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Ipsc Derived Cerebral Organoids As a Model of Tauopathy

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Ipsc Derived Cerebral Organoids As a Model of Tauopathy iPSC derived cerebral organoids as a model of tauopathy University College London Department of Neurodegenerative Diseases UCL Queen Square Institute of Neurology Christopher Edward James Lovejoy 2021 Primary Supervisor – Dr Selina Wray Secondary Supervisor – Dr Tammaryn Lashley 1 Declaration I, Christopher Edward James Lovejoy, confirm that the work presented in this thesis is my own. Where information has been derived from other sources or work has been produced through collaboration, this has been indicated in the text. This includes: 2D neuronal cultures generated by Dr Charlie Arber and Argyro Alatza. Aβ assays conducted by Dr Charlie Arber and Dr Jamie Toombs. Forebrain organoids generated by Argyro Alatza. Forebrain PCR and forebrain immunoblot in section 5 generated by Argyro Alatza. This thesis is not substantially the same as any other that may have been submitted at any other university or similar institution. 2 Acknowledgments First and foremost I would like to thank Professor Selina Wray for the amazing opportunity to not only work with her and her lab, but also work on an incredible, cutting edge, and fascinating project. For letting me develop ideas, both good and bad, and listening to them, as well as the support for my scientific writing, which has been most appreciated. Also her valuable career advice which has helped me path out my future endeavours. Dr Charlie Arber has been instrumental in my journey, answering numerous questions and helping in many a protocol, without which a lot of the work in this thesis would not be possible. I would also like to thank all members of the Wray lab, both past and present, for help they have provided during my project, as well as the fun and laughs throughout. My friends in London, Wales, and around the world for their support. Always willing to listen to me babble on about some cool new results, a beautiful ICC image I’ve taken, or of course complain about my one billionth day in tissue culture. All this social support really meant a lot to me, especially in 2020. Finally my family who always support my efforts and decisions. Especially my grandparents, who were very proud of my PhD studies, but unfortunately never got to see the fruits of that. I know you would be very proud. Finally I would like to thank Phil Bunker, the donor of the Joy Bunker scholarship and subsequently now good friend. Without Phil’s generous donation and this scholarship, this work would not have been possible. Not only has Phil allowed this exciting work to be carried out, he has allowed me to continue my studies and gaining experience. I will be forever grateful of this, and thank him deeply for the opportunity. 3 Papers arising from this thesis Engineered Cerebral Organoids Recapitulate Adult Tau Expression and Disease-relevant Changes in Tau Splicing - Christopher Lovejoy, Argyro Alatza, Charles Arber, Grant Galasso, Teisha Y. Bradshaw, An Verheyen, Tammaryn Lashley, Tamas Revesz, John Hardy, Celeste M. Karch & Selina Wray. Preprint: https://www.researchsquare.com/article/rs-37620/v1 Familial Alzheimer’s disease mutations in PSEN1 lead to premature human stem cell neurogenesis. Charles Arber, Christopher Lovejoy, Lachlan Harris, Nanet Willumsen, Argyro Alatza, Jackie Casey, Georgie Lines, Caoimhe Kerins, Anika K. Mueller, Henrik Zetterberg, John Hardy, Natalie S Ryan, Nick C Fox, Tammaryn Lashley, Selina Wray. In press at Cell Reports, 2020. Familial Alzheimer’s disease patient-derived neurons reveal distinct mutation-specific effects on amyloid beta - Charles Arber, Jamie Toombs, Christopher Lovejoy, Natalie S. Ryan, Ross W. Paterson, Nanet Willumsen, Eleni Gkanatsiou, Erik Portelius, Kaj Blennow, Amanda Heslegrave, Jonathan M. Schott, John Hardy, Tammaryn Lashley, Nick C. Fox, Henrik Zetterberg & Selina Wray. In press at Molecular Psychiatry, 2019. Stem cell models of Alzheimer’s disease: progress and challenges – Charlie Arber, Christopher Lovejoy, & Selina Wray. In press at Alzheimer’s Research and Therapy, 2017 Full text available in Appendix. 