Fig. 3: PD neurotoxin induced ATF4 activation promotes neuronal apoptosis.

A Mesencephalic neurons derived from ATF4-wildtype and ATF4-null littermates were treated with MPP+ (25 µM) or 6-OHDA (5 µM) for 48 h and then immunostained for the dopaminergic neuron marker tyrosine hydroxylase. Representative images showing increased numbers of residual dopaminergic neurons (TH+, green) identified by arrows in ATF4−/− mesencephalic cultures as compared to ATF4+/+ cultures following treatment with MPP+ or 6-OHDA. Scale bar = 100 μm. Quantification of the total number of TH+ neurons in ATF4+/+ and ATF4−/− mesencephalic cultures treated with MPP+ (25 µM) or 6-OHDA (5 µM) for 48 h. TH+ cell counts are reported as a percentage of untreated neurons from the same culture. Data represents the mean ± SEM and statistical differences were determined by ANOVA (n = 4; *p < 0.05). B ATF4+/+ and ATF4−/− cortical neurons were untreated or treated with MPP+ (50 μM) or 6-OHDA (10 μM) and the fraction of apoptotic cells was determined by Hoechst 33342 staining at 0, 24, and 48 h following drug treatment. Data represents the mean ± SEM and statistical differences were determined by two-way ANOVA (n = 5; *p < 0.01 between ATF4+/+ and ATF4−/− neurons treated with MPP+ or 6-OHDA). Representative images of Hoechst 33342 stained neurons captured at 48 h following exposure to neurotoxins showing elevated numbers of nuclei exhibiting chromatin condensation and/or nuclear fragmentation in ATF4+/+ cultures as compared to ATF4−/− cultures, scale bar = 50 μm. C Cleaved Caspase-3 levels were assessed at 24 h by western blot analysis and quantified by densitometry (n = 3; two-way ANOVA *p < 0.05). D Neuronal survival was assessed by Calcein-AM/ Ethidium-Homodimer (live/dead) staining at 48 h (n = 4; two-way ANOVA *p < 0.05). Representative images of MPP+ and 6-OHDA treated ATF4-wildtype and ATF4-deficient neurons at 48 h using live (green)/dead (red) assay, scale bar = 50 μm.