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The PPAR-γ agonist pioglitazone protects cortical neurons from inflammatory mediators via improvement in peroxisomal function.

Gray E, Ginty M, Kemp K, Scolding N, Wilkins A - J Neuroinflammation (2012)

Bottom Line: To assess the influence of peroxisomal activation on nitric oxide mediated neurotoxicity, we investigated the effects of the peroxisomal proliferator activated receptor (PPAR) gamma agonist, pioglitazone in primary cortical neurons that were either exposed to a nitric oxide donor or co-cultured with activated microglia.Moreover, cortical neurons treated with this compound showed a significant increase in the protein and gene expression of PPAR-gamma, which was associated with a concomitant increase in the enzymatic activity of catalase.In addition, the protection of neurons and axons against hydrogen peroxide-induced toxicity afforded by pioglitazone appeared to be dependent on catalase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Multiple Sclerosis and Stem Cell Group, Burden Centre, Institute of Clinical Neurosciences, Frenchay Hospital, University of Bristol, Bristol BS16 1JB, UK. elizabeth.gray@bristol.ac.uk

ABSTRACT

Background: Inflammation is known to play a pivotal role in mediating neuronal damage and axonal injury in a variety of neurodegenerative disorders. Among the range of inflammatory mediators, nitric oxide and hydrogen peroxide are potent neurotoxic agents. Recent evidence has suggested that oligodendrocyte peroxisomes may play an important role in protecting neurons from inflammatory damage.

Methods: To assess the influence of peroxisomal activation on nitric oxide mediated neurotoxicity, we investigated the effects of the peroxisomal proliferator activated receptor (PPAR) gamma agonist, pioglitazone in primary cortical neurons that were either exposed to a nitric oxide donor or co-cultured with activated microglia.

Results: Pioglitazone protected neurons and axons against both nitric-oxide donor-induced and microglia-derived nitric oxide-induced toxicity. Moreover, cortical neurons treated with this compound showed a significant increase in the protein and gene expression of PPAR-gamma, which was associated with a concomitant increase in the enzymatic activity of catalase. In addition, the protection of neurons and axons against hydrogen peroxide-induced toxicity afforded by pioglitazone appeared to be dependent on catalase.

Conclusions: Collectively, these observations provide evidence that modulation of PPAR-gamma activity and peroxisomal function by pioglitazone attenuates both NO and hydrogen peroxide-mediated neuronal and axonal damage suggesting a new therapeutic approach to protect against neurodegenerative changes associated with neuroinflammation.

