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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 PPAR-γ expression and catalase activity in primary cortical neurons. (a) Photomicrograph showing immunoreactivity for PPAR-γ (red), βIII tubulin (green) and DAPI (blue). PPAR-γ immunoreactivity shows a distinct punctuate nuclear staining pattern. PPAR-γ mRNA levels were measured by RT-PCR in (b) primary cortical neurons exposed to serum free minimal medium (MIN), minimal medium plus DETANONOate (0.1 mM; MIN NO), pioglitazone (1 μM) (MIN PIO) or DETANONOate (0.1 mM) plus pioglitazone (1 μM; MIN NO PIO). The fold difference relative to the average in control treated cells (MIN) was calculated for each treatment by the 2-ΔΔCt method, with 18 S and neuron-specific enolase as endogenous controls. Plots depict the levels of transcript for PPAR-γ in cortical neurons following exposure to experimental conditions for 1, 2 and 4 hours (*P < 0.05 compared to MIN for MIN NO, MIN PIO and MIN NO PIO at 2 hours, Student's t-test). (c) Immunoblotting of PPAR-γ and loading control GAPDH in primary cortical neurons exposed to serum free minimal medium (MIN), minimal medium plus DETANONOate (0.1 mM; MIN NO), pioglitazone (1 μM) (MIN PIO) or DETANONOate (0.1 mM) plus pioglitazone (1 μM; MIN NO PIO) for 24 and 48 hours. (d) Western blot densitometric analysis of PPAR- γ expression in cortical neurons derived from experimental conditions outlined in (c) Data are given using arbitrary units of integrated density relative to MIN control (***P < 0.001, **P < 0.01 compared to MIN for MIN PIO and MIN NO PIO respectively at 48 hours, Student's t-test). (e) Effect of pioglitazone (Pio; 0.1 to 10 μM) on catalase enzymatic activity in cortical neurons exposed to DETANONOate (0.1 mM for 24 hours; MIN NO) (*P < 0.05 compared to MIN and *P < 0.05, **P < 0.01, ***P < 0.001 compared to MIN NO, Student's t-test). (f) Effect of pioglitazone (Pio; 0.1 to 10 μM) on catalase enzymatic activity in cortical neurons exposed to serum free minimal medium (MIN) (*P < 0.05, compared to MIN, Student's t-test). Data are expressed as mean ± SEM from at least three separate experiments. DAPI, 4',6-diamidino-2-phenylindole; PPAR-γ, peroxisomal proliferator activated receptor γ; SEM, standard error of the mean. Furthermore, we examined whether pioglitazone up-regulated the activity of catalase, a specific peroxisomal enzyme, using a commercial assay kit. Neurons treated with DETANONOate show a significant decrease in activity levels. However, pre-treatment with pioglitazone (10 μM, 1 μM and 0.1 μM) in the presence of DETANONOate (0.1 mM) elicited a significant increase in catalase activity compared to treatment with DETANONOate (0.1 mM) alone (Figure 3e). Furthermore, pioglitazone exposure alone (1 μM) elicited a significant increase in catalase levels (Figure 3f).
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Figure 3: Effect of pioglitazone on PPAR-γ expression and catalase activity in primary cortical neurons. (a) Photomicrograph showing immunoreactivity for PPAR-γ (red), βIII tubulin (green) and DAPI (blue). PPAR-γ immunoreactivity shows a distinct punctuate nuclear staining pattern. PPAR-γ mRNA levels were measured by RT-PCR in (b) primary cortical neurons exposed to serum free minimal medium (MIN), minimal medium plus DETANONOate (0.1 mM; MIN NO), pioglitazone (1 μM) (MIN PIO) or DETANONOate (0.1 mM) plus pioglitazone (1 μM; MIN NO PIO). The fold difference relative to the average in control treated cells (MIN) was calculated for each treatment by the 2-ΔΔCt method, with 18 S and neuron-specific enolase as endogenous controls. Plots depict the levels of transcript for PPAR-γ in cortical neurons following exposure to experimental conditions for 1, 2 and 4 hours (*P < 0.05 compared to MIN for MIN NO, MIN PIO and MIN NO PIO at 2 hours, Student's t-test). (c) Immunoblotting of PPAR-γ and loading control GAPDH in primary cortical neurons exposed to serum free minimal medium (MIN), minimal medium plus DETANONOate (0.1 mM; MIN NO), pioglitazone (1 μM) (MIN PIO) or DETANONOate (0.1 mM) plus pioglitazone (1 μM; MIN NO PIO) for 24 and 48 hours. (d) Western blot densitometric analysis of PPAR- γ expression in cortical neurons derived from experimental conditions outlined in (c) Data are given using arbitrary units of integrated density relative to MIN control (***P < 0.001, **P < 0.01 compared to MIN for MIN PIO and MIN NO PIO respectively at 48 hours, Student's t-test). (e) Effect of pioglitazone (Pio; 0.1 to 10 μM) on catalase enzymatic activity in cortical neurons exposed to DETANONOate (0.1 mM for 24 hours; MIN NO) (*P < 0.05 compared to MIN and *P < 0.05, **P < 0.01, ***P < 0.001 compared to MIN NO, Student's t-test). (f) Effect of pioglitazone (Pio; 0.1 to 10 μM) on catalase enzymatic activity in cortical neurons exposed to serum free minimal medium (MIN) (*P < 0.05, compared to MIN, Student's t-test). Data are expressed as mean ± SEM from at least three separate experiments. DAPI, 4',6-diamidino-2-phenylindole; PPAR-γ, peroxisomal proliferator activated receptor γ; SEM, standard error of the mean. Furthermore, we examined whether pioglitazone up-regulated the activity of catalase, a specific peroxisomal enzyme, using a commercial assay kit. Neurons treated with DETANONOate show a significant decrease in activity levels. However, pre-treatment with pioglitazone (10 μM, 1 μM and 0.1 μM) in the presence of DETANONOate (0.1 mM) elicited a significant increase in catalase activity compared to treatment with DETANONOate (0.1 mM) alone (Figure 3e). Furthermore, pioglitazone exposure alone (1 μM) elicited a significant increase in catalase levels (Figure 3f).

