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UC Irvine UC Irvine Electronic Theses and Dissertations Title Functional, Safe, and Effective Mitigation of Radiation-Induced Cognitive Deficits Using Stem Cell-Derived Extracellular Vesicles in a Wild-Type Rodent Model Permalink https://escholarship.org/uc/item/1585t77j Author Leavitt, Ron Joshua Publication Date 2019 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, IRVINE DISSERTATION TITLE: Functional, Safe, and Effective Mitigation of Radiation-Induced Cognitive Deficits Using Stem Cell-Derived Extracellular Vesicles in a Wild-Type Rodent Model Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Environmental Health Sciences by Ron Joshua Leavitt Dissertation Committee: Professor Charles Limoli, Chair Professor Ulrike Luderer Professor Robert Phalen Associate Professor Mathew Blurton-Jones Associate Professor Kim Green Defended: December 12 th , 2019 Introduction © 2018 Taylor & Francis Group LLC. DEDICATION For Alex Of course it is happening inside your head, Harry, but why on earth should that mean that it is not real? JK Rowling Harry Potter and The Deathly Hallows ii TABLE OF CONTENTS Page LIST OF FIGURES v LIST OF TABLES vii ACKNOWLEDGMENTS viii CURRICULUM VITAE ix ABSTRACT OF THE DISSERTATION x INTRODUCTION AND LITERATURE REVIEW 1 Extracellular Vesicles 4 EV as Therapeutic Agents in Regenerative Medicine 12 CHAPTER 1: EV Study in Wild-Type Mice with Retro-orbital Delivery 17 Introduction 17 Cognitive Behavioral Testing Data 21 EV Tracking 28 Immunohistochemical Staining 30 Discussion 33 CHAPTER 2: EV Study Molecular Analyses and EV Cargo Analysis 38 Introduction 38 RNA Sequencing 41 EV Cargo Analysis 51 Discussion 53 CHAPTER 3: miR-124 Overexpression Study 56 Introduction 56 Cognitive Behavioral Testing Data 58 Immunohistochemical Staining 61 Expression Analysis 63 Discussion and Ongoing Research 66 CHAPTER 4: Methodology 69 SUMMARY AND CONCLUSIONS 79 REFERENCES 82 APPENDIX A: ZetaView® Particle Measurements 92 iii APPENDIX B: Additional Behavioral Testing Figures 101 APPENDIX C: Raw Cognitive Behavioral Testing Data 103 APPENDIX D: PSD-95 Staining Results 129 APPENDIX E: RNA-seq Data Tables 130 APPENDIX F: Primer Design and Efficiency Testing 131 APPENDIX G: miRNA Detection qPCR Data 134 iv LIST OF FIGURES Page Figure 1 EV treatment in cranially-irradiated athymic nude rats ameliorated radiation-induced neuroinflammation and damage to neuronal structure. 13 Figure 1.1 Stem cell-derived EV protect against radiation-induced cognitive dysfunction at 5 weeks and 6 months post irradiation. 23 Figure 1.2 EV-treated mice exhibit decreased anxiety at 5 weeks and 6 months post irradiation 25 Figure 1.3 Fear extinction training 26 Figure 1.4 Stem cell-derived EV tracked to the host hippocampus following retro-orbital or intra-hippocampal injections 29 Figure 1.5 Stem cell-derived EV treatment reduces neuroinflammation in the hippocampus following irradiation 31 Figure 1.6 Model for Radiation-Induced Brain Injury and Rescue 36 Figure 2.1 Principal Component Analysis (PCA) is not able to separate experimental groups 41 Figure 2.2 Hierarchical Clustering shows little similarity between samples of each experimental group 42 Figure 2.3 Gene Ontology enrichment suggests that inflammatory processes may be important factors in radiation-induced cognitive deficits 43 Figure 2.4 Relative expression of miR-124 target genes is decreased in hippocampal tissue overexpressing miR-124 50 Figure 3.1 miR-124 overexpression in vivo shows functional mitigation of radiation-induced cognitive dysfunction 59 Figure 3.2 CD68 staining volume decreased in both irradiated groups compared to the unirradiated ones 62 Figure 3.3 Select expression profiles of whole hippocampal tissues from wild-type mice from the miR-124 overexpression study 65 v LIST OF TABLES Page Table 1 miRNAs associated with EV from specific cell types and known functions based on peer-reviewed literature. 11 Table 2 The advantages and disadvantages of stem cell therapy versus EV therapy. 