Effect of glutamate and riluzole on manganese-induced apoptotic cell signaling in neuronally differentiated mouse P19 Cells

Abstract

Excess exposure to Mn causes a neurological disorder known as manganism which is similar to dystonic movements associated with Parkinson's disease. Manganism is largely restricted to occupations in which high atmospheric levels are prevalent which include Mn miners, welders and those employed in the ferroalloy processing or related industrial settings. T1 weighted MRI images reveal that Mn is deposited to the greatest extent in the globus pallidus, an area of the brain that is presumed to be responsible for the major CNS associated symptoms. Neurons within the globus pallidus receive glutamatergic input from the subthalamic nuclei which has been suggested to be involved in the toxic actions of Mn. The neurotoxic actions of Mn and glutamate are similar in that they both affect calcium accumulation in the mitochondria leading to apoptotic cell death. In this paper, we demonstrate that the combination of Mn and glutamate potentiates toxicity of neuronally differentiated P19 cells over that observed with either agent alone. Apoptotic signals ROS, caspase 3 and JNK were increased in an additive fashion when the two neurotoxins were combined. The anti-glutamatergic drug, riluzole, was shown to attenuate these apoptotic signals and prevent P19 cell death. Results of this study confirm, for the first time, that Mn toxicity is potentiated in the presence of glutamate and that riluzole is an effective antioxidant which protects against both Mn and glutamate toxicity.

Fig. 1

The effect of riluzole on manganese/glutamate toxicity in neuronally differentiated P19 cells. Neuronally differentiated P19 cells were treated with Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs. Toxicity was measured by trypan blue dye exclusion assay compared to the control samples treated with only riluzole/DMSO. Data is the mean ± S.E. of at least three independent experiments; ^a,c,ep < 0.001 differences from vehicle only control; ^b,d,fp < 0.001 differences from Mn and/or glutamate treatment alone.

Fig. 2

The fluorescence intensity of intracellular ROS was analyzed by DCFH-DA staining using flow cytometer upon treatment with Mn, glutamate and/ or riluzole in neuronally differentiated P19 cells. Neuronally differentiated P19 cells were treated for two hrs. with Mn (0.3 mM), glutamate (G; 5 mM) and/ or riluzole (R; 10μM). Results for ROS generation in treated cells were compared to the control incubations containing DMSO or riluzole. (A) Scatter plot showing the gated region of cells that were selected for analysis. Along with the histogram plots showing the spectral shift of the fluorescence curve to the right after treatment with Mn, glutamate or the two together. Decrease in the spectral shift is observed in samples incubated with riluzole. M1 represents the percentage of cells positive for the DCFH-DA dye. (B) Graphical representation of fluorescence intensities from three separate experiments. Results are presented as mean ± SE; Mn vs. control^a - p < 0.05; G vs. control^b - n.s., not significant; Mn + G vs. control^c - p < 0.001; Mn vs. Mn + G^d - p < 0.001; G vs. Mn + G^e - p < 0.001; Mn vs. Mn + R^f - p < 0.05; G vs. G + R^g - n.s., not significant; Mn + G vs. Mn + G + R^h - p < 0.001. Experiments shown are the mean ± S.E. of three independent experiments.

Fig. 2

The fluorescence intensity of intracellular ROS was analyzed by DCFH-DA staining using flow cytometer upon treatment with Mn, glutamate and/ or riluzole in neuronally differentiated P19 cells. Neuronally differentiated P19 cells were treated for two hrs. with Mn (0.3 mM), glutamate (G; 5 mM) and/ or riluzole (R; 10μM). Results for ROS generation in treated cells were compared to the control incubations containing DMSO or riluzole. (A) Scatter plot showing the gated region of cells that were selected for analysis. Along with the histogram plots showing the spectral shift of the fluorescence curve to the right after treatment with Mn, glutamate or the two together. Decrease in the spectral shift is observed in samples incubated with riluzole. M1 represents the percentage of cells positive for the DCFH-DA dye. (B) Graphical representation of fluorescence intensities from three separate experiments. Results are presented as mean ± SE; Mn vs. control^a - p < 0.05; G vs. control^b - n.s., not significant; Mn + G vs. control^c - p < 0.001; Mn vs. Mn + G^d - p < 0.001; G vs. Mn + G^e - p < 0.001; Mn vs. Mn + R^f - p < 0.05; G vs. G + R^g - n.s., not significant; Mn + G vs. Mn + G + R^h - p < 0.001. Experiments shown are the mean ± S.E. of three independent experiments.

Fig. 3

Effect of manganese, glutamate and/or riluzole on caspase-3 activity in neuronally differentiated P19 cells. Cells were treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs. Fifty microliters of the normalized lysate from each sample was added to the 96 well plates and incubated with 2X substrate working solution containing Z-DEVD-AMC substrate for 30 minutes at room temperature. Protein concentration for all the conditions was normalized. Fluorescence was measured using the CytofluorII™ Multiplate Reader with excitation set at 380 nm and emission set at 460 nm; Mn vs. control ^a - n.s., not significant; G vs. Control^b - n.s., not significant; Mn + G vs. control^c - p < 0.05; Mn vs. Mn + R^d - n.s., not significant; G vs. G + R^e - p < 0.05; Mn +G vs. Mn + G + R^f - p < 0.05; Mn vs. Mn + G^h- n.s., not significant; G vs. Mn + G^g - n.s., not significant. Data shown are the mean ± S.E. of three independent experiments.

Fig. 4

Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control^a- p < 0.05; Mn vs. Mn + R^b - n.s., not significant; G vs. control^c - p < 0.05; G vs. G + R^d - p < 0.05; Mn + G vs. control^e - p < 0.001; Mn + G vs. Mn + G + R^f - p<0.001; Mn vs. Mn + G^g - p < 0.001; G vs. Mn + G^h - p < 0.05. Data shown are the mean ± S.E. of four independent experiments.