Title Page
Contents
Abstract 11
I. INTRODUCTION 14
1. Neurodegenerative disease 14
2. Autophagy 17
3. Apoptosis 21
4. Antidepressants 24
II. MATERIALS AND METHODS 27
2.1. Antibodies and reagents 27
2.2. Cell culture 28
2.3. Transient transfection 28
2.4. Assessment of cell viability: lactate dehydrogenase (LDH) assay 29
2.5. Assessment of cell viability: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 29
2.6. NP-40-soluble and SDS-soluble fractionations 30
2.7. Western blot analysis 30
2.8. Immunoprecipitation 31
2.9. Immunofluorescence confocal microscopy 31
2.10. Animal treatment 32
2.11. Rotarod test 32
2.12. Immunohistochemistry 33
2.13. Statistical analysis 33
III. RESULTS 34
Part I. Amitriptyline interferes with autophagy-mediated clearance of protein aggregates via inhibiting autophagosome maturation in neuronal cells. 34
1. Amitriptyline increases protein aggregates in neuronal cells and mice brain 34
2. Amitriptyline aggravates a MG132-induced accumulation of aggregates 40
3. Amitriptyline interferes with autophagy turnover 47
4. Amitriptyline activates the PI3K/AKT/mTOR pathway 53
5. Amitriptyline induces Beclin 1 acetylation and interaction with Rubicon 57
6. Amitriptyline increases the interaction between Arl8 and SKIP 61
Part II. Fluoxetine protects neuronal cells by inducting autophagy and inhibiting apoptosis. 66
1. Fluoxetine does not effect on the accumulation of aggregates compared to other antidepressants. 66
2. Fluoxetine activates the autophagy induction. 71
3. Fluoxetine downregulates the accumulation of aggregates. 73
4. Fluoxetine protects the cell viability against 6-hydroxydopamine. 75
5. Apoptosis induced by 6-OHDA is blocked by fluoxetine. 77
6. Translocation of p53 in cytosol to nucleus by fluoxetine. 79
7. Activation of AMPK and ULK1 by nucleus p53. 81
IV. Discussion 83
REFERENCES 92
ABSTRACT IN KOREAN 105
Figure 1. autophagy 18
Figure 2. Signaling pathways of autophagy. 20
Figure 3. Intrinsic and extrinsic pathways of apoptosis. 23
Figure 4. Effect of amitriptyline on protein accumulation in neuronal cells. 36
Figure 5. Effect of amitriptyline on cytotoxicity. 37
Figure 6. Effect of amitriptyline on protein accumulation in in vivo model. 38
Figure 7. Effect of amitriptyline on MG132-induced accumulation of aggregates. 44
Figure 8. Effect of amitriptyline on recovery of agrregates induced by MG132. 45
Figure 9. Effect of amitriptyline on cell viability after recovery. 46
Figure 10. Inhibition of autophagy turnover by amitriptyline. 49
Figure 11. Effect of amitriptyline on the fusion of autophagosome and lysosome. 52
Figure 12. Activation of PI3K/AKT/mTOR pathway by amitriptyline. 55
Figure 13. Inhibition of autophagy through PI3K/mTOR/AKT pathway... 56
Figure 14. Effect of amitriptyline on acetylation of Beclin 1 by p300. 59
Figure 15. Effect of amitriptyline on autophagosome maturation. 60
Figure 16. Effect of amitriptyline on Arl8/SKIP/Kinesin complex. 63
Figure 17. Effect of amitriptyline on LAMP2 distribution. 64
Figure 18. Effect of amitriptyline on LAMP1 and mTOR distribution. 65
Figure 19. Schematic diagram showing the effect of amitriptyline on... 65
Figure 20. Effect of antidepressants on protein aggregates in neuronal cells. 68
Figure 21. Activation of autophagy induction by antidepressants. 70
Figure 22. Induction of autophagy by fluoxetine. A and B 72
Figure 23. Inhibition of protein accumulation by fluoxetine. 74
Figure 24. Effect of fluoxetine on cell viability against 6-OHDA toxicity. 76
Figure 25. Effect of fluoxetine on apoptosis induced by 6-OHDA. 78
Figure 26. Translocation of p53 by fluoxetine. 80
Figure 27. Increase of the levels of p-AMPK and p-ULK1 by fluoxetine. 82