Role of Microglia in Myelin Turnover Dissertation for the award of the degree “Doctor of Philosophy” (Ph.D.) Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen submitted by Shima Safaiyan from Esfahan, Iran Goettingen 2015 Member of the Thesis Committee: Prof. Dr. Mikael Simons, Reviewer Department of Cellular Neuroscience, Max Planck Institute for Experimental Medicine Department of Neurology, University of Goettingen Prof. Dr. Alexander Fluegel, Reviewer Department of Neuroimmunology, Institute for Multiple Sclerosis Research, University Medical Center Goettingen Prof. Dr. Holger Reichardt, Department of Cellular and Molecular Immunology, University Medical Center Goettingen AffidavitDate of the oral examination: 21 September, 2015 ii Affidavit I hereby declare that this Doctoral thesis entitled “Role of microglia in myelin turnover” has been written independently with no other aids or sources than quoted. Shima Safaiyan July, 2015 Goettingen, Germany iii Contents Contents Contents iv List of tables ........................................................................................................................................ ix List of figures ....................................................................................................................................... ix Abbreviations xi Acknowledgements xii Abstract xiii Introduction 1 1.1 Microglia .................................................................................................................................... 1 1.1.1 Microglia origin and development ..................................................................................... 1 1.1.2 Microglia phenotype in central nervous system ................................................................ 1 1.1.3 Microglia function in central nervous system .................................................................... 1 1.1.3.1 Microglia in normal brain ............................................................................................ 2 1.1.3.2 Microglia in the diseased brain ................................................................................... 3 1.1.4 Microglia in aging ............................................................................................................... 4 1.1.4.1 Aging phenotype of microglia ..................................................................................... 4 1.1.4.2 Basal activation state of microglia in aging ................................................................. 5 1.1.4.3 The functionality of microglia in aging ........................................................................ 5 1.1.4.4 Why microglia become aged? ..................................................................................... 6 1.1.4.4.1 Lysosomal storage; Lipofuscin .............................................................................. 6 1.2 Myelin ........................................................................................................................................ 7 1.2.1 Myelin structure ................................................................................................................. 7 1.2.2 Myelin composition ............................................................................................................ 8 1.2.3 Myelination in the CNS ....................................................................................................... 8 1.2.4 Myelin turnover .................................................................................................................. 9 1.2.5 Myelin plasticity ................................................................................................................. 9 iv Contents 1.2.6 Myelin in aging ................................................................................................................. 10 1.3 Demyelinating animal models ................................................................................................. 11 1.3.1 Cuprizone mouse model .................................................................................................. 11 1.3.1.1 Cuprizone-induced demyelination ............................................................................ 11 1.3.1.2 Immunopathology of cuprizone mouse model ......................................................... 12 1.3.2 Shiverer mouse ................................................................................................................. 12 1.3.3 PLP overexpressing transgenic mice, a model for Pelizaeus-Merzbacher disease .......... 12 1.3.3.1 Microglia activation in PLP overexpressing transgenic mice..................................... 13 1.4 Endocytic pathway .................................................................................................................. 13 1.4.1 Endosome biogenesis ....................................................................................................... 13 1.4.2 Lysosome biogenesis, Role of Rab7.................................................................................. 14 1.5 Question and hypothesis of the project .................................................................................. 15 Materials and Methods 16 2.1 Materials ................................................................................................................................. 16 2.1.1 Antibodies ........................................................................................................................ 16 2.1.2 Commercial kits ................................................................................................................ 17 2.1.3 Primers ............................................................................................................................. 17 2.1.4 Software ........................................................................................................................... 18 2.1.5 Solutions and Media ......................................................................................................... 18 2.1.6 Mouse lines ...................................................................................................................... 19 2.2 Methods .................................................................................................................................. 19 2.2.1 Generation and characterization of conditional Rab7 KO mice ....................................... 19 2.2.1.1 Generation and breeding of mutant mice................................................................. 19 2.2.1.2 Tamoxifen induction of conditional deletion of Rab7 gene ...................................... 21 2.2.1.3 Detection of Cre recombinase activity using reporter mice ..................................... 21 2.2.1.4 Cre recombinase-mediated deletion of Rab7 gene in microglia ............................... 21 2.2.1.5 Genotyping ................................................................................................................ 22 v Contents 2.2.1.6 Real-time quantitative PCR ....................................................................................... 23 2.2.2 Histology ........................................................................................................................... 25 2.2.2.1 Perfusion and tissue preparation .............................................................................. 25 2.2.2.2 Immunohistochemistry ............................................................................................. 25 2.2.3 Lipofuscin autofluorescent reduction .............................................................................. 26 2.2.3.1 Chemical treatment ................................................................................................... 26 2.2.3.2 Dye separation .......................................................................................................... 27 2.2.4 Imaging ............................................................................................................................. 27 2.2.4.1 Confocal microscopy ................................................................................................. 27 2.2.4.2 Electron microscopy .................................................................................................. 27 2.2.4.3 In vivo time-lapse imaging ......................................................................................... 28 2.2.5 Image processing and analysis ......................................................................................... 29 2.2.5.1 Cell counting .............................................................................................................. 29 2.2.5.2 Lipofuscin volume measurement .............................................................................. 29 2.2.5.3 Microglia-myelin contact ........................................................................................... 29 2.2.6 Isolation of lysosomal inclusions from microglia ............................................................. 30 2.2.6.1 Microglia isolation ..................................................................................................... 30 2.2.6.2 Sarkosyl-insoluble fractionation of microglia ............................................................ 31 2.2.6.3 Myelin isolation and purification .............................................................................. 31 2.2.7 In vivo endocytosis