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Calcium-mediated regulation of autophagy and programmed cell death in adult hippocampal neural stem cells: A story of calpains, ryanodine receptors, and presenilins

Calcium-mediated regulation of autophagy and programmed cell death in adult hippocampal neural stem cells: A story of calpains, ryanodine receptors, and presenilins
Chung, Kyung Min
DGIST Authors
Chung, Kyung MinYu, Seong Woon
Yu, Seong Woon
Kim, Seong Who
Issued Date
Awarded Date
2016. 8
Neural stem cellsAutophagyProgrammed cell deathCalpainRyanodine receptorsPreseniln-2
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. ⓒ 2016 DGIST
Table Of Contents
CHAPTER 1: General Introduction 15--
1.1 Adult Neurogenesis and Neural Stem Cells 15--
1.1.1 Adult Neurogenesis: The Discovery 15--
1.1.2 Adult Neural Stem Cells 17--
1.2 Programmed Cell Death 19--
1.2.1 PCD: Classifications and Features 19--
1.2.2 PCD in the Brain 22--
1.3 Autophagy 24--
1.3.1 Autophagic Cell Death: Insulin Withdrawal Model in Hippocapal Neural Stem Cells 26--
1.3.2 Autophagy in Neurodegeneration 29--
CHAPTER 2: General Materials and Common Techniques 32--
2.1 Cell Culture 32--
2.2 Western Blot Analysis 32--
2.3 Immunohistochemistry 33--
2.4 Immunocytochemistry 33--
2.5 Real-Time Quantitative PCR 34--
2.6 Cell Death Assay 34--
2.7 Transfection for Delivery of DNAs or siRNAs 34--
2.8 Flow Cytometry 34--
2.9 Statistical Analysis 35--
CHAPTER 3: Calpain Determines the Propensity of Adult HCN Cells to ACD Following Insulin Withdrawal 36--
3.1 Introduction 36--
3.2 Materials and Methods 38--
3.2.1 Antibodies and Reagents 38--
3.2.2 Plasmids and siRNAs 38--
3.2.3 Immunocytochemistry 38--
3.2.4 Intracellular Calcium Imaging 38--
3.2.5 Calpain Activity Assay 39--
3.3 Results 40--
3.3.1 Calpain 1 and 2 are Differentially Expressed in HCN Cells 40--
3.3.2 Calpain 2 Inhibition Potentiates Autophagic Death of I(-) HCN Cells 40--
3.3.3 Ectopic Expression of Calpain 1 Induces Apoptosis in HCN Cells Following Insulin Withdrawal 42--
3.3.4 Degradation of Calpain 2 Is Achieved via UPS, Not Autophagy 45--
3.3.5 Proteasome Inhibition Elevates the Concentration of Intracellular Calcium and Activates Calpain in I(-) HCN Cells 48--
3.3.6 Lactacystin Switches the Default Autophagic Death of I(-) HCN Cells to Apoptosis 51--
3.4 Discussion 56--
CHAPTER 4: Mediation of ACD by RyR3 in Adult HCN Cells 60--
4.1 Introduction 60--
4.2 Materials and Methods 65--
4.2.1 Pharmacological Reagents 65--
4.2.2 Immunofluorescnce-based Ca2+ Imaging 65--
4.2.3 Autophagic Flux Assay 65--
4.2.4 Generation of CRISPR/Cas9-mediated RYR3 Knockout HCN Cells 66--
4.3 Results 67--
4.3.1 ER-to-Cytosol Ca2+ efflux is Increased Following Insulin Withdrawal in HCN Cells 67--
4.3.2 RyR3 is the Major RyR Isoform Expressed in HCN Cells and its Expression is Elevated Following Insulin Withdrawal 67--
4.3.3 A RyR Agonist Caffeine Further Promotes ACD in I(-) HCN Cells 69--
4.3.4 ACD Induction by Caffeine is Precluded in Autophagy-Defective HCN Cells Depleted of Atg7 69--
4.3.5 Autophagy is Diminished by Pharmacological or Genetic RyR Inhibition in I(-) HCN Cells 73--
4.3.6 Knockout of RyR3 Gene Occludes ER Ca2+ Release and Thereby Prevents ACD in I(-) HCN Cells 76--
4.4 Discussion 81--
CHAPTER 5: A Novel Function of Presenilin-2 in Regulation of Autophagic Death of HCN Cells 86--
5.1 Introduction 86--
5.2 Materials and Methods 89--
5.2.1 Antibodies and Reagents 89--
5.2.2 Plasmids and siRNAs 89--
5.2.3 Organotypic Hippocampal Slice Culture 89--
5.2.4 Generation of CRISPR/Cas9-mediated Presenilin Knockout HCN Cell Lines 90--
5.3 Results 91--
5.3.1 Presenilin-2 Exhibits Distinct Expression Pattern in HCN Cells 91--
5.3.2 Expression of Presenilin-2 in HCN Cells is Significantly Upregulated Upon Insulin Withdrawal 91--
5.3.3 Genetic Depletion of PS2 Prevents Induction of ACD by Insulin Withdrawal in HCN Cells 91--
5.3.4 PS2 Expression Potentiates ACD in I(-) HCN Cells, but Not in Atg7-Deficient I(-) HCN Cells 94--
CHAPTER 6: General Discussion 97
Brain and Cognitive Sciences
Related Researcher
  • 유성운 Yu, Seong-Woon 뇌과학과
  • Research Interests Molecular mechanisms of neuronal cell death and neurodegeneration
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Department of Brain Sciences Theses Ph.D.


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