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. 1998 Jan 15;18(2):687-97.
doi: 10.1523/JNEUROSCI.18-02-00687.1998.

Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction

J N Keller  1 , M S Kindy, F W Holtsberg, D K St Clair, H C Yen, A Germeyer, S M Steiner, A J Bruce-Keller, J B Hutchins, M P Mattson
Affiliations

Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction

J N Keller et al. J Neurosci. .

Abstract

Oxidative stress is implicated in neuronal apoptosis that occurs in physiological settings and in neurodegenerative disorders. Superoxide anion radical, produced during mitochondrial respiration, is involved in the generation of several potentially damaging reactive oxygen species including peroxynitrite. To examine directly the role of superoxide and peroxynitrite in neuronal apoptosis, we generated neural cell lines and transgenic mice that overexpress human mitochondrial manganese superoxide dismutase (MnSOD). In cultured pheochromocytoma PC6 cells, overexpression of mitochondria-localized MnSOD prevented apoptosis induced by Fe2+, amyloid beta-peptide (Abeta), and nitric oxide-generating agents. Accumulations of peroxynitrite, nitrated proteins, and the membrane lipid peroxidation product 4-hydroxynonenal (HNE) after exposure to the apoptotic insults were markedly attenuated in cells expressing MnSOD. Glutathione peroxidase activity levels were increased in cells overexpressing MnSOD, suggesting a compensatory response to increased H2O2 levels. The peroxynitrite scavenger uric acid and the antioxidants propyl gallate and glutathione prevented apoptosis induced by each apoptotic insult, suggesting central roles for peroxynitrite and membrane lipid peroxidation in oxidative stress-induced apoptosis. Apoptotic insults decreased mitochondrial transmembrane potential and energy charge in control cells but not in cells overexpressing MnSOD, and cyclosporin A and caspase inhibitors protected cells against apoptosis, demonstrating roles for mitochondrial alterations and caspase activation in the apoptotic process. Membrane lipid peroxidation, protein nitration, and neuronal death after focal cerebral ischemia were significantly reduced in transgenic mice overexpressing human MnSOD. The data suggest that mitochondrial superoxide accumulation and consequent peroxynitrite production and mitochondrial dysfunction play pivotal roles in neuronal apoptosis induced by diverse insults in cell culture and in vivo.

