Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington's disease mouse model

Abstract

The present study describes evaluation of epigenetic regulation by a small molecule as the therapeutic potential for treatment of Huntington's disease (HD). We identified 5-allyloxy-2-(pyrrolidin-1-yl)quinoline (APQ) as a novel SETDB1/ESET inhibitor using a combined in silico and in vitro cell based screening system. APQ reduced SETDB1 activity and H3K9me3 levels in a HD cell line model. In particular, not only APQ reduced H3K9me3 levels in the striatum but it also improved motor function and neuropathological symptoms such as neuronal size and activity in HD transgenic (YAC128) mice with minimal toxicity. Using H3K9me3-ChIP and genome-wide sequencing, we also confirmed that APQ modulates H3K9me3-landscaped epigenomes in YAC128 mice. These data provide that APQ, a novel small molecule SETDB1 inhibitor, coordinates H3K9me-dependent heterochromatin remodelling and can be an epigenetic drug for treating HD, leading with hope in clinical trials of HD.

Keywords: Histone H3K9me3-specific transferase; Huntington’s disease; SETDB1; medium spiny neuron; motor function.

Graphical abstract

Figure 1.

Structures of recently reported SETDB1 inhibitors, nogalamycin and VH06.

Figure 2.

Primary screening of our compounds via cell-based and dot blot assay. (a) A diagram representing a generation of Tet-inducible SETDB1 cell line. (b, c) SETDB1 induction by doxycycline (Doxy) increased H3K9me3 level. (d) SETDB1 inhibitor candidates were determined by the dot blot assay. Inhibitors were treated to cells (in that SETDB1 was already induced by Doxy for 24 h) for 12 h. Histone protein was isolated by acidic extraction method and H3K9me3 level.

Scheme 1.

Synthesis of APQ. Reagents and conditions: (a) NaH, allyl bromide, 0–23 °C, 40 min, 92%. (b) (i) mCPBA, CH₂Cl₂, 0–23 °C, 2 h; (ii) POCl₃, CH₂Cl₂, 50 °C, 7 h, 39%. (c) Pyrrolidine (neat), 90 °C, 12 h, 76%.

Figure 3.

APQ ligand interaction diagram inside the SET domain. (a) SET domain represented in ribbon format. APQ and SAM showed by space-filling cpk presentation in magenta and blue colour, respectively. The hydrogen bond donor–acceptor surface area has shown around APQ. (b) APQ in magenta stick format interacts with Tyr1265, Ala855, Tyr832, Val1232, Asp851, and Asn1266. Binding site residues showed in white stick format. Pi-pi stacking, pi-sigma, pi-alkyl, and hydrophobic interactions are shown in pink, purple, light pink, and light green colour, respectively. (c) Schematic representation of SETDB1 domain organisation.

Figure 4.

APQ reduces H3K9me3 level in HD striatal cells. (a) APQ decreases the level of H3K9me3 in HD (Q111/Q111) striatal cells. (b, c) APQ reduces the immunoreactivity of H3K9me3 (Q111/Q111) striatal cells. (d) FRET imaging analysis showing that APQ effectively modulates FRET/ECFP ratios of H3K9 methylation in Q111. APQ represses the transcriptional activity of Setdb1/Eset in a dose-dependent manner. The data represents the average of three separate experiments. Significantly different at *p< .05, **p< .001.

Figure 5.

APQ improves motor behaviour and neuronal atrophy in HD transgenic (YAC128) mice. (a) A scheme showing APQ administration and rotarod test schedule. (b) Rotarod test showed that APQ administration improves motor function in YAC128 mice. Significantly different from WT at *p<.05; **p<.01. Significantly different from YAC128 mice at ^#p<.05. (c) Nissl staining showed that APQ restored the size of medium spiny neurons in the striatum of YAC128 mice. (d) Quantitative analysis showed that APQ significantly increased the size of medium spiny neurons in the striatum of YAC128 mice. Significantly different from WT at **p<.01. Significantly different from YAC128 mice at ^##p<.01.

Figure 6.

SETDB1 inhibitor (APQ) reduces H3K9me3 immunoreactivity and level in the striatum of HD transgenic (YAC128) mice. (a) APQ administration significantly decreased medium H3K9me3 immunoreactivity in the striatum of YAC128 mice (n = 5) compared to vehicle-treated YAC128 mice (n = 5). (b) APQ administration significantly reduced H3K9me3 intensity in medium spiny neurons in the striatum of YAC128 mice. Significantly different from WT at **p<.01. Significantly different from YAC128 mice at ^##p<.01. (c) Acid histone extraction and Western blot analysis showed that APQ selectively decreased H3K9me3 level but not H3K9me1 and H3K9me2 levels in the striatum of YAC128 mice compared to vehicle-treated YAC128 mice. (d) Densitometry analysis showed that APQ significantly reduced H3K9me3 level in the striatum of YAC128 mice. H3K9me3 levels were normalised to histone H3 levels. Significantly different from WT at **p<.01. Significantly different from YAC128 mice at ^#p<.05.

Figure 7.

SETDB1 inhibitor (APQ) modulates medium spiny neuronal activity in the striatum of HD transgenic (YAC128) mice. (a, b) Confocal microscopy showed that APQ administration restored DARPP32 and calbindin immunoreactivity in the striatum of YAC128 mice. (c) Densitometry analysis showed that APQ significantly improved DARPP32 level in the striatum of YAC128 mice. Significantly different from WT at **p<.01. Significantly different from YAC128 mice at ^##p<.01. (d) Densitometry analysis showed that APQ significantly improved calbindin level in the striatum of YAC128 mice. (e) DAPI staining showed that APQ rescued the nuclei size in the striatum of YAC128 mice. (f) Densitometry analysis showed that APQ significantly improved the nuclei size in the striatum of YAC128 mice. Significantly different from WT at **p<.01. Significantly different from YAC128 mice at ^##p<.01.

Figure 8.

SETDB1 inhibitor (APQ) modulates H3K9me3-landscaped epigenomes in the striatum of HD transgenic (YAC128) mice. (a) A scheme illustrating H3K9me-ChIP sequencing analysis in APQ-administered YAC128 mice. (b) Volcano plots presented that H3K9me-landscaped epigenomes are altered in APQ-administered YAC128 mice. (c) Pie graphs exhibited that APQ affects the landscaping of H3K9me in the promoter regions of epigenomes in YAC128 mice. (d) Network analysis showed that APQ modulates remodelling of H3K9me-landscaped epigenomes (red nodules) that are associated with chromatin modification, transcription regulation, and nervous system development pathway in YAC128 mice. (e) Immunohistochemistry showed that APQ reduces SUV39H1 immunoreactivity (brown) in the striatal neurons of YAC128 mice. The nuclei (blue) were counterstained with haematoxylin. (f) Densitometry analysis showed that APQ significantly reduced SUV39H1 level in the striatal neurons of YAC128 mice. Significantly different from WT at **p<.01. Significantly different from YAC128 mice at ^##p<.01.