Fig 2: OCN Function Is Blocked by RbAp48 Inhibition(A) Images of the DG showing accurate targeting of OCN (100 ng) 15 min after injection of mStrawberry alone (top) or fusion mStrawberry-OCN (bottom). Total OCN (green) staining was performed as control (scale bar, 100 µm).(B and C) Novel object recognition (NOR) test after injection of PBS or OCN in the DG of ST and DT mice (n = 16 mice per group). No differences were detected in total exploration time between the familiarization and the testing phase (three-way ANOVA, post hoc Bonferroni) (B), and discrimination index for novel object (C). OCN had an effect only on ST mice compared to DT OCN-treated mice (two-way repeated-measures [RM] ANOVA comparisons between OCN or PBS and ST versus DT; post hoc Bonferroni, p < 0.0001).(D) Schematic contextual fear discrimination task.(E-H) Contextual fear discrimination in ST or DT mice injected with OCN in the DG/CA3c (F and H). Both genotypes were able to discriminate between the two contexts after PBS injection (E and G), but only ST were able to discriminate the two contexts after OCN injection (F) (two-way RM ANOVA, post hoc Bonferroni; three-way RM ANOVA, post hoc Bonferroni for treatment × contexts in ST mice, p = 0.0003, and treatment x contexts in DT mice; n = 16 mice per group).(I) Discrimination index of ST and DT mice injected in the DG with OCN or PBS 10 days after the end of contextual fear discrimination. OCN-injected ST mice were still able to discriminate better than the ST PBS or DT OCN mice (one-way ANOVA, post hoc Bonferroni, p = 0.0029 and p = 0.0194).All data represent means ± SEM. *p < 0.05, **p<0.01, ***p < 0.001.

Fig 3: RbAp48 Controls GPR158 and OCN Signaling(A) Distribution of RbAp48 binding sites in the mouse genome.(B) Top ten biological processes for genes that RbAp48 binds to.(C) Peaks in the mouse genome for RbAp48 binding compared with the input DNA for BDNF and GPR158 loci.(D-F) Quantitative real-time PCR of ChIP assays from dentate gyrus (DG) lysates for the GPR158 promoter (D), the GPR158 intron (E), and P5 BDNF promoter (F) compared to input DNA (n = 6 mice per group). Fold enrichment of RbAp48 binding in the respective genomic regions (one-way ANOVA treatment between columns post hoc Bonferroni; p = 0.0129, p = 0.0275, p = 0.0036, respectively).(G) Western blot images of GPR158, transgenic Flag-tagged DN-RbAp48, and tubulin proteins from hippocampal lysates from control (ST) and dominant-negative RbAp48 overexpression mice (DT).(H) Quantification of GPR158 protein levels (unpaired t test, p = 0.0038; n = 6 mice per group).(I) Representative images of the DG of DT and ST mice stained for GPR158 and RbAp48 (scale bar, 20 µm).(J) Quantitative real-time PCR of GPR158 mRNA from the DG/CA3c of ST or DT mice injected with PBS or OCN. GPR158 mRNA is downregulated in the DG of DT mice compared to ST controls injected with PBS (one-way ANOVA treatment between columns post hoc Bonferroni; n = 4–5 mice per treatment, respectively; p = 0.0113).All data represent means ± SEM. *p < 0.05, **p < 0.01.

Fig 4: OCN Increases RbAp48 in the DG and Enhances Fear Expression during Context Re-exposure (Contextual Fear Conditioning)(A) Images of the DG stained for RbAp48 (red) 24 hr after administration of PBS (top) or OCN (bottom) in the DG (scale bar, 100 µm).(B) OCN injection increases RbAp48 24 hr after an injection (unpaired t tests; n = 8 mice per group; p = 0.0103).(C) Schematic representation of contextual fear conditioning (CFC) in c-FosCreERT2 × tdTomato mice.(D) Images from DG of c-FosCreERT2/tdTomato mice stained for mCherry (red) PBS (left) or OCN (right) (scale bar, 100 µm).(E) c-FosCreERT2 × tdTomato mice were administered a three-shock CFC paradigm. OCN administration did not affect fear acquisition behavior, but it resulted in increased fear expression during context re-exposure (unpaired t tests; n = 6 mice per group; p = 0.016).(F) Images from c-Fos+ dendrites in the outer molecular layer (OML), OCN (right) and PBS (left) (scale bar, 10 µm).(G) Quantification of spines in c-Fos-+ dendrites in the OML, OCN (right) and PBS (left). OCN increased spine density in c-Fos+ neurons (unpaired t tests; n = 3 mice per group; p = 0.0146; <35 dendrites per animal).All data represent means ± SEM. *p < 0.05.

Fig 5: OCN Signaling in the DG Upregulates RbAp48 and Ameliorates Age-Related Memory Loss(A) Images of the DG from 16-month-old wild-type (WT) mice stained for RbAp48 (red) 48 hr after injection of PBS (20 ng) (top) or OCN (bottom) (scale bar, 100 µm).(B) Quantification of RbAp48 protein after injection of OCN or PBS in the DG of 16-month-old WT mice. OCN increases RbAp48 levels (unpaired t tests, p = 0.0179; n = 4 mice per group).(C) Images of the DG from 16-month-old WT mice stained for CBP (green) and BDNF (red), 48 hr after injection of PBS (20 ng) (top) or OCN (bottom) (scale bar, 100 µm).(D and E) Quantification of CBP (D) and BDNF (E) protein levels after injection of OCN or PBS in the DG of 16-month-old WT mice. OCN increases CBP and BDNF levels (unpaired t tests, p < 0.0001 and p = 0.034, respectively; n = 6 mice per group).(F) No differences were observed between the genotypes in total exploration time in the familiarization or the testing phase.(G) Discrimination index for novel object after injections of PBS or OCN in the DG of 16-month-old WT mice (two-way RM ANOVA comparisons, post hoc Bonferroni, n = 16 mice per group treatment, p = 0.0036, Fam versus Nov, p = 0.0008). Aged mice injected with OCN in the DG were able to discriminate between the novel and familiar object.(H and I) Contextual fear discrimination in 16-month-old mice injected with PBS (H) or OCN (I) in the DG/CA3c. Aged mice injected with OCN in the DG were able to discriminate between the two contexts (three-way RM ANOVA, post hoc Bonferroni for OCN or PBS and CTXA and CTXB, three-way RM ANOVA for treatment and context × treatment, p < 0.0001; n = 16 mice per group).(J) Contextual fear discrimination at day 24 in 16-month-old mice injected with OCN or PBS. Aged mice injected with OCN in the DG were still able to discriminate between the two contexts (unpaired t test, p = 0.0395; n = 16 mice per group).(K) Freezing levels of mice injected with PBS or OCN in CA3c 30 s after one 1.5-mA foot-shock.(L) Freezing levels during 1–3 min of exposure to context D 48 hr after injection of OCN or PBS in CA3c (two-way RM ANOVA, post hoc Bonferroni, p = 0.04 for first minute; n = 16 mice per group).All data represent means ± SEM. *p < 0.05, **p<0.01, ***p < 0.001.