4 Abstract Insoluble, hyper-phosphorylated aggregates of tau are a pathological hallmark of a range of clinically diverse neurodegenerative diseases termed tauopathies, of which Alzheimer’s disease (AD) is the most common. The mechanisms linking neuronal death and tau dysfunction are not fully understood, but mutations uncovered in microtubule associated protein tau (MAPT) that cause aggressive frontotemporal dementia confirm a causative relationship between tau dysfunction and neurodegeneration. The tau protein exists as multiple protein isoforms in the adult human central nervous system (CNS), generated by alternative splicing of the MAPT gene. Disruptions to tau splicing are associated with a number of tauopathies, however, in vitro and in vivo models to understand the consequences of disrupted tau splicing have been lacking, due in part to species differences in tau splicing and the developmental regulation of tau in human neurons. Recently, the development of induced pluripotent stem cells (iPSC) has enabled the derivation of limitless numbers of human neurons with disease associated mutations of interest. The use of this system to model tauopathy has been challenging, in part due to the developmental regulation of tau splicing, with extended culture periods required for mature tau expression in iPSC derived neurons. Cerebral organoids are 3D based iPSC derived neuronal cultures, which help capture the heterogeneity and key aspects of architecture of the developing brain, such as distinct progenitor zones and lamination of neurons into distinct layers. We hypothesised that this may allow neurons to mature at a faster rate, resulting in earlier expression of all 6 isoforms of tau without extensive culture times. We investigated the utility of iPSC-derived cerebral organoids to model key aspects of tau biology. Cerebral organoids showed high variability in neuronal content and tau expression. To reduce this heterogeneity, we generated engineered cerebral organoids (enCORs), which utilise a floating scaffold to increase the efficiency of neural induction and reduce heterogeneity. We show that enCORs provide a robust and reproducible in vitro system for the analysis of tau expression and splicing in a 3D model. To investigate the effect of tau mutations, we generated enCORs from an isogenic series of iPSC with the MAPT 10+16 and P301S mutations. The presence of tau splicing mutations results in disease-associated alterations in tau expression, specifically a dose-dependent increase in 4R tau isoforms in the presence of the MAPT 10+16 variant. While the developmental regulation of tau splicing is conserved, maturation of tau splicing is accelerated in 3D cultures compared to 2D cultures. Finally, enCORs with coding mutations in MAPT are able to produce seed- 5 competent tau species, suggesting enCORs recapitulate early features of tau pathology. In summary, enCORs provide a novel, robust in vitro system for the study of tau in development and disease. 6 Impact Statement Affecting 50 million people globally, dementia is one of the most serious health issues facing our aging world. With dementia suffers predicted to hit 152 million worldwide by 2050, marking a 204% increase, this health crisis is set to get worse without treatment options. Modelling complex disorders of the human brain is challenging, with culminating research in additional models needed to investigate mechanisms of disease. Although extremely valuable, current 2D human iPSC derived models of tauopathies are arguably reductionist in approach and struggle to recapitulate MAPT expression, an essential part of tau aggregation disorders. 3D human iPSC derived neuronal cultures potentially offer a solution to this problem. These 3D models also help recapitulate simplified neuronal architecture of the human brain, increasing cell to cell interaction and neuronal maturity. Within this project we show how Aβ processing can be investigated using both 2D and 3D iPSC models, revealing mutation specific Aβ species secretion. With paired patient CSF showing similar Aβ profiles. This type of iPSC based patient matched research allows investigation of possible mutation or Aβ species specific treatments to be trialled in these models. With identification of mature MAPT splicing within 3D cultures, and favourable comparison to human brain samples, research in to tau aggregation utilising all tau isoforms could illuminate tau aggregation mechanics. 3D analysis may even allow pinpointing the origin of tau isoform expression over time, potentially even cell type specific tau isoform expression. This kind of data could again be utilised for drug and antibody screening in similar ways to anti-Aβ antibody trials, with potentially MAPT mutation specific therapies. Further to this, other groups are expanding on 3D organoid research, utilising assembloids for multi- brain region models, investigating neuronal migration and circuit development. Other organoid avenues include investigating blood brain barrier mechanics, drug accessibility with the human brain, and CSF production in choroid plexus organoids. Also with refinement of 3D organoid models, high throughput imaging and drug screening can be utilised for further results in a 3D neuronal model. Through continued development of 3D brain models complimenting other model data, for example animals, further discoveries about tau aggregation and dynamics are possible. 7 Table of contents Abstract ..................................................................................................................................................
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