HDAC4 reduction improves the electrophysiological characteristics of MSNs and corticostriatal synaptic function.

<p>(A–H) Box and whisker plots of (A, E) input resistance (R_m), (B, F) RMP, (C, G) rheobase current, and (D, H) spike amplitude from corticostriatal slices at (A–D) 7–8 and (E–H) 12 wk of age. Box and whisker plots: +, mean; box, interquartile range; whisker, 10–90 percentile; outliers, closed circles. (I) Measurement of evoked EPSCs following cortical stimulation showed that the R6/2 mice show lower basal transmission compared to WT or <i>Hdac4</i>HET and that this is restored in the Dble::R6/2 mice at 7–12 wk of age. (J) R6/2 MSNs have a higher paired-pulse ratio than WT (interstimulus interval, 20 ms), indicating reduced glutamate release probability. This is fully restored in Dble::R6/2 mice at 12 wk of age. (K) Representative traces of miniature EPSCs (mEPSCs) at 8 wk. R6/2 mice show strongly depressed mEPSC frequency, which is significantly rescued in the Dble::R6/2 mice. (L, M) Average cumulative plot of mEPSC interevent interval (0.1 s bins) (L) or amplitude (1 pA bins) (M); <i>n</i> = 11–12 for all four genotypes. Statistical analysis was performed by (A–H and J) one-way ANOVA with Tukey's multiple comparison test, (I) two-way ANOVA with Bonferroni multiple comparison test, and (L, M) Kolmogorov–Smirnov (KS) test. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

HDAC4 knock-down does not rescue global transcriptional dysregulation.

<p>(A) Affymetrix arrays were used to determine the effect of <i>Hdac4</i> knock-down on the cortical transcription profile of WT and R6/2 mice at 9 and 15 wk of age (<i>n</i>≥8 per genotype per time point). The number of genes that were significantly altered between genotypes with a fold-change of >30% for each pairwise comparison is noted. Statistical significance was determined after FDR-correction at a stringency of <i>p</i>≤0.05. (B) Taqman qPCR validation of the genes that were predicted to be differentially expressed between R6/2 and Dble::R6/2 cortex at 9 wk of age. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001717#pbio.1001717.s006" target="_blank">Table S3</a> for gene abbreviation definitions. (C) Cortical <i>Bdnf</i> mRNA levels for promoter transcripts 1, 2a, 4, and 5 as well as the coding exon (B) were assessed by Taqman qPCR at 15 wk of age. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: <i>Atp5b</i>, <i>Canx</i>, and <i>Rpl13a</i>. Error bars are S.E.M (<i>n</i> = 8). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; NS, not significant.</p

<i>Hdac4</i> reduction does not alter the expression levels of <i>HTT</i> exon 1, endogenous <i>Htt</i>, or other <i>Hdacs</i>.

<p>(A) Breeding scheme used to reduce <i>Hdac4</i> levels in both R6/2 and heterozygous <i>Hdh</i>Q150 mice. WT, wild type; <i>Hdac4</i>HET, <i>Hdac4</i>KO heterozygotes; Dble::R6/2, R6/2 mice heterozygous for <i>Hdac4</i>KO; Dble::<i>Hdh</i>Q150, <i>Hdh</i>Q150 mice heterozygous for <i>Hdac4</i>KO. (B) <i>Hdac</i>4 transcript levels were decreased in <i>Hdac</i>4HET, Dble::R6/2, and Dble::<i>Hdh</i>Q150 mice as measured by Taqman qPCR. (C) Taqman qPCR showed that <i>HTT</i> exon 1 transgene levels did not differ between R6/2 and Dble::R6/2 mice. (D) Taqman qPCR showed that the expression of endogenous <i>Htt</i> was equivalent between WT and <i>Hdac4</i>HETs and did not change when <i>Hdac4</i> was knocked down in R6/2 or <i>Hdh</i>Q150 mice. (E) The transcript levels of other <i>Hdacs</i> were equivalent to WT levels in <i>Hdac</i>4HET, R6/2, and Dble::R6/2 mice as determined by Taqman qPCR. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: <i>Atp5b</i>, <i>Canx</i>, and <i>Rpl13a</i>. Error bars are SEM using Student's <i>t</i> test (<i>n</i> = 8). **<i>p</i><0.01; ***<i>p</i><0.001.</p

HDAC4 knock-down delays aggregate formation in R6/2 and <i>Hdh</i>Q150 mouse models of HD.

