Open Research Online The Open University’s repository of research publications and other research outputs The Role Of The Parkinson’s Disease-Related Protein LRRK2 In Autophagy And Calcium Signalling In Cellular Models Thesis How to cite: Azeggagh, Sonia (2021). The Role Of The Parkinson’s Disease-Related Protein LRRK2 In Autophagy And Calcium Signalling In Cellular Models. PhD thesis The Open University. For guidance on citations see FAQs. c 2020 Sonia Azeggagh https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.000125c8 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk School of Life, Health and Chemical Sciences The role of the Parkinson’s disease-related protein LRRK2 in autophagy and calcium signalling in cellular models Sonia Azeggagh, BSc, MSc A Thesis submission to The Open University for the degree of Doctor of Philosophy October 2020 Declaration I declare that the work presented in this thesis is my own original work and does not contain any material submitted for a degree, diploma or any other qualification at the Open University or any other University. Contributions made by other researchers are fully acknowledged in relevant parts of the thesis. Abstract Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, affecting 3% of individuals of >75 years of age. The disease is relentless and incurable, and the need to understand its causes and develop new treatments is overwhelming. Mutations in the LRRK2 gene are the most common cause of familial PD, and strongly influence the risk of sporadic PD. This study investigates the impact of LRRK2 enzymatic activities and LRRK2 pathogenic and protective variants on autophagy and calcium signalling to gain insight into the early impairments involved in PD pathogenesis. To this end, the initial approach was to develop isogenically matched cell lines using the CRISPR/Cas9 gene-editing technique, which was unsuccessful. Alternative strategies to study LRRK2 were therefore pursued. The LRRK2 kinase and GTPase activities were modulated with pharmacological inhibitors, and the effects of LRRK2 mutations were investigated by transfection of wild-type and mutant LRRK2 constructs. Autophagy assays combined the quantification of GFP-LC3 punctae and endogenous WIPI2 punctae numbers using fluorescence and electron microscopy, whilst intracellular calcium signals were measured using ratiometric Fura-2 imaging following treatment with ATP, CPA or ionomycin. Overall, this project revealed a cell-type specific action of LRRK2 in autophagy, where LRRK2 appears to be pro-autophagic and involved in a kinase and GTPase activity-dependent manner in the early stages of this process in HEK293 GFP-LC3 cells, whilst data from RAW264.7 macrophages do not support a role for Lrrk2 in autophagy. Calcium-related findings indicated that Lrrk2 is involved in the regulation of IP3-R mediated calcium signals in WT RAW264.7 macrophages and suggest that this role for Lrrk2 may be as a scaffolding protein. The data also indicated that the function of LRRK2 in autophagy induction in HEK293 GFP-LC3 cells is unlikely to require intracellular calcium, and LRRK2 was not found to participate in the regulation of lysosomal calcium storage in either cell line. Additional findings revealed that the calcium indicators Fura-2 and Cal-590, as well as BAPTA and the BAPTA analogues di-bromo-BAPTA and di-fluoro-BAPTA, displayed significant off-target effects on autophagy, and indicated that BAPTA is an unsuitable tool to explore the involvement of calcium signals in autophagy. Taken together, this study yields new and important data about the role of LRRK2 kinase and GTPase activities in the early stages of autophagy, and sheds light on the relevance of LRRK2 to the regulation of intracellular calcium signalling. IV Acknowledgments and dedications This thesis is the result of several years of hard work, and would not have been possible without the help and support of the following people. First, I would like to thank my supervisors Dr Daniel Berwick, Dr Martin Bootman and Dr Kerry Murphy for their support throughout my PhD. In particular, I would like to express my sincere appreciation to Dan, who guided and encouraged me, helped me improve my critical thinking skills, and whose insight and expertise was invaluable. Thank you for helping me become a better researcher. I would like to also thank Cheryl Hawkes for her help, wisdom and our interesting discussions. I would like to thank Guarantors of Brain for supporting my attendance to an incredible Society for Neuroscience Conference with a travel grant, as well as The Open University for