TRPM2 ion channel trafficking and its role in mitochondrial fragmentation and cell death Nada Khaled S Abuarab Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biomedical Sciences December 2015 Declaration The candidate confirms that the work submitted is her own, except where work which has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated in the text. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2015 The University of Leeds and Nada Khaled S Abuarab I Publications . Rucha Karnik, Melanie J, Ludlow, Nada Abuarab, Andrew J. Smith, Matthew E. L. Hardy, David J. S. Elliott, Asipu Sivaprasadarao, Endocytosis of hERG is clathrin-independent and involves Arf6. Plos One, 2013. 8(12). Paul T Manna, Tim S Munsey, Nada Abuarab, Fangfang Li, Aruna Asipu, Gareth Howell, Alicia Sedo, Wei Yang, Jacqui Naylor, David J Beech, Lin-Hua Jiang, Asipu Sivaprasadarao, TRPM2 mediated intracellular Zn2+ release triggers pancreatic beta cell death. Biochem J, 2015. 466:537-546. Manuscript under revision . Fangfang Li, Nada Abuarab, Asipu Sivaprasadarao, Reciprocal regulation of actin cytoskeleton remodelling and cell migration by calcium and zinc: role of TRPM2 channels. 2015. Manuscript in preparation . Nada Abuarab, Tim S Munsey, Lin-Hua Jiang, Jing Li and Asipu Sivaprasadarao, Zn2+ entry through mitochondrial TRPM2 channels promotes Drp-1 recruitment and mitochondrial fragmentation. II Acknowledgements First and foremost I offer my sincerest gratitude to my supervisor, Professor Asipu Sivaprasadarao, for his valuable suggestions during the planning and development of this research work, his willingness to give his time so generously and his intellectual support. One simply could not wish for a better or friendlier supervisor. I would like to express my gratitude to Dr. Jing Li and Dr. Melanie Ludlow for all their valuable advice and friendly help during my lab work. I would also like to thank all of the Professor Sivaprasadarao research group members both past and present, Dr. Judith Hynes, Mr. Tim Munsey, Mrs. Hong-Lin Rong and Dr. Fangfang Li for their assistance and support during my project. I would also like to thank Dr. Lin-Hua Jiang for being my second supervisor. I also humbly thank Dr. Sreenivasan Ponnambalam for being my assessor. I also wish to thank Dr. Jamel Mankouri for providing many fantastic antibodies. My PhD would not be possible without the generous scholarship from King Saud bin Abdulaziz University for Health Sciences–National Guard Hospital and the UK-Saudi Bureau in London. Most of all, I am indebted to my family. Gratitudes in Arabic. ﻷبي الغالي خالد أبوعرب ، أعظم إنسان عرفته ، ياأنقى قلب وياأصدق الخلق. كنت وﻻزلت دوما تاج على رأسي أفتخر به. إليك أهدي هذا الجهد.. ليزداد فخرك بي. ﻷمي الحبيبة الغالية ومعلمتي فتحيه ولي ، علمتني كيف أكتب وكيف أتعلم وكيف أتحمل ، فشكرا لك لكل حرف علمتني أن أكتبه في دفتري الصغير فساعدني أن أكتب هذا الدفتر الكبير.. شكرا لحبك وعطاءك وتشجيعك الﻻ محدود. ﻷبني الغالي ومهجة قلبي فيصل ، شكرا لكل اللحظات التى أضحكتني فيها في وسط ضيق الحياة .. آسفه على كل الساعات واﻷيام التى قضيتها بعيده عنك .. أحبك جداً جداً. III ﻷختي الكبرى يسرى، كنتي دائما سنداً لي وﻹبني في غربتي.. شكرا لعطاءك ودعمك وحبك. ﻹخواتي شهد ورغد وأخي إبراهيم .. شكرا لدعمكم ودعواتكم وحبكم .. على الود نلتقي قريبا إن شاءهللا. الخميس 10 ديسمبر 2015 ندى خالد أبوعرب ليدز- المملكة المتحدة IV Abstract Mitochondria play a central role in oxidative stress-induced cell death. By increasing the production of reactive oxygen species, such as H2O2, oxidative stress causes mitochondrial fragmentation and apoptosis. It was hypothesised that Transient Receptor Potential Melastatin 2 (TRPM2) channels play a role in mitochondrial fragmentation and cell death. The rationale behind this hypothesis was the published evidence that oxidative stress stimulates TRPM2 channels, resulting in an increase in the cytosolic levels of Ca2+ and Zn2+, and that both these ions are detrimental to mitochondrial health and cell survival. To test the hypothesis, human umbilical vein endothelial cells (HUVECs) and endothelial cells isolated from wild-type and TRPM2 knock-out mice were used. TRPM2 actions were suppressed using pharmacological agents and small interfering RNA (siRNA). Fluorescent reporters were used to examine changes in intracellular ion distribution and organelle morphology. Molecular biology, biochemical and imaging techniques were used to examine the dynamics of ions and organelles. Exposure of HUVECs to H2O2 or high glucose