Mentions: To further elucidate neuroprotective mechanisms of pioglitazone, we examined whether pioglitazone up-regulates neuronal expression of PPAR-γ at the transcript and protein level. Under normal conditions, PPAR-γ was localized to the nucleus of cortical neurons and exhibited a punctuate staining pattern (Figure 3a). Cortical neurons were incubated with pioglitazone (1 μM) in the presence or absence of DETANONOate (0.1 mM) for 1, 2 and 4 hours. Incubation with pioglitazone induced a significant increase in PPAR-γ mRNA levels at 2 hours, and also in neurons that were also exposed to DETANONOate. Interestingly, exposure to DETANONOate alone (0.1 mM) also elicited a significant increase in PPAR-γ levels with no apparent additive effect of DETANONOate and pioglitazone (Figure 3b). These changes were independent of the 18 S neuron specific enolase levels, used as endogenous controls. The effects of pioglitazone on PPAR-γ mRNA levels were paralleled by a significant rise in PPAR-γ protein levels over a 48-hour period, as evaluated by western blot analysis (Figure 3c, d).


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 PPAR-γ expression and catalase activity in primary cortical neurons. (a) Photomicrograph showing immunoreactivity for PPAR-γ (red), βIII tubulin (green) and DAPI (blue). PPAR-γ immunoreactivity shows a distinct punctuate nuclear staining pattern. PPAR-γ mRNA levels were measured by RT-PCR in (b) primary cortical neurons exposed to serum free minimal medium (MIN), minimal medium plus DETANONOate (0.1 mM; MIN NO), pioglitazone (1 μM) (MIN PIO) or DETANONOate (0.1 mM) plus pioglitazone (1 μM; MIN NO PIO). The fold difference relative to the average in control treated cells (MIN) was calculated for each treatment by the 2-ΔΔCt method, with 18 S and neuron-specific enolase as endogenous controls. Plots depict the levels of transcript for PPAR-γ in cortical neurons following exposure to experimental conditions for 1, 2 and 4 hours (*P < 0.05 compared to MIN for MIN NO, MIN PIO and MIN NO PIO at 2 hours, Student's t-test). (c) Immunoblotting of PPAR-γ and loading control GAPDH in primary cortical neurons exposed to serum free minimal medium (MIN), minimal medium plus DETANONOate (0.1 mM; MIN NO), pioglitazone (1 μM) (MIN PIO) or DETANONOate (0.1 mM) plus pioglitazone (1 μM; MIN NO PIO) for 24 and 48 hours. (d) Western blot densitometric analysis of PPAR- γ expression in cortical neurons derived from experimental conditions outlined in (c) Data are given using arbitrary units of integrated density relative to MIN control (***P < 0.001, **P < 0.01 compared to MIN for MIN PIO and MIN NO PIO respectively at 48 hours, Student's t-test). (e) Effect of pioglitazone (Pio; 0.1 to 10 μM) on catalase enzymatic activity in cortical neurons exposed to DETANONOate (0.1 mM for 24 hours; MIN NO) (*P < 0.05 compared to MIN and *P < 0.05, **P < 0.01, ***P < 0.001 compared to MIN NO, Student's t-test). (f) Effect of pioglitazone (Pio; 0.1 to 10 μM) on catalase enzymatic activity in cortical neurons exposed to serum free minimal medium (MIN) (*P < 0.05, compared to MIN, Student's t-test). Data are expressed as mean ± SEM from at least three separate experiments. DAPI, 4',6-diamidino-2-phenylindole; PPAR-γ, peroxisomal proliferator activated receptor γ; SEM, standard error of the mean. Furthermore, we examined whether pioglitazone up-regulated the activity of catalase, a specific peroxisomal enzyme, using a commercial assay kit. Neurons treated with DETANONOate show a significant decrease in activity levels. However, pre-treatment with pioglitazone (10 μM, 1 μM and 0.1 μM) in the presence of DETANONOate (0.1 mM) elicited a significant increase in catalase activity compared to treatment with DETANONOate (0.1 mM) alone (Figure 3e). Furthermore, pioglitazone exposure alone (1 μM) elicited a significant increase in catalase levels (Figure 3f).
