Zinc in the Retinal Pigment Epithelium and Choriocapillaris Interface A Thesis Submitted to University College London for the degree of Doctor of Philosophy Sabrina Cahyadi BSc Department of Ocular Biology and Therapeutics Institute of Ophthalmology University College London 2012 1 Declaration I, Sabrina Cahyadi confirm that the work presented in the thesis titled “Zinc in the Retinal Pigment Epithelium and Choriocapillaris Interface” is my own work. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. ______________ Sabrina Cahyadi Date: 14 November 2011 2 Acknowledgements “For wisdom will enter your heart and knowledge will be pleasant to your soul; discretion will guard you, understanding will watch over you.” First and foremost, I would like to thank my supervisors Professor Phil Luthert and Dr. Imre Lengyel for accepting me as their student, and the enormous support, help and guidance they have provided. I would also like to thank the Dorothy Hodgkin Postgraduate Award, the Mercer Fund and Professor Alan Bird for their very generous studentship which enabled me to do this PhD. The funding I receive from the Special Trustees of Moorfields Eye Hospital allowed me to do my day-to-day lab works. I thank Peter Marshall who ensured that the funds reach me in time. I must also thank Dr. Peter Munro, Cynthia Langley, Alexandra Boss, and Dr. Virginia Calder for all the help and moral supports I have received. I cannot believe how much have happened in the past three years. The people at the Institute of Ophthalmology have supported me through all the amazing, good, bad, ho-hum, and heartbreaking episodes of my PhD. My fellow lab members Neda Barzegar-Befroei and Ashraf Gango, who provided so much help and advice.The UCL students whom I have had the greatest privilege to work with, Bhavika Patel, Yemsrach Tadesse, Anishka Wilwaraachachii and Alexia Phedonos. Thank you for all of your help with the cell biology studies and supports throughout. I thank members of the Ohnuma, Bhattacharya and Shima lab, Asma Aslam and Meihua Ju for showing the very clueless student that I was when I started how to manage and run a lab without glitches. I thank Margaret Dellett, Wanzhou Hu, Vasia Papadaki and Michelle Kim for constantly telling me to fight for my PhD and providing shoulders to cry on (literally speaking). I thank Amna Shah, Anna Rose, Sancy Low, Wendy Mustill, Liza Sharmini, and Giovanna Alfano for teaching me all the cloning aspects, for all the encouragement and for what seemed to be an infinite cake supply during my time in their office. To all my King’s girls, Saleha Hassan, Rebecca Alade, Appitha Arulappu, Natasha Khalife, and Mehnaz Hossain, need I say more, you guys are truly the best. 3 Special thanks go to Prof. Christer Hogstrand from King’s College London who took me as his student for my final year undergraduate project. It was also because of your recommendation that this PhD is possible. Tom Carroll, Josef Rasinger and Valentina Reffatto, former Hogstrand Lab PhD students who instilled the confidence in me that I am worthy enough of a PhD.I am grateful for my friends Vania Kusworo and Marco Japutra for all the 12 years that I have known you both. I thank our collaborators Dr. Elod Kortvely, Matteo Gorza and Jennifer Behler from Helmholtz Zentrum Munchen for useful discussions, sheltering me for a month in your lab and pointing out a crucial error in my experimentation which would have otherwise been detrimental. I thank Professor Richard Thompson for taking some of his valuable time to read the drafts of the manuscript and for some very useful discussions about zinc stoichiometry. I am indebted to the people closest to me, my parents who have been my ardent supporters for all 23 years of my life, my brother and my sisters for their emotional and financial support during my undergraduate and PhD degree. To my sister Janice, thank you for all the frozen Bolognese sauce tubs and banana cakes you made when cooking seemed to be the last thing on my mind. I also thank Winardi Suhali for five years of endless support, and encouragement. You have seen me at my best and at my worst, and thank you for sticking around for me. Finally, I thank my examiners Prof. John Greenwood and Dr. Kathryn Taylor for their interest in my thesis and a valuable opportunity to discuss it with them. 4 List of Posters and Publications Barzegar-Befroei, Neda, Cahyadi, Sabrina , Gango, Ashraf, Peto, Tunde, and Lengyel, Imre 2011. Zinc in Eye Diseases. Zinc in Human Health . Amsterdam: IOS Press. Sabrina Cahyadi , Bhavika J. Patel, Meihua Ju, Alexia Phedonos, Anishka Wilwaraarachchi, and Imre Lengyel. Effect of Zinc on Fenestrae Formation in Cultured Endothelial Cells.Poster presentation at the Association of Research in Vision and Ophthalmology (ARVO) annual