Pleiotropic Mechanisms of Statin Action in Alzheimer's Disease
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PLEIOTROPIC MECHANISMS OF STATIN ACTION IN ALZHEIMER’S DISEASE By STEPHEN MARC OSTROWSKI Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Gary Landreth Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY January, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________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 TABLE OF CONTENTS .............................................................................................- 1 - LIST OF FIGURES ......................................................................................................- 3 - ACKNOWLEDGEMENTS .........................................................................................- 4 - ABSTRACT...................................................................................................................- 5 - CHAPTER1: INTRODUCTION................................................................................- 7 - ALZHEIMER’S DISEASE ................................................................................................ - 8 - Clinical and Pathological Features of AD. ............................................................- 8 - Aβ and the Amyloid Hypothesis. ...........................................................................- 14 - Production of Aβ...................................................................................................- 16 - Pathogenic Mechanisms of Aβ..............................................................................- 20 - STATINS ..................................................................................................................... - 23 - Development of statins as choleseterol lowering agents. .....................................- 24 - Efficacy, safety and specificity of statins. .............................................................- 28 - Cholesterol, Statins, and AD.................................................................................- 30 - Cholesterol-independent effects of statin action...................................................- 35 - Statins, Protein Isoprenylation, and Ras Superfamily GTPases...........................- 38 - RESEARCH GOALS ..................................................................................................... - 44 - REFERENCES ......................................................................................................... - 47 - CHAPTER 2: STATINS REDUCE AMYLOID-BETA PRODUCTION THROUGH INHIBITION OF PROTEIN ISOPRENYLATION................................................- 76 - ABSTRACT.............................................................................................................. - 77 - INTRODUCTION .................................................................................................... - 79 - EXPERIMENTAL PROCEDURES ......................................................................... - 83 - RESULTS ................................................................................................................. - 87 - DISCUSSION........................................................................................................... - 97 - FIGURES................................................................................................................ - 105 - REFERENCES ....................................................................................................... - 128 - CHAPTER 3: MONITORING STATIN INHIBITION OF PROTEIN ISOPRENYLATION WITH 2D GEL ELECROPHORESIS ..............................- 137 - ABSTRACT............................................................................................................ - 138 - INTRODUCTION .................................................................................................. - 138 - EXPERIMENTAL PROCEDURES ....................................................................... - 140 - RESULTS ............................................................................................................... - 142 - DISCUSSION......................................................................................................... - 143 - FIGURES................................................................................................................ - 145 - REFERENCES ....................................................................................................... - 157 - CHAPTER 4: DISCUSSION ..................................................................................- 159 - RESEARCH CONCLUSIONS............................................................................... - 160 - - 1 - FUTURE DIRECTIONS ........................................................................................ - 163 - REFERENCES ....................................................................................................... - 174 - BIBLIOGRAPHY.....................................................................................................- 182 - - 2 - LIST OF FIGURES CHAPTER 1 – Introduction Figure 1 48 Figure 2 49 Figure 3 50 CHAPTER 2 - Statins Reduce Amyloid-Beta Production through Inhibition of Protein Isoprenylation Figure 1 108 Figure 2 110 Figure 3 112 Figure 4 114 Figure 5 116 Figure 6 118 Figure 7 120 Figure 8 122 Supplemental Figure A 124 Supplemental Figure B 126 Supplemental Figure C 128 CHAPTER 3 – Monitoring Statin Inhibition of Protein Isoprenylation with 2D Gel Electrophoresis Table 1 148 Figure1 149 Figure2 151 Figure3 153 Figure4 155 Figure5 157 - 3 - ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Gary Landreth. His enthusiasm for science is infectious. I would like to thank the members of my thesis committee: Dr. Sophia Sundararajan, Dr. Evan Deneris, and Dr. Robert Miller. Their enthusiasm and support has been greatly appreciated. The Landreth lab is truly a special and exciting place to work. I would like to thank each and every member of the lab, past and present, for making the environment a great place. It has been a joy and an honor to work with such a wonderful group of people. To my Mom and Dad, and my brothers Mike and Bill, your love and encouragement means more to me then you will ever know. I would also like to thank all my friends that have been with me through the good times and bad times while I’ve been in Cleveland. -4- Pleiotropic Mechanisms of Statin Action in Alzheimer’s Disease Abstract by STEPHEN MARC OSTROWSKI Epidemiological evidence suggests that long term treatment with the cholesterol- lowering HMG-CoA reductase inhibitors, or statins, decreases the risk for developing Alzheimer’s Disease (AD). However, statin-mediated AD protection cannot be fully explained by reduction of cholesterol levels. In addition to their cholesterol lowering effects, statins act pleiotropically to lower the concentrations of isoprenoid intermediates, such as geranylgeranyl pyrophosphate and farnesyl pyrophosphate. The Rho and Rab family small G-proteins require addition of these moieties for normal protein localization and function. In neuroblastoma cell lines, treatment with statins at high doses inhibits the membrane localization of Rho and Rab proteins, without affecting cellular cholesterol levels. Importantly, we show for the first time that at low, physiologically relevant, doses statins preferentially inhibit the isoprenylation of only a subset of GTPases. In addition, -5- we employ 2D gel electrophoresis to directly monitor the isoprenylated versus non- isoprenylated forms of the proteins, a technique that will be useful for further studies of statin inhibition of protein isoprenylation. The amyloid precursor protein (APP) is proteolytically cleaved to generate beta- amyloid (Aβ), which is the major component of senile plaques found in AD. We show that inhibition of protein isoprenylation by statins causes the accumulation of APP within the cell through inhibition of protein geranylgeranylation. Toxin A, a specific and robust inhibitor of Rho family GTPases, has no effects on APP trafficking, suggesting that statins act to inhibit the function of Rho family proteins. In addition, effects of statins on APP trafficking and Aβ production are strikingly similar to those reported previously after the inhibition of Rab1b. Moreover, inhibition of Rho family protein function reduces levels of APP C- terminal fragments (CTFs), resulting in decreased Aβ secretion. We demonstrate that the reductions of CTFs is due to novel regulaton of lysosomal dependent degradation. In summary, we show that statins selectively inhibit GTPase isoprenylation at clinically relevant doses, leading to reduced Aβ production in an isoprenoid-dependent manner. These studies provide insight into the mechanisms by which statins may reduce AD pathogenesis. -6- CHAPTER1: INTRODUCTION - 7 - Alzheimer’s Disease Alzheimer’s Disease (AD) is the leading cause of dementia in the elderly. While it is not only a devastating condition