Scoparone이 PC12 세포의 neurite outgrowth와 dopamine 생합성에 미치는 영향 = Effects of Scoparone on Neurite Outgrowth and Dopamine Biosynthesis in PC12 Cells

다국어 초록 (Multilingual Abstract)

The effects of scoparone, isolated from the stem bark of Liriodendron tulipifera, on neurite outgrowth, dopamine biosynthesis and dopamine release were investigated in PC12 cells. Scoparone at 200 uM markedly induced the outgrowth of neurites on its own and enhanced the outgrowth of neurites from cells in the presence of nerve growth factor (NGF, 2 ng/ml). The levels of intracellular cyclic AMP and concentrations of Ca2+ were also increased by 200 ?M scoparone. In addition, scoparone at 200 ?M increased the activities of extracellular signal-regulated protein kinase (ERK), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC) and Ca2+/calmodulin kinase II (CaMK II). However, scoparone-induced neurite outgrowth was blocked by a mitogen-activated protein kinase inhibitor (U0126), a PKA inhibitor (H89), a PKC inhibitor (GF109203X) and a CaMK II inhibitor (KN62). These kinase inhibitors also reduced the scoparone-induced neurite outgrowth associated with NGF. These results suggest that scoparone can induce neurite outgrowth by stimulating the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells. In addition, the effects of scoparone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Treatments of PC12 cells with scoparone showed 136% increase of dopamine content at a concentration of 200 uM for 24 h. Tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities were increased during 6 h - 48 h after exposure of 200 uM scoparone. Intracellular cyclic AMP levels and Ca2+ concentration were also increased by scoparone. Under the same conditions, scoparone increased PKA, PKC and CaMK II activities. TH phosphorylation was increased by scoparone in short-term period (5-120 min). In addition, TH mRNA levels and phosphorylated CREB levels were significantly increased by scoparone in long-term period (3-48 h). Scoparone at 200 uM also reduced L-DOPA (20-100 uM)-induced cytotoxicity. These results suggest that scoparone enhances dopamine biosynthesis by regulating TH activity and TH gene expression, and also protects the cell from L-DOPA-induced cytotoxicity in PC12 cells. Finally, the effects of scoparone on dopamine release and K+-induced dopamine release in PC12 cells were investigated. Scoparone significantly increased dopamine release at 30 and 60 min. Scoparone also phosphorylated synapsin I by activation of CaMK II and PKA through the increase of Ca2+ and cyclic AMP, respectively. Scoparone enhanced K+-induced dopamine release. Under the same conditions, scoparone maintained Ca2+ concentration and increased CaMK II phosphorylation to higher levels than K+ alone. These results suggest that scopoarone releases dopamine by synapsin I through activation of CaMK II and PKA, and enhances K+-induced dopamine release in PC12 cells. Taken together, scoparone increased the release of dopamine at 30-60 min and dopamine biosynthesis at 3-48 h, respectively. On the other hand, scoparone reduced L-DOPA-induced cytotoxicity. Furthermore, scoparone induced neurite outgrowth in long-time (2-7 days). These results suggest that scoparone shows a unique combination of dopaminergic, neuroprotective and neuritogenic properties and may be taken as a lead compound for the development of novel therapeutic strategies for neurodegenerative disease including Parkinson's disease.

목차 (Table of Contents)

• LIST OF FIGURES ⅵ
• LIST OF SCHEMES ⅹ
• ABBREVIATIONS ⅹⅰ
• ABSTRACT ⅹⅲ
• Ⅰ. INTRODUCTION 1
• 1. Scoparone 1
• 2. Neurite outgrowth 3
• 3. Catecholamines and their biosynthetic pathway 6
• 4. Regulation of TH 9
• 5. Regulation of dopamine release 13
• 6. Neurodegenerative diseases 16
• 7. PC12 cells 18
• 8. Objective 19
• Ⅱ. MATERIALS AND METHODS 20
• 1. Chemicals 20
• PAGE
• 2. Separation of scoparone 20
• 3. Cell culture 21
• 4. Assay for neurite outgrowth 21
• 5. Western blot analysis 23
• 6. Assay for PKA kinase activity 24
• 7. Assay for PKC kinase activity 25
• 8. Assay for CaMK II kinase activity 25
• 9. Determination of intracellular dopamine content 26
• 10. Determination of the release of dopamine 26
• 11. Assay for TH activity 27
• 12. Assay for AADC activity 27
• 13. Measurement of intracellular Ca2+ concentration 28
• 14. Measurement of cyclic AMP levels 28
• 15. Reverse transcriptase polymerase chain reaction (RT-PCR) 29
• 16. Preparation of nuclear extracts 30
• 17. Measurement of cell viability: MTT assay 30
• 18. Statistical analysis 31
• PAGE
• Ⅲ. RESULTS 32
• 1. Effects of scoparone on neurite outgrowth in PC12 cells 32
• 1.1. Identification of scoparone 32
• 1.2. Neurite outgrowth 32
• 1.3. Morphology 32
• 1.4. ERK activity 35
• 1.5. Cyclic AMP levels and PKA activity 35
• 1.6. Intracellular Ca2+ concentration 39
• 1.7. PKC and CaMK II activities 39
• 1.8. Scoparone-induced neurite outgrowth 39
• 1.9. NGF-induced neurite outgrowth 44
• 2. Effects of scoparone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells 53
• 2.1. Dopamine content 53
• 2.2. TH and AADC activities 53
• 2.3. Intracellular cyclic AMP levels 53
• PAGE
• 2.4. Intracellular Ca2+ concentration 61
• 2.5. PKA, PKC and CaMK II activities 61
• 2.6. TH phosphorylation 61
• 2.7. TH mRNA gene expression 67
• 2.8. Transcription factor CREB phosphorylation 67
• 2.9. Cell viability 67
• 2.10. L-DOPA-induced cytotoxicity 67
• 3. Effects of scoparone on the release of dopamine in PC12 cells 73
• 3.1. Dopamine release 73
• 3.2. Cyclic AMP levels and Ca2+ influx 73
• 3.3. CaMK II and PKA activities 73
• 3.4. p-Synapsin phosphorylation 77
• 3.5. K+-induced dopamine release 77
• 3.6. K+-induced Ca2+ influx 82
• 3.7. K+-induced CaMK II activity 82
• Ⅳ. DISCUSSION 85
• 1. Effects of scoparone on neurite outgrowth in PC12 cells 85
• PAGE
• 2. Effects of scoparone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells 88
• 3. Effects of scoparone on the release of dopamine in PC12 cells 93
• Ⅴ.CONCLUSION 99
• Ⅵ. REFERENCES 100
• SUMMARY IN KOREAN 127
• ACKNOWLEDGEMENTS 130