Gene expression changes in mouse brain following chronic HDAC inhibitor treatment.

<p>(<b>a</b>) Heatmaps illustrating transcript expression changes in mouse brain following chronic HDAC inhibitor treatment for 10 days. Cpd-60 (45 mg/kg, i.p.) significantly upregulated (red) or downregulated (blue) a similar number of transcripts in prefrontal cortex (PFC), nucleus accumbens (NAc), and hippocampus (HIP). Expression changes following SAHA treatment (25 mg/kg, i.p.) were predominantly localized to HIP. (<b>b</b>) Venn diagrams illustrate that only 1–10 genes were similarly regulated by Cpd-60 and SAHA treatment depending on brain region. Genes included in heatmaps and Venn diagrams have ≥1.2-fold expression change compared to vehicle (ANOVA p<0.05 with Tukey’s HSD <i>post hoc</i> test). (<b>c</b>) qPCR validation of a subset of genes with significantly altered expression following Cpd-60 treatment as determined by microarray analysis. *p<0.05, t-test of Cpd-60 or SAHA compared to vehicle.</p

<i>In vitro</i> and <i>in vivo</i> characterization of two structural classes of HDAC inhibitors.

<p>(<b>a</b>) Chemical structure of SAHA and Cpd-60. (<b>b</b>) <i>In vitro</i> IC₅₀ (µM) for HDAC 1-9 by SAHA and Cpd-60 using recombinant human HDAC enzymes and HDAC class-specific substrates. Inhibitor and substrate were incubated for 60 min (HDAC4-9) or 180 min (HDAC1-3)^a to control for HDAC1-3 inhibition by slow-binding test compounds. (<b>c</b>) <i>In vitro</i> binding affinity (K_i) and kinetics (half-life ‘T_1/2′ in minutes) for HDAC 1, 2 and 3 incubated with SAHA or Cpd-60 (10 µM). (<b>d</b>) H4K12 acetylation levels in HEK293 cells following 24 hr ‘constant’ exposure to DMSO, SAHA (20 µM) or Cpd-60 (20 µM) and 6 hr after drug ‘washout’ (media change) with tubulin loading control. (<b>e</b>) Dose response plots for induction of histone H4K12 acetylation in cultured primary mouse neuronal cells by SAHA or Cpd-60 for 24 hr. Cells with histone acetylation signals above an intensity threshold of >99.5% (“bright green cells”) are plotted as a percentage normalized to DMSO control. EC₅₀ values for H4K12 acetylation were 0.60 µM and 72 µM for SAHA and Cpd-60, respectively. (<b>f</b>) <i>In vivo</i> mouse brain pharmacokinetics following acute systemic administration of SAHA (25 mg/kg, i.p.) or Cpd-60 (45 mg/kg, i.p.).</p

Chromatin immunoprecipitation (ChIP) of nucleus accumbens from Cpd-60 treated mice.

<p>(<b>a</b>) Schematic of experimental design with 10-day administration of Cpd-60 (45 mg/kg, i.p.). (<b>b</b>) Chromatin was immunoprecipitated with an anti-histone H4K12ac-antibody followed by qPCR targeting regions 1.0 or 0.2 kB upstream or 0.5 kB downstream from the transcription start site (TSS). Transcripts upregulated by Cpd-60 treatment had increased histone acetylation at promoter regions upstream, but not downstream, of the TSS in Cpd-60 treated tissue compared to vehicle. *p<0.05, **p<0.01, t-test of Cpd-60 versus vehicle.</p

Effects of HDAC inhibitors on histone acetylation in mouse brain.

<p>Chronic SAHA (25 mg/kg, i.p.) or Cpd-60 (45 mg/kg, i.p.) significantly increased acetylation of histone H2B(tetra-acetylated), H3K9 and H4K12 in cortex, ventral striatum and hippocampus one hour after the 10^th daily treatment (arbitrary units, relative to vehicle control). Representative western blots are shown with total levels of histone H3 (H3pan) and histone H4 (H4pan) used as loading controls. *p<0.05, t-test of Cpd-60 or SAHA versus vehicle. <i>n = </i>6 mice/group.</p

Effect of Cpd-60 treatment on mood-related behaviors in mice.

<p>(<b>a</b>) Timecourse of locomotor activity in response to and (<b>b</b>) total locomotor activity summed over 80 min following acute amphetamine challenge (3.5 mg/kg, i.p.; Time ‘0’ indicated by arrow). Hyperlocomotion in response to amphetamine was significantly reduced in mice chronically treated with Cpd-60 (45 mg/kg, i.p.) but not with SAHA (25 mg/kg, i.p.). (<b>c</b>) Time spent immobile in the forced swim test was significantly decreased in mice treated chronically with Cpd-60 but not SAHA compared to vehicle treated control mice. *p<0.05, **p<0.01, ANOVA with Least Significant Difference <i>post hoc</i> test.</p