Human disease variants in the ryanodine receptor have effects in vivo for Caenorhabditis elegans neuromuscular function Brittany Laura Graham Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biology February 2020 ii The candidate confirms that the work submitted is her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Brittany Laura Graham to be identified as Author of this work has been asserted by Brittany Laura Graham in accordance with the Copyright, Designs and Patents Act 1988. iii Acknowledgements I would like to express my appreciation to my supervisor Ian Hope; for providing the opportunity, for all the scientific advice, for his feedback during my write-up and for his overall support through this process. Thank you for training me to think like a scientist. I would also like to thank my co-supervisor Marie-Anne Shaw. Her encouragement, comments and different point of view have helped widen my research and made me a better scientist. I must also acknowledge the White Rose DTP, the BBSRC and the FBS graduate office at the University of Leeds without whom this project would not have been possible. My special thanks are extended to NemaMetrix for hosting me on my internship and their help in generating the variant strains used in this thesis. Additional thanks go to David Pertab and Alex Jubb, for generating two further variant strains used in this research. I also appreciate the advice and many discussions had with Elpi Kalogeropoulou, Célia Ferreira and Ros Clifford in the Hope lab. I would like to recognise the help provided by the Cohen group at the University of Leeds, particularly Omer Yuval and Rob Holbrook. The multi-worm tracker and guidance on coding and software were vital to the crawling analysis in this research. My friends in the postgrad office: Zatul, Ellie, Francis, Dayah, Rosie, Nok, Victoria, Jens, Jake and Brad, provided an enjoyable environment to work for which I will always be thankful. To all my friends, wherever in the world you are, thank you for being there for me. A special mention made to Elpi and Rosie whose friendships have meant the world to me. Tanner, I am forever grateful for your endless reassurance, positivity and belief in me. Finally, to my family, I would not be where I am without your unconditional love and encouragement. I am thankful to have your support in anything I decide to do. I could not end without thanking my mum for the cups of tea that kept me going, and for everything else. Having a strong and proud woman as a role model made me believe I could do and be anything. iv I would like to dedicate this thesis to women in science. “I was taught that the way of progress was neither swift nor easy.” – Marie Curie The women before me have made my way easier, and I hope to do the same for the future women in science. v Abstract The ryanodine receptor is a key intracellular calcium ion channel in nerve and muscle cells. A number of links have been made between the ryanodine receptor and age- related muscular and neurodegeneration. Variants of the RYR1 gene are associated with malignant hyperthermia, which is a hypermetabolic reaction to inhalational anaesthetics. I used eight genome-edited Caenorhabditis elegans strains expressing ryanodine receptors with modifications equivalent to known RYR1 disease variants to characterise the consequences of these variants on neuromuscular function in vivo. Animals expressing variant ryanodine receptors exhibited increased sensitivity to halothane when both homozygous and heterozygous for the variants. This mirrors the situation in humans where malignant hyperthermia is inherited in an autosomal dominant pattern. Novel, subtle, locomotion defects were found in animals expressing variant ryanodine receptors even in the absence of halothane. In-depth analysis of crawling suggested that these variant channels release excessive calcium, resulting in increased muscle contraction. Exaggerated age-related degeneration of locomotion was observed in variant strains, which may be a result of excessive calcium release via variant ryanodine receptors throughout the lifespan resulting in cellular damage. Cholinergic pharmacological agents were used to characterise if the consequences of the variants were pre and/ or post-synaptic. A range of complex phenotypes were found, reflecting the complexity of regulatory inputs to the ryanodine receptor. The altered properties of variant ryanodine receptors and effects of long-term calcium mishandling found here are anticipated to have consequences for human carriers, which may be in the nerve cells. Assessment of these phenotypes in a whole organism was important to fully appreciate the significant effects of variant ryanodine receptors. vi Table of Contents Acknowledgements ................................................................................................ iii Abstract ................................................................................................................... v Table of Contents ...................................................................................................... vi List of Tables ............................................................................................................ x List of Figures .......................................................................................................... xi List of Abbreviations .............................................................................................. xiii Chapter 1 General introduction ................................................................................ 1 1.1. The ryanodine receptor ............................................................................................ 1 1.1.1. Ryanodine receptor isoforms and evolution ............................................................................. 2 1.1.2. Structure of the ryanodine receptor.......................................................................................... 5 1.1.3. Control of ryanodine receptor activation .................................................................................. 9 1.1.4. Ryanodine receptor related diseases ...................................................................................... 12 1.1.5. Malignant hyperthermia and its triggering agents .................................................................. 15 1.2. Caenorhabditis elegans as a model ......................................................................... 18 1.2.1. C. elegans as a model for malignant hyperthermia ................................................................. 23 1.3. Focus of this research ............................................................................................. 26 1.3.1. Modelling RyR1-related disease in C. elegans ......................................................................... 26 1.3.2. Pre- and postsynaptic effects of RYR1 gene variants .............................................................. 26 1.4. Thesis outline ......................................................................................................... 27 Chapter 2 General Methods ................................................................................... 28 2.1. C. elegans maintenance and strains ........................................................................ 28 2.1.1. C. elegans maintenance ........................................................................................................... 28 2.1.2. C. elegans strains ..................................................................................................................... 28 2.2. Generating strains through CRISPR-Cas9 genome editing ......................................... 29 2.2.1. Genotyping ryanodine receptor variant strains ...................................................................... 33 2.3. Synchronisation and ageing .................................................................................... 34 2.3.1. Generating an age-synchronised population of C. elegans ..................................................... 34 2.3.2. Ageing C. elegans ..................................................................................................................... 35 2.3.3. Statistical and graphical analysis ............................................................................................. 35 Chapter 3 Confirming a malignant hyperthermia related phenotype in genome- edited RYR1 variant C. elegans: modelling malignant hyperthermia in the worm .... 38 3.1. Introduction ........................................................................................................... 38 3.1.1. Aim of this chapter ................................................................................................................... 38 3.1.2. A malignant hyperthermia related phenotype in C. elegans .................................................. 38 vii 3.2. Methods................................................................................................................. 39 3.2.1. Halothane and thrashing assays .............................................................................................. 39 3.2.2. L1 thrashing assay ...................................................................................................................