The Role of DHHC5-Mediated Palmitoylation of δ-Catenin in Cadherin Stability and Synapse Plasticity

.... .................................................................................................................................... ii Preface...... ................................................................................................................................... iii Table of Contents .......................................................................................................................... v List of Tables ............................................................................................................................... viii List of Figures ............................................................................................................................... ix List of Abbreviations ................................................................................................................... xii Acknowledgements ..................................................................................................................... xiv Chapter 1: Introduction ............................................................................................................... 1 1.1 The Hippocampus as a Model System ............................................................................ 1 1.2 Excitatory Synapses of the Central Nervous System ...................................................... 3 1.2.1 Composition of the Presynaptic Compartment ........................................................... 4 1.2.2 Composition of the Postsynaptic Compartment .......................................................... 8 1.2.2.1 AMPA Receptors .............................................................................................. 10 1.2.2.2 NMDA Receptors .............................................................................................. 11 1.2.3 Dendritic Spines ........................................................................................................ 14 1.2.3.1 Spine Morphology ............................................................................................. 15 1.2.3.2 Structure–Function Relationship ....................................................................... 18 1.2.3.3 Spine Abnormalities and Neuropsychiatric Disease ......................................... 20 1.3 Inhibitory CNS Synapses .............................................................................................. 22 1.4 Synapse Plasticity .......................................................................................................... 25 1.4.1 Long-Term Potentiation ............................................................................................ 26 1.4.1.1 Functional LTP .................................................................................................. 27 1.4.1.2 Structural LTP ................................................................................................... 28 1.4.2 Long-Term Depression ............................................................................................. 31 1.4.2.1 Functional LTD ................................................................................................. 31 1.4.2.2 Structural LTD .................................................................................................. 32 1.4.3 LTP and LTD in Learning and Memory ................................................................... 33 1.5 Cadherin-Catenin Adhesion Complexes at CNS Synapses ........................................... 35 1.5.1 Cadherins in Synapse Formation ............................................................................... 36 1.5.1.1 Cadherin Functions at Presynaptic Compartments ........................................... 37 1.5.1.2 Cadherin Functions at Postsnaptic Compartments ............................................ 39 1.5.2 Regulation of Cadherin-Based Adhesion at the Synapse .......................................... 43 1.6 δ-Catenin at the Synapse ............................................................................................... 46 1.6.1 Postsynaptic Signaling Pathways of δ-Catenin ......................................................... 46 1.6.2 Aberrant δ-Catenin Function and Cognitive Performance ........................................ 49 1.7 Neuronal Protein Palmitoylation ................................................................................... 50 1.7.1 Palmitoyl Acyl-Transferases: The DHHC Family of Proteins .................................. 53 1.7.2 DHHC Proteins in Cognitive Function and Neurological Disease ........................... 57 vi 1.7.3 Dynamic Palmitoylation of Synaptic Substrates ....................................................... 59 1.8 Rationale and Hypothesis .............................................................................................. 61 Chapter 2: Palmitoylation of δ-Catenin by DHHC5 Mediates Activity-Induced Synapse Plasticity .................................................................................................................. 63 2.1 Introduction ................................................................................................................... 63 2.2 Materials and Methods .................................................................................................. 65 2.2.1 Antibodies and cDNA Constructs ............................................................................. 65 2.2.2 Contextual Fear Conditoning .................................................................................... 68 2.2.3 Cell Cultures .............................................................................................................. 68 2.2.4 Neuronal Activation .................................................................................................. 69 2.2.5 Immunoblot Assay .................................................................................................... 69 2.2.6 Palmitoylation Assay ................................................................................................. 70 2.2.7 Immunocytochemistry ............................................................................................... 70 2.2.8 Confocal Imaging ...................................................................................................... 71 2.2.9 Fluorescence Recovery After Photobleaching (FRAP) ............................................ 71 2.2.10 Image Analysis and Quantification ........................................................................... 72 2.2.11 Electrophysiology ...................................................................................................... 73 2.2.12 Statistical Analysis .................................................................................................... 73 2.3 Results ........................................................................................................................... 74 2.3.1 Activity-Dependent Palmitoylation of δ-Catenin in Neurons ................................... 74 2.3.2 Palmitoylation of δ-Catenin Regulates its Binding to N-Cadherin ........................... 78 2.3.3 Palmitoylated and Cadherin-Binding Residues of δ-Catenin .................................. 82 2.3.4 Palmitoylated δ-Catenin Stabilizes N-Cadherin at Synapses .................................... 85 2.3.5 Palmitoylated δ-Catenin Regulates Spine Remodeling ............................................ 89 2.3.6 Palmitoylated δ-Catenin Mediates Changes in Synapse Efficacy ............................ 92 2.3.7 δ-Catenin Palmitoylation Increases after Acqusition of Contextual Fear Memory .. 98 2.3.8 DHHC5 is Required for Palmitoylation of δ-Catenin ............................................. 100 2.4 Discussion ................................................................................................................... 107 Chapter 3: Activity-Mediated Trafficking of the Palmitoyl Acyl-Transferase DHHC5 .. 112 3.1 Introduction ................................................................................................................. 112 3.2 Materials and Methods ................................................................................................ 114 3.2.1 Antibodies and cDNA Constructs ........................................................................... 114 3.2.2 Cell Cultures ............................................................................................................ 115 3.2.3 Neuronal Activation ................................................................................................ 115 3.2.4 Acyl-Biotin Exchange (ABE) Assay ....................................................................... 116 3.2.5 Biotinylation Assay ................................................................................................. 116 3.2.6 Immunoprecipitation (IP) ........................................................................................ 116 3.2.7 Western Blot Analysis ............................................................................................. 117 3.2.8 Immunocytochemistry ............................................................................................. 117 3.2.9 Confocal Imaging .................................................................................................... 118 3.2.10 Image Analysis and Quantification ......................................................................... 118 3.2.11 Statistical Analysis .................................................................................................. 119 vii 3.3 Results ......................................................................................................................... 120 3.3.1 Activity-Induced Mobilization of DHHC5 ............................................................. 120 3.3.2 Activity-Induced Trafficking of DHHC5 ................................................................ 123 3.3.3 DHHC5 Binds δ-Catenin and Mediates its Synaptic Recruitment ......................... 126 3.3.4 Activity-Induced Endocytosis of DHHC5 and Trafficking on Recycling Endosomes ............................................................................................................... 128 3.4 Discussion ................................................................................................................... 133 Chapter 4: Conclusion ............................................................................................................. 137 4.1 Formation of a Stable Synaptic Adherens Junction .................................................... 138 4.2 Linkage of the Cadherin-Adhesion Complex with the AMPAR-Scaffold Complex by Palmitoylated δ-Catenin .............................................................................................. 141 4.3 Transsynaptic Strengthening and Synaptic Tagging by Palmitoylated δ-Catenin ...... 142 4.4 Activity-Regulated Trafficking of DHHC Proteins .................................................... 144 4.5 Significance and Limitations ....................................................................................... 145 Bibliography .............................................................................................................................. 147