Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity

Abstract

Neuronal nitric oxide synthase (nNOS) neurons kill adjacent neurons through the action of NMDA-glutamate receptor activation, although they remain relatively resistant to the toxic effects of NMDA and NO. The molecular basis of the resistance of nNOS neurons to toxic insults is unknown. To begin to understand the molecular mechanisms of the resistance of nNOS neurons, we developed a pheochromacytoma-derived cell line (PC12) that is resistant to the toxic effects of NO. We found through serial analysis of gene expression (SAGE) that manganese superoxide dismutase (MnSOD) is enriched in the NO-resistant PC12 cell-derived line (PC12-R). Antisense MnSOD renders PC12-R cells sensitive to NO toxicity and increases the sensitivity to NO in the parental, NO-sensitive PC12 line (PC12-S). Adenoviral transfer of MnSOD protects PC12-S cells against NO toxicity. We extended these studies to cortical cultures and showed that MnSOD is enriched in nNOS neurons and that antisense MnSOD renders nNOS neurons susceptible to NMDA neurotoxicity, although it has little effect on the overall susceptibility of cortical neurons to NMDA toxicity. Overexpression of MnSOD provides dramatic protection against NMDA and NO toxicity in cortical cultures, but not against kainate or AMPA neurotoxicity. Furthermore, nNOS neurons from MnSOD -/- mice are markedly sensitive to NMDA toxicity. Adenoviral transfer of MnSOD to MnSOD-/- cultures restores resistance of nNOS neurons to NMDA toxicity. Thus, MnSOD is a major protective protein that appears to be essential for the resistance of nNOS neurons in cortical cultures to NMDA mediated neurotoxicity.

Fig. 1.

Characterization of PC12-R cells: resistance to NO toxicity and increased expression of MnSOD. A, Susceptibility of PC12-S and PC12-R cells to the NO donors sodium nitroprusside (SNP), diethylenetriamine nitric oxide adduct (DETA/NO), and 3-morpholino-sydnonimine hydrochloride (SIN-1). Cells were exposed to the NO donors for 5 min, and cytotoxicity was assayed 24 hr later by trypan blue exclusion. The wild-type PC12 line (PC12-S) is remarkably sensitive to the toxic effects of NO. An NO-resistant PC12 cell line (PC12-R) was generated by treating parental PC12-S cells with 100 μm SNP for 5 min, allowing the surviving cells to grow to confluence followed by successive retreatments with incremental doses of SNP until a cell line (PC12-R) was generated that was resistant to 1 mm SNP. B, Predominant differentially expressed SAGE tag in PC12-R compared with PC12-S cell populations corresponds to MnSOD. This specific tag sequence, its abundance in each tag library analyzed, and its location in the MnSOD cDNA sequence is indicated. C, Western blot and catalytic activity analyses of MnSOD in PC12-S and PC12-R cells indicate that MnSOD levels are increased in PC12-R when compared with PC12-S. For Western blot analysis, 10 μg total protein was loaded in each lane and electrophoresed under denaturing conditions on a 12% polyacrylamide gel. MnSOD was detected as an apparent 23 kDa protein band with a rabbit polyclonal antibody raised against MnSOD (Oberley et al., 1990). MnSOD activity was measured using the nitroblue tetrazolium method ofOberley and Spitz (1985) as later modified. Data represents the mean ± SEM of two to three independent experiments. Western blots are representative of two to three independent experiments.

Fig. 2.

