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Characterisation of a novel Rab18 mouse model for Warburg Micro syndrome Sarah Marie Carpanini Thesis submitted for the degree of Doctor of Philosophy The University of Edinburgh 2013 This thesis is composed of original research undertaken by myself, and where the work of others is included their contributions have been duly acknowledged. Sarah Marie Carpanini November 2013 ii Acknowledgements Firstly, I wish to thank Irene for taking me on as a student and starting the project. You have had a very difficult few years but have still always been there to provide support and guidance whenever needed. It has been an honour to work with you and continue this project. Thank you to Ian for allowing me to become part of your lab; it has been a fantastic group to work for. Thank you for your support and enthusiasm in the project and interesting discussions and ideas after every new result. Thank you to Mark for your help and guidance and answering questions and queries; including those starting with “I know this is a stupid question but...” Thank you for your passion in the RAB protein field and science in general, you have showed me how great work in this field can be. Thank you to the Jackon lab for adopting me and making me feel like part of the family. Thanks for all your help and support and sharing reagents and antibodies. In particular, thanks to Lisa for all your help with phenotyping and maintenance of the mouse colony. Thanks to Lisa and Shalini for all your help with sectioning and characterisation of the mouse eye phenotype. Thanks to everyone in C204 office, thanks for all the gossips and chats over the last few years, and for being a great distraction exactly when needed. Thank you to Tom Gillingwater for also taking me in and making me part of your lab. Thank you for your interest, enthusiasm and ideas for taking this project forward and always making time for me either for brainstorming sessions, paper writing meetings or just general discussions. Thank you to Tom Wishart for help with analysis of the proteomics and IPA. Thanks to all members of the Gillingwater lab, in particular, thank you to Eilidh, Sarah and Sophie for teaching the nerve and muscle dissections. Thanks to Derek for coming across to perform the sciatic nerve ligation surgery, twice! Thanks to Sarah and Ann for processing the nerves and sectioning for EM. Thank you to Mike Cousin and Sarah Gordon for your help with the synaptic vesicle recycling experiments. Thank you to Paul Perry and Matt Pearson for microscope help and support. Thanks to Allyson Ross for help with histology. Thank you to Ian McCall and all technical staff at the BRF for managing the mouse colony. Thank you to Professor Francis Barr for the GFP-RAB18 and GFP-TBC1D20-RA constructs. Thanks also to Harris Morrison for performing the OPT processing and imaging. Thank you to Dad, Mum, Helen, Bex and Grandma for your continued support and patience throughout my studies, you can all breathe a sigh of relief the studies are almost over! Thank you for listening to me rant and stress and I hope to spend more weekends at home with you all, when this is all completed. iii And last but by no means least, I wish to thank Mike. Thanks for your help with illustrator. Thanks for your support and patience over the last eight years, it has meant more to me than you could ever know. Thank you for knowing exactly when I needed you there and when I needed peace, and for trying to keep quiet when Cardiff City were playing. And most of all, thank you that after all this you still want to marry me! iv Dedications I dedicate this thesis in loving memory of Nonno, who was so proud that there could one day be a Dr Carpanini but never lived to see it happen. And to Mike, my fiancé, my best friend and my rock, I love you. v Abstract Warburg Micro syndrome is a severe autosomal recessive condition characterised by abnormalities affecting the ocular, neurological and endocrine systems. Previous studies have identified causative loss-of-function mutations in four members of the RAB protein network; RAB3GAP1, RAB3GAP2, RAB18 and TBC1D20, causing clinically indistinguishable phenotypes. RAB3GAP1 and RAB3GAP2 form a heterodimeric complex specifically regulating the RAB3 family of proteins in calcium mediated exocytosis of hormones and