title page

Contents

Abbreviations 14

ABSTRACT 16

I. Introduction 19

1. Scoparone 19

2. Neurite outgrowth 21

3. Catecholamines and their biosynthetic pathway 24

4. Regulation of TH 27

5. Regulation of dopamine release 31

6. Neurodegenerative diseases 34

7. PC12 cells 36

8. Objective 37

II. Materials and methods 38

1. Chemicals 38

2. Separation of scoparone 38

3. Cell culture 39

4. Assay for neurite outgrowth 39

5. Western blot analysis 41

6. Assay for PKA kinase activity 42

7. Assay for PKC kinase activity 43

8. Assay for CaMK II kinase activity 43

9. Determination of intracellular dopamine content 44

10. Determination of the release of dopamine 44

11. Assay for TH activity 45

12. Assay for AADC activity 45

13. Measurement of intracellular Ca2+ concentration(이미지참조) 46

14. Measurement of cyclic AMP levels 46

15. Reverse transcriptase polymerase chain reaction (RT-PCR) 47

16. Preparation of nuclear extracts 48

17. Measurement of cell viability : MTT assay 48

18. Statistical analysis 49

III. Results 50

1. Effects of scoparone on neurite outgrowth in PC12 cells 50

1.1. Identification of scoparone 50

1.2. Neurite outgrowth 50

1.3. Morphology 50

1.4. ERK activity 53

1.5. Cyclic AMP levels and PKA activity 53

1.6. Intracellular Ca2+ concentration(이미지참조) 57

1.7. PKC and CaMK II activities 57

1.8. Scoparone-induced neurite outgrowth 57

1.9. NGF-induced neurite outgrowth 62

2. Effects of scoparone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells 71

2.1. Dopamine content 71

2.2. TH and AADC activities 71

2.3. Intracellular cyclic AMP levels 71

2.4. Intracellular Ca2+ concentration(이미지참조) 79

2.5. PKA, PKC and CaMK II activities 79

2.6. TH phosphorylation 79

2.7. TH mRNA gene expression 85

2.8. Transcription factor CREB phosphorylation 85

2.9. Cell viability 85

2.10. L-DOPA-induced cytotoxicity 85

3. Effects of scoparone on the release of dopamine in PC12 cells 91

3.1. Dopamine release 91

3.2. Cyclic AMP levels and Ca2+ influx(이미지참조) 91

3.3. CaMK II and PKA activities 91

3.4. p-Synapsin phosphorylation 95

3.5. K+-induced dopamine release(이미지참조) 95

3.6. K+-induced Ca2+ influx(이미지참조) 100

3.7. K+-induced CaMK II activity(이미지참조) 100

IV. Discussion 103

1. Effects of scoparone on neurite outgrowth in PC12 cells 103

2. Effects of scoparone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells 106

3. Effects of scoparone on the release of dopamine in PC12 cells 111

V. Conclusion 117

VI/V. REFERENCES 118

SUMMARY IN KOREAN 145

Acknowledgment 148

Fig. 1. Chemical structure of scoparone 20

Fig. 2. Biosynthetic pathway of catecholamines. Dopamine, norepinephrine and epinephrine are derived from the multistep processing of tyrosine in catecholaminergic neurons.... 25

Fig. 3. Regulation of TH. (A) Regulatory domains in the N terminus of TH. Short-term regulation of TH enzyme activity occurs through the phosphorylation of at least four serine residues in the first forty acino acids of... 28

Fig. 4. Schematic illustration about the phosphorylation of serine 133 of CREB. Increased cyclic AMP levels by activation of G protein-coupled receptors (R), and adenylyl cyclase (AC) phosphorylate PKA, and lead to its dissociation and... 30

Fig. 5. Schematic diagram of the regulation of synapsin I phosphorylation in nerve terminals. In the basal state, dephosphorylated synapsin I is bound to synaptic vesicles, which anchors the vesicles to the nerve terminal cytoskeleton.... 33

Fig. 6. The concentration-response curve for scoparone on neurite outgrowth in PC12 cells. Cells were plated on poly-L-lysine at a low density (ca. 1 × 10⁴ cells/㎠) and were treated with scoparone on 0, 2, 4 and 6 days.... 51

Fig. 7. The time course curve for scoparone on neurite outgrowth in PC 12 cells. Cells were plated on poly-L-lysine at a low density (ca. 1 × 10⁴ cells/㎠) and were treated with scoparone on 0, 2, 4 and 6 days.... 52

Fig. 8. Phase-contrast photomicrographs of PC12 cells after various treatments for 6 days. (A) Untreated control culture. (B) Culture treated with 200 μM scoparone only.... 54

Fig. 9. Effects of scoparone on expression of ERK in PC12 cells. (A) PC12 cells were stimulated with scoparone (200 μM) for the indicated times and harvested for Western blot analysis using the antibodies against phosphorylated ERK (top)... 55

