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The Role of Phospholipase D1 in Trafficking and Processing Of The role of phospholipase D1 in trafficking and processing of amyloid precursor protein Kimberly S. Point du Jour Submitted in the partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2013 1 © 2013 Kimberly S. Point du Jour All Rights Reserved ABSTRACT The role of phospholipase D1 in trafficking and processing of amyloid precursor protein Kimberly S. Point du Jour Growing evidence indicates that intracellular signaling lipids control the trafficking and processing of amyloid precursor protein (APP) with major implications for pathological and behavioral manifestations associated with Alzheimer's disease (AD). One such lipid is phosphatidic acid (PA), which mediates membrane trafficking and is produced by a variety of enzymes, including phospholipase D (PLD). We previously demonstrated that a PLD isoform, PLD2, is required for the synaptotoxic and memory-impairing actions of Aβ in AD mouse models. The role that the other PLD isoform, PLD1, plays in AD is unclear, although cell culture studies from other groups have suggested it modulates the trafficking of APP and presenilin 1, the catalytic subunit of -secretase. Here, we investigate the role of PLD1 in the biology of APP as well as in an AD mouse model. We report that silencing Pld1, unlike Pld2, causes a dramatic decrease in brain levels of PA, indicating that PLD1 is a major source of PA in the brain. Additionally, Pld1 ablation in primary neurons causes a redistribution of APP from endosomes, a primary station for amyloidogenesis, to the Golgi complex, while PLD1 overexpression produces the converse phenotype. Pld1 null mice harboring familial AD-linked (FAD) APP mutations exhibited decreased brain amyloid and an accumulation of APP COOH-terminal fragments. This finding was particularly evident in Pld1 null membrane rafts, whose lipidome was profoundly altered. Consistent with a regulatory role of the lipid raft environment on γ-secretase, a complex highly active in membrane rafts, acute inhibition of PLD1 reduced APP processing by this complex. Finally, Pld1 nullizygosity rescued cognitive deficits in the transgenic model. Thus PLD1 and its product PA control the metabolism of APP and emerge as potential drug targets for Alzheimer’s disease therapy. Table of Contents List of Figures iv Acknowledgements vi Chapter 1 General Introduction 1 1.1 Alzheimer’s Disease 1 Clinical characteristics of AD 2 AD management in the clinic 3 Pathological characteristics of AD 5 1.2 AD: the lipid and membrane trafficking connection 9 Lipid associated genetic risk factors in AD 9 APP trafficking and processing 12 Aβ and lipid signaling 15 Lipidomics in AD 18 1.3 Phospholipase D 19 PLD1 in Physiology 21 PLD1 in Pathology 23 FIGURES 27 Chapter 2 Characterization of PLD activity in the Pld1 KO mouse 34 2.1 PLD1 contributes the majority of PA in murine forebrain 34 2.2 Effect of PLD1 ablation on murine forebrain lipidome 36 FIGURES 38 Chapter 3 The interaction and subcellular distribution of PLD1 and APP 43 i 3.1 PLD1 localization in neurons 43 3.2 PLD1 controls the subcellular localization of endogenous APP in neurons 44 FIGURES 46 Chapter 4 The role of phospholipase D1 in AD pathology in an FAD model 49 4.1 Effect of hAPP gene expression in murine forebrain lipidome 49 4.2 Effect PLD1 ablation on hAPP murine forebrain lipidome 51 4.3 PLD1 ablation decreases the amyloidogenic processing of APP 52 FIGURES 54 Chapter 5 Molecular characterization of phospholipase D1 activity in lipid rafts in an FAD model 59 5.1 Lack of PLD1 disrupts APP processing and the lipidome in membrane rafts 59 5.2 Pharmacological inhibition of PLD1 decreases γ-secretase cleavage of APP 59 FIGURES 62 Chapter 6 Neurobehavioral and synaptic effects of phospholipase D1 65 FIGURES 67 Chapter 7 Discussion 69 7.1 PLD activity and APP trafficking 69 7.2 PLD activity and lipid rafts 71 7.3 Future Directions 73 Summary Diagram 76 Chapter 8 Materials and Methods 77 References 85 Appendix 93 ii Dataset 1: Molecular and lipid composition of murine WT and hAPP model forebrain 92 Dataset 2: Molecular and lipid composition of murine WT and hAPP forebrain lipid rafts 113 iii List of Figures Figure 1.1 Rising number of patients with Alzheimer’s disease Figure 1.2 Hallmark lesions of Alzheimer’s disease Figure 1.3 APP Intracellular trafficking Figure 1.4 Lipids and APP processing Figure 1.5 PLD1: Structure and Function Figure 1.6 PA within the lipid bilayer Figure 2.1 Generation of the Pld1 KO mouse Figure 2.2 PLD activity in vitro Figure 2.3 PLD activity in Pld null animals Figure 2.4 Disparate PEtOH production in Pld1 vs Pld2 KO Figure 2.5 Effect of PLD1 ablation on murine forebrain lipidome Figure 3.1 PLD1 colocalizes with APP in Hela cells Figure 3.2 PLD1 colocalizes with APP in primary hippocampal neurons Figure 3.3 PLD1 overexpression drives APP traffic Figure 3.4 PLD1 ablation