<p>(A) Seprion ligand ELISA was used to quantify aggregate load in the cortex of R6/2 and Dble::R6/2 mice at 4, 9, and 15 wk of age. Values for the Dble::R6/2 mice were plotted as a percentage of R6/2 aggregate load (<i>n</i> = 6). (B) TR-FRET was used to determine the levels of soluble exon 1 HTT in the cortex of R6/2 and Dble::R6/2 mice at 4, 9, and 15 wk of age (<i>n</i> = 6). (C) Seprion ligand ELISA was used to quantify aggregate load in the striatum, cortex, and cerebellum of <i>Hdh</i>Q150 and Dble::<i>Hdh</i>Q150 mice at 6 and 10 mo of age. Values for the Dble::<i>Hdh</i>Q150 mice were plotted as a percentage of aggregate load of <i>Hdh</i>Q150 mice (<i>n</i>≥7). (D) Representative S830 immunoblot of cortical lysates showing the difference in soluble and aggregated exon 1 HTT between R6/2 and Dble::R6/2 (Dble) mice and how this change occurs with age. (E) Comparison of HDAC4 levels in the nuclear and cytoplasmic fractions of R6/2 and Dble::R6/2 (Dble) brains by western blot. The purity of the fractions is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001717#pbio.1001717.s002" target="_blank">Figure S2D</a>. (F) Western blot of detergent-insoluble high molecular weight (HMW) aggregates isolated from the nuclear and cytoplasmic fractions of R6/2 and Dble::R6/2 (Dble) brains, resolved by agarose gel electrophoresis (AGERA), and immunodetected with the S830 antibody (representative of three experiments) (<i>n</i> = 8). The purity of the fractions is shown by western blotting with α-tubulin and histone H3. (G) Western blot of HDAC4 in the cytoplasmic fraction of R6/2 and Dble::R6/2 (Dble) brains at 9 wk of age. HDAC4 levels were measured by densitometry and calculated relative to α-tubulin. Error bars are SEM. <i>p</i> values were calculated using Student's <i>t</i> test.</p

HDAC4 interacts with mutant huntingtin <i>in vitro</i> and <i>in vivo</i> and colocalizes with cytoplasmic inclusions.

<p>(A) GST pull-down assays revealed that HDAC4 interacts with mutant (53Q) but not WT (20Q) exon 1 HTT. In contrast HDAC5 weakly interacts with both mutant and WT exon 1 HTT. The coomassie stained gel shows the exon 1 HTT GST fusion proteins that were used to pull-down ³⁵S-methioine labelled recombinant HDAC4 or HDAC5. (B) Western blot probed for HTT (MAB2166 or MW1) after immunoprecipitation with HDAC4 (DM-15) from brain tissue from 8-wk-old WT and <i>Hdh</i>Q150 heterozygous and homozygous mice (representative picture of three independent experiments). (C) Western blot probed for mutant HTT (MW1) after immunoprecipitation with HDAC4 (H-92) from brain tissue from 8-wk-old WT, <i>Hdh</i>Q20, and <i>Hdh</i>Q80 homozygous mice. (D) Western blot probed for mutant HTT (MAB2166 or MW1) after immunoprecipitation with HDAC5 (ab56929) from brain tissue from 8-wk-old WT and <i>Hdh</i>Q80 homozygous mice. (E) Representative immunofluorescence images of cortex from 14-wk-old R6/2 and 23-mo-old <i>Hdh</i>Q150 mice immunostained for mutant HTT (S830) and HDAC4 (CS2072) and counterstained with DAPI. A similar pattern of cytoplasmic co-localisation was also seen in the striatum and hippocampus. Scale bar, 15 µm. IP, immunoprecipitation; ID, immunodetection.</p

HDAC4 knock-down improves neurological phenotypes and extends survival.

<p>(A) Reduction in HDAC4 results in a pronounced delay in impaired motor coordination as determined by rotarod performance (<i>n</i>≥13/genotype). (B) Assessment of neurological phenotypes via a modified SHIRPA protocol (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001717#pbio.1001717.s005" target="_blank">Table S2</a> for details). Of the nine parameters that distinguished R6/2 and WT mice, all were improved in the Dble::R6/2 mice. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001717#pbio.1001717.s009" target="_blank">Video S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001717#pbio.1001717.s010" target="_blank">Video S2</a>. (C) Kaplan–Meier analysis showed that knock-down of HDAC4 significantly increased R6/2 survival (<i>n</i>≥13/genotype). (D) Reduction of HDAC4 did not result in an overall improvement in the failure of R6/2 mice to gain weight, although Dble::R6/2 mice were significantly heavier than R6/2 mice at 15 wk of age (<i>n</i>≥13/genotype). (E) There was a slight but statistically significant increase in the brain weight of Dble::R6/2 as compared to R6/2 mice at 4 and 9 wk of age (<i>n</i>≥10/genotype). Statistical analysis was performed by GLM-ANOVA with Greenhouse Geisser <i>post hoc</i> analysis (B and D), by multiple comparisons using Bonferroni post hoc test (B and D), and by log-rank test (C).</p