Phenotypic Analysis of Motor Defects in Neuronal Ostm1 Conditional Loss of Function Milhem Hajj Hassan Master of Science Faculty of Graduate Studies Division of Experimental medicine McGill University Montreal, Quebec, Canada April 2020 A thesis submitted To McGill University in partial fulfillment of the requirements of the degree of Master of Science ©Copyright Milhem Hajj Hassan 2020 all rights reserved. TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................................... vi LIST OF FIGURES .................................................................................................................................... vii ABSTRACT ................................................................................................................................................. ix RÉSUMÉ ...................................................................................................................................................... x ACKNOWLEGDMENTS ........................................................................................................................... xi CONTRIBUTION OF AUTHORS ............................................................................................................. xii ABBREVIATIONS ....................................................................................................................................xiii Introduction ................................................................................................................................................... 1 Chapter 1 ....................................................................................................................................................... 3 Literature Review .......................................................................................................................................... 3 1.1 The Nervous System ........................................................................................................................... 4 1.1.1 Central Nervous System (CNS) ................................................................................................... 4 1.1.2 Peripheral Nervous System (PNS) ............................................................................................... 4 1.2 Types of Nerves .................................................................................................................................. 4 1.3 Cells of the Nervous System ............................................................................................................... 5 1.3.1 Neurons ........................................................................................................................................ 5 1.3.2 Glial Cells .................................................................................................................................... 5 1.4 Neuromuscular Junction ..................................................................................................................... 6 1.5 Macroscopic Bone Structure ............................................................................................................... 6 1.6 Microscopic Bone Structure................................................................................................................ 7 1.7 Bone Remodeling ................................................................................................................................ 7 1.7.1 Osteoblast ..................................................................................................................................... 7 1.7.2 Osteoclast ..................................................................................................................................... 8 1.8 Osteopetrosis ....................................................................................................................................... 9 1.8.1 Role of Osteoclast in Osteopetrosis ............................................................................................. 9 ii 1.8.2 Forms of Osteopetrosis in Human ............................................................................................. 12 1.9 Mutations in ARO ............................................................................................................................. 14 1.9.1 TCIRG1 ...................................................................................................................................... 14 1.9.2 CLCN7 ....................................................................................................................................... 15 1.9.3 OSTM1 ....................................................................................................................................... 16 1.10 Ostm1 Partners ................................................................................................................................ 17 1.11 Osteopetrosis and the Central Nervous System .............................................................................. 18 1.11.1 Case Studies ............................................................................................................................. 18 1.12 Grey lethal mouse model ................................................................................................................ 20 1.13 PU.1 Transgenic Mice .................................................................................................................... 22 1.14 Autophagy ....................................................................................................................................... 24 1.5.2 Rescue of Neurodegeneration in Double Transgenic Mice ....................................................... 26 Hypothesis and Approach ....................................................................................................................... 28 Chapter 2 ..................................................................................................................................................... 29 Materials and Methods ................................................................................................................................ 29 2.1 Mice .................................................................................................................................................. 30 2.1.1 Grey-Lethal Mice ....................................................................................................................... 30 2.1.2 Ostm1 Floxed Mice .................................................................................................................... 30 2.1.3 SYNAPSIN-1 Cre Mice ............................................................................................................. 30 2.1.4 Genotyping ................................................................................................................................. 30 2.1.6 Crosses ....................................................................................................................................... 31 2.2 Molecular Analysis ........................................................................................................................... 31 2.2.1 DNA Expression and Recombination Percentage. ..................................................................... 31 2.2.2 RNA Expression ........................................................................................................................ 32 2.3 Biochemical Analysis ....................................................................................................................... 33 2.3.1 Antibodies .................................................................................................................................. 33 iii 2.3.2 Protein Extraction and Quantification ........................................................................................ 33 2.3.3 Western Blots ............................................................................................................................. 33 2.4 Histological Analysis ........................................................................................................................ 34 2.4.1 Tissue Embedding and Sectioning ............................................................................................. 34 2.4.2 Cytoarchitectural Studies ........................................................................................................... 34 2.4.3 Immunohistochemistry............................................................................................................... 35 2.4.4 Immunofluorescence .................................................................................................................. 35 2.5 Ultrastructural Analysis .................................................................................................................... 36 2.6 Myofiber Staining ............................................................................................................................. 37 2.7 Statistics ............................................................................................................................................ 37 Chapter 3 ....................................................................................................................................................