Targeting acetyl-CoA metabolism attenuates the formation of fear memories through reduced activity-dependent histone acetylation

Abstract

Histone acetylation is a key component in the consolidation of long-term fear memories. Histone acetylation is fueled by acetyl-coenzyme A (acetyl-CoA), and recently, nuclear-localized metabolic enzymes that produce this metabolite have emerged as direct and local regulators of chromatin. In particular, acetyl-CoA synthetase 2 (ACSS2) mediates histone acetylation in the mouse hippocampus. However, whether ACSS2 regulates long-term fear memory remains to be determined. Here, we show that Acss2 knockout is well tolerated in mice, yet the Acss2-null mouse exhibits reduced acquisition of long-term fear memory. Loss of Acss2 leads to reductions in both histone acetylation and expression of critical learning and memory-related genes in the dorsal hippocampus, specifically following fear conditioning. Furthermore, systemic administration of blood-brain barrier-permeable Acss2 inhibitors during the consolidation window reduces fear-memory formation in mice and rats and reduces anxiety in a predator-scent stress paradigm. Our findings suggest that nuclear acetyl-CoA metabolism via ACSS2 plays a critical, previously unappreciated, role in the formation of fear memories.

Keywords: epigenetics; fear conditioning; histone acetylation; learning and memory; mass spectrometry.

Fig. 1.

Generation and characterization of a constitutive Acss2^KO mouse. (A) Diagram showing CRISPR strategy to KO Acss2 in mouse. Guide RNAs are shown as red hairpins; stop codons as yellow stars. (B) Representative Western blot of ACSS2 in brain (cerebellum), showing age-matched WT, Acss2^Het, and Acss2^KO. (C) Western blot of ACSS2 expression in liver. (D) Acss2 mRNA levels shown as normalized transcript counts mapped to Acss2 exons. (E) University of California, Santa Cruz, genome browser tracks showing dose-dependent reduction at the Acss2 locus (Left) but unchanged neighboring gene (Gss7, Right). (F) Bar charts showing messenger RNA (mRNA) expression levels of Acly and Pdha1, the catalytic subunit of the PDC complex. (G) Bar charts of Acss1 and Acss3 mRNA levels. (H) OLM assay showing object positions during training (Left) and recall (Right). (I) Bar graph showing OLM discrimination score for WT and Acss2^KO mice. WT: n = 5; KO: n = 5. *P = 0.011, unpaired t test. All data presented as mean ± SEM. cDNA, complementary DNA; Het, heterozygous; Norm., normalized.

Fig. 2.

Loss of ACSS2 impairs FC. (A) FC schematic, showing training/acquisition (Left), cued/auditory recall (Middle), and contextual (ctx.) recall (Right). (B) Fear response during training/acquisition reflected as fold change over freezing within the pretone interval. Periods binned by period: pretone, intertone interval (ITI), tone (T), or tone-shock pairing (T/S). P = ns, 2-way ANOVA. (C) Fear response during cued/auditory recall reflected as fold change over pretone interval. *P = 0.0208, 2-way ANOVA, post hoc Fisher's least significant difference. (D) Fear response during contextual recall reflected as percent time freezing, averaged over the entire recall period (5 min). P = 0.0305, unpaired t test. Data are presented as mean ± SEM. Habit., habituated; ns, not significant; CS, conditioned stimulus.

Fig. 3.

ACSS2 KO mice exhibit reduced histone acetylation in an activity-dependent context. (A) Schematic of paired histone acetylation and RNA-seq study design. (B) Bar graphs showing histone acetylation levels expressed as fold-change over WT baseline (abundance per average baseline abundance). One-way ANOVA, post hoc Fisher's least significant difference. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. LC-MS/MS, liquid chromatography–tandem mass spectrometry.

Fig. 4.

Loss of ACSS2 negatively impacts the transcription of activity-dependent genes in a FC model. (A) Volcano plot showing gene expression changes between fear-conditioned WT and ACSS2KO mice 30 min after acquisition. Significant genes (P_adj < 0.05) denoted by colored points. WT FC 30′: n = 5; KO FC 30′: n = 4). (B) Volcano plot for gene expression changes between home-cage–housed WT and ACSS2KO mice. WT baseline: n = 5; KO baseline: n = 4. (C) Top 10 GO terms (Database for Annotation, Visualization and Integrated Discovery [DAVID], biological process [BP]) enriched in WT FC 30′ (DAVID, BP) compared with KO FC 30′. (D) Top 10 GO terms enriched in KO FC 30′ (DAVID, BP) compared with WT FC 30′. (E) Bar plots of 12 well-characterized IEGs showing normalized transcript counts across all four conditions. WT BL, n = 5; WT FC 30′, n = 3, KO BL, n = 5; KO FC 30′, n = 4. (F) Heat map of top 80 DEGs in WT FC 30′ versus baseline (RNA-seq; top 80 genes determined by P_adj value). *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. ns, not significant; rRNA, ribosomal RNA.

Fig. 5.

