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Regulation of autophagic cell death by GSK3B in adult hippocampal neural stem cells following insulin withdrawal

Regulation of autophagic cell death by GSK3B in adult hippocampal neural stem cells following insulin withdrawal
Alternative Title
GSK3Β 에 의한 성체해마신경줄기세포의 자가포식 세포사멸 연구
Hye Young Ryu
DGIST Authors
Yu, Seong-WoonKim, Seong WhoRyu, Hye Young
Seong Who Kim
Issued Date
Awarded Date
Neural stem cells, Autophagy, Programmed cell death, GSK3B, ULK1, UCHL5
신경줄기세포 NSC)는 퇴행성 뇌 질환 치료에 큰 잠재력을 갖고 있다. 현재, 신경줄기세포에 영향을 미치는 예정세포사멸 (programmed cell death)의 메커니즘은 거의 알려지지 않고 있다. 최근 우리의 연구결과에서 성체해마신경줄기세포 (hippocampal neural stem cells)가 인슐린 결핍으로 인한 자가포식 세포사멸 (autophagic cell death)을 겪는다고 보고했다. 하지만 이 같은 활동의 정확한 분자생물학적 기전에 대한 연구는 여전히 부족한 상태이다. 본 연구에서는 글리코겐 싱카제 키나제 3B (glycogen synthase kinase 3B)가 인슐린이 결핍된 HCN세포에서 ACD를 매개한다는 것을 입증하였다. GSK3Β 의 약리학적, 유전적 비활성화 모두 ACD를 크게 감소시켰다. 이와는 대조적으로 GSK3A의 활성감소는 ACD에 거의 영향을 미치지 않았다. 종합적으로 볼 때, GSK3Β 가 인슐린 결핍상황에서 HCN 세포에서 ACD의 핵심 규제 자임을 보여준다.
ULK1 (Unc-51 like kinase)은 세린/트레오닌 키나아제이며, ATG1 동족체로도 알려져 있으며, 자가자용에 필수적인 역할을 한다. 영양소 및 성장인자 신호에서 ULK1 활동과 안정성은 인산화, 아세틸화, 편재 등에 의해 조절된다. 우리는 GSK3Β 를 HCN 세포에서 인슐린 결핍의 상위 조절기임을 확인하였다. 또한 인슐린 결핍에 따라 GSK3Β 가 GABARAP (Atg8 상동 단백질) 상호작용과ULK1의S405 및 S415의 인산화를 통해 ULK1과 직접 상호작용하고 활성화한다고 보고한다. 이러한 인산화 작용은 ULK1을 안정화하고 GABARAPL1과의 우선적 상호작용을 촉진한다. ULK1의 인산화 돌연변이는 GABARAPL1과 상호작용하지 못하고 리소좀의 흐름 (autophagic flux)을 저하했으며, UCHL5의 제어 하에 프로테아좀에 의해 분해되었다. 또한 다른 세포 유형에서 영양결핍 상황에서도 ULK1의 S405와 S415의 인산화 작용이 중요하다는 것을 밝혀내 ULK1 기능에 대한 일반적인 중요성을 나타냈다. 게다가, ULK1의 S405와 S415의 높은 인산화 수준이 인간 췌장암 세포 라인에서 관찰되었다. 우리의 결과는 성장 인자나 영양소의 결핍 상태에서 GSK3B를 매개로 한 ULK1 인산화에 따른 신규 기전을 규명하였고, 나아가 성체줄기세포의 생존과 사멸 조절의 분자생물학적인 새로운 역할을 제시 하였다.
|Neural stem cells (NSCs) hold great potential for the treatment of neurodegenerative diseases. However, programmed cell death (PCD) provoked by the harsh conditions evident in the diseased brain greatly undermines the potential of NSCs. Currently, the mechanisMaster of PCD that effect NSCs remain largely unknown. We have previously reported that hippocam-pal neural stem (HCN) cells derived from the adult rat undergo autopahgic cell death (ACD) following insulin withdrawal without hallmarks of apoptosis despite their normal apoptotic capabilities. In this study, we demonstrate that glycogen synthase kinase 3 beta (GSK3B) mediates ACD in insulin-deprived HCN cells. Both pharmacological and genetic inactivation of GSK3B significantly decreased ACD, while activation of GSK3B increased autophagic flux and caused more cell death without inducing apoptosis following insulin withdrawal. In contrast, knockdown of GSK3 alpha (GSK3A) barely affected ACD. Collectively, these data demonstrate that GSK3B is a key regulator of ACD in HCN cells following insulin with-drawal. The absence of apoptotic indices in GSK3Β-induced cell death in insulin-deprived HCN cells corroborates the notion that HCN cell death following insulin withdrawal repre-sents the genuine model of ACD in apoptosis-intact mammalian cells and identifies GSK3B as a key negative effector of NSC survival downstream of insulin signaling.
ULK1 (unc-51-like kinase), a mammalian homologue of the yeast Atg1 kinase, plays an essential role in autophagy induction. In nutrient and growth factor signaling, ULK1 activ-ity and stability are regulated by various post-translational modifications including phosphory-lation, acetylation, and ubiquitination. Here, we report that, following insulin withdrawal, GSK3B directly interacts with and activates ULK1 through phosphorylation of S405 and S415 within the GABARAP-interacting region. Phosphorylation of these residues stabilizes ULK1 and facilitates its preferential interaction with GABARAPL1. Phosphorylation-defective mutants of ULK1 failed to interact with GABARAPL1 and induce autophagy flux, and were degraded by the proteasome under the control of the deubiquitinase ubiquitin C-terminal hydrolase L5. We also found that phosphorylation of S405 and S415 is important for starvation-induced autophagy in other cell types, indicating the general importance of these sites for ULK1 function. Furthermore, high phosphorylation levels of ULK1 at S405 and S415 were observed in human pancreatic cancer cell lines, all of which are known to exhibit high autophagy activity. Our results reveal the importance of GSK3B mediated phosphoryla-tion for ULK1 regulation and autophagy induction in response to nutrient or growth factor deprivation, and potentially for tumorigenesis.
Table Of Contents
Chapter 1: General introduction 1
1.1 Adult neurogenesis and neural stem cells 1
1.2 Programmed cell death 3
1.3 Autophagy 5
1.4 Autophagic cell death (ACD) 7

