The localization of c-Abl in Alzheimer’s disease by Zheng Jing BS, Beijing University, 2001 Submitted to the Graduate Faculty of School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2008 gg UNIVERSITY OF PITTSBURGH School of Medicine This dissertation was presented by Zheng Jing It was defended on July 14, 2008 and approved by Dr. Teresa Hastings, Associate Professor, Department of Neurology Dr. Donald DeFranco, Professor, Department of Pharmacology Dr. Patrick Card, Professor, Department of Neuroscience Dr. Ruth Perez, Assistant Professor, Department of Neurology Dr. Mark Smith, Professor, Department of Pathology Case Western Reserve University Dissertation Advisor: Dr. Robert Bowser, Associate Professor, Department of Pathology ii Copyright © by Zheng Jing 2008 iii The localization of c-Abl in Alzheimer’s disease Zheng Jing, PhD University of Pittsburgh, 2008 The two major hallmarks of Alzheimer’s disease (AD) are amyloid plaques and neurofibrillary tangles (NFTs). Evidence suggests that the main component of amyloid plaques, β-amyloid peptide (Aβ) facilitates tau pathology via activation of specific kinases. Both glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (cdk5) have been demonstrated to be activated by Aβ and contribute to tau hyperphosphorylation. Recently, c-Abl has been implicated in Aβ-facilitated tau pathology by in vitro model systems. Alvarez et al. reported that c-Abl could be activated by Aβ in primary cultured neurons (Alvarez et al. 2004), and Derkinderen et al. found a novel phosphorylation site in paired helical filament tau (Tyr 394) that could be phosphorylated by c-Abl (Derkinderen et al. 2005). Moreover, Aβ has been shown to bind integrin receptors on the cell surface and transduce a signal from the extracellular space to the cell interior, regulating the cytoskeleton and/or gene transcription (Caltagarone, Jing et al. 2007). c-Abl can also be activated by integrin activation. Therefore, we hypothesize that c-Abl is associated with Aβ-facilitated tau phosphorylation via integrin binding and activation, contributing to the generation of AD pathology. We tested this hypothesis by examining the expression and distribution of c-Abl in the human hippocampus and by characterizing c-Abl interacting proteins in AD brain. We discovered that the activation state of c-Abl was altered during AD progression and c-Abl was associated with phospho-tau during AD. Preliminary co-immunoprecipitation data iv also suggested a possible association of c-Abl with another integrin signaling protein, paxillin. This study is the first to examine the expression and localization of c-Abl in healthy control and AD hippocampus, which contributes to our understanding of the functional role for c-Abl in AD pathogenesis. Interestingly, c-Abl was localized to granulovacuolar degeneration bodies (GVDs) during late-stage AD, a novel discovery that identifies a new protein component of GVDs in AD. v TABLE OF CONTENTS 1.0 INTRODUCTION................................................................................................................ 1 1.1 ALZHEIMER’S DISEASE (AD) ............................................................................... 1 1.1.1 Epidemiology .................................................................................................... 1 1.1.2 Clinical features................................................................................................ 3 1.1.3 Diagnosis ........................................................................................................... 4 1.1.4 Neuropathology ................................................................................................ 5 1.1.4.1 Plaques................................................................................................... 6 1.1.4.2 Neurofibrillary tangles (NFT) ............................................................. 7 1.1.4.3 Granulovacuolar degeneration bodies (GVD) ................................... 8 1.1.4.4 Hirano bodies ........................................................................................ 8 1.1.4.5 Synaptic loss and neuronal cell death ................................................. 9 1.1.4.6 Cell loss ................................................................................................ 10 1.1.5 Genetics of AD................................................................................................ 10 1.1.5.1 APP mutations .................................................................................... 11 1.1.5.2 Presenilin mutations ........................................................................... 12 1.1.5.3 APOE polymorphisms........................................................................ 13 1.1.5.4 Others genetic risk factors of AD...................................................... 14 1.1.6 Pathogenic mechanisms of AD...................................................................... 14 vi 1.1.6.1 DNA damage ....................................................................................... 14 1.1.6.2 Cell cycle reentry ............................................................................... 15 1.1.6.3 Mitochondria....................................................................................... 16 1.1.6.4 Oxidative stress and excitoxicity ....................................................... 17 1.1.6.5 Inflammation....................................................................................... 18 1.1.7 Current therapeutics and drug targets ........................................................ 20 1.2 β-AMYLOID PEPTIDE IN AD PATHOGENESIS............................................... 23 1.2.1 Metabolism, assembly and clearance of β-amyloid peptide....................... 23 1.2.1.1 Biogenesis of β-amyloid peptide ........................................................ 24 1.2.1.2 Catabolism and clearance of β-amyloid peptide.............................. 27 1.2.1.3 Assembly of β-amyloid peptide ........................................................ 28 1.2.2 Aβ peptide is considered as a causative factor for AD pathogenesis......... 30 1.2.2.1 Aβ cascade hypothesis ........................................................................ 30 1.2.2.2 Intra- versus extra- Aβ....................................................................... 30 1.2.2.3 The molecular mechanisms of Aβ toxicity........................................ 31 1.2.2.4 Aβ induced synaptic dysfunction ...................................................... 33 1.3 TAUOPATHY IN AD PATHOGENESIS............................................................... 35 1.3.1 Background of tau.......................................................................................... 35 1.3.1.1 Tau phosphorylation .......................................................................... 39 1.3.1.2 Tau Dephosphorylation...................................................................... 40 1.3.1.3 Mechanism for pathological tau phosphorylation........................... 41 1.3.1.4 Tauopathy & Aβ ................................................................................. 43 2.0 INTRODUCTION FOR C-ABL....................................................................................... 50 vii 2.1.1 Background of c-Abl...................................................................................... 50 2.1.1.1 The structure and localization of c-Abl ............................................ 50 2.1.1.2 The activation and regulation of c-Abl ............................................. 53 2.1.2 Function of c-Abl in dividing cells ................................................................ 59 2.1.2.1 Nuclear c-Abl ...................................................................................... 60 2.1.2.2 Cytoplasmic c-Abl............................................................................... 64 2.1.3 c-Abl in the nervous system........................................................................... 67 2.1.3.1 c-Abl in neurodevelopment and synaptic plasticity......................... 67 2.1.3.2 c-Abl’s function in neurodegeneration ............................................. 70 3.0 THESIS GOALS ................................................................................................................ 74 4.0 MATERIALS AND METHODS ...................................................................................... 75 4.1 SUBJECTS................................................................................................................. 75 4.2 WESTERN BLOT ANALYSIS................................................................................ 78 4.3 IMMUNOHISTOCHEMISTRY (IHC)................................................................... 79 4.4 IMMUNOFLUORESCENT LASER SCANNING CONFOCAL MICROSCOPY (LSCM)................................................................................................................................ 80 4.5 QUANTIFICATION OF IHC AND STATISTICAL ANALYSIS ....................... 81 4.6 CO-IMMUNOPRECIPITATION (CO-IP)............................................................. 83 4.7 PRIMARY CORTICAL NEURON CELL CULTURE ........................................ 83 4.8 Aβ FIBRIL PREPARATION, CELL TREATMENT AND CYTOTOXICITY ASSAYS............................................................................................................................... 84 4.9 CELLULAR FRACTIONATION