14 Table 2.1 Top 30 differentially expressed genes in the control group compared to the IRR group 46 Table 2.2 Top 30 differentially expressed genes in the EV-treated groups compared to the IRR group 47 Table 2.3 Top 30 differentially expressed genes in the control group compared to the EV-treated groups 48 vi ACKNOWLEDGMENTS I would like to express huge thanks and appreciation to my committee chair, Professor Charles Limoli. He took me in during the darkest hour of my graduate student experience when no one else would and provided me with a lab environment where I could learn, grow, and thrive. None of this would have been possible without his kindness and generosity, not to mention his direction and mentorship. I would like to thank my “outside” committee members, Associate Professors Mathew Blurton-Jones and Kim Green for valuable insights on the project, both during the advancement meeting and in helping design the miR-124 AAV project. I would like to also thank Professor Ulrike Luderer, who not only recruited me to UC Irvine, but had been the Environmental Health Sciences program director for most of my time here. She has been an invaluable source of institutional knowledge and advice. I would like to thank Dr. Lianghao Ding and Dr. Michael Story from University of Texas Southwestern Medical Center for running the miRNA microarray experiment. I would like to thank the entire Limoli lab for help, showing me where things are located, occasional junk food, and support throughout the process. Specifically, I would like to thank lab members Dr. Janet Baulch and Assistant Professor Munjal Acharya for giving so much of their precious time to an annoying grad student and playing an indispensable role in his training. Finally, I would like to thank my friends and family for sticking with me for this long (extremely long) and difficult process. This especially includes my brand new wife Alexandria who has suffered through five years of this and in many ways put her life on hold so that I could finish my degree. I also thank Taylor & Francis Online for permission to include the Introduction of my dissertation, which was originally published in International Journal of Radiation Biology. This work was supported by the Defense Threat Reduction Agency (HDTRA 1-13-1-0022, to Charles Limoli), NIH NINDS R01 NS074388 (to Charles Limoli), California Institute for Regenerative Medicine (DISC1-10079, to Janet Baulch), UCI Research Seed Funding Program (to Janet Baulch). vii CURRICULUM VITAE Ron Joshua Leavitt 2007 B.S. in Molecular, Cell, and Developmental Biology, University of California, Los Angeles 2008-13 Research Associate, Zymo Research Corp., Irvine, CA 2019 Ph.D. in Environmental Health Sciences, University of California, Irvine FIELD OF STUDY Radiotherapy and Cognitive Function PUBLICATIONS Leavitt, R.J., Limoli, C.L., & Baulch, J.E. (2018). miRNA-based therapeutic potential of stem cell-derived extracellular vesicles: a safe cell-free treatment to ameliorate radiation-induced brain injury. International Journal of Radiation Biology, 25:1-8. Than, N.G., Romero, R., Tarca, A.L., Kekesi, K.A., Xu, Y., Juhasz, K., Bhatti, G., Leavitt, R.J., … & Papp, Z. (2018). Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia. Frontiers in Immunology, 9:1661. Chamorro-Garcia, R., Diaz-Castillo, C., Shoucri, B.M., Käch, H., Leavitt, R., Shioda, T., & Blumberg B. (2017). Ancestral perinatal obesogen exposure results in a transgenerational thrifty phenotype in mice. Nature Communications, 8(1):2012. Than, N. G., Romero, R., Xu, Y., Erez, O., Xu, Z., Bhatti, G., Leavitt, R., … & Bohn, H. (2014). Evolutionary origins of the placental expression of Chromosome 19 cluster galectins and their complex dysregulation in preeclampsia. Placenta, 35(11):855–865. Ashktorab, H., Daremipouran, M., Goel, A., Varma, S., Leavitt, R., Sun, X., & Brim, H. (2014). DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia. Epigenetics, 9(4):503–512. viii ABSTRACT OF THE DISSERTATION Functional, Safe, and Effective Mitigation of Radiation-Induced Cognitive Deficits Using Stem Cell-Derived Extracellular Vesicles in a Wild-Type Rodent Model By Ron Joshua Leavitt Doctor of Philosophy in Environmental Health Sciences University of California, Irvine, 2019 Professor Charles Limoli, Chair Radiation-induced learning and memory problems are reported in more than half of survivors of cancers treated with cranial radiotherapy, representing an unmet clinical need in a growing population. Cell-free therapy employing extracellular vesicles (EV) derived from stem cells has been pioneered to address this problem in an immunocompromised rodent model. However, no study has yet demonstrated efficacy of this cell-free treatment in wild-type animals or determined the mechanism. This research has sought to address both of those shortcomings, as well as introduce a more clinically palatable delivery method to improve safety while maintaining efficacy. A large cohort of wild-type mice was used to determine the efficacy of post-irradiation EV treatment in wild-type animals and retro-orbital sinus injection. At five weeks and six months post-irradiation, animals were subjected to a battery of cognitive behavioral tests. Follow up studies analyzed tissues via RNA-seq, RT-qPCR, and immunohistochemical staining. Behavioral testing