Analysis of Myelin Membrane Growth in Oligodendrocytes Dissertation for the award of the degree „Doctor rerum naturalium (Dr.rer.nat.)“ of the Georg-August University Göttingen within the doctoral program Biology of the Georg-August University School of Science (GAUSS) submitted by Sebastian Schmitt born in Oldenburg, Germany Göttingen, 2014 Thesis Committee: Prof. Dr. Dr. Hannelore Ehrenreich Clinical Neurosciences Max-Planck-Institute of Experimental Medicine Prof. Dr. Nils Brose Molecular Neurobiology Max-Planck-Institute of Experimental Medicine Dr. Mikael Simons Department of Neurology, Max-Planck-Institute of Experimental Medicine Members of the Examination Board Reviewer: Prof. Dr. Dr. Hannelore Ehrenreich Clinical Neurosciences Max-Planck-Institute of Experimental Medicine Second Reviewer: Prof. Dr. Nils Brose Molecular Neurobiology Max-Planck-Institute of Experimental Medicine Further members of the examination board: Dr. Mikael Simons Department of Neurology, University of Göttingen Max-Planck-Institute of Experimental Medicine Prof. Dr. Klaus-Armin Nave Department of Neurogenetics Max-Planck-Institute of Experimental Medicine Dr. Manuela Schmidt Emmy-Noether research group Somatosensory Signaling Max-Planck-Institute of Experimental Medicine Prof. Dr. Ralf Heinrich Cellular Neurobiology, Schwann-Schleiden-Research Center University of Göttingen Date of the oral examination: 12.12.2014 Contents Contents Contents ........................................................................................................................ i Affidavit .........................................................................................................................iii Abbreviations ............................................................................................................... iv Acknowledgments ......................................................................................................... v Abstract ........................................................................................................................ 1 Chapter I: Introduction .................................................................................................. 2 1.1 Biological membranes serve as permeable barriers ............................................ 2 1.2 Regulated transport is the basis of nerve cell conduction .................................... 2 1.3 Myelination accelerates nerve cell conduction ..................................................... 3 1.4 Oligodendrocytes are crucial for the function of the brain .................................... 5 1.5 Myelin is highly enriched in lipids ......................................................................... 6 1.6 Myelin has a specific protein composition ............................................................ 7 1.6.1 Proteolipid protein ......................................................................................... 8 1.6.2 Myelin Basic Proteins .................................................................................... 8 1.6.3 Cyclic nucleotide phosphodiesterase ............................................................ 9 1.6.4 Myelin-oligodendrocyte glycoprotein ............................................................. 9 1.6.5 Myelin-associated glycoprotein ................................................................... 10 1.6.6 Minor myelin proteins .................................................................................. 10 1.7 Recent views on myelin protein composition ..................................................... 11 1.8 Differentiation of OPCs and myelin formation .................................................... 12 1.8.1 Migration and proliferation of OPCs ............................................................ 13 1.8.2 Inhibitory signals keep OPCs in the precursor state .................................... 14 1.8.3 Chromatin remodeling is a first step towards differentiation of OPCs .......... 15 1.8.4 Intrinsic Factors actively promote oligodendrocyte differentiation ................ 15 1.8.5 Myelination – Contact formation, wrapping, trophic support ........................ 18 1.9 Oligodendrocyte differentiation in vitro ............................................................... 19 1.10 Aims of this study ............................................................................................ 20 Chapter II: Materials and Methods .............................................................................. 21 2.1 General consumables........................................................................................ 21 2.2 Animal work ....................................................................................................... 21 2.2.1 Fixation by intra-cardial perfusion ............................................................... 21 2.2.2 Brain and spinal cord slice preparation ....................................................... 22 2.2.3 Immuno-histochemistry (IHC) ...................................................................... 22 2.2.4 Light microscopy of tissue slices ................................................................. 23 2.2.5 Electron microscopy .................................................................................... 23 2.3 Tissue culture methods ..................................................................................... 25 2.3.1 Handling of cell-lines ................................................................................... 25 2.3.2 Cryo-preservation of mammalian cell lines .................................................. 26 2.3.3 Glial mixed cultures ..................................................................................... 26 2.3.4 Primary oligodendrocyte precursor cells ..................................................... 26 2.3.5 Astrocyte cultures ....................................................................................... 27 2.3.6 Microglia cultures ........................................................................................ 27 2.3.7 Cortical neurons .......................................................................................... 27 2.3.8 Preparation of mouse embryonic fibroblasts ............................................... 28 2.3.9 Treatment of cell cultures ............................................................................ 28 2.3.10 Transfection of cell cultures....................................................................... 28 2.3.11 Immunocytochemisty ................................................................................ 28 2.3.12 Light microscopy of cell cultures ............................................................... 29 2.4 Molecular biology methods ................................................................................ 30 i Contents 2.4.1 Isolation of genomic DNA from mouse tail tips ............................................ 30 2.4.2 Polymerase chain reaction for genotyping................................................... 30 2.4.3 Agarose gel electrophoresis ........................................................................ 31 2.4.4 High-fidelity polymerase chain reaction for cloning ...................................... 31 2.4.5 Enzymatic digestion of PCR products and plasmids ................................... 31 2.4.6 Ligation ....................................................................................................... 31 2.4.6 Transformation of competent E. coli and plasmid preparation ..................... 31 2.5 RNA methods .................................................................................................... 32 2.5.1 Preparation of TRIzol-Lysates ..................................................................... 32 2.5.2 Isolation of total RNA .................................................................................. 32 2.5.3 Denaturating agarose electrophoresis ......................................................... 33 2.5.4 Reverse-transcription quantitative real-time PCR ........................................ 34 2.5.5 RNA-extraction, generation of the cDNA libraries and Illumina RNA- Sequencing .......................................................................................................... 35 2.6 Protein biochemistry methods ........................................................................... 37 2.6.1 Preparation of lysates for SDS-PAGE ......................................................... 37 2.6.2 SDS-PAGE ................................................................................................. 37 2.6.3 Western Blot Analysis ................................................................................. 38 2.64 Proteomic analysis and data processing ...................................................... 38 2.6.5 Fc-fusion protein generation and purification ............................................... 39 2.6.6 Binding assay ............................................................................................. 40 2.6.7 Adhesion assay .......................................................................................... 40 Chapter III: Results ..................................................................................................... 41 3.1 Generation of highly pure primary cell cultures .................................................. 41 3.2 Workflow for proteomic and transcriptomic analysis .......................................... 42 3.3 Adult mouse brain proteome.............................................................................