Title Page

Contents

ABBREVIATIONS 11

ABSTRACT 12

1. Introduction 14

1.1. Physiology and pathophysiology of α-synuclein 14

1.2. Mutations of α-synuclein 17

1.3. Histone acetyltransferase (HAT) and α-synuclein 18

1.4. Oxidative stress and autophagy and α-synuclein 19

1.5. Purpose of this study 20

2. Methods and Materials 21

2.1. Chemicals 21

2.2. Cell culture and treatment 22

2.3. Transfection 22

2.4. Cell viability and nitric oxide assay 23

2.5. DPPH free radical scavenging assay 24

2.6. DNA fragmentation analysis (DNA ladder) 24

2.7. DCFDA assay 24

2.8. Mitochondrial membrane potential by JC-10 assay 25

2.9. Mitochondria/cytosol isolation 25

2.10. Nuclear/cytosol fraction preparation 26

2.11. Immunofluorescence 26

2.12. Reverse transcription-PCR (RT-PCR) 26

2.13. Western blot analysis 28

2.14. RNA interference 29

2.15. Co-Immunoprecipitation assays 29

2.16. Identification of dioscin in DN extract using HPLC 29

2.17. Statistical analysis 30

3. Result 30

3.1. Overexpression of αS and different synucleinopathy-related biomarkers characterisation 30

3.1.1. Plasmid transfection 30

3.1.2. Confirmation of transfection in neuronal cells 32

3.1.3. αS overexpression reduces cell viability 34

3.1.4. αS inhibiting H3 histone acetylation 38

3.1.5. αS and mutants are aggregated after transfection 39

3.1.6. αS and mutants promoting apoptotic markers 42

3.2. Regulation of αS in neuronal cells apoptosis via GCN5 46

3.2.1. αS interacting with endogenous GCN5 46

3.2.2. GCN5 overexpression regulating endogenous αS level 47

3.2.3. GCN5 transcriptionally regulates BIM-mediated apoptosis 48

3.2.4. Overexpression of αS transcriptionally upregulates BIM 49

3.2.5. Co-overexpression of GCN5 rescuing neuronal cells from apoptosis 51

3.2.6. Co-overexpression of GCN5 ameliorating neuronal cell viability 55

3.3. Regulation of αS in oxidative stress and autophagy 55

3.3.1. αS-mediating mitochondrial impairment and ROS production 55

3.3.2. Changes in mitochondrial autophagy regulating markers 56

3.4. A novel herbal compound as therapeutic of PD through autophagic regulation 58

3.4.1. DN is protecting microglial cells from LPS-induced viability 61

3.4.2. DN pretreatment suppressing inflammatory mediators following LPS treatment 62

3.4.3. DN-mediated amelioration of MAPK phosphorylation 63

3.4.4. Identification of dioscin in DN extract 67

3.4.5. Dioscin protects against neurotoxicity 68

3.4.6. Dioscin dose-dependently rescues autophagic function impaired by MPP+[이미지참조] 71

3.4.7. Dioscin dose-dependently upregulates autophagosome formation 72

3.4.8. Dioscin dose-dependently downregulates apoptotic markers 76

3.4.9. Dioscin dose-dependently increases TH cells and neurotrophic factors 78

4. Discussion 81

4.1. Characterisation of αS in synucleinopathy-related biomarkers 81

4.2. αS negatively regulates GCN5 in the process of neuronal apoptosis 83

4.3. Targeting herbs might ameliorate αS-mediated autophagy dysregulation and apoptosis 86

5. Future study 92

5.1. Conclusion 95

References 96

ABSTRACT (in Korean) 121

Table 1. Name of genes and specific sequences used for RT-PCR. 27

Fig 1. Physiological roles of αS. Although the exact physiological roles of αS are yet to be fully known in normal conditions αS regulates SNARE complexes, pre-synaptic... 15

Fig 2. Schematic of αS protein structure and positions of its familial mutations. The N-terminus of αS (1–60), the non-amyloidogenic component (NAC) region (61-... 18

Fig 3. Plasmid and transfection kit concentration confirmation. HEK293T cells were transfected using different doses of lipofectamine 3000 (3.75 and 7.5 µl) to... 31

Fig 4. Cell morphology on exposure to different doses of αS plasmid and lipofectamine 3000. (A-C) SH-SY5Y cells were exposed to a ratio of the plasmid to... 31

Fig 5. Morphology of puromycin post-treated cells on 72 hr. (A-C) SH-SY5Y cells were transfected using 1:1 of plasmid and lipofectamine (3.75-7.5 µl) and treated with... 33

Fig 6. Post-selection expression of αSwt and mutant A53T. This expression is from the first passage of stably transfected cells. To confirm the expression of...[이미지참조] 34

Fig 7. Protein expression of αS and 2 mutants (A53T and A30P). Post-selection protein expression of stably over-expressed cells. The densitometric calculation was... 34

Fig 8. αS and mutations are translocating into the nucleus after overexpression. (A) nuclear or cytoplasmic fractions were isolated from non-transfected SH-SY5Y... 35

Fig 9. Cell viability assay in non-neuronal cells. HEK293T cells were transiently transfected and cell viability was measured 48 h after transfection by (A) MTT, (B)... 36

Fig 10. Cell viability assay in neuronal cells. SH-SY5Y cells were transiently transfected for 48 hr and viability was measured using a DAPI staining assay; the... 37

