Title Page

Contents

Abbreviations 11

Abstract 13

I. General introduction 16

1. Catecholamines 16

2. Tetrahydroisoquinoline derivatives and tetraphydropapaveroline 18

3. Parkinson's disease and L-DOPA-induced neurotoxicity 19

4. Naive PC12 cells and differentiated PC12 cells 21

5. Apoptosis and role of intracellular signaling kinases 22

6. Objectives 24

II. Inhibitory effects of THP on dopamine biosynthesis in PC12 cells 25

1. Introduction 25

2. Materials and Methods 27

1) Chemicals 27

2) Cell culture 27

3) Determination of dopamine content 27

4) Assay for TH activity 28

5) Assay for AADC activity 29

6) Bovine adrenal TH 29

7) Measurement of protein content 30

8) Statistical analysis 30

3. Results 31

1) Intracellular dopaimne content and TH activity by THP in PC12 cells 31

2) Time courses of dopamine contents by THP 31

3) Reduction of TH activity by THP in bovine adrenal 36

4) Inhibition of THP on L-DOPA-induced increase in dopamine content and its protection by NAC 36

4. Discussion 46

III. Effects of THP on L-DOPA-induced neurotoxicity in PC12 cells 49

1. Introduction 49

2. Materials and Methods 51

1) Chemicals 51

2) Assessment of cell viability 51

3) Flow cytometric analysis of apoptotic cells 52

4) TUNEL assay for apoptotic DNA fragmentation 52

5) Nuclear staining with PI and Hoechst 33342 53

6) Measurement of intracellular reactive oxygen species (ROS) formation 54

7) ATP assay 54

8) Statistical analysis 54

3. Results 55

1) Inhibition of cell viability by THP and protection by NAC against THP-induced cytotoxicity 55

2) Interaction of THP with L-DOPA-induced apoptosis 59

3) ROS formation and depletion of intracellular ATP levels by THP 68

4. Discussion 72

IV. Effects of THP on dopamine content and L-DOPA-induced neurotoxicity in differentiated PC12 cells 76

1. Introduction 76

2. Materials and Methods 78

1) Chemicals 78

2) Cell culture 78

3) Determination of dopamine content 78

4) Assessment of cell viability 79

5) Western blot analysis 79

6) Statistical analysis 80

3. Results 81

1) Inhibition of THP on L-DOPA-induced increase in dopamine content and its protection by NAC in differentiated PC12 cells 81

2) Inhibition of L-DOPA-induced cell viability by THP and protection by NAC in differentiated PC12 cells 85

3) Activation of L-DOPA-induced intracellular signaling kinases by THP in naive and differentiated PC12 cells 92

4. Discussion 100

V. Conclusion 105

VI. References 106

요약 120

Table 1. Inhibitory effects of THP on bovine adrenal TH activity. The control of TH activity, 1.10 nmol/min/mg protein, was taken as 100. The data were expressed as means ± SEM of 4-5 experiments. Significantly different from the control value: *, p〈0.05; **,... 37

Fig. 1. Biosynthetic pathway of catecholamines. TH; Tyrosine hydroxylase, AADC; Aromatic L-amino acid decarboxylase, DBH; Dopamine β-hydroxylase, PNMT; Phenylethanolamine N-methyl transferas 17

Fig. 2. Biosynthesis of dopamine, THP and salsolinol from L-DOPA. 20

Fig. 3. Inhibitory effects of THP on the intracellular dopamine content in PC12 cells. PC12 cells were treated with THP (2.5-15 μM) and incubated at 37℃ for 24 h. PC12 cells were harvested with phosphate buffered saline and dopamine content was measured... 32

Fig. 4. Effects of THP on TH activity in PC 12 cells. PC 12 cells were harvested at 24 h after the treatment of PC12 cells with 2.5-15 μM of THP and the enzyme activity was measured by an HPLC method.... 33

Fig. 5. Effects of THP on AADC activity in PC12 cells. PC12 cells were harvested at 24 h after the treatment of PC12 cells with 2.5-15 μM of THP and the enzyme activity was measured by an HPLC method.... 34

Fig. 6. Time courses of dopamine content by 10 μM THP in PC12 cells. Dopamine content of the control was 3.64± 0.21 nmol/mg protein. Results represent the means ± SE of 5-7 dishes.... 35