Antisense oligonucleotide knockdown of MnSOD in PC12 cells. A, Dose–response of MnSOD protein and activity levels to antisense oligonucleotide to MnSOD. PC12-S and PC12-R cells were exposed to increasing concentrations of antisense oligonucleotides (AS) for 24 hr. After 24 hr in the presence of AS, cells were harvested for Western blot and activity analyses. For Western blot analysis, 10 μg of total protein was loaded in each lane. Antibodies against MnSOD detected an apparent 23 kDa protein band, and antibodies against CuZnSOD detected a 16 kDa protein band. B, MnSOD protein and activity levels in PC12-R cells over 24 hr in the presence of AS oligonucleotide to MnSOD. PC12-R cells were exposed to 10 μm AS oligonucleotide and harvested at the time points indicated after addition of the oligonucleotide. For Western blot analysis, 5 μg of total protein was loaded in each lane. C, D, Downregulation of MnSOD protein and activity levels in (C) PC12-S and (D) PC12-R cells is specific to AS oligonucleotide treatment. Cells were exposed for 24 hr to either no oligonucleotide (−), antisense oligonucleotide to MnSOD (AS), sense oligonucleotide (S), or random oligonucleotide (R); 5 μmeach oligonucleotide was used to treat PC12-S cells, and 10 μm each oligonucleotide was used for PC12-R. Ten micrograms of total protein were loaded in each lane for Western blot analysis. Western blots are representative of two to three independent experiments. Data represents the mean ± SEM of two to three independent experiments.

Fig. 3.

Antisense MnSOD increases susceptibility to NO toxicity in PC12 cells. A, Effect of oligonucleotides to MnSOD on PC12-S sensitivity to SNP. Antisense, sense, or random oligonucleotide (5 μm each) were added to PC12-S. After 24 hr, cells were treated with 10, 100, and 500 μm SNP for 5 min, and the medium replaced with fresh oligonucleotide. Twenty-four hours later, toxicity was assessed by trypan blue exclusion. B, Effect of oligonucleotides to MnSOD on PC12-R susceptibility to SNP. Experiments were performed as described for PC12-S, with the exception that 10 μm each oligonucleotide was used and the highest SNP concentration tested was 1 mm. C, Effect of oligonucleotides to MnSOD on PC12-S sensitivity to 10 μm DETA/NO. The experiment was performed as in A, with the exception that only one dose (10 μm) of DETA/NO was tested. D, Effect of oligonucleotides to MnSOD on PC12-R susceptibility to 1 mm DETA/NO. The experiment was performed as inB, with the exception that only one dose (1 mm) of DETA/NO was tested. Data represents the mean ± SEM of two to three independent experiments. *p < 0.001.

Fig. 4.

Effect of MnSOD knockdown on PC12 sensitivy to superoxide generators. A, Effect of oligonucleotides to MnSOD on PC12-S sensitivity to paraquat. Antisense, sense, or random oligonucleotides (5 μm each) were added to PC12-S. After 24 hr, cells were treated with 10, 100, and 500 μmparaquat for 5 min, and the medium was replaced with fresh oligonucleotide. Twenty-four hours later, toxicity was assessed by trypan blue exclusion. B, Effect of oligonucleotides to MnSOD on PC12-R susceptibility to paraquat. Experiments were performed as described for PC12-S, with the exception that 10 μmeach oligonucleotide was used, and the highest paraquat concentration tested was 1 mm. C, Effect of oligonucleotides to MnSOD on PC12-S sensitivity to menadione. Antisense, sense, or random oligonucleotides (5 μm each) were added to PC12-S. After 24 hr, cells were treated with 10, 100, and 500 μm and 1 mm menadione for 5 min, and the medium replaced with fresh oligonucleotide. Twenty-four hours later, toxicity was assessed by trypan blue exclusion. D, Effect of oligonucleotides to MnSOD on PC12-R susceptibility to menadione. Experiments were performed as described for PC12-S, with the exception that 10 μm each oligonucleotide was used. Data represents the mean ± SEM of two to three independent experiments. *p < 0.001.

Fig. 5.