stress led to TRPM2 activation, resulting in extracellular Ca2+ entry, lysosomal membrane permeability (LMP) and the release of lysosomal free Zn2+. Unexpectedly, this was accompanied by the accumulation of Zn2+ in the mitochondria. The rise in mitochondrial Zn2+ led to extensive mitochondrial fragmentation, mitochondrial outer membrane permeabilisation (MMP) and cell death. Silencing of TRPM2 channels with siRNA prevented intracellular Zn2+ redistribution, mitochondrial fragmentation and cell death. Endothelial cells derived from TRPM2 knock-out mice were resistant to oxidative stress-induced mitochondrial fragmentation. Biochemical and immunostaining experiments revealed an unexpected presence of TRPM2 channels in mitochondria, where they mediated mitochondrial Zn2+ uptake. Accumulation of Zn2+ in the mitochondria led to mitochondrial fragmentation by promoting the recruitment of cytoplasmic Drp1, an enzyme responsible for mitochondrial fission. Taken together, the results of this thesis revealed a novel mechanism for how oxidative stress can cause excessive mitochondrial fragmentation and cell death: the mechanism involves activation of TRPM2 channels leading to increased Ca2+ entry, V LMP and release of lysosomal Zn2+; Zn2+ thus released is taken up by the mitochondria, leading to Drp1 recruitment, mitochondrial fragmentation and finally cell death. Since mitochondrial fragmentation is associated with several age-related chronic illnesses, including neuronal (Alzheimer’s, Parkinson’s), cardiovascular (atherosclerosis, myocardial infarction) and metabolic/inflammatory (diabetes) disorders, these results suggest that the TRPM2 channel is a novel target that could be explored for therapeutic intervention of age-related illnesses. VI Abbreviations ACA N-(p-Amylcinnamoyl) anthranilic ADP Adenosine-5’-diphophate ATP Adenosine-5’-triphophate ADPR Adenosine diphosphate ribose Apaf-1 Apoptotic protease activating factor-1 Bcl-2 B-cell lymphoma 2 BPH-1 Benign prostatic hyperplasia cells CaM Calmodulin CAP Capsaicin CAT Catalase Cy3 Cyanine 3 cyt c Cytochrome c Drp1 Dynamin-related protein 1 DMSO Dulbecco’s modified eagle medium DPBS Dulbecco’s phosphate buffered saline EDTA Ethylenediamine-tetraacetic acid EGTA Ethylene glycol tetraacetic acid eNOS Endothelial nitric oxide synthase ER Endoplasmic reticulum ERAD ER-associated degradation Fis1 Fission protein 1 FITC Fluorescein isothiocyanate GFP Green fluorescent protein GPX Glutathione reductase HA Haemagglutinin A HBSS Hanks' Balanced Salt Solution HEK-MSR Human embryonic kidney cells expressing macrophage scavenger receptor HEK-TRPM2 Human embryonic kidney (HEK293) cells expressing tetracycline VII inducible human TRPM2 HeLa Line derived from cervical cancer cells taken from Henrietta Lacks HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HO Hydroxyl radical HRP Horseradish peroxidase hTRPM2 Human TRPM2 HUVECs Human Umbilical Vein Endothelial Cells H2O2 Hydrogen peroxide INS-1 Insulinoma cell LB Luria-Bertani broth LMP Lysosomal membrane permeability LAMP-1 Lysosomal associated membrane protein 1 MFF Mitochondria fission factor Mfn Mitofusin MMP Mitochondrial membrane permeability Ml Millilitre MPT Mitochondrial permeability transition pore MLSP Maximum life span potential NAD Nicotinamide adenine dinucleotide Ng Nanogram NO Nitric oxide OPA1 Optic atrophy 1 PARG Poly (ADP-ribose) glycohydrolase PARP Poly (ADP-ribose) polymerase PBS Phosphaye buffered saline PCR Polymerase chain reactions PFA Paraformaldehyde Rpm Revolutions per minutes ROS Reactive oxygen species SBS Standard Barth's Solution SDS Sodium dodecyl sulphate VIII SODs Superoxide dismutases TAE Tris-acetate-EDTA buffer Tm Primer annealing temperature TPEN N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine TRIS Tris(hydroxymethyl)aminomethane TRP Transient receptor potential TRPM2 Transient receptor potential melastatin 2 TRPV1 Transient receptor potential vanilloid 1 UV Ultraviolet light ZnT Zinc transport IX Amino acid abbreviations Amino acid Three-letter code One-letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine IIe I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V X Table of contents Declaration I Publications II Acknowledgements III Abstract V Abbreviations VII Amino acid abbreviations X List of figures XV List of tables XVIII 1 │Introduction 1 1.1 Historical overview 1 1.2 Oxidative stress 3 1.2.1 Cytoplasmic ROS and NADPH oxidase 3 1.2.2 Mitochondrial ROS 4 1.2.3