Figure 2.
Differential and region-specific expression of ATF4 mRNA in the hippocampus in human-ApoE Tg mice and human samples. A, Whole hippocampal tissue of young ApoE4 (n = 10) mice and ApoE3 (n = 8) age-matched controls was assessed for eIF2 pathway-related mRNA factors. ApoE4 mice show significantly increased ATF4 mRNA levels (***p = 2.7 × 10−6). No differences in the mRNA levels of GADD34, PKR, PERK, GCN2, CHOP, and eIF2α were observed. B, ATF4 mRNA levels in the hippocampus of ApoE3 (n = 8) and ApoE4 (n = 4) mice were compared with those in C57BL/6 (n = 4) mice. ApoE4 ATF4 mRNA levels are significantly higher than ApoE3 (***p = 0.0001) and C57BL/6 (*p = 0.02). No significant differences were observed between the two control groups. C, The mRNA levels of ATF4 are significantly increased in the hippocampus of aged ApoE3 (n = 5) mice (**p = 0.001) to a higher extent than in aged ApoE4 mice (*p = 0.03), compared with strain-matched controls. There are no significant differences in ATF4 mRNA levels between young ApoE4 and aged ApoE3 hippocampus (p = 0.46). D, eIF2α mice (4 months; n = 11) have significantly decreased mRNA levels of ATF4 in the hippocampus compared with age-matched eIF2α-WT controls (n = 9; *p = 0.03). E, RNA ISH was performed on coronal hippocampal sections using digoxigenin-labeled RNA probes against ATF4 mRNA. Two representative images are shown. Arrowheads point to the CA1 region of the hippocampus where a difference in ATF4 mRNA expression is observed between the two mouse strains. F, Quantification of the mean intensity of ATF4 mRNA staining in cell body layers of the DG, CA1, and CA3 subfields of the hippocampus are reported as arbitrary units (a.u.) of reciprocal intensity. ATF4 mRNA distribution across hippocampal layers varies between the DG, CA1, and CA3, in both ApoE3 (n = 13) and ApoE4 (n = 13) (#p < 0.0001). CA1 ATF4 mRNA staining is significantly increased in ApoE4 (*p = 0.044). G, ATF4 mRNA relative quantities (RQ) in cDNA from postmortem brains are increased in ApoE4 carriers (n = 11) versus noncarriers (n = 31, age range 67–98 years) (*p = 0.04). gusb and ipo8 were used as endogenous controls. RQ was calculated for each sample using the 2^−ΔCt formula. Data are mean RQ/au ± ΔCt/SEM. *p < 0.05. **p < 0.01. ***p < 0.001. A, D, G, t test. B, One-way ANOVA with Scheffé's post hoc comparisons. C, Two-way ANOVA with Tukey pairwise comparisons. F, Repeated-measures ANOVA.