Alternative cell death of Apaf1-deficient neural progenitor cells induced by withdrawal of EGF or insulin |
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Authors: | Hiroshi Shiraishi Hideaki Okamoto Hiromitsu Hara Hiroki Yoshida |
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Institution: | 1. Division of Medical Biochemistry, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga 849-8501, Japan;2. Division of Molecular and Cellular Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan |
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Abstract: | BackgroundVarious forms of cell death, such as apoptotic, autophagic and non-lysosomal types, are implicated in normal physiological processes. Apoptotic protease activating factor 1 (Apaf1) is an important component of the intrinsic apoptotic pathway. Deficiency of Apaf1 results in an accumulation of neural progenitor cells (NPCs) in the developing central nervous system and thus, in perinatal lethality. A small percentage of the mutant mice, however, are viable and grow to maturity. The occurrence of such normal mutants implicates alternative cell death pathways during neurogenesis.MethodsNPCs prepared from wild-type or Apaf1-deficient embryos were cultured in growth factor-deprived medium and examined for cell death, caspase activation and morphological alterations. Generation of reactive oxygen species (ROS) and the effects of antioxidants were examined.ResultsWild-type NPCs underwent apoptosis within 24 hours of withdrawal of epidermal growth factor (EGF) or insulin, whereas Apaf1-deficient NPCs underwent cell death but showed no signs of apoptosis. Autophagy was not necessarily accompanied by cell death. Cell death of the Apaf1-deficient NPCs resembled necroptosis—necrosis-like programmed cell death. The necroptosis inhibitor necrostatin-1, however, failed to inhibit the cell death. ROS accumulation was detected in NPCs deprived of growth factors, and an antioxidant partially suppressed the non-apoptotic cell death of Apaf1-deficient NPCs.ConclusionsThese data indicate that after withdrawal EGF or insulin withdrawal, the Apaf1-deficient cells underwent non-apoptotic cell death. ROS generation may partially participate in the cell death.General SignificanceNon-apoptotic cell death in NPCs may be a compensatory mechanism in the developing CNS of Apaf1-deficient embryos. |
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Keywords: | Apaf1 apoptotic protease activating factor 1 NPCs neural progenitor cells ROS reactive oxygen species EGF epidermal growth factor CNS central nervous system NAC N-acetyl-L-cysteine GFP green fluorescent protein LC3 microtubule-associated protein light chain 3 NPMM neural progenitor maintenance medium N2/DMEM/F12 Dulbecco's modified Eagle's medium/F12 containing N2 supplement FGF fibroblast growth factor MEFs mouse embryonic fibroblasts HBSS Hank's balanced salt solution PI propidium iodide DAPI 4&prime 6-diamidino-2-phenylindole CM-H2DCFDA 5-(and -6)-chloromethyl-2&prime 7&prime -dichlorodihydrofluorescein diacetate Nec-1 necrostatin-1 BHA 3(2)-t-butyl-4-hydroxyanisole RIP1 receptor interacting protein 1 MMP mitochondrial membrane permeabilization mPT mitochondrial permeability transition NMDA N-methyl-D-aspartate TNF-α tumor necrosis factor alpha |
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