Fig. 7. Inhibition of bovine adrenal TH by THP added in the enzyme reaction mixture. The data were plotted by linear regression analysis. THP concentration: (a), 0 μM; (b), 200 μM. 38

Fig. 8. Effects of L-DOPA on dopamine content in PC12 cells. PC12 cells were treated with L-DOPA (10-150 μM), and then incubated at 37℃ for 24 h. The cells were harvested with PBS and dopamine content was measured by an HPLC method.... 39

Fig. 9. Effects of L-DOPA on dopamine content in PC12 cells. PC12 cells were treated with L-DOPA (10-150 μM), and then incubated at 37℃ for 48 h. The cells were harvested with PBS and dopamine content was measured by an HPLC method.... 40

Fig. 10. Inhibitory effects of THP on the increase in dopamine content induced by L­DOPA in PC12 cells after 24 h. PC12 cells were incubated in the absence or presence of L-DOPA (20 and 50 μM) for 24 h.... 42

Fig. 11. Inhibitory effects of THP on the increase in dopamine content induced by L­DOPA in PC12 cells after 48 h. PC12 cells were incubated in the absence or presence of L-DOPA (20 and 50 μM) for 48 h.... 43

Fig. 12. Effects of NAC on THP plus L-DOPA-induced decrease in dopamine content in PC12 cells after 24 h. PC12 cells were cultured in RPMI 1640 medium with 15 μM THP plus 20 or 50 μM L-DOPA alone or in association with 0.1 mM NAC.... 44

Fig. 13. Effects of NAC on THP plus L-DOPA-induced decrease in dopamine content in PC12 cells after 48 h. PC12 cells were cultured in RPMI 1640 medium with 15 μM THP plus 20 or 50 μM L-DOPA alone or in association with 0.1 mM NAC.... 45

Fig. 14. Effects of THP on PC12 cell viability. PC12 cells were exposed for 24 h (A) or 48 h (B) to different concentrations of THP (5, 10, 15 and 20 μM). Cell viability was assessed using MTT methods, in which viable cells convert the soluble dye MTT to... 56

Fig. 15. Enhancing effects of THP on L-DOPA-induced decrease in PC12 cell viability. PC12 cells in culture were incubated in the absence or presence of L-DOPA (20, 50 and 100 μM) associated with THP (5, 10 and 15 μM) for 24 h.... 57

Fig. 16. Enhancing effects of THP on L-DOPA-induced decrease in PC12 cell viability. PC12 cells in culture were incubated in the absence or presence of L-DOPA (20, 50 and 100 μM) associated with THP (5, 10 and 15 μM) for 48 h.... 58

Fig. 17. Effects of NAC on THP plus L-DOPA-induced decrease in PC12 cell viability after 24 h. PC12 cells were cultured in RPMI 1640 medium with 15 μM THP plus 50 μM L-DOPA only or in combination with 0.1 mM NAC.... 60

Fig. 18. Effects of NAC on THP plus L-DOPA-induced decrease in PC12 cell viability after 48 h. PC12 cells were cultured in RPMI 1640 medium with 15 μM THP plus 50 μM L-DOPA only or in combination with 0.1 mM NAC.... 61

Fig. 19. Induction of apoptotic degradation by THP (15 μM) alone or associated with L­DOPA (20 and 50 μM). Lane 1; DNA from control, Lane 2; DNA from L-DOPA 20 μM, Lane 3; DNA from L-DOPA 50 μM, Lane 4; DNA from THP 15 μM, Lane 5; DNA from... 62

Fig. 20. Flow cytometry histograms of control PC12 cells and PC12 cells after 48 h exposure to THP (15 μM) alone or associated with L-DOPA (20 and 50 μM). The right histogram refers to PC12 cells after 48 h exposure to15 μM THP plus 20 or 50 μM L­... 63

Fig. 21. Enhancing effects of THP on L-DOPA-induced apoptosis in PC12 cells as determined by in situ TUNEL. Fluorescence micrographs of untreated PC12 cells (A) and apoptotic PC12 cells (green or yellow green cells) after 48 h exposure to THP 15 μM (B),... 64

Fig. 22. Enhancing effects of THP on L-DOPA-induced apoptosis in PC12 cells as determined by in situ TUNEL. Fluorescence micrographs of untreated PC12 cells (A) and apoptotic PC12 cells (green or yellow green cells) after 48 h exposure to THP 15 μM (B),... 65