neurotransmitters. Rab3gap1 deficient mice have previously been generated and showed altered short term plasticity in the hippocampus and inhibition of Ca2+ mediated exocytosis of glutamate from cortical synaptosomes, but failed to recapitulate the characteristic ocular or neurological features of Warburg Micro syndrome. Mutations in TBC1D20, a GTPase activating protein (GAP) for the RAB1 family, have recently been identified in Warburg Micro syndrome patients and the bs (blind sterile) mouse model; although this model recapitulated many ocular and endocrine abnormalities of the disease any neurological abnormalities have yet to be reported. The function and localisation of RAB18 remains to be fully elucidated and its role in disease pathogenesis is still unclear. Initially, I have confirmed previous reports co- localising RAB18 with the cis-Golgi, ER and lipid droplets in mouse embryonic fibroblasts and identified a novel localisation in neuronal processes of primary hippocampal neurons. To examine the role of RAB18 in vivo a novel Rab18 genetrap mouse was generated by MRC Harwell as part of the EUMODIC screen. In this study I describe detailed histopathological and neurological characterisation of the Rab18-/- mouse model. Rab18-/- mice were viable and fertile. At eye opening they presented with dense nuclear congenital cataracts and atonic pupils recapitulating major ocular features of Warburg Micro syndrome. Analysis of embryonic eye development revealed a delay in lens development in Rab18-/- mice as early as embryonic day 12.5. From three weeks of age Rab18-/- mice developed progressive hind limb weakness indicative of neurological dysfunction. I have undertaken vi detailed neuropathological analysis of the observed hind limb weakness and identified no abnormalities in synaptic vesicle recycling and no atrophy of peripheral muscles or aberrant development or stability of neuromuscular connectivity. However, loss of RAB18 resulted in gross accumulations of neurofilament and microtubule proteins at the neuromuscular junction and disorganisation of the cytoskeleton in peripheral nerves. Investigation of global proteomic profiling in peripheral nerve of Rab18-/- mice identified alterations in core pathways regulating the axonal cytoskeleton in neurons. In summary this thesis describes a novel Rab18-/- mouse model recapitulating the characteristic ocular and neurological features of Warburg Micro syndrome. I highlight a novel mechanistic insight into Warburg Micro syndrome disease pathogenesis and a role for RAB18 in regulating cytoskeletal dynamics in neurons. vii Publications Sarah M. Carpanini, Lisa McKie, Derek Thomson, Ann K. Wright, Sarah L. Gordon, Sarah L. Roche, Mark T. Handley, Harris Morrison, David Brownstein, Thomas M. Wishart, Michael A. Cousin, Thomas H. Gillingwater, Irene A. Aligianis and Ian J. Jackson. A mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and regulation of the neuronal cytoskeleton. Submitted Human Molecular Genetics August 2013. Ryan P. Liegel, Mark Handley, Adam Ronchetti, Stephen Brown, Lars Langemeyer, Andrea Linford, Bo Chang, Deborah Morris-Rosendahl, Sarah Carpanini, Verity Harthill, Eamonn Sheridan, Ghada M. H. Abdel-Salam, Paulien A. Terhal, Francesca Faravelli, Patrizia Accorsi, Lucio Giordano, Lorenzo Pinelli, Britta Hartmann, Alison Ebert, Francis A. Barr, Irene Aligianis and Duska J. Sidjanin. Loss-of-Function mutations in TBC1D20 Cause Cataracts and Male Infertility in blind sterile Mice and Warburg Micro syndrome in Humans. American Journal of Human Genetics, in press, corrected proof, available online 14 November 2013. Handley MT, Morris-Rosendahl DJ, Brown S, Macdonald F, Hardy C, Bem D, Carpanini SM, Borck G, Martorell L, Izzi C, Faravelli F, Accorsi P, Pinelli L, Basel-Vanagaite L, Peretz G, Abdel-Salam GM, Zaki MS, Jansen A, Mowat D, Glass I, Stewart H, Mancini G, Lederer D, Roscioli T, Giuliano F, Plomp AS, Rolfs A, Graham JM, Seemanova E, Poo P, García-Cazorla A, Edery P, Jackson IJ, Maher ER, Aligianis IA. Muation Spectrum in RAB3GAP1, RAB3GAP2 and RAB18 and Genotype-Phenotype Correlations in Warburg Micro syndrome and Martsolf syndrome. Human Mutation 2013 May;34(5):686-96. Gakovic M, Shu X, Kasioulis I, Carpanini S, Moraga I, Wright AF. The role of RPGR in cilia formation and actin stability. Human Molecular Genetics. 2011 Dec 15;20(24):4840-50.