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Effect of pioglitazone on hydrogen peroxide-mediated reductions in neuronal survival in cortical neuronal culture. (a) The effect of hydrogen peroxide exposure (MIN H2O2) (250 μM) on cortical neuronal viability in vitro compared to serum free minimal media (MIN) (**P < 0.01 compared with MIN); the effect of pioglitazone (0.1 μM to 10 μM; MIN H2O2 PIO) on cortical neuronal viability exposed to H2O2 (*P < 0.05 and **P < 0.01 as compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H2O2 (**P < 0.01 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). Cultures were treated with GW9662 for 1 hour prior to incubation with pioglitazone for 1 hour followed by exposure to hydrogen peroxide. Cell viability was assessed by MTT assay. Data are expressed as percentage of cells grown in MIN medium. Statistical significance was obtained by one-way ANOVA followed by Bonferroni post-hoc test. (b) Effect of hydrogen peroxide exposure (250 μM; MIN H2O2) on cortical neuronal cell survival (number of βIII-tubulin cells per field; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on cortical neuronal survival exposed to H2O2 (***P < 0.001 compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H2O2 (**P < 0.01 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). (c) Effect of hydrogen peroxide exposure (250 μM H2O2) on axon length within neuronal cultures (determined by SMI312 staining; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on axon length in neurons exposed to H2O2 (**P < 0.01 compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced axon protection from H2O2 (***P < 0.001 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). Values represent the mean ± SEM from at least three separate experiments. ANOVA, analysis of variance; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; PPAR-γ, peroxisomal proliferator activated receptor γ; SEM, standard error of the mean. To further examine the neuroprotective properties of pioglitazone, neurons were exposed to identical test conditions and cultures were then fixed and stained for βIII tubulin, SMI312 and the nuclear marker DAPI. The numbers of viable neurons (determined by nuclear appearance) expressing βIII tubulin were counted for each condition. As expected, neuronal survival was significantly increased following exposure to pioglitazone (1 μM) (Figure 4b). In addition, we examined the influence of pioglitazone on axonal morphology in these cultures. Neurofilament phosphorylation, as identified by the antibody SMI312, was examined as a marker of axonal integrity and length. Pre-treatment with pioglitazone (1 μM) caused a significant increase in total axon survival (Figure 4c). Pre-treatment with GW9662 led to a significant reduction in the neuroprotective (Figure 4b) and axonoprotective (Figure 4c) effects of pioglitazone. To determine further the role of catalase in pioglitazone-induced neuroprotection, we pre-incubated the cultures with the catalase inhibitor 3-amino triazole (10 mM) (3-AT) for 1 hour, prior to incubation with H2O2 (250 μM). The neuroprotective and axonoprotective effects of pioglitazone were attenuated by the addition of 3-AT (10 mM) (Figure 5a-5c). Furthermore, addition of exogenous catalase (100 U/ml) to cultures exposed to H2O2 improved neuronal survival (measured by MTT assay (Figure 5a) and neuronal morphology (Figure 5b) and axonal length within cultures (Figure 5c).
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Figure 4: Effect of pioglitazone on hydrogen peroxide-mediated reductions in neuronal survival in cortical neuronal culture. (a) The effect of hydrogen peroxide exposure (MIN H2O2) (250 μM) on cortical neuronal viability in vitro compared to serum free minimal media (MIN) (**P < 0.01 compared with MIN); the effect of pioglitazone (0.1 μM to 10 μM; MIN H2O2 PIO) on cortical neuronal viability exposed to H2O2 (*P < 0.05 and **P < 0.01 as compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H2O2 (**P < 0.01 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). Cultures were treated with GW9662 for 1 hour prior to incubation with pioglitazone for 1 hour followed by exposure to hydrogen peroxide. Cell viability was assessed by MTT assay. Data are expressed as percentage of cells grown in MIN medium. Statistical significance was obtained by one-way ANOVA followed by Bonferroni post-hoc test. (b) Effect of hydrogen peroxide exposure (250 μM; MIN H2O2) on cortical neuronal cell survival (number of βIII-tubulin cells per field; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on cortical neuronal survival exposed to H2O2 (***P < 0.001 compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H2O2 (**P < 0.01 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). (c) Effect of hydrogen peroxide exposure (250 μM H2O2) on axon length within neuronal cultures (determined by SMI312 staining; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on axon length in neurons exposed to H2O2 (**P < 0.01 compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced axon protection from H2O2 (***P < 0.001 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). Values represent the mean ± SEM from at least three separate experiments. ANOVA, analysis of variance; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; PPAR-γ, peroxisomal proliferator activated receptor γ; SEM, standard error of the mean. To further examine the neuroprotective properties of pioglitazone, neurons were exposed to identical test conditions and cultures were then fixed and stained for βIII tubulin, SMI312 and the nuclear marker DAPI. The numbers of viable neurons (determined by nuclear appearance) expressing βIII tubulin were counted for each condition. As expected, neuronal survival was significantly increased following exposure to pioglitazone (1 μM) (Figure 4b). In addition, we examined the influence of pioglitazone on axonal morphology in these cultures. Neurofilament phosphorylation, as identified by the antibody SMI312, was examined as a marker of axonal integrity and length. Pre-treatment with pioglitazone (1 μM) caused a significant increase in total axon survival (Figure 4c). Pre-treatment with GW9662 led to a significant reduction in the neuroprotective (Figure 4b) and axonoprotective (Figure 4c) effects of pioglitazone. To determine further the role of catalase in pioglitazone-induced neuroprotection, we pre-incubated the cultures with the catalase inhibitor 3-amino triazole (10 mM) (3-AT) for 1 hour, prior to incubation with H2O2 (250 μM). The neuroprotective and axonoprotective effects of pioglitazone were attenuated by the addition of 3-AT (10 mM) (Figure 5a-5c). Furthermore, addition of exogenous catalase (100 U/ml) to cultures exposed to H2O2 improved neuronal survival (measured by MTT assay (Figure 5a) and neuronal morphology (Figure 5b) and axonal length within cultures (Figure 5c).