Overexpression of MnSOD confers resistance to NO toxicity in PC12 cells. A, Western blot and activity analyses demonstrate overexpression of MnSOD after infection of PC12-S and PC12-R cells with an adenovirus-derived vector containing the MnSOD gene. Cells were exposed to 10⁸ pfu/ml of either an adenoviral vector containing the MnSOD gene (Ad.MnSOD) or a control adenoviral vector containing the β-galactosidase gene (Ad.βGal). Cells were harvested 48 hr after exposure to adenovirus for Western blot and activity analyses. Total protein (10 μg) was loaded in each lane for Western blot analysis. Also at this time point, control cells infected with Ad.βGal were assayed for X-Gal staining to determine infection efficiency. The estimated infection efficiency was 90–100% with minimal cell loss. B, Effect of adenoviral-mediated overexpression of MnSOD on PC12-S susceptibility to SNP. Cells were exposed to 10⁸ pfu/ml of either Ad.MnSOD or Ad.βGal, and 24 hr later they were treated with 1 mm SNP for 5 min. Toxicity was assayed 24 hr after treatment by trypan blue exclusion. C, Effect of adenoviral-mediated overexpression of MnSOD on PC12-S susceptibility to paraquat. Cells were exposed to 10⁸ pfu/ml of either Ad.MnSOD or Ad.βGal and 24 hr later were treated with 1 mm paraquat for 5 min. Toxicity was assayed 24 hr after treatment by trypan blue exclusion. Data represents the mean ± SEM of two to three independent experiments. Western blots are representative of two to three independent experiments. *p < 0.001.

Fig. 6.

MnSOD is selectively enriched in nNOS neurons. Immunofluorescence staining of 14 d cultured rat cortical neurons indicates that nNOS neurons (A, D, G) are enriched in MnSOD (B, E, H). nNOS and MnSOD are both extranuclear proteins concentrated mostly in the neuronal cell body and processes. In contrast, nNOS (J) neurons are not enriched in CuZnSOD (K), which is expressed ubiquitously in cortical neurons. Hoffman modulation images of cells are depicted to the right of the corresponding immunofluorescent images (C, F, I, L), andarrows indicate nNOS neurons. M, MnSOD levels parallel nNOS levels after NMDA or quisqualate treatment. Western blot analysis of nNOS, MnSOD, CuZnSOD, and β-tubulin levels in primary cortical cultures after treatment with control salt solution (C), NMDA (N), or quisqualate (Q). Rat neuronal cultures were exposed for 5 min to either 500 μm NMDA or 20 μm quisqualate. Twenty-four hours later, cells were harvested for Western blot analysis. Total protein (50 μg) was loaded in each lane and electrophoresed in a denaturing 10% polyacrylamide gel. Immunofluorescent images and Western blots are representative of two to three independent experiments.

Fig. 7.

Antisense MnSOD renders nNOS neurons susceptible to NMDA toxicity. A, Western blot and activity analyses of MnSOD in primary cortical neurons after exposure to oligonucleotides. Cultures were exposed for 24 hr to either no oligonucleotide (−), antisense oligonucleotide to MnSOD (AS), sense oligonucleotide (S), or random oligonucleotide (R). All oligonucleotides were used at 10 μm concentration. Thirty micrograms of total protein were loaded in each lane. Catalytic activity data from three independent experiments were analyzed with the Student’s t test for independent means. Statistical analysis was performed by using StatView 4.0 software. MnSOD catalytic activity after antisense oligonucleotide knockdown was significantly different from MnSOD activity in untreated, sense, and random oligonucleotide-treated cells (p < 0.001).B, Effect of antisense knockdown of MnSOD on susceptibility of nNOS neurons to NMDA toxicity. Cultures were exposed to either no oligonucleotide, 10 μm antisense oligonucleotide, or 10 μm random oligonucleotide. Twenty-four hours later, cells were treated for 5 min with 0, 300, or 500 μm NMDA, and fresh oligonucleotides were added to the medium. After 24 hr, cultures were stained for nNOS neurons by NADPH–diaphorase staining. C, Susceptibility of cultures to NMDA toxicity. Treatment was performed as inA, and total cell death was estimated by trypan blue staining 24 hr after exposure to NMDA. D, The differential resistance and susceptibility of nNOS neurons to NMDA neurotoxicity in the absence and presence of antisense oligonucleotide to MnSOD, respectively, is illustrated by plotting the ratio between the percentage of nNOS neuron survival and the percentage of total neuronal survival. Data represents the mean ± SEM of two to three independent experiments. Western blots are representative of two to three independent experiments. *p < 0.001.

Fig. 8.

