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Protease Omi cleaving Hax-1 protein contributes to OGD/R-induced mitochondrial damage in neuroblastoma N2a cells and cerebral injury in MCAO mice.

Wu JY, Li M, Cao LJ, Sun ML, Chen D, Ren HG, Xia Q, Tao ZT, Qin ZH, Hu QS, Wang GH - Acta Pharmacol. Sin. (2015)

Bottom Line: In the brains of MCAO mice, the protein level of Omi was significantly increased, while the protein level of Hax-1 was decreased.Furthermore, in OGD/R-treated N2a cells, knockdown of Omi significantly increased Hax-1 protein level.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Aim: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Inhibition of Omi protease activity or overexpression of Hax-1 protects N2a cells against OGD/R-induced cell death. (A) MTT assays were performed to show N2a cell viability after OGD/R. N2a cells were transfected with EGFP or EGFP-Hax-1 for 48 h and then subjected to OGD/R, followed by 12 h of reoxygenation. Immunoblot analyses were performed to show the levels of Hax-1 and Omi. (B) MTT assays were performed to show N2a cell viability after OGD/R and UCF-101 treatment. The N2a cells were subjected to OGD for 4 h and then treated with UCF-101 (10 μmol/L), followed by reoxygenation for 12 h. Immunoblot analyses were performed to show the levels of Hax-1 and Omi. Values are the mean±SEM from three independent experiments. bP<0.05, one-way ANOVA.
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fig2: Inhibition of Omi protease activity or overexpression of Hax-1 protects N2a cells against OGD/R-induced cell death. (A) MTT assays were performed to show N2a cell viability after OGD/R. N2a cells were transfected with EGFP or EGFP-Hax-1 for 48 h and then subjected to OGD/R, followed by 12 h of reoxygenation. Immunoblot analyses were performed to show the levels of Hax-1 and Omi. (B) MTT assays were performed to show N2a cell viability after OGD/R and UCF-101 treatment. The N2a cells were subjected to OGD for 4 h and then treated with UCF-101 (10 μmol/L), followed by reoxygenation for 12 h. Immunoblot analyses were performed to show the levels of Hax-1 and Omi. Values are the mean±SEM from three independent experiments. bP<0.05, one-way ANOVA.

Mentions: As Hax-1 is an anti-apoptotic protein belonging to Bcl-2 family, we questioned whether a decrease of Hax-1 level is associated with cell death induced by OGD/R. Therefore, we overexpressed EGFP alone or EGFP-tagged Hax-1 and evaluated cell viability after OGD/R. Significant cell death was observed in EGFP alone transfected cells after OGD/R; however, overexpression of EGFP-Hax-1 significantly blocked OGD/R-induced cell death (Figure 2A).


Protease Omi cleaving Hax-1 protein contributes to OGD/R-induced mitochondrial damage in neuroblastoma N2a cells and cerebral injury in MCAO mice.

Wu JY, Li M, Cao LJ, Sun ML, Chen D, Ren HG, Xia Q, Tao ZT, Qin ZH, Hu QS, Wang GH - Acta Pharmacol. Sin. (2015)

Inhibition of Omi protease activity or overexpression of Hax-1 protects N2a cells against OGD/R-induced cell death. (A) MTT assays were performed to show N2a cell viability after OGD/R. N2a cells were transfected with EGFP or EGFP-Hax-1 for 48 h and then subjected to OGD/R, followed by 12 h of reoxygenation. Immunoblot analyses were performed to show the levels of Hax-1 and Omi. (B) MTT assays were performed to show N2a cell viability after OGD/R and UCF-101 treatment. The N2a cells were subjected to OGD for 4 h and then treated with UCF-101 (10 μmol/L), followed by reoxygenation for 12 h. Immunoblot analyses were performed to show the levels of Hax-1 and Omi. Values are the mean±SEM from three independent experiments. bP<0.05, one-way ANOVA.
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Related In: Results  -  Collection

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fig2: Inhibition of Omi protease activity or overexpression of Hax-1 protects N2a cells against OGD/R-induced cell death. (A) MTT assays were performed to show N2a cell viability after OGD/R. N2a cells were transfected with EGFP or EGFP-Hax-1 for 48 h and then subjected to OGD/R, followed by 12 h of reoxygenation. Immunoblot analyses were performed to show the levels of Hax-1 and Omi. (B) MTT assays were performed to show N2a cell viability after OGD/R and UCF-101 treatment. The N2a cells were subjected to OGD for 4 h and then treated with UCF-101 (10 μmol/L), followed by reoxygenation for 12 h. Immunoblot analyses were performed to show the levels of Hax-1 and Omi. Values are the mean±SEM from three independent experiments. bP<0.05, one-way ANOVA.
Mentions: As Hax-1 is an anti-apoptotic protein belonging to Bcl-2 family, we questioned whether a decrease of Hax-1 level is associated with cell death induced by OGD/R. Therefore, we overexpressed EGFP alone or EGFP-tagged Hax-1 and evaluated cell viability after OGD/R. Significant cell death was observed in EGFP alone transfected cells after OGD/R; however, overexpression of EGFP-Hax-1 significantly blocked OGD/R-induced cell death (Figure 2A).

Bottom Line: In the brains of MCAO mice, the protein level of Omi was significantly increased, while the protein level of Hax-1 was decreased.Furthermore, in OGD/R-treated N2a cells, knockdown of Omi significantly increased Hax-1 protein level.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Aim: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus