The neurochemical consequences of aromatic L-amino acid decarboxylase deficiency

310 Pages·2011·6.08 MB·English

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)
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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……………………………

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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.

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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
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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
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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 ............................................................................... 84
2.3 Sulforhodamine B cell proliferation assay ................................................... 85
2.3.1 SRB assay procedure................................................................................. 86
2.3.2 Validation .................................................................................................. 86
2.4 Lactate dehydrogenase release assay ............................................................ 88
2.4.1 LDH Release assay procedure .................................................................. 88
2.4.2 Validation .................................................................................................. 89
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2.5 Western Blotting ............................................................................................. 89
2.6 RNA analysis ................................................................................................... 91
2.6.1 Cell preparation for RNA analysis ............................................................ 91
2.6.2 Total RNA extraction ................................................................................ 91
2.6.3 Reverse Transcription ............................................................................... 91
2.6.4 Agarose gel electrophoresis ...................................................................... 92
2.6.5 Quantitative polymerase chain reaction (PCR) ......................................... 92
2.7 Mitochondrial Respiratory Chain Complex Assays .................................... 94
2.7.1 Complex I .................................................................................................. 94
2.7.2 Complex II/III ........................................................................................... 94
2.7.3 Complex IV ............................................................................................... 95
2.7.4 Citrate synthase ......................................................................................... 96
2.8 Total protein determination .......................................................................... 96
2.9 Statistical analysis ........................................................................................... 97
CHAPTER 3 .................................................................................................................. 98
3 AROMATIC L-AMINO ACID DECARBOXYLASE ACTIVITY ASSAY
METHOD ...................................................................................................................... 98
3.1 Background ..................................................................................................... 99
3.2 Principle .......................................................................................................... 99
3.3 Method ............................................................................................................. 99
3.3.1 Patient Plasma Samples ............................................................................ 99
3.3.2 Cell Homogenate Preparation ................................................................. 100
3.3.3 L-dopa decarboxylation .......................................................................... 100
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3.3.4 5-HTP decarboxylation ........................................................................... 100
3.4 HPLC ............................................................................................................. 101
3.4.1 Equipment ............................................................................................... 101
3.4.2 Procedure................................................................................................. 101
3.4.3 Data Analysis .......................................................................................... 105
3.5 Validation ...................................................................................................... 105
3.5.1 Plasma L-dopa decarboxylation .............................................................. 105
3.5.2 SH-SY5Y cell samples L-dopa decarboxylation .................................... 109
3.5.3 5-HTP decarboxylation cell samples ...................................................... 110
CHAPTER 4 ................................................................................................................ 116
4 THE EFFECT OF 5-HTP AND SEROTONIN ON CELL VIABILITY,
ANTIOXIDANT STATUS AND MITOCHONDRIAL FUNCTION .................... 116
4.1 Introduction .................................................................................................. 117
4.2 Aims ............................................................................................................... 119
4.3 Methods ......................................................................................................... 120
4.3.1 Treatment solutions ................................................................................. 120
4.3.2 Cell culture .............................................................................................. 120
4.3.3 Measurement of Indoleamines ................................................................ 120
4.3.4 AADC activity ........................................................................................ 120
4.3.5 Western Blotting ..................................................................................... 120
4.3.6 RT-PCR ................................................................................................... 120
4.3.7 Measurement of GSH .............................................................................. 120
4.3.8 SRB cell proliferation assay .................................................................... 120
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4.3.9 LDH Release assay ................................................................................. 121
4.3.10 Mitochondrial respiratory chain complex assays and citrate synthase ... 121
4.4 Experimental Protocol ................................................................................. 121
4.4.1 Indoleamine measurement ...................................................................... 121
4.4.2 Toxicity testing ....................................................................................... 121
4.4.3 GSH measurement .................................................................................. 122
4.4.4 Mitochondrial respiratory chain and citrate synthase ............................. 122
4.5 Results ............................................................................................................ 122
4.5.1 AADC in SH-SY5Y and 1321N1 cells ................................................... 122
4.5.2 NSD-1015 concentration for inhibition of AADC .................................. 124
4.5.3 Indoleamine metabolism ......................................................................... 124
4.5.4 5-HTP and serotonin toxicity in SH-SY5Y cells .................................... 125
4.5.5 Effect of 5-HTP and serotonin on LDH release in astrocytoma cells ..... 127
4.5.6 Intracellular GSH in SH-SY5Y cells ...................................................... 131
4.5.7 Mitochondrial respiratory chain activity following 5-HTP or serotonin
treatment ................................................................................................................ 131
4.6 Discussion ...................................................................................................... 140
4.6.1 5-HTP and serotonin metabolism ........................................................... 140
4.6.2 5-HTP and serotonin toxicity .................................................................. 142
4.6.3 Glutathione and mitochondrial respiratory chain activity ....................... 145
4.7 Conclusions ................................................................................................... 148
CHAPTER 5 ................................................................................................................ 149
5 THE EFFECT OF L-DOPA AND DOPAMINE ON CELL VIABILITY,
GLUTATHIONE HOMEOSTASIS AND MITOCHONDRIAL FUNCTION ..... 149
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5.1 Introduction .................................................................................................. 150
5.2 Aims ............................................................................................................... 152
5.3 Methods ......................................................................................................... 152
5.3.1 Treatment solutions ................................................................................. 152
5.3.2 Cell culture .............................................................................................. 152
5.3.3 Measurement of catecholamines ............................................................. 152
5.3.4 Measurement of GSH .............................................................................. 152
5.3.5 SRB cell proliferation assay .................................................................... 153
5.3.6 LDH Release assay ................................................................................. 153
5.3.7 Mitochondrial respiratory chain complex assays and citrate synthase ... 153
5.4 Experimental Protocol ................................................................................. 153
5.4.1 Catecholamine measurement .................................................................. 153
5.4.2 Toxicity testing ....................................................................................... 153
5.4.3 GSH measurement .................................................................................. 154
5.4.4 Mitochondrial respiratory chain .............................................................. 155
5.5 Results ............................................................................................................ 155
5.5.1 Effect of NSD-1015 on catecholamine metabolism in human
neuroblastoma cells ............................................................................................... 155
5.5.2 Effect of L-dopa and dopamine on neuroblastoma cell survival ............ 156
5.5.3 Intracellular GSH neuroblastoma cells ................................................... 160
5.5.4 Intracellular GSH astrocytoma cells ....................................................... 169
5.5.5 GSH Release from human astrocytoma cells .......................................... 169
5.5.6 GSH Release from human neuroblastoma cells ...................................... 170
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