Regulation of Folate Transport at the Blood-Brain Barrier: A Novel Strategy for the Treatment of Childhood Neurological Disorders Associated with Cerebral Folate Deficiency by Camille Alam A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmaceutical Sciences University of Toronto © Copyright by Camille Alam, 2020 Abstract Regulation of Folate Transport at the Blood-Brain Barrier: A Novel Strategy for the Treatment of Childhood Neurological Disorders Associated with Cerebral Folate Deficiency Camille Alam Doctor of Philosophy Graduate Department of Pharmaceutical Sciences University of Toronto 2020 Folates are crucial for the maintenance of central nervous system homeostasis. Folate transport is mediated by three major pathways, i.e., folate receptor alpha (FRα), proton-coupled folate transporter (PCFT) and reduced folate carrier (RFC), known to be regulated by transcription factors. Brain folate delivery occurs predominantly at the choroid plexus through concerted actions of FRα and PCFT; inactivation of these transport systems causes cerebral folate deficiency resulting in early childhood neurodegeneration. Increasing brain folate permeability or finding alternative routes for brain folate transport could lead to therapeutic benefits. The overall goal of this PhD thesis was to characterize folate transport at the blood-brain barrier (BBB) by examining the role and regulation of folate transporters (i.e., RFC) in brain folate uptake. The objectives of this thesis were to: i) determine RFC functional expression in several in vitro (primary or immortalized cultures of human/rodent brain microvessel endothelial cells) and ex vivo (isolated mouse brain capillaries) BBB models, ii) investigate the role of transcription factors, particularly vitamin D receptor (VDR) and nuclear respiratory factor 1 (NRF-1), in the regulation of RFC in ii various BBB model systems, and iii) examine in vivo, using Folr1 (FRα) knockout mice, the relative contribution of RFC in overall brain folate uptake. We initially demonstrated that RFC is functionally expressed in in vitro systems representative of human (hCMEC/D3 cells) and rodent (mouse brain capillaries) BBB, and that activation of VDR by its ligand, 1,25-dihydroxyvitamin D3 or calcitriol, significantly increased RFC mRNA and protein expression as well as function. We further showed in vivo, using Folr1 knockout mice, that loss of FRα substantially decreased folate delivery to the brain, but transport was restored through calcitriol administration. Additionally, we provided in vitro and in vivo evidence that RFC expression and transport activity is inducible by another transcription factor, NRF-1. These findings demonstrate that augmenting RFC functional expression through interaction with specific transcription factors could constitute a novel strategy for enhancing brain folate delivery. Modulating folate uptake at the BBB may have clinical significance due to the lack of established optimal therapy for neurometabolic disorders caused by loss of FRα or PCFT function. iii Acknowledgments The studies presented in this thesis were conducted at the Department of Pharmaceutical Sciences, University of Toronto, with financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) awarded to Dr. Reina Bendayan. I am also very grateful for the studentship support from the Ontario Graduate Scholarship (OGS), Centre for Pharmaceutical Oncology (CPO), Pfizer Canada Graduate Fellowship, Mary Gertrude L’Anson Scholarship, Benjamin Cohen Bursary Fund, Shaping Student Life and Learning Fund, and the Department of Pharmaceutical Sciences. I would like to acknowledge everyone who contributed to this thesis, with special thanks to the following individuals: Dr. Reina Bendayan, my thesis supervisor, for her mentorship, guidance, and motivation throughout my graduate studies. Thank you for giving me the opportunity to pursue doctoral studies under your supervision and for entrusting me with this research project. I am grateful for your trust and support. Dr. Deborah L. O’Connor, co-author and member of my advisory committee, for allowing me to work in her laboratory and providing training and resources to complete the in vivo portion of this thesis. Dr. Carolyn Cummins, a member of my advisory committee, for sharing her expertise in transporter regulation by transcription factors and providing helpful advice in the design of in vitro and in vivo experiments. Dr. Shinya Ito, a member of my advisory committee, for sharing his knowledge on cellular drug transport and providing helpful feedback on in vitro functional assays. Dr. I. David Goldman, a co-author and collaborator at the Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, for his tremendous support and guidance during my research thesis. Thank you for sharing your scientific and clinical knowledge of folate transporters and for providing antibodies and inhibitors for the in vitro experiments. Dr. Richard H. Finnell, a co-author and collaborator at the Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, for all his input on this project and for generously providing breeding pairs of Folr1 knockout mice. Dr. Robert Steinfeld, a collaborator at the Department of Pediatric Neurology, University Children’s Hospital Zürich, for his initial insights on this work. Dr. Susanne Aufreiter, a co-author and collaborator at the Translational Medicine Program of the Hospital for Sick Children, for sharing her expertise in folate quantification by LC-MS/MS. Thank you for helping develop the protocol for folate extraction and quantification from mouse tissues. Dr. Tozammel Hoque, a co-author and research associate in the laboratory of Dr. R. Bendayan, for providing initial laboratory training and for assisting with immunoblotting and iv immunocytochemical analysis of transporter expression in human and rodent in vitro blood-brain barrier models. Dr. Bogdan Wlodarczyk at the Baylor College of Medicine, for providing excellent advice on establishing the breeding colonies of Folr1 knockout mice. All the past and present members of the Bendayan Lab for creating a positive and friendly work environment: Amy Kao, Billy Huang, Julian Gilmore, Wanying Dai, Steven Choi, Nareg Kara- Yacoubian, Olanrewaju Kayode, Olena Kis, and Tamima Ashraf. I would like to specifically thank Amila Omeragic and Sana-Kay Whyte-Allman for all the laughs and words of encouragement; the past five years would not have been the same without you both. I would also like to acknowledge the amazing graduate and undergraduate students that made significant contributions to the fulfillment of this research project: Adrian Turner, Constantine J. Georgiou, Marc Li, and Misaki Kondo. My sincerest gratitude to my family and friends for their unwavering love and support. To my parents, Apolinar and Dinah Alam, and my sister Diane, thank you for inspiring me to choose my own path and pursue my goals. I am forever grateful for your trust, understanding, and guidance. Lastly, I would like to thank my significant other, Richy Seto, for being my constant source of happiness and strength. You have taught me to be resilient and patient during challenging situations. This would not have been possible without your unconditional love and dedication. v Table of Contents Abstract .......................................................................................................................................... ii Acknowledgments ........................................................................................................................ iv Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Appendices ...................................................................................................................... xiv List of Abbreviations ...................................................................................................................xv Chapter 1 ........................................................................................................................................1 Introduction ...............................................................................................................................1 1.1 Folate Family of Compounds ............................................................................................3 1.1.1 Folate Derivatives ....................................................................................................3 1.1.2 Physiological Importance of Folates ........................................................................5 1.1.3 Global Folate Status .................................................................................................6 1.2 Folate Transport Pathways .............................................................................................10 1.2.1 Folate Receptors (FRs; FOLR) ..............................................................................12 1.2.2 Reduced Folate Carrier (RFC; SLC19A1) ..............................................................14