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C:\Documents and Settings\lproctor.ULL\Local Settings\Temporary Internet Files\OLK36\Copyright - thesis.doc MOLECULAR STUDY OF CELL CULTURE MODELS OF PARKINSON’S DISEASE AND HUNTINGTON’S DISEASE by Michael Orth, MD Department of Clinical Neurosciences Royal Free and University College Medical School University of London A thesis submitted, in fulfilment, for the degree of Doctor of Philosophy September 2004 1 UMI Number: U593085 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U593085 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The discovery of the genetic basis of neurodegenerative disorders has enabled the generation of models to study their pathogenesis. In part one, human embryonic kidney cells with inducible expression of wild-type or mutant G209A a-synuclein modelled increased a-synuclein expression and a familial form of Parkinson’s disease, respectively. Both wild-type and mutant a- synuclein were localised to vesicles, some of which were catecholaminergic. Over­ expression of wild-type or mutant (G209A) a-synuclein alone did not reduce cell viability, cause oxidative stress or impair mitochondrial function. However, mutant a- synuclein expression enhanced the susceptibility to dopamine toxicity causing increased oxidative stress and cell death. This effect was similar to that of reserpine, an inhibitor of vesicular monoamine uptake, in controls. These results suggest that a-synuclein may play a role in dopamine compartmentalisation. Loss of function conferred by the G209A mutation could therefore increase cytoplasmic dopamine concentrations with subsequent cell damage or death. In part two, myoblast cell lines were established, and characterised, from the R6/2 mouse model of Huntington’s disease (HD). Mutant N-terminal huntingtin transgene over-expression was associated with significantly greater numbers of myotubes suggesting a role of huntingtin in muscle differentiation. In long-term culture, differentiated R6/2 myotubes, but not controls, formed nuclear huntingtin inclusions. Inclusion number depended upon culture medium conditions suggesting that environmental factors might be relevant. This model of HD in non-neuronal post­ mitotic cells may be useful to study the pathophysiology of, and possibly the effect of therapeutics on, huntingtin aggregate formation. The third part examined the suggestion that codon 129 homozygosity of the prion protein (PrP) gene may predispose to sporadic inclusion body myositis (sIBM). 2 Codon 129 zygosity in 41 sIBM muscle biopsies was not significantly different to results published in population studies in several Western countries suggesting sIBM is not linked to homozygosity at codon 129 of the PrP gene. 3 Declaration All of the experimental work in this thesis was performed by the author and the majority of this thesis is published (see Publications). 4 Acknowledgements There are many people who I am indebted to in one way or another. These include my supervisors, Tony Schapira and Mark Cooper, who have inspired and encouraged me each in their own way. I am grateful to Tony particularly for helping me to keep an eye on the “bigger picture” beyond the work necessary for this degree and for his ongoing support. To Mark I am grateful for teaching me patiently about scientific thinking and critical appraisal of my own work. Many insights came only after years on this educational path on which he led with great spirit and always in good humour. This work would have been very difficult without the help of Sarah Tabrizi. Not only did she welcome me with a warm heart but also she had laid some of the foundations for some of this work. And let’s not forget the encouraging “tannoy- DAAARLINGS” that brightened up the day! A special thanks has to go to Jan-Willem Taanman for always being there to answer questions and give advice, without him I would have struggled more! And then there were the “fellow strugglers” competing in the number of tissue culture plates and incubator space needed such as Paul Hart and Sion Williams, and later Pras and Chris. Thanks also to Ross King, Michelle and Jane W for help and their companionship, and to the secretary team who are always there to help and always have a friendly word. Last but not least I want to thank my family and friends, some far away “in Europe”, for their encouragement, and most of all Maria for her love and support even though this “thing” was taking up a sheer endless amount of time and energy. Without her I would most certainly have become even more of a recluse towards the end. Moltes gracies! 5 Dedication To my family; my parents, Hans and Heidi, and my sister, Meike Index page Abstract 2 Declaration 4 Acknowledgements 5 Dedication 6 Table of contents 7 List of Tables 16 List of Figures 17 Abbreviations 20 CHAPTER 1. General introduction 1.1. Introduction 23 1.1.1. Mitochondrial function 24 1.1.2. Types of cell death 26 1.1.2.1. Necrosis 26 1.1.2.2. Programmed cell death 27 1.1.3. Free radicals and cellular defence systems 31 1.1.4. Cellular consequences of oxidative stress 33 1.1.5. Excitotoxicity 35 1.1.6. The ubiquitin-proteasome system (UPS) 36 1.1.7. Models of neurodegenerative diseases 37 1.1.7.1. Toxin models 38 1.1.7.2. Knock-out models 38 1.1.7.3. Transgenic animal models 39 1.1.7.4. Cell culture models 39 7 CHAPTER 2. Material and Methods 2.1. Materials 42 2.2. Cell lines and cell culture 44 2.2.1. EcR 293 cell line (Invitrogen, UK) 44 2.2.2. NT2 cells (Ntera2/D1 )(Stratagene, UK) 44 2.2.3. SH-SY5Y neuroblastoma cells 45 2.2.4. R6/2 transgenic mice and myoblast cell lines 45 2.2.5. Myotube desmin stain and myotube counts 46 2.3. Cell culture 47 2.3.1. Cell freezing and defrosting 47 2.4. Molecular biology 48 2.4.1. The Ecdysone Mammalian Expression System 48 2.4.2. Generation of vectors 49 2.4.3. Transfection of cDNA 50 2.4.4. Ring cloning 51 2.4.5. DNA extraction from cells 52 2.4.6. DNA extraction from muscle tissue 51 2.4.7. Estimation of DNA concentration and purity 53 2.4.8. Polymerase chain reaction (PCR) 53 2.4.8.1. Amplification of cDNA in pIND constructs 54 2.4.8.2. Prion protein gene ( PRNP) PCR 54 2.4.9. PCR clean-up and sequencing 55 2.4.10. DNA digestion by restriction endonucleases 55 2.4.11. Detection of DNA 55 2.5.1. Cytochemical staining 56 2.5.1.1. Haematoxylin and Eosin 56 2.5.1.2. Modified Gomori trichrome 56 8 2.5.1.3. Modified Congo red 57 2.5.2. Immunocytochemistry 57 2.5.3. Immunohistochemistry 58 2.5.4. Antibodies 59 2.5.5. Catecholamine stain 59 2.5.6. Image analysis and photography 60 2.6. Protein assays 60 2.6.1. Protein extraction 60 2.6.2. Protein determination 61 2.6.2.1. The bicinchoninic acid copper assay (BCA) 61 2.6.2.2. The BioRad protein determination kit 61 2.6.3. Protein separation 62 2.6.4. Staining of protein gels 62 2.6.5. Immunostaining of blots 62 2.7. Enzyme analysis 63 2.7.1. Preparation of mitochondrial enriched fractions (MEFs) 64 2.7.2.