Chapter 2: General material and methods 9
2.1 Cell culture 9
2.2 Cell death assay 10
2.3 Western blot analysis 10
2.4 Annexin V staining 11
2.5 CRISPR/Cas9 11
2.6 DNA plasmids and transfection 12
2.7 Generation of phosphospecific antibodies 12
2.8 Generation of recombinant proteins 13
2.9 GFP-LC3 puncta assay 13
2.10 Immunocytochemistry (ICC) 14
2.11 Immunoprecipitation assay (IP) 14
2.12 In vitro kinase assay 14
2.13 Reagent and antibodies 15
2.14 Stable knockdown 16
2.15 Statistical analysis 16

Chapter 3: Regulation of autophagic cell death by GSK3B in adult
hippocampal neural stem cells following insulin withdrawal 17
3.1 Introduction 17
3.2 Results 21
3.2.1 GSK3B is activated in HCN cells following insulin withdrawal 21
3.2.2 Pharmacological and genetic inhibition of GSK3B decreases ACD in insulin-deprived HCN cells 26
3.2.3 Over-expression of GSK3B up-regulates autopahgic flux and ACD, but not apoptosis, following insulin withdrawal in HCN cells 32
3.3 Discussion 40

Chapter 4: GSK3B induces autophagy by phosphorylating and stabilizing ULK1 44
4.1 Introduction 44
4.2 Results 50
4.2.1 GSK3B interacts with ULK1 50
4.2.2 Phosphorylation of S405 and S415 is important for ULK1
activation and autophagosome formation in response to insulin withdrawal 57
4.2.3 GSK3B phosphorylates ULK1 at S405 and S415 69
4.2.4 Phosphorylation of S405 and S415 promotes interaction of ULK1 with GABARAPL1 74
4.2.5 GSK3B-dependent ULK1 phosphorylation regulates starvation- induced autophagy in various cell types 79
4.2.6 GSK3B-dependent ULK1 phosphorylation is important for its stability 83
4.2.7 Deubiquitinase UCHL5 regulates ULK1 degradation 86
4.2.8 ULK1 phosphorylation at S405 and S415 is enhanced in human pancreatic cancer cell lines 88
4.3 Discussion 90

Chapter 5: Conclusion 95
Chapter 6: Summary 국문요약 106
Department of 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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