IN VITRO RESILIENCE AND NANOTOXICITY IN 3D BRAIN MODELS by Georgina Harris A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy. Baltimore, Maryland July, 2018 © Georgina Harris 2018 All rights reserved ABSTRACT Current neurotoxicity testing does not meet the needs to protect human health from potential neurotoxicants. The increase in incidence of neurological disorders has shown that environmental exposures may pose a risk in conjunction with genetic factors. Pesticide exposure and aging are associated with increased Parkinson’s disease (PD) risk. To date, in vitro research focuses on apical endpoints from high-dose acute exposures. We propose to study cellular recovery and resilience in vitro, to challenge current acute toxicity testing and question (a) whether dopaminergic cells can recover from low-dose exposures and (b) how they respond to a subsequent toxicant hit. To address the current needs, we developed and characterized an in vitro human dopaminergic 3D brain model using LUHMES (Lund Human Mesencephalic cell line). Taking advantage of the fact that our model is cultured in suspension, we analyzed not only acute but also delayed response to the pesticide rotenone after compound withdrawal and 7 days recovery. Rotenone quantification demonstrated it was effectively removed from media after wash-out. We further assessed viability after second exposures to test our resilience hypothesis. Molecular and functional assays were used to assess toxicity and recover. Dopaminergic neurons were able to recover functionally (neurite outgrowth and electrical acitivty) from low-dose acute rotenone effects, however other endpoints (complex I inhibition, gene expression) were permanently altered and pre-exposed cells were resilient to a second hit indicating long-term molecular memory after wash-out. Repeated low-dose exposures to rotenone upregulated PD-related genes. Finally, 3D LUHMES and iPSC-derived BrainSphere model ware applied to study internalization and toxicity of nano-delivery particles (AuSC, AuPEG and PLA). Effects on viability, mitochondrial membrane potential and oxidative response genes were observed. Our results present a different approach to studying toxicity in vitro, with the use of 3D models and compound wash-out to better understand whether acute effects are reversible (more similar to in vivo exposures). Genetic or epigenetic factors could lead to altered recovery and drive disease development. Furthermore, advances in nanotechnology require new testing strategies to assess the safety for novel drug delivery systems. ii THESIS COMMITTEE Prof. Thomas Hartung Professor Advisor Department of Environmental Health and Engineering Johns Hopkins Bloomberg School of Public Health Prof. Daniele Fallin Chair, Professor Department of Mental Health Johns Hopkins Bloomberg School of Public Health Prof. Marsha Wills-Karp Chair, Professor Department of Environmental Health and Engineering Johns Hopkins Bloomberg School of Public Health Dr. Anne Hamacher-Brady Assistant Professor W. Harry Feinstone Department of Molecular Microbiology and Immunology Johns Hopkins Bloomberg School of Public Health Dr. Sin-ichi Kano Assistant Professor Department of Psychiatry and Behavioral Sciences School of Medicine Dr. Wan-Yee Tang Associate Professor Department of Environmental Health Sciences Johns Hopkins Bloomberg School of Public Health Dr. Jiou Wang Associate Professor Department of Biochemistry and Molecular Biology Johns Hopkins Bloomberg School of Public Health iii PREFACE The Center for Alternatives to Animal testing (CAAT) is dedicated to the promotion, development and education of the 3Rs (reduction, refinement and replacement) in toxicology. Since 1981, it has supported the creation, development, validation, and use of alternatives to animals in research and product safety testing by providing up to date information and acting as a hub for pharma, industry and regulatory scientists to engage in discussions about the needs in toxicology. In line with the goals of bodies such as the Organization for Economic Co-operation and Development (OECD), Environmental Protection Agency (EPA), Food and Drug administration (FDA), ICCVAM and ECVAM, the center aims to develop approaches which are more predictive of toxicity to protect human health. Animal testing is costly, time consuming and has been shown to not be highly predictive of human adverse outcomes. Moreover, for chronic diseases, which are driven by gene-environment interactions, animal models fail to reproduce human pathology. Therefore, using novel 3D in vitro models which more closely represent human organ regions with close cell-cell interactions, we can define molecular mechanisms upon toxicant exposure and how different genetic backgrounds may impact neuronal degeneration. Once the 3D dopaminergic model was developed, we attempted to study neuronal recovery. Most in vitro research to date has been performed at high concentrations, measuring acute