Fig. 1: PD neurotoxins MPP+ and 6-OHDA cause sustained activation of ATF4 and ATF4-dependent induction of pro-death genes Chop. Trib3, and Puma in neurons.

A Mesencephalic neuron cultures (7 DIV) were treated with increasing concentrations of MPP+ or 6-OHDA for 48 h and then immunostained for the DA neuron specific marker TH and the pan-neuronal marker MAP2. Representative images of DA neurons (TH, red) and non-DA neurons (MAP2, green) showing preferential loss of DA neurons following treatment with MPP+ and 6-OHDA, scale bar = 100 μm. Survival was determined as the fraction of TH+ (or MAP2+) cells remaining in treated wells as compared with vehicle treated wells and reported as a percentage (n = 4; 2-way ANOVA, *p < 0.001). B Cortical neurons (7 DIV) were treated with MPP+ (50 µM) or 6-OHDA (10 µM) and at the indicated timepoints ATF4 protein levels were assessed by western blot analysis. Protein levels were quantified by densitometry and ATF4 expression is reported as fold increase over untreated controls following normalization to Cyclophilin B levels (n = 3; one-way ANOVA *p < 0.05). C Mesencephalic neuron cultures (7 DIV) were treated with MPP+ (25 μM) or 6-OHDA (5 μM) for 12 h and then immunostained for TH and ATF4 and counterstained with Hoechst 33342. Representative images showing induction of ATF4 expression (red) in the nucleus of TH+ dopaminergic neurons (green) following treatment with PD neurotoxins, scale bar = 10 μm. Confocal images were acquired and the fluorescence intensity of ATF4 in the nucleus of TH+ neurons was quantified from a minimum of 36 dopaminergic neurons for each treatment group from three independent experiments (one-way ANOVA, *p < 0.01). D Mesencephalic neurons derived from ATF4+/+ and ATF4−/− littermates were treated with vehicle, MPP+ (25 μM) or 6-OHDA (5 μM) for 12 h and then probed for Chop, Trib3, and Puma mRNA levels using RNAscope fluorescence multiplex in situ hybridization and then immunostained for tyrosine hydroxylase. Representative images showing PD neurotoxin induced expression of Chop, Trib3, and Puma mRNA puncta in the soma and nucleus of a wildtype dopaminergic neuron but not in ATF4-null dopaminergic neurons, scale bar = 7.5 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 represent the mean ± SEM number of indicated mRNA transcript per DA neuron (ANOVA, *p < 0.001). E Cortical neurons derived from ATF4-wildtype and ATF4-null littermates were treated with MPP+ (50 μM) for 16 h or 6-OHDA (10 μM) for 12 h and mRNA levels of indicated transcripts were determine by quantitative RT-PCR. Expression was normalized to S12 mRNA levels and is reported as fold increase over untreated neurons (n = 4; unpaired t-test *p < 0.01).