Luis Carlos Morales Burbano
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Lipid pathways in Huntington’s disease by Luis Carlos Morales Burbano A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pharmacology University of Alberta © Luis Carlos Morales Burbano, 2018 Abstract Huntington’s disease (HD) is a monogenic neurodegenerative disorder characterized by progressive choreic movements, dystonia, motor incoordination, cognitive decline and behavioural changes. HD is caused by an abnormal increase in the number of CAG repeats in the exon 1 of the huntingtin (HTT) gene, leading to an expanded polyglutamine stretch in the HTT protein. Mutant huntingtin (muHTT) is prone to misfold into toxic conformations and to aggregate, impairing vesicular transport, mitochondrial metabolism, cell signalling and gene transcription, and leading to neuronal dysfunction and death in various brain regions. Our laboratory and others have shown that two distinct lipid biosynthetic pathways, the mevalonate pathway and the pathway for the synthesis of gangliosides, are also impaired in HD, potentially contributing to disease pathogenesis. The mevalonate pathway is responsible for the production of cholesterol and isoprenoids. Isoprenoids are used for the prenylation of small GTPases, a crucial post-translational modification that regulates small GTPases activity and a wide range of downstream cell processes, including vesicular traffic and autophagy. Several studies have investigated the synthesis of cholesterol in HD models, but whether impairment of the mevalonate pathway could also lead to defective protein prenylation was not known, and has been investigated in the first part of this thesis. I showed that steady-state prenylation of various representative small GTPases is not affected in a wide range of cell and animal HD models, in spite of downregulation of the mevalonate pathway. However, levels of one of these small GTPases, RAB7, a protein involved in vesicular transport and selective autophagy, were significantly decreased across models of HD. Together with cholesterol, gangliosides are another class of lipids that are highly enriched in the brain and are crucial for neuronal functions. Studies in our laboratory showed that gangliosides ii levels are decreased in HD models and that administration of one ganglioside in particular, GM1, has profound therapeutic effects in HD mouse models. The underlying mechanisms were in part, investigated in this thesis. I demonstrated that GM1 decreases accumulation of protein aggregates in HD cells, as well in a second model of proteotoxic stress represented by pharmacological inhibition of the ubiquitin- proteasome system, thus alleviating cellular stress. Surprisingly, the beneficial effects of GM1 were not mediated by an increase in autophagy, a cellular process known to degrade muHTT aggregates and to be modulated by gangliosides in other model systems. Instead, I showed that administration of GM1 promotes the secretion of muHTT, as well as components of the aggresome, within extracellular vesicles (EVs). These are membrane-delimited vesicles that form within the multivesicular bodies (MVB) or that bud from the plasma membrane, carrying a wide array of proteins and nucleic acids, including toxic misfolded proteins. These data suggest that the neuroprotective activity of GM1 in HD models might be mediated, at least partially, by the secretion of toxic muHTT towards the extracellular space, thus relieving intracellular proteotoxic load in susceptible neurons. iii Preface This thesis is an original work by Luis Morales. Part of the data shown in Chapter 3 was generated in collaboration with Dr. Amany Mohamed, a former Ph.D. student of the Department of Pharmacology, under the supervision of Dr. Elena Posse de Chaves (Department of Pharmacology, University of Alberta). Data presented in chapter 6 have been generated in collaboration with Ms. Vaibhavi Kadam, a current student of the graduate program in Neuroscience (Neuroscience and Mental Health Institute, University of Alberta), under the supervision of Dr. Simonetta Sipione. Additionally, some of the analyses performed by electron microscopy were done by Dr. Nasser Tahbaz (Department of Cell Biology, University of Alberta). Part of the data presented in Appendix I was generated in collaboration with Mrs. Alba Di Pardo, a former student in Dr. Sipione laboratory, and will be submitted for publication as a co-authored paper, which is currently in preparation. I contributed with experimental data, as well as with the preparation of the manuscript. Chapters 4, 5 and 7 are my original work. iv Acknowledgements First, I would like to thank my Ph.D. supervisor, Dr. Simonetta Sipione, the person who has guided me through this long journey that has finally come to an end. I sincerely appreciate all the hard work you have been doing as my mentor during all these years. Your advice and feedback have helped me to become a better researcher every day. I am immensely grateful for giving me the opportunity to work with you. For sure, you will not be forgotten. I would also like to thank Dr. Elena Posse de Chaves. More than once you have been emotional support, as well as the voice of reason. To Dr. Luc Berthiaume, for your advice, comments and suggestions. To all the current and past members of the Sipione laboratory, especially to Diego Ordóñez who worked by my side in order to standardize new techniques and methodologies that were the basis of much of this thesis. Of course, I’d like to thank Melanie Alpaugh, Sabine Schmelz, Mel Horkey, Danny Galleguillos, Mel Horkey and Qian Wang. I would also like to say thank you to our younger members of the laboratory, Vaibhavi Kadam, John Monyror and Aislinn Maguire. You have given me the opportunity to be part of your growth as future researchers. To my little family here in Canada, Lina Parra, Libardo Estupiñán, and many other amazing people who have contributed to my happiness in one way or another. Hundreds of stories and adventures will be remembered forever. To my parents who have given me everything I ever needed and more. Mom and Dad who are the pillars of my life, and my brother, who has been my role model since my infancy. v To my friends Edwin Insuasty and Heler Romero, two wonderful people I had the opportunity to grow with, both professionally and personally. In the case I did not say it before, I want to express how proud I am of both of you. Thanks for teaching me that friendship is forever. Importantly, I’d like to thank Colciencias and the government of Colombia for providing me with the financial support for my Ph.D. studies. And finally, to Adriana Briones, the person whose existence made all this possible. I will never have enough words to express my gratitude towards you. vi Table of contents Chapter 1: General background ...................................................................................................... 1 1.1. General introduction ............................................................................................................. 2 1.2. Epidemiology ....................................................................................................................... 3 1.3. Clinical Features ................................................................................................................... 4 1.3.1. Diagnosis ....................................................................................................................... 4 1.3.2. Motor abnormalities ...................................................................................................... 5 1.3.3. Non-motor abnormalities ............................................................................................... 6 1.4. Neuropathology .................................................................................................................... 8 1.4.1. Neuronal pathways of the basal ganglia ........................................................................ 9 1.4.2. Macroscopic alterations ............................................................................................... 10 1.4.3. Microscopic findings ................................................................................................... 12 1.5. Huntingtin functions and mutant huntingtin dysfunctions ................................................. 16 1.5.1. The Huntingtin gene and protein ................................................................................. 16 1.5.2. Function(s) of Huntingtin protein ................................................................................ 19 1.5.3. Pathophysiology of HD: The loss and gain-of-function of mutant HTT .................... 21 1.5.3.1. Cleavage, misfolding and aggregation of muHTT ............................................... 22 1.5.3.2. Mitochondrial dysfunction .................................................................................... 25 1.5.3.3. Transcriptional dysregulation ............................................................................... 27 1.5.3.4. Impairment in protein clearance systems .............................................................. 29 1.6. Animal and cell models of HD ........................................................................................... 33 1.6.1. Non-mammalian models of HD .................................................................................. 33 1.6.2. Mouse models of HD ..................................................................................................