Role of Slitrk Family Members in Neurodevelopment François Beaubien Integrated Program in Neuroscience Montreal Neurological Institute McGill University Montreal, Quebec, Canada April 2012 A thesis dissertation submitted to the Department of Graduate and Postdoctoral Studies of McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy in Neurological Sciences © François Beaubien, 2012 TABLE OF CONTENTS Abstract .........................................................................................................................................5 Résumé..........................................................................................................................................6 List of figures ...............................................................................................................................7 List of abbreviations ...................................................................................................................9 Acknowledgements .................................................................................................................. 12 Author contributions ............................................................................................................... 14 Chapter 1: Literature Review 1. General introduction ......................................................................................................... 15 2. Synaptogenesis 2.1. Historical perspective ................................................................................................ 16 2.2. Example of the neuromuscular junction ................................................................ 18 2.2.1. Comparison between nerve terminals of neuromuscular junctions and central synapses ................................................................................................. 21 2.3. CNS synaptogenesis 2.3.1. Introduction ....................................................................................................... 22 2.3.2. The four steps of synaptogenesis .................................................................... 24 2.3.3. Synaptogenic proteins ....................................................................................... 25 2.3.3.1. Secreted factors 2.3.3.1.1. Neuronally derived priming factors ........................................... 27 2.3.3.1.1.1. Wnt ....................................................................................... 27 2.3.3.1.1.2. FGF ...................................................................................... 28 2.3.3.1.2. Glial-derived priming factors ...................................................... 29 2.3.3.1.3. Neuronal pentraxin family ........................................................... 30 2.3.3.2. Synaptic adhesion complexes 2.3.3.2.1. Cadherins ....................................................................................... 32 2.3.3.2.2. Nectins ........................................................................................... 33 2.3.3.2.3. NCAM ............................................................................................ 34 2.3.3.2.4. Neurexins and Neuroligins .......................................................... 35 2.3.3.2.5. Tripartite complexe neurexin/Cbln1/GluRδ2 ......................... 39 2.3.3.2.6. Neurexins and Dystroglycans ..................................................... 40 2.3.3.2.7. Neurexins and Neurexophilins ................................................... 40 2.3.3.2.8. Neurexins and LRRTMs .............................................................. 41 2.3.3.2.9. SALMs ............................................................................................ 42 2.3.3.2.10. SynCAMs..................................................................................... 43 2.3.3.2.11. NGLs ........................................................................................... 44 2.3.3.2.12. Ephrins and Eph Receptors ..................................................... 46 2.3.3.2.13. FLRTs .......................................................................................... 47 2.3.3.2.14. IL1RAPL1 and IL-1RAcP ........................................................ 48 2.3.4. Role of Leucine-Rich Repeat proteins in synaptogenesis ............................ 48 2 3. The Slitrk family 3.1. Identification............................................................................................................... 49 3.2. Genomic organization of the mouse and human Slitrks ...................................... 50 3.3. Phylogeny analysis for Slitrk1 ................................................................................... 50 3.4. Slitrks protein structure ............................................................................................. 52 3.5. Expression of Slitrks in the nervous system .......................................................... 54 3.6. Relationship to Slits and Trks proteins ................................................................... 55 3.7. Functions of the Slitrks 3.7.1. Slitrks and neuropsychiatric disorders 3.7.1.1. Tourette’s syndrome ............................................................................... 56 3.7.1.2. Other potential implications in neuropsychiatric disorders .............. 58 3.7.2. Slitrks and their CNS functions 3.7.2.1. Early experiments with cell lines and primary neurons ..................... 60 3.7.2.2. Interaction between Slitrk1 and the 14-3-3 proteins ......................... 60 3.7.2.3. Characterization of Slitrks knockout mice .......................................... 61 4. Rationale and objectives ................................................................................................... 63 Chapter 2: Differential Expression of Slitrk Family Members in the Mouse Nervous System 1. Preface ................................................................................................................................. 64 2. Abstract ............................................................................................................................... 65 3. Introduction ........................................................................................................................ 65 4. Experimental Procedures ................................................................................................. 67 5. Results 5.1. Specificity of the cRNA probes used in this study ................................................ 68 5.2. Slitrks expression in the olfactory system ............................................................... 72 5.3. Slitrks expression in the developing cerebral cortex ............................................. 74 5.4. Slitrks expression in the hippocampal region ........................................................ 76 5.5. Expression of Slitrk6 in the diencephalon ............................................................. 78 5.6. Slitrks expression in the cerebellum ........................................................................ 79 5.7. Slitrks expression in the embryonic spinal cord .................................................... 83 5.8. Slitrks expression in the trigeminal ganglion .......................................................... 85 5.9. Slitrks expression during eye development ............................................................ 87 6. Discussion ........................................................................................................................... 89 Chapter 3: Slitrk1 and Slitrk2 Promote Excitatory Synapse Development 1. Preface ................................................................................................................................. 91 2. Abstract ............................................................................................................................... 92 3. Introduction ........................................................................................................................ 92 3 4. Experimental Procedures ................................................................................................. 94 5. Results 5.1. Slitrk1 and Slitrk2 are enriched in synaptosomal and postsynaptic density fractions ....................................................................................................................... 97 5.2. Slitrk1 and Slitrk2 induce presynaptic differentiation in a mixed-culture assay100 5.3. Overexpression of Slitrk1 and Slitrk2 in neurons induces excitatory presynaptic differentiation ........................................................................................................... 105 5.4. Slitrk1 and Slitrk2 do not induce postsynaptic differentiation in a mixed-culture assay ........................................................................................................................... 108 5.5. Application of Slitrk1 antibodies reduces synapse number in hippocampal neuron cultures ......................................................................................................... 110 6. Discussion ......................................................................................................................... 114 Chapter 4: General Discussion 1. Original contributions .................................................................................................... 117 2. Slitrks as synaptogenic proteins ...................................................................................