Fig. 6: The eIF2α kinase inhibitor C16 inhibits ATF4 activation and protects against MPP+ and 6-OHDA induced neuronal death.

A Cortical neurons were treated with MPP+ (50 μM) or 6-OHDA (10 μM) for 8 h in the presence of vehicle or C16 (2 μM) and the level of ATF4 was assessed by western blot analysis and quantified by densitometry. Data represents mean ± SEM and statistical differences determined by one-way ANOVA (n = 3; *p < 0.05). B Mesencephalic neurons were treated with MPP+ (25 μM) or 6-OHDA (5 μM) in the presence of C16 (2 μM) or vehicle for 12 h and then immunostained for ATF4 and tyrosine hydroxylase. Representative images showing reduced ATF4 (red) expression in the nucleus of dopaminergic (TH+/green) neurons following treatment with PD neurotoxins in the presence of C16, scale bar = 10 μm. Confocal images were acquired and ATF4 fluorescence intensity in the nucleus of TH+ neurons was quantified in a minimum of 30 dopaminergic neurons from three independent experiments (paired t-test, *p < 0.01). C Mesencephalic neurons were treated with MPP+ (25 µM) or 6-OHDA (5 µM) for 12 h in the presence of C16 (2 μM) or vehicle and then immunostained for TH and probed for Chop, Trib3, and Puma mRNA levels using RNAscope fluorescence multiplex in situ hybridization. Representative images showing that C16 attenuates MPP+ and 6-OHDA induced expression of Chop, Trib3, and Puma mRNA (puncta) in the soma and nucleus of dopaminergic neurons, scale bar = 5 μm. The number of transcripts for each target was counted in a minimum of 30 dopaminergic neurons for each treatment group from three independent experiments and data represents the mean ± SEM number of indicated mRNA transcript per DA neuron (two-way ANOVA, *p < 0.05). D Mesencephalic neurons were treated with MPP+ (25 µM) or 6-OHDA (5 µM) for 48 h in the presence of C16 (2 μM) or vehicle and then immunostained for tyrosine hydroxylase (TH). The number of TH+ neurons for each treatment was counted and is reported as the percentage of TH+ cells in corresponding untreated neuronal cultures (two-way ANOVA, n = 6; *p < 0.05). E Cortical neurons were treated with MPP+ (50 μM) or 6-OHDA (10 μM) in the presence of C16 (2 μM) or vehicle and the levels of p-eIF2α and total eIF2α was assessed at 4 h by western blot. P-eIF2α and total eIF2α levels were quantified by densitometry and data is represented as the ratio of p-eIF2α/ total eIF2α (ANOVA, n = 4, *p < 0.05). F Mesencephalic neurons were transduced at 25-MOI with Ad-GFP or Ad-GFP-ATF4 for 24 h and then treated with MPP+ (25 μM) or 6-OHDA (5 μM) in the presence of C16 (2 μM) or vehicle for 48 h. Neurons were then immunostained for ATF4 and tyrosine hydroxylase (TH). Representative images showing elevated levels of ATF4 (red) expression in mesencephalic cultures transduced with Ad-ATF4 as compared to Ad-GFP following treatment with PD neurotoxins in the presence of C16, scale bar = 25 μm. Images also show fewer residual TH+ neurons (indicated by arrowheads) in cultures transduced with Ad-ATF4 as compared to Ad-GFP indicating that ectopic expression of ATF4 overrides the neuroprotective effects of C16. The number of TH+ neurons was counted for each treatment and is reported as a percentage of the number of TH+ neurons in cultures transduced with Ad-GFP-ATF4 or Ad-GFP-ATF4ΔRK but not treated with PD neurotoxins (two-way ANOVA, n = 4; *p < 0.05).