Epigenetic Activation of ASCT2 in the Hippocampus Contributes to Depression-Like Behavior by Regulating D-Serine in Mice

Abstract

The roles of D-serine in depression are raised concerned recently as an intrinsic co-agonist for the NMDA receptor. However, the mechanisms underlying its regulation are not fully elucidated. ASCT2 is a Na⁺-dependent D-serine transporter. We found that decreased D-serine and increased hippocampal ASCT2 levels correlated with chronic social defeat stress (CSDS) in mice. Lentivirus-mediated shRNA-mediated knockdown of ASCT2 and the administration of exogenous D-serine in the hippocampus alleviated CSDS-induced social avoidance and immobility. In vivo and in vitro experiments revealed that upregulation of ASCT2 expression in CSDS was regulated through histone hyper-acetylation, not DNA methylation in its promoter region. Immunohistochemistry demonstrated the co-localization of ASCT2 and D-serine. Uptake of D-serine by ASCT2 was demonstrated by in vivo and in vitro experiments. Our results indicate that CSDS induces ASCT2 expression through epigenetic activation and decreases hippocampal D-serine levels, leading to social avoidance, and immobility. Thus, targeting D-serine transport represents an attractive new strategy for treating depression.

Keywords: ASCT2; D-serine; SLC1A5; chronic social defeat stress; depression; mouse.

Figure 1

D-serine levels in the hippocampus define depression-like behavior in response to chronic social defeat stress (CSDS) in mice. (A) Relative levels of L-serine, D-serine, and L-glutamine in the hippocampus after CSDS (n = 6, ^*p < 0.05, control vs. CSDS mice; unpaired t-test). (B) Relative levels of L-serine, D-serine, and L-glutamine of hippocampal intercellular fluid after CSDS (n = 4–5, ^*p < 0.05, control vs. CSDS mice; unpaired t-test) (C) Social interaction score and (D) immobility time in the forced swim tests (FST) after acute D-serine administration (20 min prior to the test) into the hippocampus (n = 10–12; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001; two-way ANOVA). (E) Re-testing of social interaction 48 h after D-serine administration (n = 5, ^*p < 0.05, 20 min vs. 48 h; paired t-test). (F) The location of injection sites in the hippocampus.

Figure 2

Increased ASCT2 expression in the mouse hippocampus after CSDS. (A) Relative Asct2 mRNA expression (n = 5, ^*p < 0.05, CSDS vs. control mice; unpaired t-test). (B,C) Western blot analysis of ASCT2; representative immunoblot (B); quantification of ASCT2 protein expression (C) (n = 7, ^*p < 0.05, CSDS vs. control mice; unpaired t-test). (D) The distribution of ASCT2 in the hippocampus presented by immunostaining. (E,F) Immunostaining of ASCT2 in the CA1 (upper image) and CA3 (lower image) regions of the hippocampus in control (E) and CSDS (F) mice. (G,H) Quantification of ASCT2-specific immunostaining (n = 5). The data are expressed as mean ± S.E.M. of the mean gray value (^*p < 0.05 for CA1 and ^**p < 0.01 for CA3, CSDS vs. control mice; unpaired t-test).

Figure 3

ASCT2 downregulation in the hippocampus alleviated depression-like behavior in CSDS mice. (A) mRNA expression of Asct2 after being infected by lentiviruses carrying Asct2-specific (Lenti-535, Lenti-1127, and Lenti-1234) (shASCT2) or scrambled (shSCR) GFP-labeled shRNA, in HT22 hippocampal cells (n = 3, ^**p < 0.01, shASCT2 vs. shSCR; unpaired t-test). (B) Infected region of shASCT2-1127 in the hippocampus. (C,D) Effects of shASCT2 on depression-like behavior in Control (Con) and CSDS mice: the social interaction (C) and forced swim test (D) (n = 7–10; ^*p < 0.05, ^**p < 0.01; two-way ANOVA).

Figure 4

Asct2 mRNA expression was regulated by histone acetylation but not DNA methylation in the promoter. (A–C) Acetylation of histones H3K4 (A), H3K9 (B), and H3K27 (C) in three different regions of the Asct2 promoter (triplicate in RT-qPCR repeat, ^*p < 0.05, CSDS vs. control mice; unpaired t-test). (D) Percentage of methylated clones at different CpG sites in the Asct2 promoter (black in every circle represents the percentage of methylation in each CpG; TSS, transcription start site). (E) Relative Asct2 mRNA expression in HT22 hippocampal cells treated with 5 or 10 mM suberoylanilide hydroxamic acid (SAHA) (triplicate in RT-qPCR repeat, ^*p < 0.05, one-way ANOVA). (F) H3K9 acetylation at the P2 Asct2 promoter region in SAHA-treated HT22 cells (^*p < 0.05 and ^***p < 0.001, 10 mM SAHA vs. vehicle; unpaired t-test). (G) A diagram shows the locations of the primers used in CHIP assay and sequenced part in bisulfite sequencing.

Figure 5

D-serine level in the hippocampus was regulated by ASCT2. (A–C) Co-localization of D-serine and ASCT2 in the hippocampus: D-serine (A); ASCT2 (B); Merge (C). (D) Relative levels of L-serine, D-serine, and L-glutamine in the hippocampus of control (Con) and CSDS mice infected by lentiviruses carrying Asct2-specific shRNA (shASCT2) or scrambled shRNA (shSRC) (n = 4, ^*p < 0.05, two-way ANOVA). (E) mRNA expression of Asct2 in HT22 cells after being infected by lentiviruses carrying ASCT2, shASCT2, or shSRC [triplicate in RT-qPCR repeat, ^**p < 0.01, control vs. over-expressed (OverExp); ^*p < 0.05, control vs. down-expressed (DownExp); unpaired t-test]. (F) Cell uptake of L-serine, D-serine, and L-glutamine after being infected by lentiviruses carrying ASCT2, shASCT2, or shSRC. (n = 3, ^***p < 0.0001, control vs. OverExp and control vs. DownExp; unpaired t-test). (G–I) SRR expression unchanged after CSDS. Relative Srr mRNA expression (n = 5; unpaired t-test) (G); Representative immunoblot (H); quantification of Srr protein expression (n = 8, unpaired t-test) (I).

Figure 6

A timeline diagram of different experiments. (A) The timeline of molecular determinations: the first, third, fourth, fifth, and eighth batches of mice were manipulated accordingly. (B) The timeline of microdialysis: the second batch of mice was manipulated accordingly. (C) The timeline of D-serine administration: the sixth batch of mice was manipulated accordingly. (D) The timeline of lentivirus administration: the seventh batch of mice was manipulated accordingly.