Fig. 4: ATF4 transcriptional activity is required for PD neurotoxin induced dopaminergic neuronal death.

A Schematic of adenovirus constructs Ad-GFP-ATF4 and Ad-GFP-ATF4ΔRK that enable bicistronic expression of GFP and either ATF4 or ATF4ΔRK that contains a mutated DNA binding ___domain rendering it transcriptionally inactive. B ATF4-null cortical neurons were transduced at 50-MOI with Ad-GFP-ATF4 or Ad-GFP-ATF4ΔRK for 48 h and the expression of GFP and ATF4 was confirmed by western blot. C ATF4−/− cortical neurons were transduced at 50-MOI with Ad-GFP-ATF4 or Ad-GFP-ATF4ΔRK for 36 h. Neurons were then treated with MPP+ (50 μM) or 6-OHDA (10 μM) for 12 h and the mRNA levels of Chop, Trib3 and Puma were determined by qRT-PCR. Expression was normalized to S12 mRNA levels and is reported as fold increase over control (n = 4; paired t-test *p < 0.05). D Mesencephalic neurons derived from ATF4−/− mice were transduced at 25-MOI with Ad-GFP-ATF4 or Ad-GFP-ATF4ΔRK for 24 h. Neurons were then treated with MPP+ (25 μM) or 6-OHDA (5 μM) and after 48 h neurons were immunostained for ATF4 and the dopaminergic neuron marker tyrosine hydroxylase (TH). Representative images showing Ad-GFP-ATF4 and Ad-GFP-ATF4ΔRK mediated expression of ATF4 (red) in the majority of ATF4-deficient neurons. Images also show decreased numbers of residual dopaminergic neurons (TH+/green) identified by arrows in ATF4−/− mesencephalic cultures transduced with Ad-GFP-ATF4 as compared to Ad-GFP-ATF4ΔRK following treatment with MPP+ or 6-OHDA, scale bar =  50 μm. 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. Data represent the mean ± SEM and statistical differences were determined by two-way ANOVA (n = 4; *p < 0.01).