Mentions: During inflammation, a major function of the peroxisomal enzyme catalase is to convert hydrogen peroxide (H2O2) into water. Therefore, having demonstrated induction of catalase activity by pioglitazone we wished to determine whether the pioglitazone could also protect cortical neurons against H2O2-mediated injury. Neuronal cultures were incubated for 1 hour in the presence of increasing concentrations of pioglitazone (0.1 to 10 μM) prior to exposure to hydrogen peroxide (250 μM). Neuronal viability was significantly reduced when compared to control after hydrogen peroxide exposure (250 μM for 24 hours; Figure 4a). In the presence of pioglitazone (10 μM, 1 μM and 0.1 μM), neuronal viability was significantly increased during exposure to hydrogen peroxide (Figure 4a). Furthermore, the neuroprotective effects of pioglitazone were prevented by the concomitant presence of GW9662 (1 μM) suggesting that PPAR-γ activation is necessary for the pioglitazone-induced neuroprotection of cortical neurons against the toxic effects of hydrogen peroxide.


The PPAR-γ agonist pioglitazone protects cortical neurons from inflammatory mediators via improvement in peroxisomal function.

Gray E, Ginty M, Kemp K, Scolding N, Wilkins A - J Neuroinflammation (2012)

Effect of pioglitazone on hydrogen peroxide-mediated reductions in neuronal survival in cortical neuronal culture. (a) The effect of hydrogen peroxide exposure (MIN H2O2) (250 μM) on cortical neuronal viability in vitro compared to serum free minimal media (MIN) (**P < 0.01 compared with MIN); the effect of pioglitazone (0.1 μM to 10 μM; MIN H2O2 PIO) on cortical neuronal viability exposed to H2O2 (*P < 0.05 and **P < 0.01 as compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H2O2 (**P < 0.01 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). Cultures were treated with GW9662 for 1 hour prior to incubation with pioglitazone for 1 hour followed by exposure to hydrogen peroxide. Cell viability was assessed by MTT assay. Data are expressed as percentage of cells grown in MIN medium. Statistical significance was obtained by one-way ANOVA followed by Bonferroni post-hoc test. (b) Effect of hydrogen peroxide exposure (250 μM; MIN H2O2) on cortical neuronal cell survival (number of βIII-tubulin cells per field; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on cortical neuronal survival exposed to H2O2 (***P < 0.001 compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H2O2 (**P < 0.01 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). (c) Effect of hydrogen peroxide exposure (250 μM H2O2) on axon length within neuronal cultures (determined by SMI312 staining; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on axon length in neurons exposed to H2O2 (**P < 0.01 compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced axon protection from H2O2 (***P < 0.001 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). Values represent the mean ± SEM from at least three separate experiments. ANOVA, analysis of variance; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; PPAR-γ, peroxisomal proliferator activated receptor γ; SEM, standard error of the mean. To further examine the neuroprotective properties of pioglitazone, neurons were exposed to identical test conditions and cultures were then fixed and stained for βIII tubulin, SMI312 and the nuclear marker DAPI. The numbers of viable neurons (determined by nuclear appearance) expressing βIII tubulin were counted for each condition. As expected, neuronal survival was significantly increased following exposure to pioglitazone (1 μM) (Figure 4b). In addition, we examined the influence of pioglitazone on axonal morphology in these cultures. Neurofilament phosphorylation, as identified by the antibody SMI312, was examined as a marker of axonal integrity and length. Pre-treatment with pioglitazone (1 μM) caused a significant increase in total axon survival (Figure 4c). Pre-treatment with GW9662 led to a significant reduction in the neuroprotective (Figure 4b) and axonoprotective (Figure 4c) effects of pioglitazone. To determine further the role of catalase in pioglitazone-induced neuroprotection, we pre-incubated the cultures with the catalase inhibitor 3-amino triazole (10 mM) (3-AT) for 1 hour, prior to incubation with H2O2 (250 μM). The neuroprotective and axonoprotective effects of pioglitazone were attenuated by the addition of 3-AT (10 mM) (Figure 5a-5c). Furthermore, addition of exogenous catalase (100 U/ml) to cultures exposed to H2O2 improved neuronal survival (measured by MTT assay (Figure 5a) and neuronal morphology (Figure 5b) and axonal length within cultures (Figure 5c).