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Figure 3: Effect of pioglitazone on PPAR-γ expression and catalase activity in primary cortical neurons. (a) Photomicrograph showing immunoreactivity for PPAR-γ (red), βIII tubulin (green) and DAPI (blue). PPAR-γ immunoreactivity shows a distinct punctuate nuclear staining pattern. PPAR-γ mRNA levels were measured by RT-PCR in (b) primary cortical neurons exposed to serum free minimal medium (MIN), minimal medium plus DETANONOate (0.1 mM; MIN NO), pioglitazone (1 μM) (MIN PIO) or DETANONOate (0.1 mM) plus pioglitazone (1 μM; MIN NO PIO). The fold difference relative to the average in control treated cells (MIN) was calculated for each treatment by the 2-ΔΔCt method, with 18 S and neuron-specific enolase as endogenous controls. Plots depict the levels of transcript for PPAR-γ in cortical neurons following exposure to experimental conditions for 1, 2 and 4 hours (*P < 0.05 compared to MIN for MIN NO, MIN PIO and MIN NO PIO at 2 hours, Student's t-test). (c) Immunoblotting of PPAR-γ and loading control GAPDH in primary cortical neurons exposed to serum free minimal medium (MIN), minimal medium plus DETANONOate (0.1 mM; MIN NO), pioglitazone (1 μM) (MIN PIO) or DETANONOate (0.1 mM) plus pioglitazone (1 μM; MIN NO PIO) for 24 and 48 hours. (d) Western blot densitometric analysis of PPAR- γ expression in cortical neurons derived from experimental conditions outlined in (c) Data are given using arbitrary units of integrated density relative to MIN control (***P < 0.001, **P < 0.01 compared to MIN for MIN PIO and MIN NO PIO respectively at 48 hours, Student's t-test). (e) Effect of pioglitazone (Pio; 0.1 to 10 μM) on catalase enzymatic activity in cortical neurons exposed to DETANONOate (0.1 mM for 24 hours; MIN NO) (*P < 0.05 compared to MIN and *P < 0.05, **P < 0.01, ***P < 0.001 compared to MIN NO, Student's t-test). (f) Effect of pioglitazone (Pio; 0.1 to 10 μM) on catalase enzymatic activity in cortical neurons exposed to serum free minimal medium (MIN) (*P < 0.05, compared to MIN, Student's t-test). Data are expressed as mean ± SEM from at least three separate experiments. DAPI, 4',6-diamidino-2-phenylindole; PPAR-γ, peroxisomal proliferator activated receptor γ; SEM, standard error of the mean. Furthermore, we examined whether pioglitazone up-regulated the activity of catalase, a specific peroxisomal enzyme, using a commercial assay kit. Neurons treated with DETANONOate show a significant decrease in activity levels. However, pre-treatment with pioglitazone (10 μM, 1 μM and 0.1 μM) in the presence of DETANONOate (0.1 mM) elicited a significant increase in catalase activity compared to treatment with DETANONOate (0.1 mM) alone (Figure 3e). Furthermore, pioglitazone exposure alone (1 μM) elicited a significant increase in catalase levels (Figure 3f).
Mentions: To further elucidate neuroprotective mechanisms of pioglitazone, we examined whether pioglitazone up-regulates neuronal expression of PPAR-γ at the transcript and protein level. Under normal conditions, PPAR-γ was localized to the nucleus of cortical neurons and exhibited a punctuate staining pattern (Figure 3a). Cortical neurons were incubated with pioglitazone (1 μM) in the presence or absence of DETANONOate (0.1 mM) for 1, 2 and 4 hours. Incubation with pioglitazone induced a significant increase in PPAR-γ mRNA levels at 2 hours, and also in neurons that were also exposed to DETANONOate. Interestingly, exposure to DETANONOate alone (0.1 mM) also elicited a significant increase in PPAR-γ levels with no apparent additive effect of DETANONOate and pioglitazone (Figure 3b). These changes were independent of the 18 S neuron specific enolase levels, used as endogenous controls. The effects of pioglitazone on PPAR-γ mRNA levels were paralleled by a significant rise in PPAR-γ protein levels over a 48-hour period, as evaluated by western blot analysis (Figure 3c, d).

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