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Title: Electron transport chain defects as a culprit in a Parkinson fly model
Other Titles: Defecten in de Electron transport keten zijn betrokken bij de ziekte van Parkinson in een vliegenmodel
Authors: Vos, Melissa
Issue Date: 22-Oct-2012
Abstract: Parkinson¬ís disease (PD) is the second most common neurodegenerative disorder affecting mostly elderly people. However, the etiology remains elusive; mitochondria seem to play an important role in PD. The first mitochondrial link was observed when injection of the mitochondrial drug MPTP that inhibits Complex I of the electron transport chain (ETC), results in Parkinsonism. Moreover, post mortem brains of sporadic PD cases exhibit reduced activity of Complex I. Despite the fact that most cases are sporadic, genes have been identified to be involved in the pathology. At least 3 of these genes, PINK1, parkin and DJ-1, have a mitochondrial link. PINK1 is a mitochondrial kinase that results in early-onset PD. Mutations in pink1 in flies result in locomotion defects and mitochondrial defects, including reduced ATP levels, disrupted mitochondrial membrane potential, reduced Complex I activity and morphological defects. Here, we performed a screen to identify dominant modifiers of pink1 mutant phenotypes. From this screen, we identified Acon that suppresses the pink1 mutant phenotypes. Reduction of Acon in pink1 mutants and over-expression of the iron chelator results in less swollen mitochondria in pink1 mutant flies. This is due to the iron regulatory function of Acon that is induced by superoxide radicals that leak from the ETC. Acon-mediated rescue of pink1 mutants suggest an important role for the ETC. Our data supports this idea, since vitamin K2 feeding and application of Low-level Laser Light therapy (LLLT) increases ATP production in pink1 mutants resulting in amitochondrial and organismal rescue of Parkinson fly models. We identified Heix, a protein involved in the synthesis of vitamin K2 in our modifier screen. Comparable to its function in bacteria, we found that in flies vitamin K2 also serves as electron transfer molecule in the energy production resulting in a rescue of mitochondrial phenotypes in PD models that show ETC defects, including parkin mutant flies and rotenone-induced PD in flies. LLLT with a wavelength of 808nm is thought to activate Complex IV of the ETC. Here, we show that in pink1 mutants LLLT results in increased ATP production rates leading to increased ATP levels and improvement in mitochondrial membrane potential. Also, mitochondrial morphology is improved both in larval muscle and dopaminergic neurons. Thus, we show that ETC defect are a major culprit in Pink1 deficient animals and that via stimulation of the ETC we can partially rescue Pink1 deficiency.<span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:<br>normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><i style="mso-bidi-font-style:normal"><i style="mso-bidi-font-style:normal"><i style="mso-bidi-font-style:normal"><w:latentstyles deflockedstate="false" defunhidewhenused="true"  <w:lsdexception="" locked="false" priority="0" semihidden="false"  
Table of Contents: Table of contents i
List of abbreviation vii
Chapter 1: Introduction 1
1.1 The brain 1
1.2 Parkinson’s disease 1
1.3 Loss of dopaminergic neurons in Parkinson’s disease 3
1.3.1 Dopamine 3
1.3.2 Nigrostriatal pathway 4
1.4 Treatment of Parkinson’s disease 8
1.4.1 Pharmacological treatment of Parkinson’s disease 8
1.4.2 Surgical treatment of Parkinson’s disease 9
1.4.3 Physical exercise as treatment of Parkinson’s disease 10
1.5 Mitochondria play a role in Parkinson’s disease 10