meeting 2011. Sabrina Cahyadi , Bhavika Patel, Meihua Ju and Imre Lengyel Effect of zinc on fenestra formation in cultured endothelial cells. Poster presentation at the Young Researcher Vision Camp in Liebertingen June 25-27 2010. Zinc in the RPE-Choroid Interface Presentation at the Zinc-UK meeting 2010 5 Abbreviations AE – Acrodermatitis Enteropathica AD – Alzheimer’s Disease AKT – Serine Threonine Kinase AMD – Age-Related Macular Degeneration AMG - Autometallography APS – Ammonium Persulfate AREDS – Age-Related Eye Disease Study BCA – Bichinconinic Assay BSA – Bovine Serum Albumin CDK4 – Cell division kinase 4 CRALBP - Cellular retinaldehyde binding protein CFH – Complement Factor H DNA – Deoxyribonucleic Acid ERK - Extracellular-signal-Regulated Kinases E-Cadherin – Epithelial Cadherin FAM - 6-carboxyfluoroscein FGF – Fibroblast Growth Factor GABA - Gamma-Aminobutyric Acid GAPDH - Glyceraldehyde 3-Phosphate Dehydrogenase GDP – Guanosine Diphosphate GFP – Green Fluorescent Protein GPx – Glutathione Peroxidase 6 GTP – Guanosine Triphosphate HMDS - Hexamethyldisilazone HRP – Horseradish Peroxidase HUVECs - Human umbilical vein endothelial cells IL6 – Interleukin 6 IP3 - Inositol 1,4,5-Trisphosphate kDa - Kilodalton KDR - Kinase insert Domain Receptor LA – Latrunculin A LB- Luria-Bertani N-Cadherin – Neural Cadherin nM – Nano molar MDCK – Madin-Darby Canine Kidney cell line MMLV- Moloney Murine Leukemia Virus MT – Metallothionein MTF - Metal Responsive Element Binding Transcription Factor 1 NEAA – Non-Essential Amino Acid NO – Nitric Oxide NK – Natural Killer PEDF – Pigment Epithelium Derived Factor PDGF - Platelet- Derived Growth Factor PCR – Polymerase Chain Reaction PFA – Paraformaldehyde 7 PI3K - phosphoinositol-3-Kinase PV-1 – Plasmalemma Vesicle Associated Protein PVDF - Polyvinylidene Fluoride QPCR- Quantitative Polymerase Chain Reaction RNA – Ribonucleic Acid RPE- Retinal Pigment Epithelium RT-PCR – Reverse Transcription Polymerase Chain Reaction SOC – Super Optimal Broth with glucose SDS- Sodium Dodecyl Sulfate TER – Transepithelial Resistance TBS – Tris-Buffered Saline TBST- Tris- Buffered Saline Tween TPEN - N,N,N',N'-tetrakis (2 pyridylmethyl) ethylenediamine TEMED - ,,′, ′-Tetramethylethylenediamine TGF-b - Transforming Growth Factor – beta UBC – Ubiquitin C protein VEGF – Vascular Endothelial Growth Factor YWHAZ - Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein ZIP – ZRT/IRT-like proteins ZNT – also known as cation diffusion facilitator proteins ZO-1 – Zona Occludin 1 protein ZONAB – ZO-1 associated nucleic acid binding protein 8 Abstract The highest concentration of zinc in human tissues is found in the retinal pigment epithelium (RPE)-choroid complex. Despite the association of zinc deficiency with age-related macular degeneration (AMD) and the widespread use of zinc supplementation to slow the progression ofAMD, very little is known about how zinc affects the RPE and the choroid. Molecular and cell biology techniques were used to uncover how changes in zinc levels could play a role in regulating the RPE- choroid complex. First, QRT-PCR was used to assess the expressions of all 24 known zinc transporters in cadaveric human RPE, cultured RPE cells and cells isolated from other parts of the retina, ZIP12 was identified as a potentially important transporter to regulate zinc levels at the RPE-choroid interface. As there is very little published about ZIP12, bioinformatics and data mining were used to understand how this protein might function. Confirmation of these predictions was achieved through the cloning and expression of V5-tagged ZIP12 protein in different cell lines. Based on these experiments, we concluded that ZIP12 is a plasma membrane transporter that mediates zinc influx. In parallel, we tested the hypothesis that extracellular zinc levels in Bruch’s membrane might be involved in regulating both the RPE as well as the fenestrated choroidal capillaries using cultures of ARPE19 and bEND5 cells respectively. The presence of extracellular zinc in the growth media affected the characteristics of ARPE19 cells as well as fenestrae formation in bEND5 cells. In summary, the range of zinc transporter at the RPE-choroid interface was defined and properties of one particular transporter, ZIP12 which may have a specific role at this site, were elucidated. Using cellular systems some of the effects of zinc on the RPE-choroid complex were investigated. Future studies are required to elucidate the role of zinc in the AMD pathogenesis. 9 Table of Contents Declaration ................................................................................................................. 2 Abstract .....................................................................................................................