Small-molecule inhibitor of ACSS2 reduces histone acetylation in vitro, is bioavailable in rodents, and disrupts LTM formation in vivo. (A) Western blot on NT2 cell lysates for H3K9ac, H3, and GAPDH loading following treatment with cACSS2i. Quantification (Right; n = 2) shows a reduction in H3K9ac/H3 ratio (one-way ANOVA F_6,7 = 30.9, P = 0.0001, post hoc Dunnett test comparing each concentration with DMSO P_adj ≤ 0.0002). (B) Pharmacokinetic analysis of cACSS2i following IV administration in rat brain homogenate (n = 3 animals at time [t] 0.083 and t1; n = 1 at t4). cACSS2i is not detected (ND) at 8 h. (C) cACSS2i in mouse hippocampal lysate up to 30 min following IP injection (n = 2 for each time point). (D) Open-field assessment in mouse indicates no difference in basal locomotion following IP injection of DMSO or cACSS2i (DMSO, n = 8; cACSS2i, n = 7). (E) Long-term memory formation is disrupted in cACSS2i-injected animals in the OLM assay (DMSO, n = 6; cACSS2i, n = 7; P = 0.0091, unpaired Student’s t test). (F) Freezing behavior in mouse during acquisition with cACSS2i treatment reflected as %time freezing/total time. Freezing during habituation period was averaged; the remaining time was binned into 30-s intervals. Timing of CS-US pairings indicated by transparent red bars. P = ns, two-way ANOVA. (G) Freezing behavior during cued recall in mice treated with cACSS2i or DMSO at acquisition plotted as %time freezing/total time, binned into 30-s intervals. Onset of tone marked by black arrow. Significant time points are observed at the onset of cue. Two-way ANOVA, Fisher’s LSD, **P = 0.0308, ***P = 0.0319. (H) Freezing behavior in rats during acquisition with cACSS2i plotted as % time freezing/total time. Freezing during habituation period was averaged, remaining time binned into 30-s intervals. Time of CS-US pairings indicated by transparent red bars. Two-way ANOVA, Fisher’s LSD, **P = 0.0375, 0.0208. (I) Freezing behavior during cued recall in rats treated with cACSS2i or DMSO at acquisition plotted as %time freezing/total time, binned into 30-s intervals. Onset of tone is marked by a black arrow. Significant time points are observed late in cue presentation. Two-way ANOVA, Fisher’s LSD, *P = 0.0285, ***P = 0.0003, *P = 0.0070. (J) Freezing behavior in mouse during acquisition with cACSS2i treatment reflected as % time freezing/total time. Freezing during habituation period was averaged, remaining time was binned into 30-s intervals. Timing of CS-US pairings is indicated by transparent red bars. P = ns, two-way ANOVA. (K) Freezing behavior during cued recall in mice treated with cACSS2i or DMSO at acquisition plotted as %time freezing/total time, binned into 30-s intervals. Timing of CS indicated by transparent red bars. Two-way ANOVA, Fisher’s LSD, *P < 0.05. (L) Fear response during contextual recall reflected as %time freezing, averaged over the entire recall period (5 min). P = 0.0305, unpaired t test. Data presented as mean ± SEM. Habit., habitualized; CS, conditioned stimulus; US, unconditioned stimulus; ITI, intertone interval; T/S, tone-shock pairing; ns, not significant.

Fig. 6.

cACSS2i treatment impairs PSS-induced fear-memory consolidation in rats. (A) Outline of the PSS assay. Rats are exposed to PSS (soiled cat litter) or sham-PSS (fresh cat litter) after they were injected with cACSS2i or DMSO. One week later, anxiety level in animals is measured through EPM and acoustic startle response. The following day, animals are re-exposed to the cat litter stimulus and freezing behavior is measured. A second reminder exposure takes place 1 wk later. (B) PSS-exposed, DMSO-injected rats display increased startle amplitude relative to sham-exposed DMSO-injected rats. cACSS2i-treated, PSS-exposed rats exhibit a reduced startle amplitude. (C) PSS-exposed, DMSO-injected rats have an increased Anxiety Index relative to sham-exposed DMSO-injected rats. cACSS2i-treated, PSS-exposed rats have a lower Anxiety Index. (D) Total entries into open and closed arms of the EPM apparatus to indicate total activity in the assay. No differences between treatment groups were detected. (E) PSS-exposed, DMSO-injected rats display increased freezing relative to sham-exposed DMSO-injected rats upon first reminder. No increase observed in cACSS2i-treated, PSS-exposed rats. (F) Effects observed at first reminder persist 1 wk later at a second reminder session. PSS-exposed, DMSO-injected rats display increased freezing relative to sham-exposed, DMSO-injected rats at the first reminder session. No increase observed in cACSS2i-treated, PSS-exposed rats. Two-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Data presented as mean ± SEM. AU, arbitrary units.

Fig. 7.

A small-molecule inhibitor of ACSS2 reduces histone acetylation in vitro, is bioavailable in rodents, and disrupts LTM formation in vivo. (A) Western blot on NT2 cell lysates for H3K9ac, H3, and GAPDH following treatment with nACSS2i. Quantification (Right; n = 2) shows a reduction in H3K9ac/H3 (one-way ANOVA F_{(6, 7)} = 17.38, P = 0.0007; post hoc Dunnett each concentration compared with DMSO, P_adj ≤ 0.0013). (B) Pharmacokinetic analysis of nACSS2i following IV administration in rat brain homogenate (n = 3). (C) Mouse freezing behavior during acquisition with nACSS2i treatment reflected as %time freezing/total time. Freezing during habituation period was averaged; the remaining time was binned into 30-s intervals. Time of CS-US pairings indicated by transparent red bars. P = ns, two-way ANOVA. (D) Freezing behavior during cued recall in mice treated with nACSS2i or DMSO at acquisition plotted as % time freezing/total time, binned into 30-s intervals. Tone onset indicated by black arrow. Significant time points observed late in cue presentation. Two-way ANOVA, Fisher’s LSD, **P = 0.0408. Habit, habituated; CS, conditioned stimulus, US, unconditioned stimulus.