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TETRAHYDROBIOPTERIN DEFICIENCY AND BRAIN NITRIC OXIDE METABOLISM By Michael Peter Brand A thesis submitted as partial fulfilment for the degree of Doctor of Philosophy in the Faculty of Science at the University of London. March 1997 Institute of Neurology Department of Neurochemistry Queen Square London WCIN 3BG ProQuest Number: 10055824 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. uest. ProQuest 10055824 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract. Tetrahydrobiopterin is an essential cofactor for the aromatic amino acid mono­ oxygenase group of enzymes. Inborn errors of tetrahydrobiopterin metabolism result in hyperphenylalaninaemia and impaired catecholamine and serotonin turnover. Tetrahydrobiopterin is also a cofactor for all known isoforms of nitric oxide synthase (NOS). The effect of tetrahydrobiopterin deficiency on brain nitric oxide metabolism has to date been given little consideration. In this thesis the effect of tetrahydrobiopterin deficiency on brain nitric oxide metabolism has been studied using a mouse model of tetrahydrobiopterin deficiency, the hph-1 mouse. Tetrahydrobiopterin was measured in 10 and 30 day old mice in whole brain and cerebellum. At both age points there was a significant -50% reduction in tetrahydrobiopterin content for whole brain and cerebellum in the hph-1 mouse compared to corresponding control mice. NOS activity was measured in whole brain from 10 and 30 day old hph-1 and control mice. No difference was observed in enzyme activity when tetrahydrobiopterin was included in the incubation medium. However, omission of tetrahydrobiopterin from the reaction buffer resulted in significantly lower NOS activity in the hph-1 mouse group compared to controls. Tetrahy- drobiopterin was also shown to have a potent effect on the affinity of brain NOS for arginine. The for arginine was virtually identical for the control and hph-1 mouse when tetrahydrobiopterin was included in the reaction buffer. In the absence of cofactor, the for arginine was three fold greater for control and five fold higher for hph-1 preparations. The accumulation of cGMP from slices prepared from the cerebellum was measured in both groups of mice at both 10 and 30 days using the glutamate analogue, kainate. In the 10 day old hph-1 rhouse there was a significant 50% reduction in cGMP levels under basal and stimulated conditions. In the 30 day old hph-1 mouse there was a significant 30% reduction in cGMP accumulation in both basal and activated states. W hole brain amino acid levels were measured. In the 10 day old hph-1 mouse confounding hyperphenylalaninaemia may affect the availability of the NOS substrate, arginine. In the 30 day old hph-1 mouse, which has normal phenylalanine levels, reduced citrulline levels may indicate reduced NOS activity. Reduced levels of tetrahydrobiopterin and also arginine, have been shown to lead to superoxide formation by nitric oxide synthase. Superoxide can react with nitric oxide to form the oxidising species, peroxynitrite, which has been shown to damage of the mitochondrial electron transport chain. Mitochondrial function together with the anti­ oxidant, glutathione, were analysed in both hph-1 and control mice at 10 and 30 days to measure oxidative stress. However, no differences were observed between the two groups. In summary, partial deficiency of tetrahydrobiopterin appears to lead to impaired brain NOS function leading to an impairment of the nitric oxide/cGMP pathway. Acknowledgements. I would like to thank my two supervisors. Dr Simon Heales and Professor John Clark for allowing me this opportunity to study for a PhD and for their advice, encouragement and support during the duration of this project. As one of the grantholders for this project I would like to thank Dr John Land for his help, guidance and being available whenever I needed his advice. I would like to thank all the members of the Department of Neurochemistry for their support during the last three years but I would particularly like to thank Dr Jane Barker and Dr Roger Hurst. This thesis could not have been prepared with out the expert assistance of June Smalley who helped with the more complicated figures. I would like to extend my gratitude to the staff of the Denny Brown Laboratories, especially John Frogley, Janet Cunningham and Wendy Rooke. I must thank Mr Tony Briddon and Mr Robert Courtney for their expertise and help in the analysis of whole brain amino acids and brain slice morphology respectively. Finally, I must thank Dr Isabel Smith, Dr Mark Goss-Sampson and Dr David Müller for setting me on the right path. I am indebted to the Brain Research Trust for their generous funding of this project. Contents. Page Title P a g e............................................................................................................................. 1 Abstract ...............................................................................................................................2 Acknowledgements ...........................................................................................................4 List of Figures and Tables ...............................................................................................9 Abbreviations ................................................................................................................ 13 Chapter 1. Introduction. 14 1.1. Tetrahydrobiopterin ......................................................................................... 15 1.1.1. P teridines .......................................................................................................... 15 1.1.2. Biological importance of pterins .................................................................. 15 1.1.3. Biosynthesis of tetrahydrobiopterin ............................................................. 19 1.1.4. Regulation of biosynthesis p athw ay ................................................................ 23 1.1.5. Catabolism of tetrahydrobiopterin ...................................................................28 1.1.6. Cofactor role in tetrahydrobiopterin dependent mono-oxygenases ..............28 1.1.7. Other functions of tetrahydrobiopterin ............................................................32 1.2. Hyperphenylalaninaemia due to phenylalanine hydroxylase deficiency . 37 1.3. Inborn errors of tetrahydrobiopterin metabolism .......................................... 37 1.3.1. GTP cyclohydrolase I deficiency ..................................................................... 39 1.3.2. 6 -pyruvoyl-tetrahydropterin deficiency ............................................................39 1.3.3. Dihydropteridine reductase deficiency ........................................................... 40 1.3.4. Pterin-4a-carbinolamine dehydratase deficiency ............................................ 41 1.3.5. L-Dopa responsive dystonia .............................................................................41 1.4. Tetrahydrobiopterin deficiency in other diseases ..........................................42 1.4.1. Alzheimer's disease ............................................................................................42 1.4.2. Parkinson's disease ........................................................................................... 43 1.5. Nitric oxide and nitric oxide synthase ........................................................... 43 1.5.1. Historical perspective ......................................................................................... 43 1.5.2. The role of nitric oxide .................................................................................... 44 1.5.3. Nitric oxide sy nthase ......................................................................................... 46 1.5.4. Neuronal nitric oxide synthase (Type I N O S ) ............................................... 47 5 1.5.5. Endothelial nitric oxide synthase (Type III NOS) ....................................... 48 1.5.6. Inducible nitric oxide synthase (Type II NOS) ............................................ 48 1.5.7. Biosynthesis of nitric oxide ............................................................................... 48 1.5.8. The role of tetrahydrobiopterin in the NOS complex .................................. 51 1.6. Aims .................................................................................................................... 54 Chapter 2. Materials and Methods. 55 2.1. Chem icals.............................................................................................................56 2.2. A n im als ................................................................................................................57 2.3. Tetrahydrobiopterin an aly sis .............................................................................57 2.4. Glutathione analysis ............................................................................................60 2.5. Nitric oxide synthase activity .........................................................................

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