Histone deacetylase inhibitor MS-275 restores social and synaptic function in a Shank3-deficient mouse model of autism

Abstract

Autism is a neurodevelopmental disorder characterized by social deficits and repetitive behaviors. Genetic screening has identified synaptic, transcriptional, and chromatin genes disrupted in autistic patients. Haploinsufficiency of Shank3, which encodes a scaffold protein at glutamatergic synapses, is causally linked to autism. Using a Shank3-deficient mouse model that exhibits prominent autism-like phenotypes, we have found that histone acetylation in the prefrontal cortex (PFC) is abnormally low, which can be reversed by MS-275 (also known as Entinostat, SNDX-275), a class I histone deacetylase (HDAC) inhibitor that is selectively potent in PFC. A brief (3-day) treatment with MS-275 (i.p.) led to the sustained (11 days) rescue of autistic social preference deficits in Shank3-deficient mice, without altering locomotion, motor coordination, anxiety, or the increased grooming. MS-275 treatment also rescued the diminished NMDAR surface expression and NMDAR function induced by Shank3 deficiency. Moreover, F-actin at synapses was restored and the transcription of actin regulators was elevated by MS-275 treatment of Shank3-deficient mice, which may contribute to the recovery of actin-based NMDAR synaptic delivery. Taken together, these results suggest that MS-275 treatment could normalize the aberrant epigenetic regulation of genes, leading to the amelioration of synaptic and social deficits associated with autism.

Fig. 1

Treatment with the HDAC inhibitor MS-275 rescues autism-like social deficits in Shank3-deficient mice. a Immunoblots and quantification analysis of the level of acetylated H3 and total H3 in the nuclear fraction of cortical slices from WT or Shank3^+/ΔC mice treated with MS-275 (5 mg/kg, i.p., 3x) or saline. Immunoblotting was performed at 3–5 days post injection. *p < 0.05, one-way ANOVA. b Bar graphs showing the time spent investigating either the social (Soc) or non-social (NS) stimulus during three-chamber sociability testing in juvenile male Shank3^+/ΔC mice or wild-type (WT) mice treated with MS-275 (5 mg/kg, i.p., 3x) or saline. ***p < 0.001, saline vs. MS-275; ⁺⁺⁺p < 0.001, Soc vs. NS, two-way ANOVA. c Bar graphs showing the preference index of the sociability testing in WT or Shank3^+/ΔC mice treated with MS-275 or saline. ***p < 0.001, two-way ANOVA. d Representative heat maps illustrating the time spent in different locations of the three chambers (blue: 0 s; red: ~20 s) from the social preference tests of all groups. Locations of Soc and NS stimuli are labeled with the circles. e Plots of social preference index in Shank3^+/ΔC mice treated with MS-275 (5 mg/kg, i.p., 3x) or saline at different time points. ***p < 0.001, saline vs. MS-275; ^###p < 0.001, pre- vs. post injection, two-way rmANOVA. f Scatter plots and summary (mean ± SEM) showing the preference index of the sociability testing in Shank3^+/ΔC mice before and after the treatment with different doses of MS-275 (0.05, 0.5, and 5 mg/kg, i.p., 3x). ***p < 0.001, paired t-test

Fig. 2

MS-275 treatment does not alter other behaviors of Shank3-deficient mice. a–d Bar graphs (mean ± SEM) showing a variety of behaviors in WT, saline-injected Shank3^+/ΔC and MS-275 (5 mg/kg, i.p., 3x)-treated Shank3^+/ΔC mice, including the number of midline crossing in locomotion tests (a), the latency to fall during two trails of rotarod tests (b), the time spent in the center in open field tests (c), and the time spent self-grooming (d). ***p < 0.001, one-way ANOVA

Fig. 3

MS-275 treatment restores NMDAR surface expression in Shank3-deficient mice. a Quantitative real-time RT-PCR data on the mRNA level of NMDAR and AMPAR subunits in PFC slices from WT mice or Shank3^+/ΔC mice treated with MS-275 (5 mg/kg, i.p., 3x) or saline. b, c Immunoblots and quantification analysis of the total (b) and surface (c) protein level of NMDAR and AMPAR subunits in PFC slices from WT or Shank3^+/ΔC mice treated with MS-275 or saline. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA

Fig. 4

MS-275 treatment restores NMDAR function in Shank3-deficient mice. a Representative traces of NMDAR-mediated ionic currents in cultured cortical neurons (DIV 21) transfected with control siRNA or Shank3 siRNA treated with MS-275, MGCD0103, or vehicle. Scale bars: 200 pA, 0.5 s. b Bar graph summary of the NMDAR current density in cortical cultures with different transfections. ***p < 0.001, t-test. c Input–output curves of NMDAR-EPSC in PFC pyramidal neurons from WT vs Shank3^+/ΔC mice treated with MS-275 (5 mg/kg, i.p., 3x) or saline. Recordings were performed at 3–5 days post injection. Inset: representative NMDAR-EPSC traces. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA

Fig. 5

MS-275 treatment of Shank3-deficient mice restores actin filaments at synaptic membrane and increases the transcription of actin regulators. a, b Immunoblots and quantification (mean ± SEM) showing actin in the total lysates, triton-soluble synaptic cytosolic fraction (G-actin) and triton-insoluble synaptic membrane fraction (F-actin) in PFC slices from WT mice or Shank3^+/ΔC mice treated with MS-275 (5 mg/kg, i.p., 3x) or saline. *p < 0.05, one-way ANOVA. c, d Quantitative RT-PCR data on the mRNA level of actin regulators in PFC slices from WT mice or Shank3^+/ΔC mice treated with MS-275 (5 mg/kg, i.p., 3x) or saline. *p < 0.05, one-way ANOVA. e PCR image showing the input (total DNA), ChIP (AcetyH3-occupied DNA), and no-template control (NTC) signals with a pair of primer designed against the promoter regions of βPIX. f ChIP assay data showing the acetylated H3 level at βPIX promoter in PFC lysates from saline-injected WT, saline-injected Shank3^+/ΔC, and MS-275-treated Shank3^+/ΔC mice. **p < 0.01, one-way ANOVA