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Figure 4: Effect of pioglitazone on hydrogen peroxide-mediated reductions in neuronal survival in cortical neuronal culture. (a) The effect of hydrogen peroxide exposure (MIN H2O2) (250 μM) on cortical neuronal viability in vitro compared to serum free minimal media (MIN) (**P < 0.01 compared with MIN); the effect of pioglitazone (0.1 μM to 10 μM; MIN H2O2 PIO) on cortical neuronal viability exposed to H2O2 (*P < 0.05 and **P < 0.01 as compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H2O2 (**P < 0.01 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). Cultures were treated with GW9662 for 1 hour prior to incubation with pioglitazone for 1 hour followed by exposure to hydrogen peroxide. Cell viability was assessed by MTT assay. Data are expressed as percentage of cells grown in MIN medium. Statistical significance was obtained by one-way ANOVA followed by Bonferroni post-hoc test. (b) Effect of hydrogen peroxide exposure (250 μM; MIN H2O2) on cortical neuronal cell survival (number of βIII-tubulin cells per field; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on cortical neuronal survival exposed to H2O2 (***P < 0.001 compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H2O2 (**P < 0.01 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). (c) Effect of hydrogen peroxide exposure (250 μM H2O2) on axon length within neuronal cultures (determined by SMI312 staining; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on axon length in neurons exposed to H2O2 (**P < 0.01 compared with MIN H2O2); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced axon protection from H2O2 (***P < 0.001 comparing MIN H2O2 Pio 1 μM GW9662 1 μM with MIN H2O2 Pio 1 μM). Values represent the mean ± SEM from at least three separate experiments. ANOVA, analysis of variance; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; PPAR-γ, peroxisomal proliferator activated receptor γ; SEM, standard error of the mean. To further examine the neuroprotective properties of pioglitazone, neurons were exposed to identical test conditions and cultures were then fixed and stained for βIII tubulin, SMI312 and the nuclear marker DAPI. The numbers of viable neurons (determined by nuclear appearance) expressing βIII tubulin were counted for each condition. As expected, neuronal survival was significantly increased following exposure to pioglitazone (1 μM) (Figure 4b). In addition, we examined the influence of pioglitazone on axonal morphology in these cultures. Neurofilament phosphorylation, as identified by the antibody SMI312, was examined as a marker of axonal integrity and length. Pre-treatment with pioglitazone (1 μM) caused a significant increase in total axon survival (Figure 4c). Pre-treatment with GW9662 led to a significant reduction in the neuroprotective (Figure 4b) and axonoprotective (Figure 4c) effects of pioglitazone. To determine further the role of catalase in pioglitazone-induced neuroprotection, we pre-incubated the cultures with the catalase inhibitor 3-amino triazole (10 mM) (3-AT) for 1 hour, prior to incubation with H2O2 (250 μM). The neuroprotective and axonoprotective effects of pioglitazone were attenuated by the addition of 3-AT (10 mM) (Figure 5a-5c). Furthermore, addition of exogenous catalase (100 U/ml) to cultures exposed to H2O2 improved neuronal survival (measured by MTT assay (Figure 5a) and neuronal morphology (Figure 5b) and axonal length within cultures (Figure 5c).
Mentions: During inflammation, a major function of the peroxisomal enzyme catalase is to convert hydrogen peroxide (H2O2) into water. Therefore, having demonstrated induction of catalase activity by pioglitazone we wished to determine whether the pioglitazone could also protect cortical neurons against H2O2-mediated injury. Neuronal cultures were incubated for 1 hour in the presence of increasing concentrations of pioglitazone (0.1 to 10 μM) prior to exposure to hydrogen peroxide (250 μM). Neuronal viability was significantly reduced when compared to control after hydrogen peroxide exposure (250 μM for 24 hours; Figure 4a). In the presence of pioglitazone (10 μM, 1 μM and 0.1 μM), neuronal viability was significantly increased during exposure to hydrogen peroxide (Figure 4a). Furthermore, the neuroprotective effects of pioglitazone were prevented by the concomitant presence of GW9662 (1 μM) suggesting that PPAR-γ activation is necessary for the pioglitazone-induced neuroprotection of cortical neurons against the toxic effects of hydrogen peroxide.