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Figures

Fig. 1.
Fig. 1.
Expression of human MnSOD in PC6 cells: localization to the mitochondria and protection against oxidative stress-induced apoptosis. A, Western blot analysis and subcellular fractionation of MnSOD and F1/Fo-ATPase levels in PC6-V and PC6-MnSOD cells are shown. W, Whole cells;Mi, mitochondrial fraction; N, nuclear fraction; Me, membrane fraction; S, soluble fraction (cytosol). Fifty nanograms of protein/lane (for both PC6-V and PC6-MnSOD cells) were separated by SDS-PAGE, transferred to a nitrocellulose sheet, and immunoreacted with antibodies to either MnSOD (upper) or the mitochondrial enzyme F1/Fo-ATPase (lower). The upper and lower blots are two separate blots using the same sample preparations; the upper blot was reacted with the MnSOD antibody, and the lower blot was reacted with the F1/Fo-ATPase antibody. Note that MnSOD is localized almost exclusively in the mitochondrial fraction. B, Cultures of PC6-V and two different lines of PC6-MnSOD cells were exposed for 24 hr to saline (vehicle), 100 μm FeSO4, 50 μm Aβ, or 100 μm SNP, and the percentages of cells exhibiting apoptotic nuclei were determined. Values are the mean ± SEM of determinations made in eight cultures; *p < 0.01 compared with the value for vehicle-treated cultures and with each value in PC6-MnSOD cells (ANOVA with Scheffe’s post hoc tests).
Fig. 2.
Fig. 2.
Mitochondrial MnSOD suppresses peroxynitrite accumulation and protein tyrosine nitration. A, Levels of DHR fluorescence were quantified 18 hr after exposure of PC6-V and PC6-MnSOD cells to vehicle, 100 μmFeSO4, 50 μm Aβ25–35, or 100 μm SNP. Values are the mean ± SEM of determinations made in six cultures; *p < 0.01 compared with the value for vehicle-treated control cultures; **p < 0.01 compared with the corresponding value in PC6-V cells.B, Levels of nitrotyrosine immunoreactivity were quantified 6 and 12 hr after exposure to vehicle, 100 μmFeSO4, 50 μm Aβ25–35, or 100 μm SNP. Values are the mean ± SEM of determinations made in six cultures. At both the 6 and 12 hr time points in PC6-V cells, the values for cultures exposed to FeSO4, Aβ25–35, and SNP were significantly greater than were the values for vehicle-treated cultures (p < 0.01). At both the 6 and 12 hr time points, the value for PC6-MnSOD cells exposed to SNP was significantly less than was the value for PC6-V cells exposed to SNP (p < 0.01). At the 12 hr time point, the values for PC6-MnSOD cells exposed to FeSO4(p < 0.05) or Aβ25–35 (p < 0.01) were significantly less than were the corresponding values in PC6-V cells (ANOVA with Scheffe’spost hoc tests). C, Confocal laser scanning microscope images of nitrotyrosine immunoreactivity in cultured PC6-V and PC6-MnSOD cells exposed to vehicle (Control), 50 μm Aβ25–35, or 100 μm SNP are shown. Aβ and SNP induced large increases in nitrotyrosine immunoreactivity in PC6-V cells (e.g., arrowheads) but not in PC6-MnSOD cells. Scale bar, 5 μm.
Fig. 3.
Fig. 3.
Mitochondrial MnSOD suppresses lipid peroxidation induced by apoptotic insults. A, Levels of TBARS fluorescence were quantified in PC6-V and PC6-MnSOD cells at the indicated time points after exposure to vehicle (Cont), 100 μm FeSO4, or 50 μmAβ. Values are the mean ± SEM of determinations made in eight cultures. At each time point, the value for PC6-MnSOD cells exposed to FeSO4 or Aβ was significantly less than was the corresponding value for PC6-V cells exposed to FeSO4 or Aβ (p < 0.01; ANOVA with Scheffe’spost hoc tests). B, Cultures of PC6-V cells (control and uric acid; 200 μm uric acid) and PC6-MnSOD cells (MnSOD) were exposed for 6 hr to either vehicle or 100 μm SNP. Levels of TBARS fluorescence were quantified, and the values represent the mean ± SEM of determinations made in eight separate cultures; *p < 0.01 compared with the value for vehicle-treated cultures. C, Cells were exposed for 12 hr to vehicle (Cont), 100 μm FeSO4, or 50 μmAβ25–35. The cells were then immunostained with HNE antibody, and confocal laser scanning microscope images of cellular immunofluorescence were acquired. Note that both FeSO4 and Aβ induced a large increase in HNE immunoreactivity in PC6-V cells but not in PC6-MnSOD cells.
Fig. 4.
Fig. 4.
A caspase inhibitor and antioxidants prevent apoptosis induced by FeSO4, Aβ25–35, and SNP. Cultures of PC6-V cells were pretreated for 2 hr with 50 μm zVAD-fmk (ZVAD), 2 mmglutathione ethyl ester (GSH), 50 μm propyl gallate (PG), or 200 μm uric acid (UA). Cultures were then exposed for 24 hr to 100 μm FeSO4, 50 μm Aβ25–35, or 100 μm SNP, and percentages of cells with apoptotic nuclei were quantified. Values are the mean ± SEM of determinations made in six cultures; *p < 0.001 compared with the value for vehicle-treated (Cont) cultures and with the values for cultures pretreated with ZVAD, GSH,PG, and UA (ANOVA with Scheffe’spost hoc tests).
Fig. 5.
Fig. 5.
Evidence that peroxynitrite-induced mitochondrial dysfunction mediates oxidative stress-induced apoptosis.A, PC6-V and PC12-MnSOD cells were exposed to vehicle, 100 μm FeSO4, 50 μmAβ, or 100 μm SNP for 12 hr, and levels of JC-1 fluorescence were quantified. Values are the mean ± SEM of determinations made in six separate cultures; *p < 0.01 compared with the value for vehicle-treated PC6-V cells and with each value in PC6-MnSOD cells. B, Cultures were pretreated for 2 hr with vehicle or cyclosporin A and were then exposed for 20 hr to 100 μm FeSO4, 50 μm Aβ25–35, or 100 μm SNP. Values are the mean ± SEM of determinations made in six cultures; *p < 0.01 compared with the control value; **p < 0.01 compared with the corresponding vehicle value.
Fig. 6.
Fig. 6.
Characterization of MnSOD expression in brain tissue of MnSOD transgenic mice. A, Northern blot analysis of MnSOD mRNA in brain tissue from Tg and wild-type (NTg) mice is shown. Poly(A+)RNA from the brains of Tg and NTg mice was separated on a 1.1% formaldehyde–agarose gel and, after transfer to nylon membrane, probed with a 32P-labeled human MnSOD cDNA.B, Relative levels of MnSOD immunoreactivity in cerebral cortex were quantified in brain tissue sections (see Materials and Methods). Values are the mean ± SEM (n = 4 mice/group); *p < 0.01 compared with the corresponding WT value.
Fig. 7.
Fig. 7.
Levels of cellular injury and lipid peroxidation are reduced in MnSOD Tg mice after cerebral ischemia. A, Cortical infarct volumes were quantified 24 hr after MCA occlusion in WT and MnSOD Tg mice. Values are the mean ± SEM (n = 6 mice in each group); *p< 0.05 compared with the WT value. B, TBARS levels were quantified in infarcted cortical tissue 24 hr after MCA occlusion in WT and MnSOD Tg mice. Values are the mean ± SEM (n = 4 mice in each group); *p< 0.01 compared with the corresponding WT value; **p < 0.01 compared with the WT control value.
Fig. 8.
Fig. 8.
Ischemia-induced protein tyrosine nitration is reduced in MnSOD Tg mice. Bright-field micrographs of nitrotyrosine immunoreactivity in coronal brain sections from mice killed 24 hr after sham surgery (A, wild-type; C, MnSOD Tg) or after MCA occlusion ischemia (B, wild-type;D, MnSOD Tg). Note the great increase in nitrotyrosine immunoreactivity in the injured cortex of the wild-type mouse (B, arrow) relative to the MnSOD Tg mouse that showed less of an increase in nitrotyrosine immunoreactivity (D, arrow). Similar differences were observed in each of four wild-type and four MnSOD Tg mice examined.
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