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Characterization of Synaptic Alterations and the Effect of Genetic Background in a Mouse Model of Spinal Muscular Atrophy

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Characterization of Synaptic Alterations and the Effect of Genetic Background in a Mouse Model of Spinal Muscular Atrophy Characterization of synaptic alterations and the effect of genetic background in a mouse model of Spinal Muscular Atrophy Mehdi Eshraghi Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Doctorate in Philosophy degree in Neuroscience Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa © Mehdi Eshraghi, Ottawa, Canada, 2017 Abstract Spinal muscular atrophy (SMA) is a genetic disorder characterized by muscle weakness and atrophy and death of motor neurons in humans. Although almost all cases of SMA occur due to mutations in a gene called survival motor neuron 1 (SMN1), SMA patients present with a wide range of severities of the symptoms. The most severe cases never achieve any developmental motor milestone and die within a few years after birth. On the other hand, mild cases of SMA have a normal life span and show trivial motor deficits. This suggests the role of other factors (rather than the function of SMN1) in the outcome of the disease. Indeed, the copy number of an almost identical gene, called SMN2, is the main determining factor for the severity of SMA. In addition, a few other genes (e.g. Plastin 3) are proposed as disease modifiers in SMA. SMN1 is a housekeeping gene, but due to unknown reasons the most prominent pathologies in SMA are atrophy of myofibers and death of motor neurons. However, recent studies showed that some other cell types are also affected in the course of SMA disease. We investigated the alterations of central synapses in Smn2B/- mice, a model of SMA. We did not observe any degeneration of central synapses in these mice until a post symptomatic stage. However, mass spectrometry (MS) analysis on isolated synaptosomes from spinal cords of these animals revealed widespread alterations in the proteome of their central synapses at a presymptomatic stage. Functional cluster analysis on MS results suggested that several molecular pathways are affected within synapses of spinal cords of Smn2B/- mice prior to onset of any obvious pathology in their motor units. The affected molecular pathways are involved in basic cell biological functions including energy production, protein synthesis, cytoskeleton regulation and intracellular trafficking. We showed that the levels of several proteins involved in actin cytoskeleton regulation are altered in synaptosomes isolated from spinal cords of Smn2B/- mice. ii More investigations are required to determine the exact functional abnormalities of affected pathways in central synapses of these mice. We also generated congenic Smn2B/- mice in two different mouse genetic backgrounds; FVB and BL6. Using a systematic approach, we showed that congenic Smn2B/- mice in the FVB background show a more severe SMA phenotype than Smn2B/- mice in a BL6 background. Smn2B/- mice in the FVB background had a shorter survival, higher rate of weight loss, earlier and more severe pathologic changes compared to Smn2B/- mice in the BL6 background. We investigated the levels of several actin binding proteins in spinal cords of these animals and found higher induction of plastin 3 in Smn2B/- mice in BL6 background. More investigations are underway to determine the role of plastin 3 in severity of the phenotype of Smn2B/- mice, and to find other possible SMA modifier genes in these animals. iii TABLE OF CONTENTS List of Tables ............................................................................................................................................ viii LIST OF FIGURES ................................................................................................................................... ix List of Abbreviations ................................................................................................................................ xii Authorization ........................................................................................................................................... xiv Acknowledgments ..................................................................................................................................... xv Author Contributions .............................................................................................................................. xvi Chapter 1 : General Introduction ............................................................................................................. 1 Spinal Muscular Atrophy .......................................................................................................................... 2 Clinical presentations of SMA .................................................................................................................. 4 Type I SMA (OMIM number 253300) .................................................................................................... 6 Type II SMA (OMIM number 253550) ................................................................................................... 6 Type III SMA (OMIM number 253400) .................................................................................................. 6 Type IV SMA (OMIM number 271150) .................................................................................................. 7 Atypical SMA ......................................................................................................................................... 7 Genetics of SMA ....................................................................................................................................... 7 SMN gene .............................................................................................................................................. 7 Etiology of SMA ..................................................................................................................................... 9 Structure of SMN protein ........................................................................................................................ 11 Mouse models of SMA ........................................................................................................................... 15 Smn2B/- mice ........................................................................................................................................ 16 Functions of SMN ................................................................................................................................... 16 The role of SMN in biogenesis of small nuclear ribonucleoproteins (snRNPs) .................................... 19 The role of SMN in assembly of ribonucleoproteins (RNPs) ................................................................ 20 The role of SMN in transcription and translation ................................................................................ 21 The role of SMN in inhibition of apoptosis .......................................................................................... 21 Motor neuron specific functions of SMN............................................................................................. 21 Pathologic alterations due to depletion of SMN ..................................................................................... 23 Pathologic changes of motor units in SMA ......................................................................................... 26 Pathologic changes of neuromuscular junctions (NMJs) in SMA ........................................................ 28 Pathologic changes of central synapses in SMA ................................................................................. 32 Pathologic changes of peripheral neuronal tissues in SMA ................................................................ 33 iv Pathologic changes of non-neuronal tissues in SMA .......................................................................... 34 Signalling pathways affected in SMA .................................................................................................. 34 Effect of genetic background on the severity of SMA phenotype .......................................................... 36 Modifier genes of SMA phenotype ......................................................................................................... 40 SMN2 ................................................................................................................................................... 41 Plastin 3 ............................................................................................................................................... 41 RhoA .................................................................................................................................................... 43 Rationale ................................................................................................................................................. 44 Hypothesis............................................................................................................................................... 45 Aims and Goals ....................................................................................................................................... 45 Chapter 2 : Materials and Methods ........................................................................................................ 46 Mouse maintenance and handling ........................................................................................................... 47 Generation of congenic
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