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Title Page
ABSTRACT
Contents
List of Abbreviations 11
CHAPTER 1: General Introduction 14
1.1. Adult Neurogenesis and Neural Stem Cells 14
1.1.1. Adult Neurogenesis: The Discovery 14
1.1.2. Adult Neural Stem Cells 16
1.2. Programmed Cell Death 18
1.2.1. Programmed Cell Death: Classifications and Features 18
1.2.2. Programmed Cell Death in the Brain 21
1.3. Autophagy 23
1.3.1. Autophagic Cell Death: Insulin Withdrawal Model in Hippocampal Neural Stem Cells 25
1.3.2. Autophagy in Neurodegeneration 28
CHAPTER 2: General Materials and Common Techniques 31
2.1. Cell Culture 31
2.2. Western Blot Analysis 31
2.3. Immunohistochemistry (IHC) 32
2.4. Immunocytochemistry (ICC) 32
2.5. Real-Time Quantitative PCR 33
2.6. Cell Death Assay 33
2.7. Transfection for Delivery of DNAs or siRNAs 33
2.8. Flow Cytometry 33
2.9. Statistical Analysis 34
CHAPTER 3: Calpain Determines the Propensity of Adult HCN Cells to ACD Following Insulin Withdrawal 35
3.1. Introduction 35
3.2. Materials and Methods 37
3.2.1. Antibodies and Reagents 37
3.2.2. Plasmids and siRNAs 37
3.2.3. Immunocytochemistry 37
3.2.4. Intracellular Calcium Imaging 37
3.2.5. Calpain Activity Assay 38
3.3. Results 39
3.3.1. Calpain 1 and 2 are Differentially Expressed in HCN Cells 39
3.3.2. Calpain 2 Inhibition Potentiates Autophagic Death of I(-) HCN Cells 39
3.3.3. Ectopic Expression of Calpain 1 Induces Apoptosis in HCN Cells Following Insulin Withdrawal 41
3.3.4. Degradation of Calpain 2 Is Achieved via UPS, Not Autophagy 44
3.3.5. Proteasome Inhibition Elevates the Concentration of Intracellular Calcium and Activates Calpain in I(-) HCN Cells 47
3.3.6. Lactacystin Switches the Default Autophagic Death of I(-) HCN Cells to Apoptosis. 50
3.4. Discussion 55
CHAPTER 4: Mediation of ACD by RyR3 in Adult HCN Cells 59
4.1. Introduction 59
4.2. Materials and Methods 64
4.2.1. Pharmacological Reagents 64
4.2.2. Immunofluorescence-based Ca2+ Imaging(이미지참조) 64
4.2.3. Autophagic Flux Assay 64
4.2.4. Generation of CRISPR/Cas9-mediated RYR3 Knockout HCN Cells 65
4.3. Results 66
4.3.1. ER-to-Cytosol Ca2+ Efflux is Increased Following Insulin Withdrawal in HCN Cells(이미지참조) 66
4.3.2. RyR3 is the Major RyR Isoform Expressed in HCN Cells and its Expression is Elevated Following Insulin Withdrawal 66
4.3.3. A RyR Agonist Caffeine Further Promotes ACD in I(-) HCN Cells 68
4.3.4. ACD Induction by Caffeine is Precluded in Autophagy-Defective HCN Cells Depleted of Atg7 68
4.3.5. Autophagy is Diminished by Pharmacological or Genetic RyR Inhibition in I(-) HCN Cells 72
4.3.6. Knockout of RyR3 Gene Occludes ER Ca2+ Release and Thereby Prevents ACD in I(-) HCN Cells(이미지참조) 75
4.4. Discussion 80
CHAPTER 5: A Novel Function of Presenilin-2 in Regulation of Autophagic Death of HCN Cells 85
5.1. Introduction 85
5.2. Materials and Methods 88
5.2.1. Antibodies and Reagents 88
5.2.2. Plasmids and siRNAs 88
5.2.3. Organotypic Hippocampal Slice Culture 88
5.2.4. Generation of CRISPR/Cas9-mediated Presenilin Knockout HCN Cell Lines 89
5.3. Results 90
5.3.1. Presenilin-2 Exhibits Distinct Expression Pattern in HCN Cells 90
5.3.2. Expression of Presenilin-2 in HCN Cells is Significantly Upregulated upon Insulin Withdrawal 90
5.3.3. Genetic Depletion of PS2 Prevents Induction of ACD by Insulin Withdrawal in HCN Cells 90
5.3.4. PS2 Expression Potentiates ACD in I(-) HCN Cells, but Not in Atg7-Deficient I(-) HCN Cells 93
5.4. Discussion 96
CHAPTER 6: General Discussion 98
References 102
Table 1. A list of primers used for quantitative real-time qPCR analysis. 8
Figure 1. Development of newborn neurons from NSCs in the DG of adult hippocampus. 15
Figure 2. Immunofluorescent staining of neurons, astrocytes, and oligodendrocytes differentiated from adult... 17
Figure 3. Adult neurogenesis in SVZ and SGZ. 19
Figure 4. Types and characteristics of PCD. 22
Figure 5. Molecular network of autophagy induction. 24
Figure 6. Insulin signaling pathways in insulin withdrawal model of ACD. 27
Figure 7. Autophagy in human diseases. 29
Figure 8. Calpain 1 and 2 are expressed distinctly in adult rat HCN cells. 40
Figure 9. Calpain inhibition augments autophagic death of I(-) HCN cells. 42
Figure 10. Knockdown of calpain 1 does not exhibit autophagy-related alterations in HCN cells. 43
Figure 11. Calpain 1 overexpression in I(-) HCN cells switches the mode of cell death from ACD to apoptosis. 45
Figure 12. Calpain 2 does not undergo protein degradation via autophagy. 46
Figure 13. Calpain 2 undergoes proteasome-dependent protein degradation in I(-) HCN cells. 48
Figure 14. Proteasome inhibition elevates the concentration of intracellular calcium and activates calpain in I(-)... 49
Figure 15. Lactacystin switches the default ACD to apoptosis in I(-) HCN cells. 51
