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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

Omi processes Hax-1 after I/R in vivo and OGD/R in vitro. (A and B) Immunoblot analyses were performed to show Omi (A) and Hax-1 (B) protein levels in the penumbra after I/R. ICR mice (n=3) were subjected to a middle cerebral artery occlusion (MCAO) for 2 h, followed by 24 h of reperfusion. The bottom panels show the band intensity of Omi or Hax-1 relative to that of tubulin (A) or GAPDH (B), respectively. (C) Immunoblot analyses were performed to show Hax-1 protein levels in N2a cells that were exposed to OGD for 4 h, followed by 12 h of reoxygenation. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. (D) Real-time quantitative PCR was performed to examine Hax-1 mRNA levels in N2a cells that were subjected to OGD for 4 h, followed by 12 h of reoxygenation. The Hax-1 mRNA levels were quantified and normalized to GAPDH. (E) Immunoblot analyses were performed to show Hax-1 levels in OGD/R N2a cells in which Omi was knocked down. N2a cells were subjected to OGD for 4 h, followed by 12 h of reoxygenation. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. (F) Immunoblot analyses were performed to show Hax-1 levels in OGD/R N2a cells that were treated with MG132 (10 μmol/L) and bafilomycin A1 (1 μmol/L). The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. Values are the mean±SEM from three independent experiments. aP>0.05, bP<0.05, one-way ANOVA.
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fig1: Omi processes Hax-1 after I/R in vivo and OGD/R in vitro. (A and B) Immunoblot analyses were performed to show Omi (A) and Hax-1 (B) protein levels in the penumbra after I/R. ICR mice (n=3) were subjected to a middle cerebral artery occlusion (MCAO) for 2 h, followed by 24 h of reperfusion. The bottom panels show the band intensity of Omi or Hax-1 relative to that of tubulin (A) or GAPDH (B), respectively. (C) Immunoblot analyses were performed to show Hax-1 protein levels in N2a cells that were exposed to OGD for 4 h, followed by 12 h of reoxygenation. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. (D) Real-time quantitative PCR was performed to examine Hax-1 mRNA levels in N2a cells that were subjected to OGD for 4 h, followed by 12 h of reoxygenation. The Hax-1 mRNA levels were quantified and normalized to GAPDH. (E) Immunoblot analyses were performed to show Hax-1 levels in OGD/R N2a cells in which Omi was knocked down. N2a cells were subjected to OGD for 4 h, followed by 12 h of reoxygenation. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. (F) Immunoblot analyses were performed to show Hax-1 levels in OGD/R N2a cells that were treated with MG132 (10 μmol/L) and bafilomycin A1 (1 μmol/L). The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. Values are the mean±SEM from three independent experiments. aP>0.05, bP<0.05, one-way ANOVA.

Mentions: Previous studies have shown that Omi levels increased and Hax-1 levels decreased in the penumbra after I/R22. To explore how Omi regulates Hax-1 after I/R, we first examined the protein levels of Omi and Hax-1 in the penumbra of MCAO mouse brains 24 h after I/R. Consistent with the previous findings22, protein levels of Omi were increased (Figure 1A), while Hax-1 levels were decreased (Figure 1B). In addition, although Hax-1 protein levels were decreased in cells exposed to OGD/R (Figure 1C), transcription of Hax-1 was not altered (Figure 1D), suggesting a post-translational process of Hax-1. As we previously found that Omi cleaves Hax-124 and that Hax-1 is degraded by the ubiquitin-proteasome pathway after apoptotic stimulation26, we performed experiments using a cellular OGD/R model to address how Omi regulates Hax-1 after I/R. In N2a cells that were exposed to OGD/R, an increase of Omi and a decrease of Hax-1 were observed, however, Hax-1 protein levels were dramatically increased after Omi was knocked down (Figure 1E), suggesting that Omi possibly processes Hax-1. We further examined the Hax-1 levels in OGD/R cells with or without the administration of the proteasomal inhibitor MG132 or the lysosomal inhibitor bafilomycin A1 (BafA1) to differentiate Hax-1 degradation pathways. MG132 or BafA1 did not block the degradation of Hax-1 (Figure 1F), further supporting that Hax-1 is processed by Omi but not degraded by the proteasomal or lysosomal pathway.


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)

Omi processes Hax-1 after I/R in vivo and OGD/R in vitro. (A and B) Immunoblot analyses were performed to show Omi (A) and Hax-1 (B) protein levels in the penumbra after I/R. ICR mice (n=3) were subjected to a middle cerebral artery occlusion (MCAO) for 2 h, followed by 24 h of reperfusion. The bottom panels show the band intensity of Omi or Hax-1 relative to that of tubulin (A) or GAPDH (B), respectively. (C) Immunoblot analyses were performed to show Hax-1 protein levels in N2a cells that were exposed to OGD for 4 h, followed by 12 h of reoxygenation. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. (D) Real-time quantitative PCR was performed to examine Hax-1 mRNA levels in N2a cells that were subjected to OGD for 4 h, followed by 12 h of reoxygenation. The Hax-1 mRNA levels were quantified and normalized to GAPDH. (E) Immunoblot analyses were performed to show Hax-1 levels in OGD/R N2a cells in which Omi was knocked down. N2a cells were subjected to OGD for 4 h, followed by 12 h of reoxygenation. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. (F) Immunoblot analyses were performed to show Hax-1 levels in OGD/R N2a cells that were treated with MG132 (10 μmol/L) and bafilomycin A1 (1 μmol/L). The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. Values are the mean±SEM from three independent experiments. aP>0.05, bP<0.05, one-way ANOVA.
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Related In: Results  -  Collection

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fig1: Omi processes Hax-1 after I/R in vivo and OGD/R in vitro. (A and B) Immunoblot analyses were performed to show Omi (A) and Hax-1 (B) protein levels in the penumbra after I/R. ICR mice (n=3) were subjected to a middle cerebral artery occlusion (MCAO) for 2 h, followed by 24 h of reperfusion. The bottom panels show the band intensity of Omi or Hax-1 relative to that of tubulin (A) or GAPDH (B), respectively. (C) Immunoblot analyses were performed to show Hax-1 protein levels in N2a cells that were exposed to OGD for 4 h, followed by 12 h of reoxygenation. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. (D) Real-time quantitative PCR was performed to examine Hax-1 mRNA levels in N2a cells that were subjected to OGD for 4 h, followed by 12 h of reoxygenation. The Hax-1 mRNA levels were quantified and normalized to GAPDH. (E) Immunoblot analyses were performed to show Hax-1 levels in OGD/R N2a cells in which Omi was knocked down. N2a cells were subjected to OGD for 4 h, followed by 12 h of reoxygenation. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. (F) Immunoblot analyses were performed to show Hax-1 levels in OGD/R N2a cells that were treated with MG132 (10 μmol/L) and bafilomycin A1 (1 μmol/L). The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. Values are the mean±SEM from three independent experiments. aP>0.05, bP<0.05, one-way ANOVA.
Mentions: Previous studies have shown that Omi levels increased and Hax-1 levels decreased in the penumbra after I/R22. To explore how Omi regulates Hax-1 after I/R, we first examined the protein levels of Omi and Hax-1 in the penumbra of MCAO mouse brains 24 h after I/R. Consistent with the previous findings22, protein levels of Omi were increased (Figure 1A), while Hax-1 levels were decreased (Figure 1B). In addition, although Hax-1 protein levels were decreased in cells exposed to OGD/R (Figure 1C), transcription of Hax-1 was not altered (Figure 1D), suggesting a post-translational process of Hax-1. As we previously found that Omi cleaves Hax-124 and that Hax-1 is degraded by the ubiquitin-proteasome pathway after apoptotic stimulation26, we performed experiments using a cellular OGD/R model to address how Omi regulates Hax-1 after I/R. In N2a cells that were exposed to OGD/R, an increase of Omi and a decrease of Hax-1 were observed, however, Hax-1 protein levels were dramatically increased after Omi was knocked down (Figure 1E), suggesting that Omi possibly processes Hax-1. We further examined the Hax-1 levels in OGD/R cells with or without the administration of the proteasomal inhibitor MG132 or the lysosomal inhibitor bafilomycin A1 (BafA1) to differentiate Hax-1 degradation pathways. MG132 or BafA1 did not block the degradation of Hax-1 (Figure 1F), further supporting that Hax-1 is processed by Omi but not degraded by the proteasomal or lysosomal pathway.

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