Preserving mitochondrial function by inhibiting GRP75 ameliorates neuron injury under ischemic stroke

Abstract

Ischemic stroke is a life‑threatening disease, which is closely related to neuron damage during ischemia. Mitochondrial dysfunction is essentially involved in the pathophysiological process of ischemic stroke. Mitochondrial calcium overload contributes to the development of mitochondrial dysfunction. However, the underlying mechanisms of mitochondrial calcium overload are far from being fully revealed. In the present study, middle cerebral artery obstruction (MCAO) was performed in vivo and oxygen and glucose deprivation (OGD) in vitro. The results indicated that both MCAO and OGD induced significant mitochondrial dysfunction in vivo and in vitro. The mitochondria became fragmented under hypoxia conditions, accompanied with upregulation of the heat shock protein 75 kDa glucose‑regulated protein (GRP75). Inhibition of GRP75 was able to effectively ameliorate mitochondrial calcium overload and preserve mitochondrial function, which may provide evidence for further translational studies of ischemic diseases.

Keywords: GRP75; calcium overload; mitochondria; mitochondria‑associated endoplasmic reticulum membrane.

Figure 1.

Mitochondria fragmentation and dysfunction induced by ischemia in vivo and in vitro. (A) Infarct volumes of mouse brains from different groups of mice. (B) Electron microscopy analysis of damaged mitochondrial (arrows) in mouse brains. The number of long mitochondria per cell was determined (scale bars, 2 µm). (C) Primary neurons were cultured under different conditions in vitro and the relative ATP content (left panel), mitochondrial membrane potential (relative to red/green fluorescence, middle panel) and relative content of ROS (right panel) were determined. Representative images are provided and quantitative data are expressed as the mean ± standard error of the mean of each group from three separate experiments. **P<0.01, ****P<0.0001 vs. Ctrl. Ctrl, control; MCAO, middle cerebral artery occlusion; OGD, oxygen-glucose deprivation; long-mito, long mitochondria with a length/diameter ratio of >2; ROS, reactive oxygen species.

Figure 2.

Mitochondrial fragmentation and dysfunction induced by OGD in vitro. (A) Fluorescence imaging analysis of mitochondrial morphology of HT22 and N2a cells (scale bars, 5 µm). (B) Western blot analysis revealed mitochondrial dynamic protein expression in the N2a cells. (C) OGD impaired mitochondrial function in HT22 and N2a cells. Representative images are provided and quantitative results are expressed as the mean ± standard error of the mean of each group from three separate experiments. **P<0.01, ***P<0.001, ****P<0.0001 vs. Ctrl. Ctrl, control; OGD, oxygen-glucose deprivation; MFN, mitofision protein; MFF, mitochondria fission factor; DRP1, dynamin-1 protein; p, phosphorylated.

Figure 3.

Mitochondrial calcium overload induced by OGD. (A) OGD-stimulated calcium overload in the mitochondria of N2a cells (scale bars, 5 µm). (B and C) Western blot analysis revealed that OGD upregulated GRP75 expression in (B) HT22 and (C) N2a cells. (D) Western blot analysis revealed that OGD upregulated GRP75 expression in the MCAO tissue. (E) Western blot analysis was used to determine HIF1α expression in N2a cells. Representative images are provided and quantitative results are expressed as the mean ± standard error of the mean of each group from three separate experiments. **P<0.01, ***P<0.001, ****P<0.0001 vs. Ctrl. GRP75, 75 kDa glucose-regulated protein; Ctrl, control; OGD, oxygen-glucose deprivation; MCAO, middle cerebral artery occlusion; HIF, hypoxia-inducible factor.

Figure 4.

Inhibition of GRP75 by pharmacological agent preserves mitochondria morphology and function under OGD. (A) Western blot analysis revealed that MKT077 regulated GRP75 expression in HT22 cells. (B) Treatment with MKT077 to inhibit GRP75 mitigated the OGD-stimulated calcium overload in the mitochondria of Na2 cells. (C) Fluorescence imaging analysis of mitochondrial morphology of HT22 cells (scale bars, 5 µm). (D) Mitochondrial function in N2a cells from the different groups. Representative images are provided and quantitative data are expressed as the mean ± standard error of the mean of each group from three separate experiments. **P<0.01, ****P<0.0001 vs. Ctrl; ^#P<0.05, ^##P<0.01, ^####P<0.0001 vs. OGD group. n.s., no significance; GRP75, 75 kDa glucose-regulated protein; Ctrl, control; OGD, oxygen-glucose deprivation.

Figure 5.

Knockdown of GRP75 preserves mitochondrial morphology and function under OGD. (A) Western blot analysis confirmed siRNA-mediated knockdown of GRP75 expression in N2a cells. (B) Treatment with siRNA targeting GRP75 to inhibit GRP75 mitigated the OGD-stimulated calcium overload in the mitochondria of N2a cells. (C) Fluorescent imaging analysis of mitochondrial morphology of N2a cells (scale bars, 5 µm). The average length of mitochondria, number of long mitochondria per cell and average number of mitochondria per cell were determined. (D) Mitochondrial function in N2a cells from the different groups; the relative ATP content (left panel), mitochondrial membrane potential (relative to red/green fluorescence, middle panel) and relative content of ROS (right panel) were determined. Representative images are provided and quantitative data are expressed as the mean ± standard error of the mean of each group from three separate experiments. **P<0.01, ***P<0.001, ****P<0.0001 vs. Ctrl; ^#P<0.05, ^##P<0.01, ^####P<0.0001 vs. OGD group. Ctrl, control; OGD, oxygen-glucose deprivation; GRP75, 75 kDa glucose-regulated protein; siRNA, small interfering RNA; long-mito, long mitochondria with a length/diameter ratio of >2; ROS, reactive oxygen species.

Figure 6.

Schematic illustrating that preserving mitochondrial function by inhibiting GRP75 ameliorates neuron injury under ischemic stroke. Ischemia or OGD may also upregulate GRP75 expression to promote mitochondrial calcium overload, thus causing mitochondrial dysfunction and neuron death. Treatment with MKT077 or siRNA may inhibit GRP75 activation or expression and attenuate the ischemia- or OGD-induced mitochondrial calcium overload in neurons. GRP75, 75 kDa glucose-regulated protein; siRNA, small interfering RNA; OGD, oxygen-glucose deprivation; MCAO, middle cerebral artery occlusion.