(A) Schematic of young adult (3-month-old) versus aged (18-month-old) mice.

(B) Quantitative reverse-transcription PCR of Tet2 mRNA from hippocampal and cortical lysates of young and aged mice (n = 5 per group; t test, ∗p < 0.05; n.s., not significant).

(C) Representative slot blot and quantification of isolated hippocampal DNA probed with anti-5hmC antibodies from young and aged mice (n = 4 per group; t test, ∗p < 0.05).

(D) Representative field and quantification of 5hmC expression in the dentate gyrus (DG) of the young and aged hippocampus (n = 4 per group; scale bar, 100 μm; t test, ∗p < 0.05).

(E) Association of regions of 5hmC lost during age with genomic elements (differentially 5-hydroxymethylated regions [DhMRs] lost between 3 and 18 months; 345 DhMRs at q = 0.05). Pie chart depicts overall loss and gain of DhMRs during aging.

(F) Top gene ontology terms for genes overlapping with age-associated 5hmC loss (>2-fold enrichment; ordered by p value).

(G) Reverse-transcription qPCR of Tet2 mRNA from hippocampal lysates of aging mice (n = 5; ANOVA with Dunnett’s post hoc test, ∗∗p < 0.01).

(H and I) Neurogenesis was analyzed by immunostaining and confocal microscopy. Representative field (H) and quantification (I) of Nestin-positive, MCM2-positive, and Doublecortin (DCX)-positive cells in aging dentate gyrus (DG) at 3, 6, 12, and 18 months (n = 5; scale bar, 100 μm; ANOVA with Dunnett’s post hoc test, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001).