Figure 16. Lactacystin-mediated switch of cell death mode in I(-) HCN cells does not involve necrosis. 54
Figure 17. Schematic diagram illustrating the switch of PCD mode between autophagy and apoptosis via... 57
Figure 18. Illustration of intracellular Ca2+ dynamics between the ER and the cytosol.(이미지참조) 61
Figure 19. Insulin withdrawal increases intracellular Ca2+ levels without the involvement of extracellular Ca2+...(이미지참조) 67
Figure 20. RyR3 is the major isoform in HCN cells and its expression is upregulated following insulin... 69
Figure 21. RyR agonist caffeine potentiates ACD in I(-) HCN cells. 70
Figure 22. Caffeine-induced potentiation of ACD is prevented in Atg7 knockdown HCN cells. 73
Figure 23. RyR inhibition suppresses ACD in HCN cells. 74
Figure 24. Genetic suppression of RyR3 diminishes autophagic flux in I(-) HCN cells. 77
Figure 25. ER Ca2+ efflux is significantly inhibited in I(-) RYR3KO HCN cells.(이미지참조) 78
Figure 26. Autophagic flux in RYR3KO HCN cells is unaltered upon insulin withdrawal or caffeine treatment. 79
Figure 27. Schematic diagram illustrating the role of ER-to-cytosol Ca2+ in regulation of survival and death of...(이미지참조) 83
Figure 28. Illustration of Presenilin-2 structure. 86
Figure 29. Presenilin-2 exhibits distinct expression pattern in HCN cells. 91
Figure 30. Genetic silencing of PS2 prevents induction of ACD in HCN cells. 92
Figure 31. Ectopic expression of PS2 potentiates and rescues ACD in insulin-deprived HCN cells. 95
초록보기 더보기
Importance of proper cell death regulation has received great recognitions in biomedical field as the greater understanding of cell death can provide better solutions to treatment of various human diseases, most notably neurodegenerative diseases. However, the underlying mechanisms of programmed cell death (PCD) are largely unknown, especially in neural stem cells (NSCs). Utilizing our well-established insulin withdrawal model of autophagic cell death (ACD) in adult hippocampal neural stem (HCN) cells, we explored the functional relevance of autophagic death of NSCs to pathogenesis of Alzheimer’s disease (AD). Aberrant neuronal Ca2+ levels in brains of AD patients have given a rise to Ca2+ hypothesis of AD which states that the amyloidogenic pathway leads to ultimate cognitive impairment in affected individuals through dysregulation of neuronal Ca2+ signaling. The aberrant Ca2+ homeostasis consequently mediates the abnormal activation of calpains, the Ca2+-dependent cysteine proteases which also play an essential role in diverse cellular events including cell development, differentiation and proliferation, and cell death.
We discovered that a switch of cell death mode in HCN cells occurs along with changes in calpain activities which is regulated by the proteasome-dependent degradation of calpain proteins. The dynamic change in calpain activity through the proteasome-mediated modulation of calpain level and intracellular Ca2+ rise can be the critical contributor to the demise of NSCs. The switching mechanism of the PCD mode by calpain is a novel discovery in the field of NSCs, and thereby may provide an insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in regulation of NSC death especially in neurodegeneration. Furthermore, by examining ryanodine receptors (RyRs) and IP3 receptors (IP3Rs) – the main Ca2+ release channels located in the endoplasmic reticulum (ER) membranes known to direct various cellular events such as autophagy and apoptosis – we investigated the intracellular Ca2+-mediated regulation of survival and death of HCN cells utilizing the insulin withdrawal model of ACD. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished autophagic cell death of HCN cells. Our finding delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and PCD in NSCs which may represent pathophysiological brain states. In addition, familial AD-related gene presenilin-2 is another key regulator of intracellular Ca2+ homeostasis that is located in the ER. Stemming from previous reports showed reduced the brain insulin levels along with the markedly elevated autophagy in brains of AD patients, we hypothesized that presenilin-2 plays an active, functional role in progression of AD pathology. Our results demonstrate that presenilin-2 is a direct upstream regulator of autophagy and ACD in HCN cells. Silencing and ectopic expression of presenilin-2 reduce and increase the level of autophagy, respectively, providing a hint for possible association of autophagy in AD pathogenesis. Our findings in novel functions of presenilin-2 in neural stem cell biology will open up a highly promising revenue for further NSC research.
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