funding this project and this opportunity to make my first steps in academia. I would like to recognise the invaluable assistance and knowledge provided by the OU lab support staff Julia, Brett, George and Eduardo, and the EM Suite staff, Igor and Radka. I would also like to thank Jen, for her help with anything I needed, but also for being such a joy to be around, as well as Priya and Dee for their precious help. My time during this PhD would not have been the same without other students with whom I shared fun moments and thought-provoking conversations. In particular, I would like to thank Edu, Ester, Alexandra, Shereen, Laura, and Iwona for their friendship, their support, but especially for being the best friends/colleagues I could have asked for during this life-changing experience. We have made wonderful memories together, and hopefully, more are yet to come. I would like to extend my thanks to other students, including visiting students, who also did their part in making this environment friendly, fun and interesting: Maëva, Juan, Naiara, Sarai, Emily B, Emily C, Maurine, Marta, Ellie, Morgane, Lewis, Salah, Conor, Marcelle, Pelumi, Stephen, Tala… to name “a few”. V I would like to thank my incredible and dearest friends Namya and Linda, both amazing doctors, for their constant support and motivation that helped me through highs and lows. Thank you for believing in me. I will always be grateful that our paths crossed all those years ago. No matter the distance, our friendship continues to grow. Thanks to my other long-time friends in France, whom I always looked forward to meet when I travelled back home. Above all, I would like to give my biggest thanks to my family. It goes without saying that this thesis could not have been written without the strong support of my family throughout these challenging years. As a family, we are always together, no matter the distance. You truly inspire me to be the best version of myself and do better. To my incredible sisters Lyna, Sabina and Lilia, I am proud of you and the women you are, and seeing you thrive warms my heart. This thesis is especially dedicated to my wonderful mother, a true force of nature, my role model. Lastly, I am forever indebted to James, who has helped me in innumerable ways. I am grateful for all his encouragements, his unconditional love and support, and for getting me through these last few years. You are amazing. "Many people say that it is the intellect which makes a great scientist. They are wrong: it is character." Albert Einstein. "It does not matter how slowly you go as long as you do not stop.” Confucius. VI Conference items and publications Publications • Berwick Daniel C.; Heaton George R.; Azeggagh Sonia; Harvey Kirsten. (2019) LRRK2 Biology from structure to dysfunction: research progresses, but the themes remain the same. Molecular neurodegeneration, 14, article no. 49. doi: 10.1186/s13024-019-0344-2 • Azeggagh Sonia. (2017) Rewriting DNA: An introduction to genome editing. Explainer article published on The Open University Open Learn website. Used by the BBC/OU co-production team to directly support “Tomorrow’s World”. doi: 10.13140/RG.2.2.15807.51360 • Azeggagh Sonia. (2017) Targeted genome editing: Introducing the CRISPR/Cas9 system. Explainer article published on The Open University Open Learn website. Used by the BBC/OU co-production team to directly support “Tomorrow’s World”. doi: 10.13140/RG.2.2.29229.28642 Conference items • Azeggagh Sonia; Berwick Daniel C.; Bootman M.; Murphy K. P. S. J.; Allman Sarah A. (2018) The combined use of pathogenic and protective variants as a tool to explore the role of LRRK2 in autophagy and calcium signalling, Poster, Program No. 655.04. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience. Online • Azeggagh Sonia; Adan Abdullahi; Needs Sarah; Murphy K. P. S. J.; Allman Sarah A.; Berwick Daniel C. (2017). A comprehensive "Disease-in-a-Dish" approach to Parkinson's Disease, Poster in: British Neuroscience Association - 2017 Festival of Neuroscience, 10-13 Apr 2017, Birmingham, United Kingdom. VII Table of Contents Abstract .............................................................................................................................................................................. IV Acknowledgments and dedications ................................................................................................................................... V Conference items and publications ................................................................................................................................