1.5.1 Function of mitochondria 11
1.5.2 Energy conversion 15
1.5.3 Mitochondrial defects in Parkinson’s disease 17
1.6 Genetics in Parkinson’s disease 18
1.6.1 Autosomal dominant Parkinson’s disease 20
1.6.2 Autosomal recessive Parkinson’s disease 22
1.7 Parkin 22
1.8 PTEN-induced kinase 24
1.9 The PINK1/Parkin pathway 26
1.10 Complex I and PINK1 30
1.10.1 Complex I defects in PINK1 30
1.10.2 Complex I in parallel to the PINK1/Parkin pathway 31
1.11 Drosophila melanogaster as a model to study Parkinson’s disease 31
1.11.1 Animal models to study Parkinson’s disease 31
1.11.2 Drosophila melanogaster 32
1.11.3 Genetic fly models of Parkinson’s disease 34
1.11.4 Neurotoxic fly models of Parkinson’s disease 35
Chapter 2: General Objectives 36
Chapter 3: Results 38
3.1 Genetic screen to identify modifiers of pink1 38
3.2 Aconitase causes iron toxicity in Drosophila pink1 mutants 42
3.2.1 Abstract 43
3.2.2 Introduction 43
3.2.3 Results 46
Aconitase down regulation suppresses pink1 mutant phenotypes 46
Mitochondrial failure in pink1 mutants is due to Acon-dependent iron toxicity 56
Mitochondrial defects in Complex I-deficient dopaminergic neurons are rescued by partial loss of Acon 61
Mitoferritin rescues dopaminergic neuron mitochondrial defects in pink1B9 mutants and animals with reduced CG3683 expression 63
3.2.4 Discussion 64
3.2.5 Materials and methods 68
Drosophila stocks and maintenance. 68
Molecular Biology and biochemistry 68
Flight assay 70
ATP measurements 70
H2O2 levels 71
Mitochondria isolation 71
Superoxide production 72
Fe2+ measurements 72
Aconitase activity 72
Mitochondrial morphology in DA neurons 73
Mitochondrial morphology in flight muscles 74
3.2.6 Acknowledgements 74
3.3 Vitamin K2 is a mitochondrial electron carrier that rescues Pink1 deficiency 75
3.3.1 Abstract 76
3.3.2 Introduction 76
3.3.3 Results 78
Identification of heix 78
Heix is involved in the bio-synthesis of vitamin K2 82
Vitamin K2 rescues pink1B9 85
Vitamin K2 is an electron carrier 92
3.3.4 Discussion 99
3.3.5 Materials & methods 101
Genetics and S2 cells 101
Feeding experiments: MK-4, Q-10, E. coli and rotenone 103
Behavioral assays 105
Mitochondrial assays 105
Bacterial strains 109
Quinone isolation and RPHPLC 109
Bioinformatics and statistical analysis 110
3.3.6 Acknowledgements 110
3.4 Stimulation of the electron transfer chain via near-infrared light results in a rescue of Parkinson fly models 111
3.4.1 Abstract 112
3.4.2 Introduction 113
3.4.3 Results 115
Optimization of light stimulation 115
LLLT rescues functional defects of mitochondria 118
Mitochondrial morphology in larval muscle and dopaminergic neurons in adult pink1 mutant flies is restored 120
Functional rescue reaches its maximum prior to morphological rescue of PD-affected mitochondria 123
3.4.4 Discussion 124
3.4.5 Materials and methods 126
Fly stock and irradiation of flies 126
Flight assay 127
Mitochondrial assays 127
Bioinformatics and statistical analysis 128
3.4.6 Acknowledgements 128
Chapter 4: General discussion 129
4.1 Mitochondrial drugs induce Parkinson’s disease 130
4.2 Defects in Complex I activity have been shown in Parkinson’s disease patients 131
4.3 Oxidation of dopamine inhibits mitochondrial respiration 133
4.4 Genetic forms of Parkinson’s disease that are involved in mitochondrial respiration 135
4.5 General conclusion 139
Summary 141
Samenvatting 143
Lay summary 146
Populaire samenvatting 147
References 148
Melissa Vos * Curriculum Vitae 186
Publication status: accepted
KU Leuven publication type: TH
Appears in Collections:Department of Human Genetics - miscellaneous
Laboratory of Neuronal Communication

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