Title Page
Contents
ABSTRACT 14
Chapter 1. Mini review of NOX4 in neurodegenerative diseases 16
1. Introduction 16
1.1. Neurodegenerative diseases 16
1.2. NAPDH oxidase 4 (NOX4) 19
1.3. Additional roles for NOX in Alzheimer's disease (AD) and Parkinson's disease (PD) 21
References 26
Chapter 2. NOX4 as a critical effector mediating neuroinflammatory cytokines, myeloperoxidase and osteopontin, specifically in astrocytes in the hippocampus in Parkinson's disease 33
1. Introduction 33
2. Materials and methods 37
2.1. Animal study 37
2.2. Experimental procedures 37
2.3. Behavioral test 38
2.4. Tissue preparation and dissection of brain regions in mice and human 38
2.5. Antibodies 39
2.6. Immunohistochemistry and immunofluorescence analysis 40
2.7. Immunoblot 41
2.8. Proteome arrays 43
2.9. Human astrocytes 43
2.10. 3D images and morphological cell death 44
2.11. Mitochondrial ROS (mtROS) production 44
2.12. Statistical analysis 45
3. Results 46
3.1. Chronic MPTP treatment leads to loss of dopaminergic neurons and motor function in mice 46
3.2. NOX4 is increased in the hippocampus of PD models 57
3.3. MPO and OPN are elevated in PD models 69
3.4. NOX4 associates MPO and OPN expressions in the hippocampal astrocytes 81
3.5. MPO and OPN promote impairment of the mitochondrial metabolism and induce lipid peroxidation in human astrocytes 85
4. Discussion 96
References 102
Chapter 3. NADPH oxidase 4 (NOX4) as a novel biomarker and therapeutic target in neurodegenerative diseases 112
1. Contents 112
2. References 119
SUPPLEMENTARY DATA 123
List of Abbreviations 128
Figure 1. NOX isoforms and regulatory subunits 20
Figure 2.1. Schematic representation of the experimental procedure, drug administration and behavioral test. In an animal model of... 48
Figure 2.2. Representative images and quantification of Nissl-stained neurons in the substantia nigra of chronic MPTP intoxication mice... 49
Figure 2.3. Immunohistochemistry images and quantification of TH-positive dopaminergic neurons in substantia nigra of chronic MPTP... 50
Figure 2.4. Immunohistochemistry images and quantification of TH-positive fibers in the striatum after chronic MPTP intoxication of... 51
Figure 2.5. Measurement of body weight of mice in the control and MPTP group (n=12 per group). Body weight was measured at the... 52
Figure 2.6. Measurement of food intake per day in the control and MPTP group (n=12 per group). Food intake measured at the same... 53
Figure 2.7. Measurement of water consumption in the control and MPTP group (n=12 per group). Water consumption was measured at... 54
Figure 2.8. Representative images and quantification of immunohistochemistry for alpha-synuclein of PD patients and... 55
Figure 2.9. The effect of rotarod test in the MPTP-induced PD mice at a fixed speed at 10, 20, 30 and 40 rpm for 120 s. The animals were tested... 56
Figure 2.10. Images of NOX4 expression in different regions of the human brain (hippocampus and cerebellum). Scale bar: 125 and 300 μ... 59
Figure 2.11. The quantification of NOX4 expression in different regions of the human brain (hippocampus and cerebellum). Values are means... 60
Figure 2.12. Images of NOX4 expression in different regions of the PD mice model (striatum, hippocampus, substantia nigra, cerebellum). Scale... 61
Figure 2.13. The quantification of NOX4 expression in different regions of the PD mice model (striatum, hippocampus, substantia nigra,... 62
Figure 2.14. Representative double-immunofluorescence images of PD patients for NOX4 using Alexa flour 488 (green) in combination with... 63
Figure 2.15. Representative the quantification for cell numbers of NOX4 in combination with the astrocyte marker, GFAP or with the... 64
Figure 2.16. Representative double-immunofluorescence images of PD mouse models for NOX4 using Alexa flour 488 (green) in combination... 65
