Pharmacological Selectivity Within Class I Histone Deacetylases Predicts Effects on Synaptic Function and Memory Rescue

Abstract

Histone deacetylases (HDACs) are promising therapeutic targets for neurological and psychiatric disorders that impact cognitive ability, but the relationship between various HDAC isoforms and cognitive improvement is poorly understood, particularly in mouse models of memory impairment. A goal shared by many is to develop HDAC inhibitors with increased isoform selectivity in order to reduce unwanted side effects, while retaining procognitive effects. However, studies addressing this tack at the molecular, cellular and behavioral level are limited. Therefore, we interrogated the biological effects of class I HDAC inhibitors with varying selectivity and assessed a subset of these compounds for their ability to regulate transcriptional activity, synaptic function and memory. The HDAC-1, -2, and -3 inhibitors, RGFP963 and RGFP968, were most effective at stimulating synaptogenesis, while the selective HDAC3 inhibitor, RGFP966, with known memory enhancing abilities, had minimal impact. Furthermore, RGFP963 increased hippocampal spine density, while HDAC3 inhibition was ineffective. Genome-wide gene expression analysis by RNA sequencing indicated that RGFP963 and RGFP966 induce largely distinct transcriptional profiles in the dorsal hippocampus of mature mice. The results of bioinformatic analyses were consistent with RGFP963 inducing a transcriptional program that enhances synaptic efficacy. Finally, RGFP963, but not RGFP966, rescued memory in a mouse model of Alzheimer's Disease. Together, these studies suggest that the specific memory promoting properties of class I HDAC inhibitors may depend on isoform selectivity and that certain pathological brain states may be more receptive to HDAC inhibitors that improve network function by enhancing synapse efficacy.

Figure 1

Novel pimelic diphenylamide-based histone deacetylase (HDAC) inhibitors have differential selectivity within the class I HDACs. (a) Chemical structures of novel RGFP HDAC inhibitors. IC₅₀ values for (b) HDAC1, (c) HDAC2, and (d) HDAC3. See Supplementary Table S1 for all other HDACs.

Figure 2

The inhibition of distinct class I histone deacetylases (HDACs) has differential effects on synaptogenesis. (a) Representative images of synaptophysin (SYN)-YFP-transfected neurons. Arrows point to SYN-YFP (green) and synapsin 1 (red) puncta, respectively. (b) Schematic time line of experiment. Camera symbol indicates when the neurons were imaged. (c) Representative images of SYN-YFP-transfected cortical neurons treated with DMSO or trichostatin A (TSA; N=6 per group). (d) Normalized increase of synapse formation from DIV8 to DIV10 with TSA or DMSO treatment. Student’s t-test, *P<4E-5. (e) Normalized dose response of cortical neurons treated with RGFP963 or DMSO (N=6 per group). One-way ANOVA, *P<0.05 relative to DMSO, ^$P<0.05 relative to 0.1 μM RGFP963, ^#P<0.05 relative to 1 μM RGFP963. (f) Normalized dose response to treatment with RGFP966 or DMSO (N=6 per group). One-way ANOVA, *P<0.05. (g) Normalized dose response to treatment with RGFP233 or DMSO (N=6 per group). One-way ANOVA, *P<0.05. (h) Relative increase of synapse formation after treatment at maximum efficacy (TSA=300 nM, RGFP compounds=10μM. One-way ANOVA, *P<0.05 relative to DMSO, ^#P<0.05 relative to RGFP233, and ^$P<0.05 relative to RGFP966. A Tukey HSD post hoc test was applied where appropriate. Error bars reflect SEM. A full colour version of this figure is available at the Neuropsychopharmacology journal online.

Figure 3

The inhibition of distinct class I histone deacetylases (HDACs) has differential effects on spontaneous excitatory neurotransmission. (a) Schematic time line of experiment. (b) Representative recordings of miniature excitatory events and mean miniature excitatory postsynaptic current (mEPSC) frequency and amplitude in DMSO- (N=10) and TSA (N=7)-treated neurons. Student’s t-test, *P<0.05. (c) Representative recordings of miniature excitatory events and mean mEPSC frequency and amplitude in DMSO- (N=14) and RGFP963 (N=19)-treated neurons. Student’s t-test, ^**P<0.005. (d) Representative recordings of miniature excitatory events and mean mEPSC frequency and amplitude in DMSO- (N=10) and RGFP966 (N=9)-treated neurons. Error bars reflect SEM.

Figure 4

Simultaneous inhibition of histone deacetylase (HDAC)-1, -2, and -3 increased spine density in CA1 pyramidal neurons and activated a transcriptional program supportive of synaptogenesis. (a) Schematic time line of experiment. (b) Representative images of dendritic spines from Thy1-GFP(m) neurons from CA1 region of hippocampus and mean spine density following RGFP963 treatment (N=5 per group). (c) Representative images of dendritic spines and mean spine density following RGFP966 treatment. Student’s t-test, *P<0.05. (d) Systemic injection of RGFP963 and RGFP966 induces both overlapping and unique expression patterns in the dorsal hippocampus. Roughly 20% of regulated genes were shared in both treatment groups. N=3 per group. (e) Predicted pathways with synapse-related functions from the RGFP963- and RGFP966-regulated transcriptomes. (f) A subset of genes were selected for qPCR validation and their expression patterns closely mirrored RNA-seq findings. *P<0.05, one-way ANOVA comparing vehicle, RGFP963, and RGFP966 groups. *P<0.05, one-tailed t-test between vehicle group and RGFP963 or RGFP966 group. ^#P<0.05, two-tailed t-test between RGFP963 and RGFP966 groups. t indicates trend (0.05<P<0.10). N=3 per group, ±SEM. (g) Relative expression of the class I HDAC isoforms in dorsal hippocampus tissue. Error bars reflect SEM.

Figure 5

Simultaneous inhibition of histone deacetylase (HDAC)-1, -2, and -3, but not HDAC3 alone, rescued memory deficits in APPswe/PS1dE9 mice. (a) Schematic time line of experiment. (b) Freezing behavior of mice treated with vehicle (WT: N=10, APP/PS1: N=6) or RGFP963 (WT: N=8, APP/PS1: N=7). (c) Freezing behavior of mice treated with vehicle (WT: N=15, APP/PSI: N=15) or RGFP966 (WT: N=15, APP/PS1: N=15). Two-way ANOVA analysis with Fischer LSD post hoc, *P<0.01. Error bars reflect SEM.