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Characterisation of Human and Mouse SOD1-ALS Proteins in Vivo and in Vitro

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Characterisation of Human and Mouse SOD1-ALS Proteins in Vivo and in Vitro Characterisation of human and mouse SOD1-ALS proteins in vivo and in vitro Rachele Saccon A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy to the University of London Department of Neurodegenerative Disease Institute of Neurology University College London 1 Declaration I, Rachele Saccon, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease affecting motor neurons (MNs). It is primarily sporadic, however a proportion of cases are inherited and of these ~20 % are caused by mutations in the superoxide dismutase 1 (SOD1) gene. The work described in this thesis has focused on the characterisation of the role that the SOD1 protein plays in ALS, investigating the human and the mouse variants in vivo and in vitro. SOD1 mutations result in ALS by an unknown gain of function mechanism, although mouse models suggest that complete loss of SOD1 is also detrimental to MN function. To investigate a possible role of SOD1 loss of function in SOD1-ALS, a meta-analysis was carried out on the literature reviewing measures of SOD1 activity from patients carrying SOD1 familial ALS mutations and the phenotype of Sod1 knockout mice. The first set of experiments aimed to phenotypically characterise a novel mouse model, Sod1D83G, carrying a pathological mutation in the mouse Sod1 gene. Sod1D83G/D83G mice have no SOD1 activity, low levels of SOD1 protein, develop central MN degeneration and a distal peripheral neuropathy. Further the Sod1D83G mice were crossed with Sod1 knockout mice and mice overexpressing the human wild-type SOD1 to determine if it was possible to dissect elements of a loss of function (the peripheral axonopathy) and aspects of a gain of function (the central body degeneration). ALS mutations generally cause SOD1 to become more aggregate-prone, but it is unclear whether the human and the mouse SOD1 proteins co-aggregate in mouse models of SOD1 familial ALS. To investigate possible interactions between human and mouse SOD1 variants, recombinant proteins were produced, characterised and their spontaneous aggregation propensity was assessed in vitro. Finally a sensitised screen focused on the effect of unknown mutations on the life-span of a low copy SOD1G93A transgenic model of ALS, identified one mouse line with reduced survival, named Galahad. The phenotype of the Galahad mouse progeny was examined and a quantitative trait loci analysis was carried out to try to identify possible modifying locus/loci interacting with the SOD1 G93A mutation. The present work aims to shed light on the interaction between human and mouse SOD1 proteins and increase our understanding on the mechanism affecting central and peripheral degeneration of MNs in the context of SOD1 familial ALS mutations. 3 Acknowledgements I would like to express my deep gratitude to Professor Elizabeth Fisher for giving me the opportunity of a lifetime, for always believing and encouraging me providing guidance and support throughout my PhD. Thank you for being such and amazing mentor. Many thanks also to Dr Abraham Acevedo and Professor Linda Greensmith for giving me valuable and constructive advice every step of the way. Thank you to all past and present members of the Fisher, Isaacs and Greensmith labs all of who have played their part in making my PhD a wonderful and fun learning experience. I would also like to thank the members of staff from the UCL Department of Neurodegenerative Disease and the MRC Prion Unit who were essential for the work I have presented in this thesis. So many people have supported me that there are too many to mention individually by name, but in particular I would like to thank Dr Sarah Mizielinska, Dr Emma Clayton, Dr Rosie Bunton-Stasyshyn, Dr Phil McGoldrick, Dr Pietro Fratta, Mark Batchelor, Robin Labesse-Garbal and Cristina Venturini. Another particular whole hearted thanks go to my amazing friends Daniel, Elisa, Caterina, Alessia, Luca, Karinne and Christiane who have always encourage me and lift my spirit during this journey. Finally, my heartfelt thanks to my parents, to my grandparents and Martino, I would not be who I am and where I am without you. 4 Table of contents Declaration..............................................................................................................................2 Abstract ....................................................................................................................................3 Acknowledgements............................................................................................................4 Table of contents.................................................................................................................5 List of figures.......................................................................................................................15 List of tables.........................................................................................................................18 List of abbreviations........................................................................................................20 Chapter 1 Introduction .......................................................................................... 25 1.1 Amyotrophic lateral sclerosis (ALS) .................................................................. 25 1.1.1 Epidemiology ....................................................................................................... 25 1.1.2 ALS clinical course ............................................................................................. 25 1.1.2.1 Motor neuron degeneration .................................................................. 25 1.1.2.2 Cognitive dysfunctions .......................................................................... 26 1.1.3 Diagnosis .............................................................................................................. 27 1.1.4 Treatments ........................................................................................................... 27 1.1.5 Environmental risk factors ................................................................................ 28 1.1.6 ALS pathological features .................................................................................. 28 1.1.6.1 Degeneration of motor neurons .......................................................... 28 1.1.6.2 Gliosis ...................................................................................................... 30 1.1.6.3 Inclusion bodies ...................................................................................... 30 1.1.6.4 Muscle atrophy ........................................................................................ 31 1.1.7 Genetics of ALS .................................................................................................. 31 1.2 SOD1-fALS ................................................................................................................ 37 1.2.1 Clinical course and pathogenicity of SOD1-fALS ......................................... 37 1.2.2 SOD1 gene and inheritance pattern ................................................................. 37 1.3 SOD1 protein ............................................................................................................ 39 5 1.3.1 Biological function .............................................................................................. 40 1.3.2 Structural features and protein stability ........................................................... 41 1.3.3 Properties of mutant SOD1 proteins ............................................................... 43 1.3.3.1 Effect of mutations on protein function ............................................ 43 1.3.3.2 Effect of mutations on SOD1 biochemical properties ..................... 43 1.4 SOD1 mouse models .............................................................................................. 47 1.4.1 Why we use mouse models in ALS research .................................................. 47 1.4.1.1 Mouse models of SOD1-ALS .............................................................. 47 1.4.2 SOD1 transgenic mice ....................................................................................... 48 1.4.2.1 Transgenic SOD1G93A high copy mice ................................................. 48 1.4.2.2 Transgenic SOD1WT mice ...................................................................... 50 1.4.3 Sod1 knock out mice ........................................................................................... 51 1.4.4 Effect of background on mouse phenotypes ................................................. 51 1.4.5 Limitations of current SOD1-ALS mouse models ........................................ 54 1.5 How mutant SOD1 causes disease .................................................................... 55 1.5.1 SOD1-fALS proposed mechanisms of pathogenicity ................................... 55 1.5.1.1 Glutamate toxicity and calcium homeostasis ..................................... 55 1.5.1.2 Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) .......................................................................................................... 56 1.5.1.3 Mitochondrial dysfunction ...................................................................
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