Fig 2: 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 3: Disruption of OCN/GPR158 Signaling in Nestin+ and Nestin Cells Affect Contextual Fear Discrimination(A) Schematic of viral strategy to disrupt GPR158/OCN signaling in Nestin+ and Nestin− cells.(B) Images of DG from control and GPR158 knockdown mice, in Nestin+ or Nestin− cells showing RbAp48 (blue) co-localization with Nestin YFP (green) and GPR158 (red) (scale bar, 100 μm).(C) shGFP and Nestin− mice spent less time exploring both objects in the novel phase than the familiar phase (two-way RM ANOVA comparisons, post hoc Bonferroni; n = 9 mice per genotype; p = 0.001, Fam versus Nov, p = 0.001 for shGFP and p = 0.0126 for Nestin−. No differences were observed between the genotypes in total exploration time in the familiarization or the testing phase.(D) Discrimination index for novel object during a NOR test in control (shGFP) and Nestin+ or Nestin− GPR158 knockdown mice. Only shGFP mice were able to discriminate the novel object (two-way RM ANOVA comparisons, post hoc Bonferroni; n = 9 mice per group; p = 0.0008).(E-G) Contextual fear discrimination in control (shGFP) (E) and Nestin+ (F) or Nestin− (G) GPR158 knockdown mice. shGFP mice were able to discriminate between contexts (two-way RM ANOVA comparisons of treatment × contexts between control shGFP and GPR158 knockdown in Nestin+ and Nestin− cells; post hoc Bonferroni, p < 0.0001).(H) Discrimination index of control (shGFP) and Nestin+ or Nestin− GPR158 knockdown in the DG, 10 days after the end of contextual fear discrimination. Only shGFP mice were able to discriminate between the two contexts (one-way ANOVA, post hoc Bonferroni, p = 0.0016 and p = 0.025 and p = 0.0016).All data represent means ± SEM. *p < 0.05, **p<0.01, ***p < 0.001.

Fig 4: OCN/GPR158 Signaling in the CA3a Affects Pre-exposure-Mediated Contextual Fear Conditioning(A) Schematic of pre-exposure contextual fear conditioning.(B) Freezing levels of mice injected with PBS or OCN 30 s after one 1.5-mA foot-shock.(C) Freezing levels during 1–3 min of exposure to context D 48 hr after injection of OCN or PBS in CA3a. OCN enhances freezing after injection in CA3a (two-way RM ANOVA, post hoc Bonferroni; n = 10 mice per group; p = 0.0311 and p = 0.0013).(D) Freezing levels of ST and DT mice injected with OCN or PBS in CA3a 30 s after one 1.5-mA foot-shock.(E) Freezing levels of ST and DT mice 1–3 min during exposure to context D 48 hr after injection of OCN or PBS in CA3. OCN had an effect only in ST mice (two-way RM ANOVA for OCN versus PBS, Bonferroni post hoc, p = 0.0136 and p = 0.0295 for first minute and second minute comparison between ST OCN and ST PBS mice; three-way ANOVA for genotype x treatment effect, p = 0.0094; n = 12 mice).(F) Freezing levels of shGFP and shGPR158 mice injected in DG/CA3c 30 s after one 1.5-mA foot-shock.(G) Freezing levels of shGFP and shGPRI 58 mice in DG/CA3c during exposure to context D, 48 hr after tail injection of OCN. Tail injection of OCN in shGPRI 58 mice had an increase in freezing (two-way RM ANOVA, post hoc Bonferroni; treatment, p = 0.0409; n = 11 mice per group; p = 0.0228 for first minute).(H) Images from CA3a regions of shGFP (control, top) or shGPRI 58 (knockdown, bottom) animals injected in the DG after peripheral administration of OCN and pre-exposure-mediated contextual fear conditioning, stained for GFP (green) and OCN (red) (scale bar, 20 μm).(I) Quantification of OCN protein accumulation in the CA3a region 7 days after the end of the behavioral task (unpaired t test, p = 0.0397; n = 6 mice per group). All data represent means ± SEM.