Figure 2.17. The quantification for cell numbers of NOX4 in combination with the astrocyte marker, GFAP or with the... 66
Figure 2.18. Representative double-immunofluorescence images of PD patients and MPTP mice models for NOX4 using Alexa flour 594 (red)... 67
Figure 2.19. Immunoblot analysis and quantification of NOX4, GFAP and NeuN levels from hippocampal lysates of MPTP-induced PD mouse... 68
Figure 2.20. Representative images of the Proteome Profiler Mouse XL Cytokines Array immunoblot analysis of 111 different cytokines,... 71
Figure 2.21. Representative quantification MPO and OPN of the Proteome Profiler Mouse XL Cytokines Array immunoblot analysis of...[원문불량;p.57] 72
Figure 2.22. Representative immunoblot analysis and quantification for MPO and OPN levels in MPTP groups compared to control. GAPDH... 73
Figure 2.23. Immunofluorescence staining and quantification of MPO and OPN that were labelled with Alexa Fluor 488 (green) and Alexa... 75
Figure 2.24. Immunofluorescence staining and quantification of MPO and OPN that were labelled with Alexa Fluor 488 (green) and Alexa... 76
Figure 2.25. Representative visualization by immunofluorescence of MPO or OPN (red) expressing on astrocyte markers (GFAP) and... 77
Figure 2.26. Quantification of MPO and OPN in the hippocampal brain of MPTP mice co-localized with GFAP and NeuN in MPTP mice. Scale... 78
Figure 2.27. Representative visualization by immunofluorescence of MPO or OPN (red) expressing on astrocyte markers (GFAP) and...[원문불량;p.64] 79
Figure 2.28. Quantification of MPO and OPN in the hippocampal brain of PD patients co-localized with GFAP and NeuN in human PD... 80
Figure 2.29. Representative images of immunofluorescence staining and quantification for MPO (red) expressing on astrocyte markers (GFAP)... 82
Figure 2.30. Representative images of immunofluorescence staining and quantification for OPN (red) expressing on astrocyte markers (GFAP)... 83
Figure 2.31. Quantification for MPO or OPN expressing on astrocyte markers (GFAP) in hippocampal brain of control, MPTP, NOX4... 84
Figure 2.32. Representative images of human astrocytes transfected with MPO- or OPN- GFP-tagged plasmids. Scale bars: 20 µm. 86
Figure 2.33. Representative 3D images of control and MPO and OPN overexpression on human astrocytes. Scale bars: 20 µm. 87
Figure 2.34. Quantification of the morphological dead cells (%) in control compared to MPO and OPN overexpressing human astrocytes.... 88
Figure 2.35. Images and quantification of immunoblot analysis for five mitochondrial ETC protein levels, including Complex I (CI (NDUFB8)),... 91
Figure 2.36. Representative of images and quantification of immunoblot analysis for five mitochondrial ETC protein levels, including Complex I... 92
Figure 2.37. Representative images and quantification of immunofluorescence for 4-HNE expression (red) with astrocyte marker... 93
Figure 2.38. Immunoblot analysis images and quantification of 4-HNE protein levels of MPO and OPN overexpressing human astrocytes.... 94
Figure 2.39. mtROS levels and quantification using MitoSOX staining in human astrocytes of control (Con) and MPO and OPN overexpression.... 95
Figure 2.40. Schematic diagram of a possible pathway contributing to PD in the astrocytic hippocampus. The red arrow indicates growth.... 101
Figure 3.1. NOX4 in the brain participates in neurodegenerative disease (including AD and PD). 116
Supplementary Figure S1. Representative images of the Proteome Profiler Mouse XL Cytokines Array for immunoblot analysis of 111... 123
Supplementary Figure S2. The quantification of immunoblot analysis of the Proteome Profiler Mouse XL Cytokines Array of 111 different... 125
Supplementary Figure S3. Representative images of immunofluorescence staining and quantification of MPO (red)... 126
Supplementary Figure S4. Representative images of immunofluorescence staining and quantification of OPN (red) expression... 127