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Gene Transfer As a Potential Treatment for Tetralujdrobiopterin Deficient States

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Gene Transfer As a Potential Treatment for Tetralujdrobiopterin Deficient States Gene Transfer as a Potential Treatment for Tetralujdrobiopterin Deficient States Rickard F oxton Division of Neurochemistry Department of Molecular Neuroscience Institute of Neurology University College London Submitted November 2006 Funded by Brain Research Trust Thesis submitted for the degree of Doctor of Philosophy, University of London. I, Richard Hartas Foxton, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis.' 1 UMI Number: U592813 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 U592813 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 Tetrahydrobiopterin (BH4) is an essential cofactor for dopamine (DA), noradrenaline (NA), serotonin and nitric oxide (NO) synthesis in the brain. Inborn errors of BH4 metabolism including GTP cyclohydrolase 1 (GTP-CH) deficiency are debilitating diseases in which BH4, DA, 5-HT and NO metabolism are impaired. Current treatment for these disorders is typically monoamine replacement +/- BH4. Whilst correction of the primary defect is the ideal, BH4 treatment is problematic as it is expensive and inefficacious. One approach to treat BH4 disorders is to use gene therapy as a more permanent, effective alternative. In this thesis the potential of gene therapy in an animal model of partial BH4 deficiency, the hph-1 mouse, was examined. These mice show many neurochemical similarities associated with BH4 deficient states, including impaired BH4 (-69%), DA (-14%), NA (-23%), serotonin turnover (-55%) and NO metabolites in the brain. In cultured astrocytes from hph-1 mice BH4 was significantly lower than wild type (-53%), and produced less BH4 (- 89%) and NO metabolites (-64%) when stimulated with lipopolysaccharide (LPS) plus interferon-y (IFN-y), stimuli that increase GTP-CH and iNOS expression. When hph-1 astrocytes were infected with a recombinant adenovirus encoding human GTP cyclohydrolase (AdGCH), concentration-dependent increases in BH4 levels were observed, with just 1 virus particle per 10 cells resulting in 50-fold increases in BH4. AdGCH can upregulate the impaired NO production observed in hph-1 astrocytes following stimulation with LPS + IFN-y, although only if BH4 was increased prior to stimulation. Examination of the molecular mechanisms behind the impaired NO production in LPS + IFN-y stimulated cells revealed that iNOS dimerisation is attenuated in hph-1 astrocytes when compared wild type (-84%), and could be increased to wild type levels when cells were pre-treated with AdGCH. Analysis of total iNOS protein expression revealed no difference between wild type and hph-1. These results raise the possibility that gene therapy could be used as a corrective solution for tetrahydrobiopterin deficient states. 2 CONTENTS ABSTRACT.......................................................................................................................... 2 C o n t e n t s ..........................................................................................................................3 List of figures ...........................................................................................................1 0 LIST OF TABLES.............................................................................................................13 ABBREVIATIONS...........................................................................................................14 ACKNOWLEDGEMENTS.............................................................................................17 1. INTRODUCTION....................................................................................................... 18 1.1. Pterins and pteridines ......................................................................................19 1.2. Tetrahydrobiopterin ........................................................................................ 19 1.3. De novo biosynthesis of tetrahydrobiopterin ...............................................19 1.4. Tetrahydrobiopterin recycling and salvage pathways ................................23 1.5. Structure of the tetrahydrobiopterin biosynthetic enzymes ......................25 1.5.1. GTP cyclohydrolase (GTP-CH).........................................................25 1.5.2. 6-pyruvoyl-tetrahydropterin synthase...............................................25 1.5.3. Sepiapterin reductase.......................................................................... 25 1.6. Regulation of tetrahydrobiopterin homeostasis ......................................... 26 1.7. Functions of Tetrahydrobiopterin ................................................................ 28 1.7.1. Aromatic amino acid mono-oxygenases....................................... 29 1.7.2. Further cofactor roles o f tetrahydrobiopterin...............................31 1.7.3. Further biological roles o f tetrahydrobiopterin............................31 1.8. Nitric oxide synthase ...................................................................................... 32 1.8 .1. General structure and function o f nitric oxide synthases............ 32 1.8.2. Biosynthesis o f nitric oxide.............................................................. 34 1.8.3. Role o f tetrahydrobiopterin in nitric oxide synthesis...................36 1.9. Nitric oxide synthase isoforms ......................................................................37 1.9.1. Neuronal nitric oxide synthase........................................................37 1.9.2. Inducible nitric oxide synthase........................................................38 1.9.3. Endothelial nitric oxide synthase....................................................38 1.9.4. Mitochondrial nitric oxide synthase............................................... 39 3 1.10. Biological role of nitric oxide ..................................................................... 39 1.11. Disorders of tetrahydrobiopterin metabolism ...........................................41 1.11.1. Autosomal recessive GTP cyclohydrolase 1 deficiency........... 43 1.11.2. 6-Pyruvoyl tetrahydropterin synthase deficiency.......................44 1.11.3. Sepiapterin reductase deficiency.................................................. 44 1.11.4. Dihydropteridine reductase deficiency........................................ 45 1.11.5. Dopa responsive dystonia...............................................................45 1.11.6. Impaired nitric oxide metabolism in tetrahydrobiopterin deficiencies....................................................................................................47 1.11.7. Acquired tetrahydrobiopterin disorders...................................... 48 1.12. The hph-1 mouse ...........................................................................................49 1.13. Gene therapy ..................................................................................................49 1.14. Aims................................................................................................................49 MATERIALS AND METHODS..................................................... 50 2.1. High Performance Liquid Chromatography (HPLC) ................................51 2.1.1. Quantification of tetrahydrobiopterin by reverse-phase HPLC coupled with electrochemical detection.....................................................51 2.1.2. Method o f separation o f tetrahydrobiopterin............................... 53 2.1.3. Quantification of monoamines by reverse-phase HPLC coupled with electrochemical detection...................................................................57 2.1.4. Method o f separation o f the monoamines...................................... 57 2.2. Tissue Culture..................................................................................................61 2.2.1. Animals............................................................................................... 61 2.2.2. Preparation of primary astrocyte cultures.................................... 61 2.2.3. Shaking and plating o f cells.............................................................63 2.2.4. Treating cells..................................................................................... 64 2.3. Sample Preparation .........................................................................................64 2.3.1. Preparation o f brain cytosol fraction............................................ 64 2.3.2. Preparation of tissue for tetrahydrobiopterin and monoamine measurement..................................................................................................64 2.3.3. Harvesting and preparation o f astrocytes for tetrahydrobiopterin measurement.................................................................................................65 2.3.4. Harvesting and preparation o f astrocytes for Western blotting..61 2.4. Measurement of Tetrahydrobiopterin biosynthetic capacity ....................67
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