Essential role of caspase 9 in regulation of autophagic cell death in adult hippocampal neural stem cells
- Title
- Essential role of caspase 9 in regulation of autophagic cell death in adult hippocampal neural stem cells
- Alternative Title
- 자가포식 세포사멸 조절에서 카스페이즈 9의 필수 역할
- Author(s)
- Hyun-Kyu An
- DGIST Authors
- Hyun-Kyu An ; Seong-Woon Yu ; Ja Wook Koo
- Advisor
- 유성운
- Co-Advisor(s)
- Ja Wook Koo
- Issued Date
- 2022
- Awarded Date
- 2022/02
- Type
- Thesis
- Description
- Autophagic flux, CASP9, CK2, HCN, Mitochondria
- Table Of Contents
-
Ⅰ. General introduction 1
1.1 Programmed cell death (PCD) 1
1.1.1 The history of PCD 1
1.1.2 Apoptosis 4
1.1.3 Autophagic cell death (ACD) 7
1.1.4 Necrosis 9
1.2 Adult neurogenesis and PCD 11
1.2.1 Discovery of adult neurogenesis and neural stem cells (NSCs) 11
1.2.2 Neurodegeneration and PCD 13
1.3 Caspase 9 (CASP9) 15
1.3.1 General role of CASP9 15
1.3.2 Non apoptotic role of CASP9 17
1.3.3 Post-translational modification of CASP9 19
1.4 Casein kinase 2 (CK2) 21
ⅠI. CASP9 (caspase 9) is essential for autophagosome maturation through regulation of mitochondrial homeostasis 23
2.1 Introduction 23
2.2 Materials and methods 26
2.2.1 Reagents and antibodies 26
2.2.2 Cell cultures 26
2.2.3 Construction of CRISPR-Cas9 mediated KO cells 27
2.2.4 Cell death assay 27
2.2.5 Subcellular fractionation 28
2.2.6 Western blot analysis 29
2.2.7 CASPs activity assay 29
2.2.8 Immunocytochemistry 30
2.2.9 Live cell imaging 30
2.2.10 Quantitative real-time PCR 31
2.2.11 PCR cloning of rat CASP9 and human ATG3 31
2.2.12 Fluorescence-activated cell sorting (FACS) analysis 32
2.2.13 Transfection and expression vectors 32
2.2.14 Alignment of CASP9 isoform sequences 33
2.2.15 Statistical analysis 33
2.3 Results 34
2.3.1 CASP9 is required for autophagy in HCN and HeLa cells 34
2.3.2 Pharmacological inhibition of CASP9 suppresses autophagy flux 37
2.3.3 Knockout of CASP9 decreases autophagy flux 39
2.3.4 APAF1 (apoptotic peptidase activating factor 1) is not required for autophagy flux 45
2.3.5 Initiation and nucleation steps of autophagosome biogenesis are not affected by CASP9 KO 48
2.3.6 Autophagosome closure and maturation are impaired by CASP9 KO 51
2.3.7 Production of mitochondrial ROS is decreased in CASP9 KO cells 58
2.3.8 Mitochondrial homeostasis is disrupted by CASP9 KO 62
2.4 Discussion 69
III. Phosphorylation of CASP9 by CK2 plays as a molecular determinant in death mode of HCN cells 75
3.1 Introduction 75
3.2 Materials and methods 78
3.2.1 Antibodies, reagents and plasmid information 78
3.2.2 Cell culture 80
3.2.3 Cell death assay 80
3.2.4 Fluorescent-activated cell sorting (FACS) analysis 80
3.2.5 CASP9 activity assay 80
3.2.6 Immunoblotting 80
3.2.7 Immunocytochemistry (ICC) and subcellular organelle imaging 80
3.2.8 In-vitro kinase phosphorylation assay 81
3.2.9 Site-directed mutagenesis 82
3.2.10 Generation of lentivirus-mediated knockdown cell line 82
3.2.11 Statistical analysis 82
3.3 Results 84
3.3.1 CASP9 is activated upon CORT treatment in HCN cells 84
3.3.2 CASP9 is required for increase of autophagy flux by CORT treatment 86
3.3.3 CASP9 promotes CORT-induced autophagosome maturation 89
3.3.4 CASP9 promotes mitophagy during CORT-induced ACD 91
3.3.5 Inhibition of CK2 drives CORT-induced ACD to apoptosis 93
3.3.6 CK2 phosphorylates CASP9 95
3.3.7 CK2 phosphorylation on CASP9 restricts activation of apoptosis 97
3.4 Discussion 99
ⅠV. Conclusion 102
V. Reference 104
VⅠ. Abstract in Korean 112
- URI
-
http://dgist.dcollection.net/common/orgView/200000592767
http://hdl.handle.net/20.500.11750/16280
- Degree
- Doctor
- Department
- Brain and Cognitive Sciences
- Publisher
- DGIST
- Related Researcher
-
-
Yu, Seong-Woon
- Research Interests Molecular mechanisms of neuronal cell death and neurodegeneration
-
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- Appears in Collections:
- Department of Brain Sciences Theses Ph.D.
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