title: Protective mechanisms of CA074-me (other than cathepsin-B inhibition) against programmed necrosis induced by global cerebral ischemia/reperfusion injury in rats.
abstract: Many studies have demonstrated the key role of lysosomes in ischemic cell death in the brain and have led to the "lysosomocentric" hypothesis. In this hypothesis, the release of cathepsin-B due to a change of lysosomal membrane permeabilization (LMP) or rupture is critical, and this can be prevented by its inhibitors CA074 and CA074-me. However, the role of CA074-me in neuronal death and its effect on the change of lysosomal membrane integrity after global cerebral ischemia/reperfusion (I/R) injury is not clear, so we investigated this here. Rat hippocampal CA1 neuronal death was evaluated after 20-min global cerebral I/R injury. CA074-me (1 μg, 10 μg) were given intracerebroventricularly 1h before ischemia or 1h post reperfusion. The changes of heat shock protein 70 (Hsp70), cathepsin-B, lysosomal-associated membrane protein 1 (LAMP-1), receptor-interacting protein 3 (RIP3), and the change of lysosomal pH were evaluated respectively. Hippocampal CA1 neuronal programmed necrosis induced by global cerebral I/R injury was prevented by CA074-me both pre-treatment and post-treatment. Diffuse cytoplasmic cathepsin-B and LAMP-1 immunostaining synchronized with the pyknotic nuclear changes 2 days post reperfusion, and a rise of lysosomal pH with the leakage of DND-153, a dye of lysosomes, after oxygen-glucose deprivation (OGD) was detected. Both of these changes demonstrated the rupture of lysosomal membrane and the leakage of cathepsin-B, and this was strongly inhibited by CA074-me pre-treatment. The overexpression and nuclear translocation of RIP3 and the reduction of NAD(+) level after I/R injury were also inhibited, while the upregulation of Hsp70 was strengthened by CA074-me pre-treatment. Delayed fulminant leakage of cathepsin-B due to lysosomal rupture is a critical harmful factor in neuronal programmed necrosis induced by 20-min global I/R injury. In addition to being an inhibitor of cathepsin-B, CA074-me may have an indirect neuroprotective effect by maintaining lysosomal membrane integrity and protecting against lysosomal rupture.
authors.affiliation: Department of Neurology, Brain Medical Centre, First Affiliated Hospital, Zhejiang University School of Medicine, 89 Qingchun Road, Hangzhou 310003, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Neurology, First Affiliated Hospital, Anhui Medical University, 218 Jixi Road, Hefei 230022, China.
authors.forename: Jingye
authors.initials: J
authors.lastname: Wang
authors.affiliation: Core Facilities, Zhejiang University School of Medicine, 866 Yuhangtang Road, Hangzhou 310058, China.
authors.forename: Xinghui
authors.initials: X
authors.lastname: Song
authors.affiliation: Department of Neurology, Brain Medical Centre, First Affiliated Hospital, Zhejiang University School of Medicine, 89 Qingchun Road, Hangzhou 310003, China.
authors.forename: Ruili
authors.initials: R
authors.lastname: Wei
authors.affiliation: Department of Neurology, Brain Medical Centre, First Affiliated Hospital, Zhejiang University School of Medicine, 89 Qingchun Road, Hangzhou 310003, China.
authors.forename: Fangping
authors.initials: F
authors.lastname: He
authors.affiliation: Department of Neurology, Brain Medical Centre, First Affiliated Hospital, Zhejiang University School of Medicine, 89 Qingchun Road, Hangzhou 310003, China.
authors.forename: Guoping
authors.initials: G
authors.lastname: Peng
authors.affiliation: Department of Neurology, Brain Medical Centre, First Affiliated Hospital, Zhejiang University School of Medicine, 89 Qingchun Road, Hangzhou 310003, China. Electronic address: luobenyan@zju.edu.cn.
authors.forename: Benyan
authors.initials: B
authors.lastname: Luo
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002545:Brain Ischemia / Q000150:complications / Q000188:drug therapy* / Q000473:pathology / Q000503:physiopathology; D056547:CA1 Region, Hippocampal / Q000187:drug effects / Q000473:pathology / Q000503:physiopathology; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology*; D004195:Disease Models, Animal; D005947:Glucose / Q000172:deficiency; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D008297:Male; D009243:NAD / Q000378:metabolism; D009336:Necrosis / Q000209:etiology / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control*; D009474:Neurons / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D017207:Rats, Sprague-Dawley; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D015427:Reperfusion Injury / Q000150:complications / Q000188:drug therapy* / Q000473:pathology / Q000503:physiopathology
chemical_list: C400541:CA 074 methyl ester; D004151:Dipeptides; D018840:HSP70 Heat-Shock Proteins; D018696:Neuroprotective Agents; D009243:NAD; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C582791:Ripk3 protein, rat; D002401:Cathepsin B; C533916:Ctsb protein, rat; D005947:Glucose
keywords: CA074-me; Cathepsin-B; Heat shock protein 70; Ischemia/reperfusion injury; Lysosomal membrane permeabilization; Receptor-interacting protein 3