redistributes APP from endosomes to Golgi in neurons Figure 4.1 PLD1 ablation specifically modulates murine APP processing in vivo Figure 4.2 Effect of hAPP transgene on murine forebrain lipidome Figure 4.3 Lipidomic analysis of hAPP mice lacking PLD1 Figure 4.4 PLD1 ablation decreases amyloidogenesis in a FAD mouse model iv Figure 5.1 Lack of PLD disrupts APP processing and the lipidome in membrane rafts Figure 5.2 Pharmacologic PLD1 inhibition reduces WT and Swedish APP processing Figure 5.3 Pharmacologic PLD1 inhibition reduces APP processing through γ-secretase Figure 6.1 PLD1 ablation improves neurobehavioral deficits in the FAD hAPP model Figure 6.2 Control behavioral tests Figure 7.1 Model for roles of PLD1 in trafficking and processing of APP v Acknowledgements First I express heartfelt gratitude to my advisor, Dr. Gilbert Di Paolo for being a kind mentor, a wise educator, and a magnificent scientist. I am very grateful to members of my committee for their time, helpful suggestions, and constructive criticisms: Dr. Karen Duff, Chair, Dr. Tae-Wan Kim, Dr. Ai Yamamoto, and Dr. Samuel Gandy (Mount Sinai School of Medicine). I thank past and present members of the Di Paolo lab for constant support and for sharing their scientific prowess. I am indebted to Dr. Robin Chan for performing LC-MS analyses and guidance in processing the data. I thank Drs. Claudia Dall’Armi and Zofia Lasiecka for performing confocal microscopy, guidance in image analysis, and comments and suggestions as the work progressed. I thank Dr. Tiago Oliveira for early and guidance in the lab and contributing the in vivo PLD assay and HPLC analysis. I am grateful to Dr. Jayson Bastien for help formatting this thesis. Each member of the Di Paolo laboratory has been instrumental in my development as a scientist and I am grateful for their support through my time in the lab. I thank Drs. Guanghou Shui and Markus Wenk for contributing the LC-MS analysis of PEtOH species. I am grateful to Elizabeta Micevska for genotyping and maintenance of the mouse colony, and Dr. Ottavio Arancio and Agnieszka Staniszewski for access to equipment and technical help with behavioral experiments. I am grateful to Dr. Michael Frohman for the kind gift of the GFP-hPLD1 construct and Dr. Sung Ho Ryu for kindly providing the PLD antibody. I am thankful to Dr. Luciano D’Adamio and Dr. Eric Schon for experimental guidance and protocols. I am thankful to the MD/PhD program especially Drs. Michael Shelanski, Ron Liem, and Patrice Spitalnik for encouragement vi and mentorship through the program. This work is supported by NRSA 1F31NS073387, of which I am grateful. I am grateful to my family, especially my dear husband Dr. Joseph Point du Jour, for listening, for always believing in me, and for pointing me back to our Source of strength. My eternally supportive parents, Joan and Chris Robinson, sister, Tiffany Robinson, and aunt Jennifer Hendricks have seen me through every challenge and triumph. From my first breaths through her own struggle with Alzheimer’s disease my grandmother, Elsa Robinson, has prayed for me and helped me to keep a positive perspective. I also acknowledge Tenesha Patrick for bringing me joy when the process was difficult, and I thank scientific mentors Mrs. Carla Traister, Dr. Barbara-Anne Battelle, and Dr. David Nowack for introducing me to the scientific endeavor and encouraging me in my career. vii Dedication To God our Savior, Who alone is wise, Be glory and majesty, Dominion and power, Both now and forever. viii 1 Chapter 1 General Introduction Opening remark: This thesis encompasses a number of traditional scientific disciplines such as phospholipid metabolism, comparative neuroscience, and cell biology as well as the emerging field of lipidomics. Introductory comments will cover these broad fields as they relate to this work. First, I will describe Alzheimer’s disease and the mental, molecular and gross pathology associated with its presentation. Following this I will focus on providing background for lipid metabolism pathways related to this work. Then I will present the findings of this thesis on exploiting a murine genetic ablation model to study both the physiology of the central nervous system and the pathological state with the goal of contributing knowledge towards understanding the mechanisms of Alzheimer’s disease. 1.1 Alzheimer’s Disease Alzheimer’s disease (AD), a fatal, insidious, neurodegenerative disorder, is the most common cause of dementia in the elderly (Querfurth and LaFerla, 2010; Tanzi and Bertram, 2005). It may occur as a sporadic disease for which there is currently no precise cause or, in less than 3% of cases, it may present as an autosomal dominant disease caused by nearly 200 mutations in amyloid precursor protein (APP) or presenilin 1 (PSEN1) and presenilin 2 (PSEN2), the catalytic components of the γ- secretase complex (Tanzi and Bertram, 2005).
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