Molecular characterisation of SGCE-associated myoclonus-dystonia ...

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Results 67 3.3.6 Oxidative stress in PINK1 mutant fibroblasts Superoxide dismutase 2 (SOD2) and GSH are important antioxidants, which function in the cellular stress defence. Whereas SOD2 can only be found in mitochondria, GSH is additionally detectable in the cytosol. The cellular SOD2 level was determined relative to the complex II expression by densitometric analysis of three western blots (cp. 2.3.11) prepared with cell lysates of all available mutant samples and six controls. In comparison to the average control level the nonsense mutants expressed 36% less SOD2 (p fourfold standard deviation of the control group) (see Fig 24A). When GSH levels were chromatically measured (cp. 2.3.4) in the cell lysates of control, nonsense or missense PINK1 fibroblast cultures, GSH levels were similar. However, upon treatment with exogenous oxidizing species generated by paraquat, GSH levels were elevated in control and nonsense cultures by 66% and 65%, respectively. GSH levels were elevated to a greater extent (148%) in missense cultures, despite the cells having impaired mitochondrial function (cp. Fig 24B). Basal conditions Stressed conditions 200 A ** 180 160 p fourfold standard deviation of the control group, n – number of individuals.
Results 68 3.3.7 Mitochondrial network When the mitochondrial network was studied by use of MitoTracker Green FM controls, missense mutants and nonsense mutants showed a similar pattern. A B C Fig 25 Mitochondrial network in primary fibroblasts. MitoTracker Green FM was used to visualize the mitochondrial network. The cell nucleus is stained with DAPI. (A) Control, (B) nonsense mutant and (C) missense mutant fibroblasts with comparable mitochondrial structure. 3.3.8 Electron microscopy studies To study the mitochondrial morphology in the cells, pellets of four controls, the four nonsense mutant samples and the missense mutant sample were prepared (procedure described in 2.3.13.1). The latter sample was repeated once. Transverse sections of the pellets were cut and microscopically examined by a specialist, who was blinded to the mutational status of the samples. In a subset of the PINK1 p.V170G mutant fibroblasts the EM analysis revealed a mitochondrial pattern clearly different from the structures seen in controls. The respective mitochondria in the missense sample were swollen and markedly enlarged (see mitochondrial shape in Fig 26).
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Aus der Klinik für Neurologie (Pro
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This thesis is dedicated to my fami
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List of content II 2.2.6 Equipment
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List of content IV 4 DISCUSSION ...
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List of tables VI LIST OF TABLES Ta
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Introduction 2 The EPS has been imp
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Introduction 4 7, 9, 10, 13, and 15
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Introduction 6 chromosome contained
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Introduction 8 PD is the most commo
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Introduction 10 results in a p.W437
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Introduction 12 showed good respons
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Introduction 14 sequentially from o
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Page 57 and 58: Table 5 Demographic, clinical and g
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Page 99 and 100: Zusammenfassung 90 mutationspositiv
Page 101 and 102: References 92 8 REFERENCES Abou-Sle
Page 103 and 104: References 94 Butler AG, Duffey PO,
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Page 107 and 108: References 98 Hedrich K, Hagenah J,
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Page 111 and 112: References 102 Martin MA, Molina JA
Page 113 and 114: References 104 Piccoli C, Sardanell
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Page 125 and 126: Appendix 116 04 - 06/2006 Erasmus M
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Appendix 118 ABSTRACTS 1. Grünewal
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Appendix 120 I would like to thank
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