The neurochemical consequences of aromatic L-amino acid decarboxylase deficiency Submitted By: George Francis Gray Allen Department of Molecular Neuroscience UCL Institute of Neurology Queen Square, London Submitted November 2010 Funded by the AADC Research Trust, UK Thesis submitted for the degree of Doctor of Philosophy, University College London (UCL) 1 I, George Allen 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. Signed………………………………………………….Date…………………………… 2 Abstract Aromatic L-amino acid decarboxylase (AADC) catalyses the conversion of 5- hydroxytryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (L-dopa) to the neurotransmitters serotonin and dopamine respectively. The inherited disorder AADC deficiency leads to a severe deficit of serotonin and dopamine as well as an accumulation of 5-HTP and L-dopa. This thesis investigated the potential role of 5- HTP/L-dopa accumulation in the pathogenesis of AADC deficiency. Treatment of human neuroblastoma cells with L-dopa or dopamine was found to increase intracellular levels of the antioxidant reduced glutathione (GSH). However inhibiting AADC prevented the GSH increase induced by L-dopa. Furthermore dopamine but not L-dopa, increased GSH release from human astrocytoma cells, which do not express AADC activity. GSH release is the first stage of GSH trafficking from astrocytes to neurons. This data indicates dopamine may play a role in controlling brain GSH levels and consequently antioxidant status. The inability of L-dopa to influence GSH concentrations in the absence of AADC or with AADC inhibited indicates GSH trafficking/metabolism may be compromised in AADC deficiency. 5-HTP was demonstrated to potentially be mildly toxic to human neuroblastoma cells but not astrocytoma cells; however the concentrations required for this response are likely to be higher than pathophysiological levels in AADC deficiency. These results indicate the need for investigations addressing the effects of chronic 5-HTP exposure as only acute effects were investigated in the current study. This thesis also investigated the effect of altered availability of the AADC coenzyme pyridoxal 5‟-phosphate (PLP) on AADC activity, protein and expression. In two patients with inherited disorders of PLP metabolism reductions in plasma AADC activity were observed. Furthermore PLP- deficient human neuroblastoma cells were found to exhibit reduced levels of AADC activity and protein but not altered expression. These findings suggest maintaining adequate PLP availability may be important for optimal treatment of AADC deficiency. 3 Table of Contents THE NEUROCHEMICAL CONSEQUENCES OF AROMATIC L-AMINO ACID DECARBOXYLASE DEFICIENCY ............................................................................ 1 ABSTRACT ..................................................................................................................... 3 TABLE OF CONTENTS ................................................................................................ 4 TABLE OF FIGURES .................................................................................................. 13 LIST OF TABLES ........................................................................................................ 17 LIST OF ABBREVIATIONS ...................................................................................... 18 ACKNOWLEDGEMENTS .......................................................................................... 21 CHAPTER 1 .................................................................................................................. 23 1 INTRODUCTION ................................................................................................. 23 1.1 Monoamine Neurotransmitters ..................................................................... 24 1.1.1 Serotonin ................................................................................................... 24 1.1.2 Dopamine .................................................................................................. 25 1.2 Monoamine Metabolism ................................................................................ 25 1.2.1 Indoleamine Metabolism ........................................................................... 25 1.2.2 Catecholamine Metabolism ....................................................................... 26 1.3 Monoamine Neurotransmission .................................................................... 26 1.3.1 Vesicular Uptake ....................................................................................... 26 1.3.2 Release ...................................................................................................... 27 1.3.3 Receptors ................................................................................................... 29 1.3.4 Re-uptake .................................................................................................. 29 1.3.5 Degradation ............................................................................................... 31 4 1.4 Disorders of Aromatic Amino Acid Metabolism ......................................... 33 1.4.1 Hyperphenyalaninemia ............................................................................. 33 1.4.2 Monoamine Neurotransmitter Deficiency................................................. 33 1.4.3 Treatment .................................................................................................. 34 1.4.4 Related Conditions .................................................................................... 35 1.5 AADC deficiency ............................................................................................ 38 1.5.1 Clinical Picture .......................................................................................... 38 1.5.2 Diagnosis ................................................................................................... 39 1.5.3 Inheritance ................................................................................................. 39 1.5.4 Treatment .................................................................................................. 40 1.6 Aromatic L-amino acid decarboxylase ......................................................... 42 1.6.1 Structure and Reaction Mechanism .......................................................... 42 1.6.2 Alternative Reactions ................................................................................ 44 1.6.3 Alternative Substrates ............................................................................... 44 1.6.4 AADC Splicing ......................................................................................... 45 1.6.5 AADC Localisation ................................................................................... 46 1.6.6 AADC Regulation ..................................................................................... 48 1.7 AADC Deficiency Pathogenesis ..................................................................... 50 1.7.1 Pathogenic Mechanism ............................................................................. 50 1.7.2 Precursor Accumulation ............................................................................ 51 1.7.3 Alternative metabolic pathways for 5-HTP .............................................. 52 1.7.4 Alternative Metabolic Pathways for L-dopa ............................................. 54 1.7.5 Oxidative Stress ........................................................................................ 56 5 1.7.6 Glutathione ................................................................................................ 58 1.7.7 Mitochondrial Respiratory Chain .............................................................. 59 1.8 Hypotheses ...................................................................................................... 62 1.9 Aims ................................................................................................................. 62 CHAPTER 2 .................................................................................................................. 66 2 MATERIAL AND METHODS ............................................................................ 66 2.1 Materials .......................................................................................................... 67 2.2 High performance liquid chromatography .................................................. 69 2.2.1 Catecholamine HPLC................................................................................ 69 2.2.2 Equipment ................................................................................................. 69 2.2.3 Measurement of Indoleamines by HPLC .................................................. 70 2.2.4 GSH HPLC ............................................................................................... 74 2.2.5 Measurement of PLP by HPLC ................................................................ 75 Tissue Culture ........................................................................................................... 76 2.2.6 SH-SY5Y neuroblastoma cell culture ....................................................... 76 2.2.7 1321N1 astrocytoma cell culture .............................................................. 81 2.2.8 Cell sample collection ..............................................................................