Efficient gene transfer into primary neurons using an adenovirus vector. A, Neurons (90–100%) were infected with 10⁸ pfu/ml of an adenovirus containing the β-galactosidase reporter gene, as assessed by X-Gal staining 24 hr after exposure of cultures to the virus. B–F, The rat MnSOD gene was efficiently transferred (90–100% infection efficiency) into primary neurons via an adenovirus (Ad.MnSOD), as assessed by immunofluorescence staining of cultures 24 hr after exposure to 10⁸ pfu/ml of Ad.MnSOD. D, F, Hoffman modulation images corresponding to panels C and E, respectively. Images are representative of three to four independent experiments.

Fig. 9.

MnSOD overexpression confers resistance to NMDA and NO toxicity in primary cortical neurons. A, Western blot and activity analyses of MnSOD in rat cortical neurons afterin vitro exposure to either control salt solution (No Ad.), 10⁸ pfu/ml of MnSOD-containing adenovirus (Ad.MnSOD), or 10⁸ pfu/ml of β-galactosidase-containing adenovirus (Ad.βGal). Cells were harvested for Western blot and activity analyses 48 hr after exposure to the adenovirus. B, Susceptibility of cortical neurons to NMDA toxicity in cultures that were not exposed to adenovirus and in cultures infected with Ad.MnSOD or Ad.βGal. C, Susceptibility of cortical neurons to NMDA, kainate (KA), and AMPA after exposure to control salt solution (no virus), Ad.MnSOD, or Ad.βGal. Cultures were exposed to 10⁸ pfu/ml Ad.MnSOD or Ad.βGal, and 24 hr later treated with 0.5 mm NMDA, 0.1 mm kainate, or 0.1 mm AMPA. NMDA was applied for 5 min and then washed off. KA and AMPA were applied for 14 hr. Toxicity was assessed 24 hr after exposure to the toxic agent by trypan blue exclusion.D, Susceptibility of cortical neurons to NO donors after exposure to control salt solution (no virus), Ad.MnSOD, or Ad.βGal. Cultures were exposed to 10⁸ pfu/ml Ad.MnSOD, or Ad.βGal, and 24 hr later they were treated for 5 min with 1 mm SNP, 2 mm SIN-1, or 2 mmDETA/NO. Toxicity was assessed 24 hr after exposure to the toxic agent by trypan blue exclusion. Data represents the mean ± SEM of two to three independent experiments. Western blots are representative of two to three independent experiments. *p < 0.001.

Fig. 10.

MnSOD is required for nNOS neuron survival.A, Effect of targeted disruption of MnSOD on susceptibility of cortical neurons to NMDA toxicity. Wild-type (+/+), MnSOD^+/−, and MnSOD^−/−cultures were treated for 5 min with 100 μm NMDA, and total cell death was estimated by trypan blue staining or computer-assisted cell counting 24 hr after exposure to NMDA.B, Effect of targeted disruption of MnSOD on susceptibility of nNOS neurons to NMDA toxicity. Cultures were treated for 5 min with 100 μm NMDA, and after 24 hr cells were stained for nNOS neurons by NADPH–diaphorase staining.C, The differential resistance and susceptibility of nNOS neurons to NMDA neurotoxicity in wildtype (+/+), MnSOD^+/−, and MnSOD^−/−cultures is illustrated by plotting the ratio between the percentage of nNOS neuronal survival and the percentage of total neuronal survival.D, Susceptibility of cortical neurons to 100 μm NMDA toxicity in wild-type (+/+), MnSOD^+/−, and MnSOD^−/−cultures that were not exposed to adenovirus and in cultures infected with Ad.MnSOD or Ad.βGal. E, Susceptibility of nNOS neurons to 100 μm NMDA toxicity in wild-type (+/+), MnSOD^+/−, and MnSOD^−/−cultures that were not exposed to adenovirus and in cultures infected with Ad.MnSOD or Ad.βGal. F, The differential resistance and susceptibility of nNOS neurons to NMDA neurotoxicity in wild-type (+/+), MnSOD^+/−, and MnSOD^−/− cultures that were not exposed to adenovirus and in cultures infected with Ad.MnSOD or Ad.βGal is illustrated by plotting the ratio between the percentage of nNOS neuronal survival and the percentage of total neuronal survival. Data represent the mean ± SEM of five independent experiments. *p < 0.001.