Bottom Line: To assess the influence of peroxisomal activation on nitric oxide mediated neurotoxicity, we investigated the effects of the peroxisomal proliferator activated receptor (PPAR) gamma agonist, pioglitazone in primary cortical neurons that were either exposed to a nitric oxide donor or co-cultured with activated microglia.Moreover, cortical neurons treated with this compound showed a significant increase in the protein and gene expression of PPAR-gamma, which was associated with a concomitant increase in the enzymatic activity of catalase.In addition, the protection of neurons and axons against hydrogen peroxide-induced toxicity afforded by pioglitazone appeared to be dependent on catalase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Multiple Sclerosis and Stem Cell Group, Burden Centre, Institute of Clinical Neurosciences, Frenchay Hospital, University of Bristol, Bristol BS16 1JB, UK. elizabeth.gray@bristol.ac.uk

ABSTRACT

Background: Inflammation is known to play a pivotal role in mediating neuronal damage and axonal injury in a variety of neurodegenerative disorders. Among the range of inflammatory mediators, nitric oxide and hydrogen peroxide are potent neurotoxic agents. Recent evidence has suggested that oligodendrocyte peroxisomes may play an important role in protecting neurons from inflammatory damage.

Methods: To assess the influence of peroxisomal activation on nitric oxide mediated neurotoxicity, we investigated the effects of the peroxisomal proliferator activated receptor (PPAR) gamma agonist, pioglitazone in primary cortical neurons that were either exposed to a nitric oxide donor or co-cultured with activated microglia.

Results: Pioglitazone protected neurons and axons against both nitric-oxide donor-induced and microglia-derived nitric oxide-induced toxicity. Moreover, cortical neurons treated with this compound showed a significant increase in the protein and gene expression of PPAR-gamma, which was associated with a concomitant increase in the enzymatic activity of catalase. In addition, the protection of neurons and axons against hydrogen peroxide-induced toxicity afforded by pioglitazone appeared to be dependent on catalase.

Conclusions: Collectively, these observations provide evidence that modulation of PPAR-gamma activity and peroxisomal function by pioglitazone attenuates both NO and hydrogen peroxide-mediated neuronal and axonal damage suggesting a new therapeutic approach to protect against neurodegenerative changes associated with neuroinflammation.

Show MeSH
Related in: MedlinePlus