Fig 11. Cell viability in presence of neurotoxin MPP+ (1 mM). (A) SH-SY5Y and (B) HEK293T cells were transiently transfected using wild type αS (αS+/+) and mutant...[이미지참조] 38

Fig 12. Nuclear translocation inhibiting H3 histone acetylation. Overexpressing αS decreases H3 histone acetylation and lysine residue 9 (ac-Lys 9). 39

Fig 13. Aggregation of αS and mutant in neuronal cells after overexpression. (A) Stably transfected cells were tested for possible aggregation using αS multimer... 41

Fig 14. Aggregation of αS and mutant in neuronal cells after overexpression. Stably transfected cells were subjected to cytochemistry to analyse possible... 42

Fig 15. Impact of αS and mutant's overexpression on apoptosis. (A) Activation of caspase-3 -mediated apoptosis, correlated by (B) mitochondrial cytochrome c release. 44

Fig 16. Effect of αS and mutant's overexpression on apoptotic markers. Proteins were isolated from respective stably transfected cells, densitometric analysis was done... 45

Fig 17. αS interacting with GCN5. (A) Co-IP of GCN5 and αS in protein lysates of wildtype SH-SY5Y cells showed the interaction of GCN5-αS. (B) Immunostaining of... 47

Fig 18. GCN5 overexpression, not knockdown, regulates the endogenous level of αS (A) Pharmacological inhibition by MB-3 upregulates αS level, while activation of... 48

Fig 19. GCN5 regulates apoptotic signalling in neuronal cells. (A) Knockdown of GCN5 by siRNA significantly upregulating BIM transcription and downstream... 49

Fig 20. αS overexpression upregulates Bim transcription. (A) mRNA and (B) protein expression of BIM, E2F-1 and EGR1. (C) Co-IP probe of anti-αS and E2F-1... 50

Fig 21. Activation of caspase-3 followed by BIM upregulation. Protein lysates from αS overexpressing cells were probed for anti-caspase-3 and anti-Bcl-2 to check... 51

Fig 22. αS endogenously interacting with GCN5 and E2F-1 in neuronal cells. Co-IP of αS and GCN5 and E2F-1 using protein extracts from wild-type SHSY5Y cells. 52

Fig 23. Co-overexpression of αS-GCN5 in neuronal SH-SY5Y cells. (A) mRNA and (B) protein expression confirming co-overexpression of αS-GCN5. 53

Fig 24. mRNA expression of BIM and E2F-1 in respective neuronal cells. (A) mRNA and (B) protein expression of BIM and downstream apoptotic signalling in... 54

Fig 25. Impact of αSwt and αS+GCN5 in neuronal SH-SY5Y cells viability. Cell viability of Vec, αSwt and αS+GCN5 was analysed using conventional DAPI staining...[이미지참조] 55

Fig 26. Mitochondrial integrity assessment in αSwt and mutations. (A) ROS production was measured by DCFDA fluorescence assay, and (B) mitochondrial...[이미지참조] 56

Fig 27. Effect of αS and mutant on mitochondrial autophagy regulating markers. (A) Protein lysates from NC or overexpressed αS and its mutations in SH-SY5Y cells... 57

Fig 28. Dioscorea nipponica Makino and chemical structure of its active compound dioscin. 61

Fig 29. Dose selection and antioxidative activities of DN. (A) MTT assay and (B) NO-releasing assay showing dose-dependent protection by DN in LPS-treated BV-2... 62

Fig 30. Anti-inflammatory activities of DN in LPS-treated BV-2 cells. (A) DN dose-dependently reduced mRNA and (B) protein expression of inflammatory... 63

Fig 31. DN dose-dependently ameliorating inflammatory pathway activation. (A) Protein expression of mediators of MAPK signalling in LPS treated BV-2 cells with... 67

Fig 32. HPLC fingerprinting analysis of DN. (right) Standard pick of dioscin at 10.907 min RT (retention time), (left) DN extract at the same RT showing the presence... 67

Fig 33. Evaluation of compounds on cell viability. (A) dose-dependent cell viability after MPP+ treatment; (B) dose-dependent cell viability after chloroquine (CQ)...[이미지참조] 70

Fig 34. Dioscin dose-dependently activates autophagy. Representative data of ATG5 and p62 immunoblots of whole-cell (SH-SY5Y) lysates. ATG5 and p62 were... 72

Fig 35. Dioscin dose-dependently activates autophagosome. Representative data of LC3 immunoblots of whole-cell (SH-SY5Y) lysates. Cells were pre-treated with... 75

Fig 36. Anti-apoptotic activity of dioscin. Representative data of Bcl-2, Bax (A & B) and Cas3/cleaved caspase-3 (C & D) immunoblots of whole-cell (SH-SY5Y)... 77

Fig 37. Dioscin improves neurotrophic factors and TH cells. Representative data of TH (C & D), BDNF, CREB and pCREB (A & B) immunoblots of whole-cell (SH-... 80

Fig 38. A schematic presentation of αS-GCN5 role in neuronal cell apoptosis. In normal physiology, GCN5 maintains the BIM-Bcl-2 ratio by transcriptional... 86

Fig 39. A schematic presentation of dioscin-mediated neuroprotection. 91

Fig 40. Dioscin activity on αS and mitophagy regulating markers. (A) Different doses of dioscin downregulating αS expression in αSwt cells and (B) normalising...[이미지참조] 93

Fig 41. A schematic presentation of αS-related future studies. As αS is a native protein, from our study we prospect that a PTM in αS structure could be leading to... 94