title: Protease Omi cleaving Hax-1 protein contributes to OGD/R-induced mitochondrial damage in neuroblastoma N2a cells and cerebral injury in MCAO mice.
abstract: OBJECTIVE
  In the penumbra after focal cerebral ischemia, an increase of protease Omi is linked to a decrease of Hs1-associated protein X-1 (Hax-1), a protein belonging to the Bcl-2 family. In this study we investigated the mechanisms underlying the regulation of Hax-1 by protease Omi in cerebral ischemia/reperfusion (I/R) injury.
  
  
  METHODS
  Mouse neuroblastoma N2a cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R); cell viability was assessed with MTT assay. Mice underwent 2-h middle cerebral artery occlusion (MCAO) and reperfusion, and the infarct volume was determined with TTC staining. The expression of Omi and Hax-1 was detected using immunoblot and immunofluorescence assays. The mitochondrial membrane potential was measured using TMRM staining.
  
  
  RESULTS
  In the brains of MCAO mice, the protein level of Omi was significantly increased, while the protein level of Hax-1 was decreased. Similar changes were observed in OGD/R-treated N2a cells, but the mRNA level of Hax-1 was not changed. Furthermore, in OGD/R-treated N2a cells, knockdown of Omi significantly increased Hax-1 protein level. Immunofluorescence assay showed that Omi and Hax-1 were co-localized in mitochondria of N2a cells. OGD/R caused marked mitochondrial damage and apoptosis in N2a cells, while inhibition of Omi protease activity with UCF-101 (10 μmol/L) or overexpression of Hax-1 could restore the mitochondrial membrane potential and attenuate cell apoptosis. Moreover, pretreatment of MCAO mice with UCF-101 (7.15 mg/kg, ip) could restore Hax-1 expression, inhibit caspase activation, and significantly reduce the infarct volume.
  
  
  CONCLUSIONS
  Protease Omi impairs mitochondrial function by cleaving Hax-1, which induces apoptosis in OGD/R-treated N2a cells and causes I/R injury in MCAO mice.
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Jia-yuan
authors: JY
authors: Wu
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Mei
authors: M
authors: Li
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Li-juan
authors: LJ
authors: Cao
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Mei-ling
authors: ML
authors: Sun
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Dong
authors: D
authors: Chen
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Hai-gang
authors: HG
authors: Ren
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Qin
authors: Q
authors: Xia
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Zhou-teng
authors: ZT
authors: Tao
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Zheng-hong
authors: ZH
authors: Qin
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Qing-song
authors: QS
authors: Hu
authors: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors: Guang-hui
authors: GH
authors: Wang
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D005947:Glucose / Q000378:metabolism; D000074923:High-Temperature Requirement A Serine Peptidase 2; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism* / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D010100:Oxygen / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: C491295:Hs1bp1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D012697:Serine Endopeptidases; D000074923:High-Temperature Requirement A Serine Peptidase 2; C000615915:Htra2 protein, mouse; D005947:Glucose; D010100:Oxygen