Regulated 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.
Table Of Contents
Ⅰ. 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 11 184.108.40.206 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