Fig. 23. Micrographs of PC12 cells from untreated cultures (A) and from cultures exposed for 48 h to 50 μM L-DOPA (B), 15 μM THP (C) or 50 μM L-DOPA+15 μM THP.... 67

Fig. 24. Effects of NAC on THP- and L-DOPA-induced generation of ROS in PC12 cells loaded with DCFH2-DA. PC12 cells were cultured for 48 h in RPMI 1640 medium with 5 or 15 μM THP plus 20 or 50 μM L-DOPA only or in combination with 0.1 mM NAC.... 69

Fig. 25. Effects of THP and L-DOPA on intracellular ATP content in PC12 cells after 24 h (A) or 48 h (B). After incubation with toxic concentration of THP (15 μM) and/or L­DOPA (50 μM), PC12 cells were extracted in lysis buffer.... 71

Fig. 26. Effects of L-DOPA on the intracellular dopamine content in differentiated PC12 cells. Differentiated PC12 cells were treated with L-DOPA (20-200 μM) and incubated at 37℃ for 24 h.... 82

Fig. 27. Inhibitory effects of THP on the intracellular dopamine content in differentiated PC12 cells. Differentiated PC12 cells were treated with THP (2.5-30 μM) and incubated at 37℃ for 24 h.... 83

Fig. 28. Inhibitory effects of THP on the increase in dopamine content induced by L-DOPA in differentiated PC12 cells after 24 h. Differentiated PC12 cells were incubated in the absence or presence of L-DOPA (20, 50, 100 and 150 μM) for 24 h. Dopamine content of the control was 3.54 ± 0.25 nmol/min/mg protein.... 84

Fig. 29. Effects of NAC on THP plus L-DOPA-induced decrease in dopamine content in differentiated PC12 cells after 24 h. The cells were cultured in RPMI 1640 medium with 20 μM THP plus 50, 100 or 150 μM L-DOPA alone or in association with 0.1 mM NAC.... 86

Fig. 30. Effects of THP on differentiated PC12 cell viability. Differentiated PC12 cells were exposed for 24 h to different concentrations of THP (5-30 μM). Cell viability was assessed using MTT methods, in which viable cells convert the soluble dye MTT to... 87

Fig. 31. Effects of L-DOPA on differentiated PC12 cell viability. Differentiated PC12 cells were exposed for 24 h to different concentrations of L-DOPA (20-200 μM). Cell viability was assessed using MTT methods, in which viable cells convert the soluble dye... 89

Fig. 32. Enhancing effects of THP on L-DOPA-induced decrease in differentiated PC12 cell viability. Differentiated PC12 cells in culture were incubated in the absence or presence of L-DOPA (20, 50, 100 and 150 μM) associated with THP (5, 10 and 20 μM) for 24 h.... 90

Fig. 33. Effects of NAC on THP plus L-DOPA-induced decrease in differentiated PC12 cell viability after 24 h. PC12 cells were cultured in RPMI 1640 medium with 20 μM THP plus 50, 100 or 150 μM L-DOPA only or in combination with 0.1 mM NAC.... 91

Fig. 34. Representative blots illustrating the effects of THP treatment on activation of ERK. JNK and p38 MAPK in PC12 cells. The cells were treated with 20 IlM THP, and harvested at different time intervals for immunoblot analysis to detect phospho-MAPKs... 94

Fig. 35. Representative blots illustrating the effects of L-DOPA treatment on activation of ERK, JNK and p38 MAPK in PC12 cells. The cells were treated with 150 μM L-DOPA, and harvested at different time intervals for immunoblot analysis to detect phospho­... 95

Fig. 36. Representative blots illustrating the effects of THP and L-DOPA treatment on activation ofERK, JNK and p38 MAPK in PC12 cells. The cells were treated with 10 μM THP and 150 μM L-DOPA, and harvested at different time intervals for immunoblot... 96

Fig. 37. Representative blots illustrating the effects of THP treatment on activation of ERK, JNK and p38 MAPK in differentiated PC12 cells. The cells were treated with 30 μM THP, and harvested at different time intervals for immunoblot analysis to detect... 97

