Inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membrane in ischemic astrocytes

Xian Yong Zhou, Yu Luo, Yong Ming Zhu, Zhi He Liu, Thomas A. Kent, Jia Guo Rong, Wei Li, Shi Gang Qiao, Min Li, Yong Ni, Kazumi Ishidoh, Hui Ling Zhang

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

Our previous study and others have demonstrated that autophagy is activated in ischemic astrocytes and contributes to astrocytic cell death. However, the mechanisms of ischemia-induced autophagy remain largely unknown. In this study, we established a rat's model of permanent middle cerebral artery occlusion (pMCAO) and an in vitro oxygen and glucose deprivation (OGD) model. Autophagy was inhibited by either pharmacological treatment with 3-methyladenine (3-MA) and wortmannin (Wort) or genetic treatment with knockdown of Atg5 in primary cultured astrocytes and knockout of Atg5 in mouse embryonic fibroblast (MEF) cells, respectively. We found that pharmacological or genetic inhibition of autophagy reversed pMCAO or OGD-induced increase in LC3-II, active cathepsin B and L, tBid, active caspase-3 and cytoplastic cytochrome c (Cyt-c), and suppressed the injury-induced reduction in mitochondrial Cyt-c in ischemic cortex, in injured astrocytes and MEF cells. Immunofluorescence analysis showed that 3-MA or Wort treatment reversed OGD-induced release of cathepsin B and L from the lysosome to the cytoplasm and activation of caspase-3 in the astrocytes. Furthermore, treatment of 3-MA or Wort decreased OGD-induced increase in lysosomal membrane permeability and enhanced OGD-induced upregulation of lysosomal heat shock protein 70.1B (Hsp70.1B) in astrocytes. Inhibition of autophagy by 3-MA or Wort reduced infarction volume in rats and protected OGD-induced astrocytic cell injury. A nonselective caspase inhibitor z-VAD-fmk or a specific caspase-3 inhibitor Q-DEVD-OPh also rescued OGD-induced astrocytic cell injury. In conclusion, our presenting data suggest that inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membranes, possibly due to upregulation of the lysosomal Hsp70.1B in ischemic astrocytes.

Original languageEnglish (US)
Article numbere2618
JournalCell Death and Disease
Volume8
Issue number2
DOIs
StatePublished - 2017

ASJC Scopus subject areas

  • Immunology
  • Cellular and Molecular Neuroscience
  • Cell Biology
  • Cancer Research

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