Fig. 10. Effects of scoparone on intracellular cyclic AMP levels in PC12 cells. PC12 cells were treated with scoparone (10, 20, 40 and 60 ng/ml), and then incubated at 37℃ for 5 min and 15 min.... 56

Fig. 11. Effects of scoparone on PKA activity in PC12 cells. Scoparone (200 μM) were added to PC12 cell and incubated for 5 min - 30 min. After treatment, cells were lysed and protein extracts (10 μg) were analyzed on ELISA.... 58

Fig. 12. Effects of scoparone on intracellular Ca2+ concentration in PC12 cells. The intracellular Ca2+ concentration of scoparone-responsive PC12 cells, represented as the ratio of the single FLUO4/AM-loaded cell fluorescence at...(이미지참조) 59

Fig. 13. Effects of scoparone on PKC activity in PC12 cells. Scoparone (200 μM) were added to PC12 cells and incubated for 5, 10 and 30 min. After treatment, cells were lysed and protein extracts (10 μg) were analyzed on ELISA.... 60

Fig. 14. Effects of scoparone on CaMK II activity in PC12 cells. Scoparone (200 μM) were added to PC12 cells and incubated for 30, 45 and 60 min. Protein extracts (3 μg) were analyzed on ELISA.... 61

Fig. 15. Effect of U0126 on neurite outgrowth promoting activity of scoparone in PC12 cells. Cells were treated with U0129 (MEK inhibitor, 0.1 μM or 1 μM) and scoparone (200 μM) for 6 days.... 63

Fig. 16. Effect of H89 on neurite outgrowth promoting activity of scoparone in PC 12 cells. Cells were treated with H89 (PKA inhibitor, 0.1 μM or 1 μM) and scoparone (200 μM) for 6 days.... 64

Fig. 17. Effect of GF 109203X on neurite outgrowth promoting activity of scoparone in PC12 cells. Cells were treated with FG 109203X (PKC inhibitor, 0.1 μM or 1 μM) and scoparone (200 μM) for 6 days.... 65

Fig. 18. Effect of KN62 on neurite outgrowth promoting activity of scoparone in PC12 cells. Cells were treated with KN62 (CaMK II inhibitor, 1 μM or 10 μM) and scoparone (200 μM) for 6 days.... 66

Fig. 19. Effect of U0126 on neurite outgrowth promoting activity of scoparone in the presence of 2 ng/ml NGF in PC12 cells. Cells were treated with U0126 (MEK inhibitor, 0.1 μM or 1 μM) and scoparone (200 μM) for 6 days.... 67

Fig. 20. Effect of H89 on neurite outgrowth promoting activity of scoparone in the presence of 2 ng/ml NGF in PC12 cells. Cells were treated with H89 (PKA inhibitor, 0.1 μM or 1 μM) and scoparone (200 μM) for 6 days.... 68

Fig. 21. Effect of GF 109203X on neurite outgrowth promoting activity of scoparone in the presence of 2 ng/ml NGF in PC12 cells. Cells were treated with GF 109203X (PKC inhibitor, 0.1 μM ro 1 μM) and scoparone (200 μM) for 6 days.... 69

Fig. 22. Effect of KN62 on neurite outgrowth promoting activity of scoparone in the presence of 2 ng/ml NGF in PC12 cells. Cells were treated with KN62 (CaMK II inhibitor, 1 μM or 10 μM) and scoparone (200 μM) for 6 days.... 70

Fig. 23. Effects of scoparone on dopamine content in PC12 cells. PC12 cells were treaed with scoparone (100 - 500 μM), and then incubated for 24 h. PC12 cells were harvested and dopamine content was measured by and HPLC method.... 72

Fig. 24. Time courses of intracellular dopamine content by scoparone in PC12 cells. PC12 cells were treated with 200 μM scoparone, and incubated at 37℃ for 3-48 h.... 73

Fig. 25. Effects of scoparone on dopamine release in PC12 cells. PC12 cells were treated with 200 μM scoparone and incubated at 37℃ for 30 min or 1 h. Dopamine content in the media was measured by an HPLC method.... 74

Fig. 26. Time courses of intracellular norepinephrine content by scoparone in PC12 cells. PC12 cells were treated with 200 μM scoparone, and incubated at 37℃ for 3-48 h.... 75

Fig. 27. Time courses of TH activity by scoparone in PC12 cells. PC12 cells were treated with 200 μM scoparone, and incubated at 37℃ for 3-48 h. TH activity of the control was 3.68 ± 0.29 nmol/min/ng protein.... 76

Fig. 28. Time courses of AADC activity by scoparone in PC12 cells. PC12 cells were treated with 200 μM scoparone, and incubated at 37℃ for 3-48 h. AADC activity of the control was 31.5 ± 0.29 nmol/min/ng protein.... 77

Fig. 29. Effects of scoparone on intracellular cyclic AMP levels in PC12 cells. PC12 cells were treated with scoparone(100, 200 and 300 μM), and then incubated at 37℃ for 5-60 min.... 78

Fig. 30. Effects of scoparone on intracellular Ca2+ concentration in PC12 cells. PC12 cells were treated with scoparone(100, 200 and 500 μM). The variation of intracellular Ca2+ concentration of scoparone-responsive PC12 cells,...(이미지참조) 80

Fig. 31. Effects of scoparone on phosphorylation of PKA in PC12 cells. The data shown here are representative of a minimum of five independent experiments. Scoparone (200 μM)-treated cells were incubated for 5-180 min.... 81

Fig. 32. Effects of scoparone on phosphorylation of PKC in PC12 cells. The data shown here are representative of a minimum of five independent experiments. Scoparone (200 μM)-treated cells were incubated for 5-180 min.... 82

Fig. 33. Effects of scoparone on phosphorylation of CaMK II in PC12 cells. The data shown here are representative of a minimum of five independent experiments. Scoparone (200 μM)-treated cells were incubated for 30-180 min.... 83

Fig. 34. Effects of scoparone on phosphorylation of TH (Ser 40) in PC12 cells. (A) PC12 cells were stimulated with scoparone(200 μM) and harvested for Western blot analysis using the antibodies against phosphor-TH (upper panel),... 84

Fig. 35. Effects of scoparone on TH gene expression. PC 12 cells were treated with scoparone (200 μM) for 3 - 48 h and then harvested for RT-PCR to the mRNA of TH.... 86

Fig. 36. Time-related CREB phosphorylation (Ser 133) induced by scoparone. (A) PC12 cells were treated with scoparone (200 μM) for the indicated times and harvested for harvested for Western blot analysis as described under... 87

Fig. 37. Effects of scoparone on PC12 cell viability. PC12 cells were exposed to various concentrations of scoparone (250-1000 μM) for 24 h. Cell viability was assessed using MTT methods, in which viable cells convert the soluble dye MTT... 88

Fig. 38. Effects of scoparone on L-DOPA-induced cytotoxicity in PC12 cells. After pretreatment of scoparone (200 μM) for 6 h, PC12 cells were exposed to L-DOPA (20 μM, 50 μM, 100 μM and 200 μM) for 24 h, and the cell viability... 89

Fig. 39. Effects of scoparone on basal dopamine release from PC12 cells. PC12 cells were treated with 50,100 or 200 μM scoparone and incubated at 37℃ for 30 min or 1 h.... 92

Fig. 40. Effects of scoparone on dopamine release from PC12 cells in the absence of the extracellular Ca2+. PC12 cells were treated with 50,100 or 200 μM scoparone and incubated at 37℃ for 15 min or 30 min....(이미지참조) 93

Fig. 41. Effects of an adenylyl cyclase inhibitor on the increase of intracellular cyclic AMP levels induced by scoparone in PC12 cells. PC12 cells were pretreated with MDL-12,330 (20 μM) for 30 min, then treated with scoparone... 94

Fig. 42. Effects of scoparone on phosphorylation of CaMK II in PC12 cells. PC12 cells were treated with 100 or 200 μM scoparone and incubated at 37℃ for 60 min, 120 min or 180 min.... 96

Fig. 43. Effects of scoparone on phosphorylation of PKA in PC12 cells. PC12 cells were treated with 100 or 200 μM scoparone and incubated at 37℃ for 5 min, 15 min or 30 min.... 97

Fig. 44. Phosphorylation of synapsin induced by scoparone. (A) PC12 cells were treated with scopoarone (200μM) for 5 and 15 min and harvested for Western blot anlaysis as described under Materials and methods.... 98

Fig. 45. Effects of scoparone on K+-induced dopamine release from PC12 cells. PC12 cells were pretreated for 30min with 50, 100 or 200 μM scoparone at 37℃, and then treated with 56 mM KCl for 10-60 min....(이미지참조) 99

Fig. 46. Effect of scoparone on K+-induced desensitization in PC12 cells. The traces show representative [Ca2+]i variation in K+-responsive cells and scoparone and K+-responsive cells....(이미지참조) 101

Fig. 47. Effects of scoparone on K+-induced CaMK II phosphorylation in PC 12 cells. PC12 cells were treated with K+ alone (56 mM) or K+ associated with scoparone (200 μM) and incubated at 37℃ for 15-120 min....(이미지참조) 102

Scheme 1. Fractionation of Liriodendron tulipifera and isolation of scoparone 40

Scheme 2. Mechanisms of scoparone on neurite outgrowth 107

Scheme 3. Mechanisms of scoparone on dopamine biosynthesis and TH regulation 112

Scheme 4. Mechanisms of scoparone on dopamine release 116