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

• Published in: Cell death & disease Vol. 8; no. 2; p. e2618
• Main Authors: Zhou, Xian-Yong, Luo, Yu, Zhu, Yong-Ming, Liu, Zhi-He, Kent, Thomas A, Rong, Jia-Guo, Li, Wei, Qiao, Shi-Gang, Li, Min, Ni, Yong, Ishidoh, Kazumi, Zhang, Hui-Ling
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 16-02-2017; Springer Nature B.V; Nature Publishing Group
• Subjects: 13/106; 13/95; 14/19; 14/34; 38/1; 631/378/2596/1308; 631/443/592/75/593/15/1939; 631/80/82/39; 631/80/86; 82/51; Animals; Antibodies; Apoptosis; Astrocytes - metabolism; Astrocytes - pathology; Autophagy; Biochemistry; Biomedical and Life Sciences; Cathepsin B - metabolism; Cell Biology; Cell Culture; Immunology; Ischemia; Life Sciences; Lysosomes - metabolism; Membranes; Mice; Original; original-article; Signal Transduction; Studies
• ISSN: 2041-4889; 2041-4889
• DOI: 10.1038/cddis.2017.34

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
Atg
5 in primary cultured astrocytes and knockout of
Atg
5 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 non-selective 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.