effects. To better recapitulate life exposures, we treated a 3D dopaminergic model acutely, but washed out the compound to determine whether these effects are relevant in long-term toxicity or whether cells can cope with low-dose effects. We further studied the effects of low, repeated-dose in the 3D model determining that chronic exposure using a 3D model is more relevant to study neurodegenerative mechanisms in vitro. The work in this PhD thesis was aimed at researching (i) current neurotoxicity testing and in vitro models, (ii) evidence showing how environmental exposures are a risk factor for iv neurodegenerative diseases such as Parkinson’s and (iii) novel nanoparticle drug-delivery systems to treat neurodegenerative diseases. The scientific papers included in this thesis present Georgina Harris’ contributions to this field; the development and use of a 3D in vitro dopaminergic model to study acute, delayed and repeated-dose effects, neuronal resilience and the use of 3D models for nanotoxicity testing. During my PhD have been very fortunate to have had experienced mentors with a strong drive for alternative methods. My first and upmost gratitude is to my parents Daphne Lopez and Donald Harris who provided me with all the tools and education to reach my goals. They have always been supportive of my choices and have been there to help me when it was needed. I would never achieve any of this without you. David Pamies who lept on this adventure with me in the US, he has been the best co-worker I could have asked for and has taught me patience, persistence and how we can overcome any hurdles together. Thank you for all the support these years and for taking care of me always. My sisters Carla and Vanessa, for putting up with me and always being so caring. My family in Elche, for always checking in on me, cheering me on throughout the years, visiting us in Baltimore and making me the best meals when I am in town. Thanks to my advisor, Prof. Thomas Hartung, whom I will always admire every time I attend one of his presentations. Ever since our fist meeting he has tried to open doors for my future and enabled networking and travel to further educate me. I am forever grateful for the opportunities and advice he has provided me with. I am grateful for the International Foundation for Ethical Research who provided four years of funding to aid my PhD as well as funding for my research. As one of the few institutions that provides fnding to non-US citizens, I am very thankful for the opportunity and support to foreign students. To my thesis committee, who met with me multiple times over the past three years, thank you for your constructive feedback and for guiding me along this path. I would also like to thank my second advisor, Dr. Lena Smirnova who taught me lab techniques and allowed me to develop scientific research independence. Thank you for always v having time for me and helping me when I needed it. Shelly Odwin, for keeping our lab organized, supporting me, making me laugh, working with me in the summer and teaching me patience and that one can always achieve new goals (now it is my turn to support you). Dr. Helena Hogberg, whom the lab would not exist without. Thank you for always having the time to talk and give me advice, your PhD thesis was an inspiration for me and you kept me going when I needed support. To Mounir, Andre and Alex, thank you for your help with metabolomics and transcriptomics data, your knowledge is priceless and helped me troubleshoot data throughout the years. To fellow students whom I have trained or worked closely with, Dana, Melanie, Mariana, Johannes, Marize, Rober, Vy and Daphne, you have been my greatest inspiration and sense of achievement. Teaching you and learning from you all bring memories of the best moments of my PhD. To Ruth, Jamie and Michelle, thank you for your patience and hard work every day, your friendliness and caring attitude provides CAAT with a lovely working environment. To my collaborators, my research would not have been possible without you and your expertise. Thank you for working hard on the vision I had and the experiments I proposed. Throughout my PhD, some mentors went above and beyond to enquire about my research and provide constructive criticism. Dr. Milena Mennecozzi, Dr. Brigitte Landesmann and Prof. Anna Price are my most inspiring women in science who have believed in me and helped me grow as a scientist. Dr. Maurice Whelan, Prof. Marcel Leist, Dr. Jack Fowle, Prof. Martin Wilks and Dr. Pedro DelValle have also strongly influenced my growth and a scientist and have always had the time for me, providing me with opportunities throughout the years. I am lucky to have had such strong role models. To my friends, thank you for always being there for the good and bad times. Moving to a different continent was worth it to meet you all and share beautiful moments. Minipandi and Charm, you are my second family and I’m lucky to have your friendship.