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).