction ................................................................................................................. 107 Materials and Methods ................................................................................................ 110 Results ......................................................................................................................... 115 IDE-mediated Aβ degradation is facilitated in the presence of apoE ..................... 115 ApoE acts as a chaperone to facilitate Aβ degradation by IDE .............................. 116 Lipidation status of apoE regulates its ability to promote IDE-mediated Aβ degradation .............................................................................................................. 118 ApoE isoforms differentially facilitate Aβ degradation by IDE ............................. 120 Aβ12-28 promotes Aβ degradation by IDE through enhancing allosteric reaction 121 Discussion ................................................................................................................... 122 Figures ........................................................................................................................ 129 Chapter 4 Discussion ....................................................................................................138 Effect of cholesterol modulation on Aβ levels in vivo ............................................... 142 Cholesterol derivatives and ACAT inhibitors in Aβ degradation ............................... 145 Resident and infiltrating microglia in the clearance of Aβ ......................................... 147 Clearance of Aβ by other glial cells ............................................................................ 151 Anti-inflammatory properties of apoE ........................................................................ 152 Conclusion .................................................................................................................. 153 Figures ........................................................................................................................ 155 Chapter 5 Literatures cited ..........................................................................................159 -4- LIST OF FIGURES Figure 1.1: APP mutations associated with early-onset Alzheimer’s disease ....................59 Figure 1.2: Proteolytic processing pathways of APP .........................................................60 Figure 1.3: Model of the structure of lipid-free apoE and the formation of an α-helical hairpin conformation in apoE-HDL-like particles. ............................................................62 Figure 2.1: Facilitation of Aβ degradation is a common feature of apolipoproteins .........91 Figure 2.2: Facilitation of Aβ degradation relies on the cholesterol efflux function of apolipoproteins ...................................................................................................................92 Figure 2.3: Blocking cholesterol efflux impairs the ability of ApoE to enhance Aβ degradation .........................................................................................................................95 Figure 2.4: Reducing cellular cholesterol levels promotes Aβ degradation ......................96 Figure 2.5: Increasing cellular cholesterol levels impairs Aβ degradation ........................97 Figure 2.6: Direct manipulation