Dataset Information

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

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 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.

SUBMITTER: Zhou XY

PROVIDER: S-EPMC5386481 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Zhou Xian-Yong XY Luo Yu Y Zhu Yong-Ming YM Liu Zhi-He ZH Kent Thomas A TA Rong Jia-Guo JG Li Wei W Qiao Shi-Gang SG Li Min M Ni Yong Y Ishidoh Kazumi K Zhang Hui-Ling HL

Cell death & disease 20170216 2

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 tr ...[more]

PMID: 28206988

Similar Datasets

Project description:Receptor-interacting protein kinase 1 (RIPK1) contributes to necroptosis. Our previous study showed that pharmacological or genetic inhibition of RIPK1 protects against ischemic stroke-induced astrocyte injury. In this study, we investigated the molecular mechanisms underlying RIPK1-mediated astrocyte injury in vitro and in vivo. Primary cultured astrocytes were transfected with lentiviruses and then subjected to oxygen and glucose deprivation (OGD). In a rat model of permanent middle cerebral artery occlusion (pMCAO), lentiviruses carrying shRNA targeting RIPK1 or shRNA targeting heat shock protein 70.1B (Hsp70.1B) were injected into the lateral ventricles 5 days before pMCAO was established. We showed that RIPK1 knockdown protected against OGD-induced astrocyte damage, blocked the OGD-mediated increase in lysosomal membrane permeability in astrocytes, and inhibited the pMCAO-induced increase in astrocyte lysosome numbers in the ischemic cerebral cortex; these results suggested that RIPK1 contributed to the lysosomal injury in ischemic astrocytes. We revealed that RIPK1 knockdown upregulated the protein levels of Hsp70.1B and increased the colocalization of Lamp1 and Hsp70.1B in ischemic astrocytes. Hsp70.1B knockdown exacerbated pMCAO-induced brain injury, decreased lysosomal membrane integrity and blocked the protective effects of the RIPK1-specific inhibitor necrostatin-1 on lysosomal membranes. On the other hand, RIPK1 knockdown further exacerbated the pMCAO- or OGD-induced decreases in the levels of Hsp90 and the binding of Hsp90 to heat shock transcription factor-1 (Hsf1) in the cytoplasm, and RIPK1 knockdown promoted the nuclear translocation of Hsf1 in ischemic astrocytes, resulting in increased Hsp70.1B mRNA expression. These results suggest that inhibition of RIPK1 protects ischemic astrocytes by stabilizing lysosomal membranes via the upregulation of lysosomal Hsp70.1B; the mechanism underlying these effects involves decreased Hsp90 protein levels, increased Hsf1 nuclear translocation and increased Hsp70.1B mRNA expression.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets