title: 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.
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science; Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
authors.forename: Xian-Yong
authors.initials: XY
authors.lastname: Zhou
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science; Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science; Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
authors.forename: Yong-Ming
authors.initials: YM
authors.lastname: Zhu
authors.affiliation: Guangzhou Institute of Traumatic Surgery, Guangzhou Red Cross Hospital, Medical College, Jinan University, Guangzhou, China.
authors.forename: Zhi-He
authors.initials: ZH
authors.lastname: Liu
authors.affiliation: Stroke Outcomes Laboratory, Department of Neurology, Baylor College of Medicine, Houston, TX, USA.|Center for Translational Research on Inflammatory Diseases, Michael E DeBakey Veterans Affairs Medical Center, Houston, TX, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kent
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science; Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
authors.forename: Jia-Guo
authors.initials: JG
authors.lastname: Rong
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science; Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science; Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
authors.forename: Shi-Gang
authors.initials: SG
authors.lastname: Qiao
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science; Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science; Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Ni
authors.affiliation: Institute for Health Sciences, Division of Molecular Biology, Tokushima Bumi University, Yamashiro-cho, Tokushima City, Tokushima, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Ishidoh
authors.affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science; Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
authors.forename: Hui-Ling
authors.initials: HL
authors.lastname: Zhang
mesh_terms: D000818:Animals; D017209:Apoptosis; D001253:Astrocytes / Q000378:metabolism* / Q000473:pathology; D001343:Autophagy; D002401:Cathepsin B / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D015398:Signal Transduction
chemical_list: D002401:Cathepsin B