Parkin Promotes Mitophagic Cell Death in Adult Hippocampal Neural Stem Cells Following Insulin Withdrawal

Autophagy-dependent cell death, c-Jun, Hippocampal neural stem cells, Mitophagy, ParkinRegulated cell death (RCD) plays a fundamental role in human health and disease. Apoptosis is the best-studied mode of RCD, but the importance of other modes has recently been gaining attention. We have previously demonstrated that adult rat hippocampal neural stem (HCN) cells undergo autophagy-dependent cell death (ADCD) following insulin withdrawal. Here, I show that Parkin mediates mitophagy and ADCD in insulin-deprived HCN cells. Insulin withdrawal increased the amount of depolarized mitochondria and their colocalization with autophagosomes. Insulin withdrawal also upregulated both mRNA and protein levels of Parkin, gene knockout of which prevented mitophagy and ADCD. c-Jun is a transcriptional repressor of Parkin and is degraded by the proteasome following insulin withdrawal. In insulin-deprived HCN cells, Parkin is required for Ca2+ accumulation and depolarization of mitochondria at the early stages of mitophagy as well as for recognition and removal of depolarized mitochondria at later stages. In contrast to the prodeath role of Parkin during mitophagy, Parkin deletion rendered HCN cells susceptible to apoptosis, revealing distinct roles of Parkin depending on different modes of RCD. Taken together, these results indicate that Parkin is required for the induction of ADCD accompanying mitochondrial dysfunction in HCN cells following insulin withdrawal. Since impaired insulin signaling is implicated in hippocampal deficits in various neurodegenerative diseases and psychological disorders, these findings may help to understand the mechanisms underlying the death of neural stem cells and develop novel therapeutic strategies aiming to improve neurogenesis and survival of neural stem cells.openⅠ. Introduction 1.1 Neural stem cells (NSCs) and neurogenesis 1 1.2 NSC and mitochondria 3 1.2.1 Mitochondria in adult neurogenesis 3 1.2.2 Adult neurogenesis and neurodegeneration 6 1.3 Regulated cell death (RCD) 8 1.3.1 Apoptosis 8 1.3.2 Necrosis 10 1.3.3 Autophagy 11 1.3.3.1 Three types of autophagy 11 1.3.4 Mitophagy 13 1.4 Autophagy-dependent cell death (ADCD) 18 1.4.1 Insulin withdrawal model in hippocampal neural stem cells 18 1.5 Mitophagic cell death 20 1.6 Parkin 20 Ⅱ. Materials and Methods 2.1 Reagents and Antibodies 22 2.2 Cell culture 22 2.3 Cell Death Assay 23 2.4 Caspase 3 Activity Assay 23 2.5 TUNEL Assay 24 2.6 Western Blotting 24 2.7 Mitochondrial and Cytosolic Fractionation 25 2.8 Plasmids and Transfection 25 2.9 siRNA mediated knockdown by nucleofection 26 2.10 Park2 and Pink1 Knockout 27 2.11 qRT-PCR 27 2.12 Flow Cytometry Analysis 27 2.13 Immunocytochemistry and Quantification of Relative Fluorescence Intensity or Colocalization Coefficient 28 2.14 Promoter Activity Assay 28 2.15 Statistics 29 Ⅲ. Results 3.1 Insulin withdrawal induces ADCD with mitochondrial Alterations in HCN Cells 30 3.2 Insulin -deprived HCN cells undergo excessive mitophagy 35 3.3 Parkin is upregulated through inhibition of its transcriptional repressor c-Jun in insulin-deprived HCN Cells 36 3.4 Parkin knockdown/knockout prevents ADCD in HCN cells following insulin withdrawal 47 3.5 Parkin KO rescues mitochondrial alterations and prevents initiation of mitophagy in insulin-deprived HCN cells 55 Ⅳ. Discussion 63 Ⅴ. Reference 67 Ⅵ. 요약문 81HCN cells) 인슐린을 제거한 배지에서 오토파지 종속 세포 죽음 (Autophagy dependent cell death조절된 세포 사멸은(Regulated cell death) 인간 건강 및 질병에서 근본적인 역할을 한다. 우리는 이전에 렛의 성체해마줄기세포가 (rat hippocampal neural stem cellsADCD)을 겪고 있음을 확인했다. 이번 연구에서 파킨 (Parkin)은 인슐린이 결핍된 HCN 세포에서 mitophagy 와 ADCD 를 매개한다는 것을 보여준다. 인슐린 제거로 인해 탈분극된 미토콘드리아의 수가 증가하였으며 또한 autophagosome 과 의 공동 국소화(colocalization)를 증가시켰다. 인슐린 결핍은 또한 Parkin의 mRNA 및 단백질 수준 둘 다를 상향 조절하였고, 이의 유전자 녹아웃은 mitophagy 및 ADCD 를 방지하였다. c-Jun 은 Parkin의 전사 억제 인자이며 인슐린 제거 후 프로 프로테아좀 (proteasome)에 의해 분해된다. 인슐린이 제거된 HCN 세포에서, 파킨은 mitophagy의 초기 단계에서 미토콘드리아의 Ca2 + 축적 및 탈분극뿐만 아니라 후기 단계에서 탈분극된 미토콘드리아의 인식 및 제거를 위해 필요하다. Mitophagy동안 Parkin의 pro-death 역할과 대조적으로, Parkin 결실은 HCN 세포가 apoptosis에 취약하게 하는 등 RCD 의 세포사멸의 다른 모드에 따라 Parkin 의 독특한 역할을 나타냈다. 종합하면, 이들 결과 는 Parkin 이 인슐린 제거된 HCN 세포에서 미토콘드리아 기능 장애를 수반하는 ADCD 의 유도에 필요하다는 것을 나타낸다. 인슐린 신호전달 장애는 다양한 신경 퇴행성 질환과 관련이 있기 때문에, 이러한 발견은 신경세포줄기 세포의 사멸의 기전을 이해하고 신경 발생 및 생존을 개선시키는 것을 목표로 하는 새로운 치료 전략을 개발하는 데 도움이 될 수 있다.DoctordCollectio

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