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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

Protective effects in MCAO mice by UCF-101. (A) Immunoblot analyses were performed to show Hax-1 protein levels in the penumbra after I/R. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to I/R. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. Values are the mean±SEM from three independent experiments. bP<0.05, one-way ANOVA. (B) Immunoblot analyses were performed to show the level of cleaved caspase-3 in the penumbra after I/R. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to I/R. (C) The infarct volume of MCAO/R mice with or without UCF-101 treatment. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to MCAO/R. Twenty-four hours after MCAO/R the mice were euthanized and the mouse slices were stained with TTC. The bottom panel shows the infarct volumes. bP<0.05, one-way ANOVA.
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fig5: Protective effects in MCAO mice by UCF-101. (A) Immunoblot analyses were performed to show Hax-1 protein levels in the penumbra after I/R. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to I/R. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. Values are the mean±SEM from three independent experiments. bP<0.05, one-way ANOVA. (B) Immunoblot analyses were performed to show the level of cleaved caspase-3 in the penumbra after I/R. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to I/R. (C) The infarct volume of MCAO/R mice with or without UCF-101 treatment. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to MCAO/R. Twenty-four hours after MCAO/R the mice were euthanized and the mouse slices were stained with TTC. The bottom panel shows the infarct volumes. bP<0.05, one-way ANOVA.

Mentions: We have shown that the anti-apoptotic effects on OGD/R cells were caused by an inhibition of Omi protease activity or overexpression of Hax-1 (Figure 3 and 4). Next, we questioned whether inhibition of Omi could protect neurons against apoptosis and neurons in MCAO mice to survive. To answer it, we examined Hax-1 expression in the penumbra after I/R. As shown in Figure 5A, Hax-1 levels in the penumbra were dramatically decreased after I/R, but were significantly restored by treatment with UCF-101. We then examined the cleavage of caspase-3. The cleavage of caspase-3 was increased in the penumbra after I/R but was blocked after an administration of UCF-101 (Figure 5B). Thus, these data strongly suggest that UCF-101 inhibits Omi activity to decrease the processing of Hax-1, leading to an inhibition of caspase-3 activation in MCAO animals. Finally, we examined whether inhibition of Omi had an effect on neuronal survival. Using TTC staining, we evaluated the infarct volume of MCAO mice treated with vehicle or UCF-101 24 h after I/R. In comparison to the sham group, MCAO mice with vehicle treatment presented an average infarct volume of approximately 27% (27.22%±0.83%); however, the average infarct volume in MCAO mice treated with UCF-101 decreased to approximately 17% (17.31%±1.44%) (Figure 5C), suggesting a protective effect of UCF-101.


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)

Protective effects in MCAO mice by UCF-101. (A) Immunoblot analyses were performed to show Hax-1 protein levels in the penumbra after I/R. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to I/R. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. Values are the mean±SEM from three independent experiments. bP<0.05, one-way ANOVA. (B) Immunoblot analyses were performed to show the level of cleaved caspase-3 in the penumbra after I/R. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to I/R. (C) The infarct volume of MCAO/R mice with or without UCF-101 treatment. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to MCAO/R. Twenty-four hours after MCAO/R the mice were euthanized and the mouse slices were stained with TTC. The bottom panel shows the infarct volumes. bP<0.05, one-way ANOVA.
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fig5: Protective effects in MCAO mice by UCF-101. (A) Immunoblot analyses were performed to show Hax-1 protein levels in the penumbra after I/R. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to I/R. The bottom panel shows the band intensity of Hax-1 relative to that of GAPDH. Values are the mean±SEM from three independent experiments. bP<0.05, one-way ANOVA. (B) Immunoblot analyses were performed to show the level of cleaved caspase-3 in the penumbra after I/R. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to I/R. (C) The infarct volume of MCAO/R mice with or without UCF-101 treatment. ICR mice (n=3) were given an intraperitoneal injection of vehicle or UCF-101 (7.15 mg/kg). Two hours after injection the mice were subjected to MCAO/R. Twenty-four hours after MCAO/R the mice were euthanized and the mouse slices were stained with TTC. The bottom panel shows the infarct volumes. bP<0.05, one-way ANOVA.
Mentions: We have shown that the anti-apoptotic effects on OGD/R cells were caused by an inhibition of Omi protease activity or overexpression of Hax-1 (Figure 3 and 4). Next, we questioned whether inhibition of Omi could protect neurons against apoptosis and neurons in MCAO mice to survive. To answer it, we examined Hax-1 expression in the penumbra after I/R. As shown in Figure 5A, Hax-1 levels in the penumbra were dramatically decreased after I/R, but were significantly restored by treatment with UCF-101. We then examined the cleavage of caspase-3. The cleavage of caspase-3 was increased in the penumbra after I/R but was blocked after an administration of UCF-101 (Figure 5B). Thus, these data strongly suggest that UCF-101 inhibits Omi activity to decrease the processing of Hax-1, leading to an inhibition of caspase-3 activation in MCAO animals. Finally, we examined whether inhibition of Omi had an effect on neuronal survival. Using TTC staining, we evaluated the infarct volume of MCAO mice treated with vehicle or UCF-101 24 h after I/R. In comparison to the sham group, MCAO mice with vehicle treatment presented an average infarct volume of approximately 27% (27.22%±0.83%); however, the average infarct volume in MCAO mice treated with UCF-101 decreased to approximately 17% (17.31%±1.44%) (Figure 5C), suggesting a protective effect of UCF-101.

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