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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 OGD/R cells against apoptosis. (A) Hoechst staining was performed to show the effect of inhibition of Omi protease activity on apoptosis after OGD/R. N2a cells were subjected to OGD/R as in Figure 3C. Scale bar, 10 μm. (B) EGFP/Hoechst double staining was performed to show the effects of Hax-1 overexpression on apoptosis after OGD/R. N2a cells were subjected to OGD/R as in Figure 3D. Scale bar, 10 μm. (C) Immunoblot analyses were performed to show that inhibition of Omi protease activity or overexpression of Hax-1 decreases the level of cleaved caspase-3 in OGD/R cells.
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fig4: Inhibition of Omi protease activity or overexpression of Hax-1 protects OGD/R cells against apoptosis. (A) Hoechst staining was performed to show the effect of inhibition of Omi protease activity on apoptosis after OGD/R. N2a cells were subjected to OGD/R as in Figure 3C. Scale bar, 10 μm. (B) EGFP/Hoechst double staining was performed to show the effects of Hax-1 overexpression on apoptosis after OGD/R. N2a cells were subjected to OGD/R as in Figure 3D. Scale bar, 10 μm. (C) Immunoblot analyses were performed to show that inhibition of Omi protease activity or overexpression of Hax-1 decreases the level of cleaved caspase-3 in OGD/R cells.

Mentions: Mitochondria are important cellular organelles that are involved in neuronal cell death after I/R by releasing cytochrome c13. Because Hax-1 is decreased in OGD/R cells as well as in the brains after I/R (Figure 1), we questioned whether the increase of Omi and the decrease of Hax-1 are associated with mitochondrial damage in OGD/R cells. Omi and Hax-1 both localize to mitochondria, as shown by co-localization with mitochondrial protein Tom20 (Figure 3A and 3B). In normoxic cells, inhibition of Omi protease activity by UCF-101 did not affect the mitochondrial membrane potential (Figure 3C). In OGD/R cells, there was a significant decrease of mitochondrial membrane potential, indicated by TMRM staining (Figure 3C). However, administration of UCF-101 significantly restored the mitochondrial membrane potential (Figure 3C). Moreover, overexpression of Hax-1 also restored the mitochondrial membrane potential in OGD/R cells, showing a similar effect as UCF-101 treatment (Figure 3D). These data suggest that an inhibition of Omi protease activity or overexpression of Hax-1 is able to maintain mitochondrial integrity against OGD/R injury. Next, we examined whether an inhibition of Omi protease activity or overexpression of Hax-1 protects OGD/R cells against apoptosis. After OGD/R, a majority of the cells presented condensed nuclei (Figure 4A and 4B), whereas, only a small portion of OGD/R cells presented apoptotic features after administration of UCF-101 (Figure 4A). Additionally, overexpression of Hax-1 significantly alleviated apoptotic changes induced by OGD/R (Figure 4B). Consistent with the morphological data (Figure 4A and 4B), administration of UCF-101 (Figure 4C, left panel) or overexpression of Hax-1 (Figure 4C, right panel) in OGD/R cells decreased the levels of cleaved caspase-3. These data further suggest that Hax-1 protects OGD/R cells against apoptosis.


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 OGD/R cells against apoptosis. (A) Hoechst staining was performed to show the effect of inhibition of Omi protease activity on apoptosis after OGD/R. N2a cells were subjected to OGD/R as in Figure 3C. Scale bar, 10 μm. (B) EGFP/Hoechst double staining was performed to show the effects of Hax-1 overexpression on apoptosis after OGD/R. N2a cells were subjected to OGD/R as in Figure 3D. Scale bar, 10 μm. (C) Immunoblot analyses were performed to show that inhibition of Omi protease activity or overexpression of Hax-1 decreases the level of cleaved caspase-3 in OGD/R cells.
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fig4: Inhibition of Omi protease activity or overexpression of Hax-1 protects OGD/R cells against apoptosis. (A) Hoechst staining was performed to show the effect of inhibition of Omi protease activity on apoptosis after OGD/R. N2a cells were subjected to OGD/R as in Figure 3C. Scale bar, 10 μm. (B) EGFP/Hoechst double staining was performed to show the effects of Hax-1 overexpression on apoptosis after OGD/R. N2a cells were subjected to OGD/R as in Figure 3D. Scale bar, 10 μm. (C) Immunoblot analyses were performed to show that inhibition of Omi protease activity or overexpression of Hax-1 decreases the level of cleaved caspase-3 in OGD/R cells.
Mentions: Mitochondria are important cellular organelles that are involved in neuronal cell death after I/R by releasing cytochrome c13. Because Hax-1 is decreased in OGD/R cells as well as in the brains after I/R (Figure 1), we questioned whether the increase of Omi and the decrease of Hax-1 are associated with mitochondrial damage in OGD/R cells. Omi and Hax-1 both localize to mitochondria, as shown by co-localization with mitochondrial protein Tom20 (Figure 3A and 3B). In normoxic cells, inhibition of Omi protease activity by UCF-101 did not affect the mitochondrial membrane potential (Figure 3C). In OGD/R cells, there was a significant decrease of mitochondrial membrane potential, indicated by TMRM staining (Figure 3C). However, administration of UCF-101 significantly restored the mitochondrial membrane potential (Figure 3C). Moreover, overexpression of Hax-1 also restored the mitochondrial membrane potential in OGD/R cells, showing a similar effect as UCF-101 treatment (Figure 3D). These data suggest that an inhibition of Omi protease activity or overexpression of Hax-1 is able to maintain mitochondrial integrity against OGD/R injury. Next, we examined whether an inhibition of Omi protease activity or overexpression of Hax-1 protects OGD/R cells against apoptosis. After OGD/R, a majority of the cells presented condensed nuclei (Figure 4A and 4B), whereas, only a small portion of OGD/R cells presented apoptotic features after administration of UCF-101 (Figure 4A). Additionally, overexpression of Hax-1 significantly alleviated apoptotic changes induced by OGD/R (Figure 4B). Consistent with the morphological data (Figure 4A and 4B), administration of UCF-101 (Figure 4C, left panel) or overexpression of Hax-1 (Figure 4C, right panel) in OGD/R cells decreased the levels of cleaved caspase-3. These data further suggest that Hax-1 protects OGD/R cells against apoptosis.

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