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Understanding the Role of MLCL AT-1 and Tafazzin in Mitochondrial Function

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Understanding the Role of MLCL AT-1 and Tafazzin in Mitochondrial Function Understanding the Role of MLCL AT-1 and Tafazzin in Mitochondrial Function by Edgard Marcello Mejia A Thesis submitted to the Faculty of Graduate Studies of the University of Manitoba in partial fulfillment of the requirements of the degree of Doctor of Philosophy Department of Pharmacology and Therapeutics University of Manitoba Copyright © 2016 Edgard Marcello Mejia Abstract Cardiolipin (CL) is a phospholipid found exclusively in mitochondria and is required for normal mitochondrial function. CL biosynthesis requires a crucial remodelling step that incorporates specific acyl chains onto its molecular structure. The enzyme primarily responsible for CL remodelling is Tafazzin (TAZ), a mitochondrial protein encoded by the TAZ gene localized to chromosome Xq28.12. Mutations on the TAZ gene result in a rare yet severe disease known as Barth Syndrome (BTHS). BTHS is characterized by symptoms that include cardiomyopathies, neutropenia and skeletal myopathies. The mitochondrial enzyme Monolysocardiolipin Acyltransferase -1 (MLCL AT-1) also exhibits the ability to remodel CL with specific acyl chains. The aims of our study were to 1) determine if a relationship exists between TAZ and MLCL AT-1 , 2) determine if MLCL AT-1 expression in BTHS lymphoblasts leads to improvements in mitochondrial function, and 3) to use the Taz knockdown (KD) mouse model to get a better understanding of the phenotypes displayed by BTHS patients. Our results showed that in normal healthy lymphoblasts, expression of MLCL AT-1 was inversely dependent on TAZ expression. However, in BTHS lymphoblasts, expression of MLCL AT-1 was significantly lower compared to healthy controls. With the use of a MLCL AT- 1-carrying plasmid, we expressed MLCL AT-1 in BTHS cells. This resulted in increased MLCL AT-1 gene, protein and enzyme activity. In addition, expression of MLCL AT-1 in BTHS cells resulted in increases in CL mass, improved mitochondrial function and a reduction in reactive oxygen species (ROS) production. However, no changes were detected in mitochondrial respiratory chain supercomplex (SC) assembly in BTHS cells expressing MLCL AT-1 compared to healthy controls. SC formation was disrupted in II | P a g e the hearts and skeletal muscle, but not the liver, of the Taz KD mice compared to wild- type (WT) animals. These results correlated with an elevated generation of hydrogen peroxide (H2O2) in the heart and skeletal muscle mitochondria of Taz KD mice compared to WT. Liver mitochondria from Taz KD mice, on the other hand, generated significantly less H2O2 compared to WT mice. The results from this study and our other published work demonstrate that MLCL AT-1 expression varies depending on the health of mitochondria and is tissue specific. In addition, our results reveal that TAZ expression is essential for various aspects of mitochondrial function including SC formation and ROS production. III | P a g e Acknowledgements This thesis was made possible by the financial support provided by the Barth Syndrome Foundation of Canada/USA and the Canadian Institutes for Health Research. Edgard M. Mejia received financial assistance from the Research Manitoba in conjunction with the Children's Hospital Research Institute of Manitoba. I would like to thank my supervisor, Dr. Grant Hatch, for giving me the opportunity to pursue my PhD under his guidance. Working in his laboratory has been a privilege and an experience I will never forget. I would also like to thank all of my lab members, both past and present. Their kindness, help and support made my time here both productive and enjoyable. I would also like to thank all the members of the department of Pharmacology and Therapeutics. It takes an entire department to ensure a student successfully completes their training, and I appreciate what each and every one of you have done for me. For her contribution to my lipid mass spectrometry data, I would like to thank Dr. Genevieve Sparagna. I would also like to acknowledge my committee members which include Dr. Donald Miller, Dr. Donald Smyth and Dr. Barbara Triggs-Raine. They have provided me with invaluable guidance throughout my training and helped ensure my thesis was properly evaluated. I would like to extend a special thanks to the late William Taylor. He was a constant source of advice and helped me develop my skills in the laboratory. Bill was always patient and always willing to help. I would not have made it this far without his guidance. Last but not least, I would like to thank my friends and family. I dedicate this work to them and thank them for their unshakeable support throughout these past IV | P a g e years. To my girlfriend Ngoc, thank you for your encouragement and for always giving me great advice. You know better than anyone what I have gone through to get this far. Thank you for always being there. To my mother and father, thank you for always believing I could do this and for teaching me to never give up. I will never forget the sacrifices you have made for me. Without you, none of this would be possible. V | P a g e Table of Contents Title Page ..................................................................................................................................................... I Abstract ........................................................................................................................................................ II Acknowledgements ................................................................................................................................... IV Table of Contents ...................................................................................................................................... VI List of Tables ............................................................................................................................................ VIII List of Figures ............................................................................................................................................ IX List of Copyrighted Material ..................................................................................................................... XI Abbreviations ............................................................................................................................................ XII Introduction .................................................................................................................................................. 1 1) Barth Syndrome .................................................................................................................................... 1 2) Cardiolipin ............................................................................................................................................. 1 3) CL-Protein Interactions ......................................................................................................................... 4 4) Mitochondrial Supercomplexes ............................................................................................................ 7 5) Mitochondrial supercomplexes and reactive oxygen species production ......................................... 10 6) Cardiolipin interacts with mitochondrial supercomplexes ................................................................. 12 7) Cardiolipin Biosynthesis ...................................................................................................................... 13 8) Tafazzin ............................................................................................................................................... 17 9) Barth Syndrome - Current Knowledge ................................................................................................ 20 10) Monolysocardiolipin Acyltransferase-1 ............................................................................................ 24 Objectives .................................................................................................................................................. 27 Materials and Experimental Methods .................................................................................................... 27 1) Materials ............................................................................................................................................. 27 2) Cell Culture.......................................................................................................................................... 29 3) Doxycycline-inducible Taz knockdown mouse model ........................................................................ 31 4) Cell Transfection ................................................................................................................................. 31 5) MLCL AT-1 Plasmid Preparation ......................................................................................................... 35 6) Cell Harvesting .................................................................................................................................... 35 7) RNA Isolation & Relative Gene Expression ......................................................................................... 35 8) Lipid Mass Spectrometry .................................................................................................................... 39 VI | P a g e 9) Lipid Isolation and Sensitive Lipid Phosphorous Assay ....................................................................... 39 10) Radiolabelling
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