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.

The Lysosomal Membrane Protein Lamp2 Alleviates Lysosomal Cell Death by Promoting Autophagic Flux in Ischemic Cardiomyocytes

“…However, it has also been reported that different mechanisms to inhibit autophagy also protect against myocardial ischemia/reperfusion injury (Wu et al, 2018;Yu et al, 2018). It has been found that application of 3-MA to inhibit autophagy alleviates astrocyte injury by inhibiting LMP (Zhou et al, 2017). In the present study, we also observed that 3-MA treatment and ATG5 siRNA improved cardiomyocyte resistance against OGD stress and inhibited LMP, whereas application of CQ to impede autophagic flux further exacerbated LMP.…”

Eugenol Attenuates Cerebral Ischemia-Reperfusion Injury by Enhancing Autophagy via AMPK-mTOR-P70S6K Pathway

“…All these studies revealed that autophagy plays neuroprotective roles after cerebral I/R injury. Reports also demonstrate that autophagy is deleterious for ischemic brain (Zhou et al, 2017;Feng et al, 2018). This discrepancy might be caused by different animal strains, ischemic models, and time of ischemia.…”

Neuronal-targeted TFEB rescues dysfunction of the autophagy-lysosomal pathway and alleviates ischemic injury in permanent cerebral ischemia

“…Mounting evidence strongly indicates that ALP is induced in multiple ischemic stroke models as reflected by the accumulation of autophagosomes and activation of lysosomal function [1,2,6,7]. However, it is noteworthy that ischemic injury can trigger a defect in ALP by causing lysosomal dysfunction, thus resulting in the abnormal accumulation of autophagosomes and substrates [8][9][10][11][12]. In a 2vessel occlusion (2-VO) ischemic stroke model, treatment with chloroquine (CQ) does not further change the LC3-II levels, suggesting that ALP is impaired [8].…”