Fig. 38. Representative blots illustrating the effects of L-DOPA treatment on activation of ERK, JNK and p38 MAPK in differentiated PC12 cells. The cells were treated with 200 μM L-DOPA, and harvested at different time intervals for immunoblot analysis to detect... 98

Fig. 39. Representative blots illustrating the effects of THP and L-DOPA treatment on activation of ERK, JNK and p38 MAPK in differentiated PC12 cells. The cells were treated with 15 μM THP and 200 μM L-DOPA, and harvested at different time intervals... 99

Tetrahydropapaveroline (THP)은 dopamine에서 Pictet-Spengler type의 축합 반응에 의해 생성된 tetrahydroisoquinolines (TIQs)로 L-DOPA 요법 파킨슨씨 질환 및 알코올중독 환자의 뇨 및 혈중에서 발견된다. 또한, MPTP와의 구조적 유사성으로 endogenous neurotoxin으로 간주되고 있다. 따라서, 본 연구에서는 THP가 PC12 세포와 신경분화된 PC12 세포중의 dopamine 생합성 및 L-DOPA 유도 세포독성에 미치는 영향에 대해 검토하였다.

THP는 PC12 세포에 24 시간 혹은 48 시간 전처치하였을 때 dopamine 함량을 농도 의존적으로 유의성 있게 감소시켰고, PC12 세포내의 TH 활성 또한 농도 의존적으로 저하시켰다. 반면, L-DOPA는 PC12 세포중의 dopamine 함량을 현저히 증가시켰지만, THP의 병용 처리에 의해 L-DOPA 유도 dopamine 함량 증가 작용을 저해하였다. 또한, 감소된 dopamine 함량은 항산화제인 NAC의 사용으로 회복되었다.

또한, THP를 L-DOPA와 병용 처리하였을 때 L-DOPA 유도 세포 독성에 미치는 영향에 대해서도 검토하였다. THP 10 μM 혹은 L-DOPA 20 μM과 같은 저농도에서는 MTT 분석에 의한 세포 생존율에 영향을 미치지 않았으며, TUNEL과 flow cytometry 분석에 의한 세포사도 유발되지 않았다. 그러나, THP 15 μM 및 L-DOPA 50 μM 이상의 고농도에서는 세포 독성을 유발하였다. 또한, THP를 L-DOPA와 병용 처리시엔 L-DOPA 유도 세포 독성을 현저히 증가시켰다. THP, L-DOPA 단독이나 병용 시 세포내 ROS의 생성은 현저히 증가시켰고, ATP 수준은 감소시켜 산화적 스트레스에 의한 세포사가 유발됨을 확인할 수 있었고, 항산화제인 NAC 처리에 의해 유발되는 세포사는 강력히 억제되었다.

THP의 신경 독성 작용을 확인하기 위해 NGF로 분화시킨 PC12 세포를 이용하여 세포내 dopamine 함량 및 L-DOPA 로 유도된 세포독성에 미치는 영향에 대해 검토하였다. 분화된 PC12 세포에서는 THP에 의해 15 μM 농도에서부터 유의성 있게 dopamine 함량을 감소시켰고, L-DOPA 와 병용 처리 시 L-DOPA 유도 dopamine 함량 증가 작용을 저해하였다. MTT 분석에 의한 세포 독성은 20 μM 농도에서부터 유의성 있게 나타내었고, THP를 L-DOPA 와 병용 처리시엔 L-DOPA 유도 세포 독성을 현저히 증가시켰다. PC12 세포에서 THP에 의해서 MAPK family 중 세포 생존과 적응에 중요한 ERK는 이른 시간에 활성화되었다가 감소하는 반면, 세포사와 관련된 JNK는 보다 오래 유지됨을 확인할 수 있었다. 또한, 부분적으로 세포 생존에 관여하는 p38은 ERK와 유사한 경향을 나타냈다. 분화된 PC12 세포에서도 비슷한 경향을 나타냈으나, 특히 ERK는 이른 시간에 활성화 되었다가 오래 유지되었다.

이상의 결과를 종합하면, THP는 PC12 세포와 분화된 PC12 세포에서 모두 dopamine 함량을 감소시키고, L-DOPA 로 유도된 dopamine 함량 증가를 억제하며, L-DOPA 세포독성을 증가시키나, 분화된 PC12 세포에서 그 독성이 완화됨을 확인할 수 있었다.