<i>Hdac3</i> genetic reduction does not reverse transcriptional dysregulation in R6/2.

<p>(A) Expression of <i>Bdnf</i> transcripts from different promoters (<i>Bdnf I</i>, <i>IV</i> and <i>V</i>) and the coding region (<i>Bdnf B</i>) in the cortex are represented as a percent of WT expression levels. With the exception of a slight decrease in <i>Bdnf</i> V, <i>Hdac3</i> reduction did not affect <i>Bdnf</i> expression. (B) Expression levels of genes specifically altered in the cerebellum of R6/2 mice are represented as a percent of WT expression. No significant difference was induced by <i>Hdac3</i> genetic reduction. (C) Expression levels of genes specifically altered in the striatum of R6/2 mice are represented as a percent of WT expression. A significant decrease in the expression of <i>Cnr1</i> in non-transgenic animals was observed as well as a slightly significant increase in <i>Cnr1</i> expression in R6/2 striata. Expression of the R6/2 transgene in Dbl brains is represented as a percent of that in R6/2 brains for cortex (D), cerebellum (E) and striatum (F). <i>Hdac3</i> reduction did not induce a significant change in transgene expression. Error bars correspond to S.E.M. (n = 8) *p<0.05. The same color code (blue = WT; red = Hdac3; green = R6/2 and purple = Dbl) was used for all the graphs. <i>Bdnf I, IV V</i>, brain derived neurotrophic factor promoter I, IV, V; <i>Bdnf B</i>, brain derived neurotrophic factor coding exon B; <i>Igfbp5</i>, insulin-like growth factor binding protein 5; <i>Kcnk2</i>, potassium channel subfamily K, member 2; <i>Nr4a2</i>, nuclear receptor subfamily 4, group A, member 2; <i>Pcp4</i>, Purkinje cell protein 4; <i>Uchl1</i>, ubiquitin C-terminal hydrolase L1; <i>Cnr1</i>, cannabinoid receptor 1; <i>Darpp32</i>, dopamine and cAMP regulated neuronal phosphoprotein; <i>Drd2</i>, dopamine D2 receptor; <i>Penk1</i>, proenkephalin.</p

Hdac3 genetic reduction does not reduce HTT aggregation in R6/2 mouse brain.

<p>(A) The SEPRION ligand based ELISA assay was used to quantify HTT aggregation in the cortex, hippocampus and brain stem of 4, 9 and 15 week-old mice. The graphs represent the microtitre-plate reading of R6/2 (green) and Dbl (purple) lysates. Background readings obtained with WT and <i>Hdac3</i> lysates were comparable to water. Aggregation levels augment with age but are not modified by <i>Hdac3</i> reduction. Error bars correspond to S.E.M. (n>6). (B) Representative western blot of hippocampal lysates at 4, 9 and 15 weeks of age. The aggregated HTT fraction (stacking gel) augments with age whereas the soluble fraction decreases with age. α-tubulin was used as a loading control. (C) Quantification of (B). Soluble HTT is represented as a percentage of the soluble fraction in R6/2. Error bars correspond to S.E.M. (n = 6). The same color code (R6/2 = green; Dbl = purple) is used in (A) and (C).</p

Generation of an <i>Hdac3</i> convention knock-out allele.

<p>(A) Strategy to generate an <i>Hdac3</i> conventional knock-out allele. The genomic structure and the targeting vector are shown. The <i>Hdac</i>3 gene contains 15 exons (blue rectangles). LoxP sites (red triangles) were introduced upstream exon 11 and within exon 15. The vector contains a 5′ homology arm covering the exonic and intronic region from intron 3–4 to intron 10–11 and a 3′ homology arm covering a part of exon 15 and the 3′UTR (green rectangle). The conditional knock-out region (yellow rectangle) covers exon 11 to 14 and 5′ end of exon 15. This conditional allele was introduced by homologous recombination in ES cells. The neomycine cassette (pink rectangle) flanked by 2 LoxP sites was removed by electroporation of Cre recombinase in ES cells and the cells containing the allele corresponding to a complete deletion of exon 11 to 14 were selected. Primers used for genotyping are represented as black arrows. F1 = forward 1; F2 = forward 2; R = Reverse. (B) Representative genotyping PCR on mouse genomic DNA. Duplex PCR with F1, F2 and R primers detects both the WT (250 bp band with primers F2/R) and the knock-out (500 bp with primers F1/R) allele.</p

<i>Hdac3</i> genetic reduction does not modify R6/2 phenotypes.

<p>(A) Weight loss in males (left panel) and females (right panel) are shown between 4 and 15 weeks of age. <i>Hdac3</i> genetic reduction did not induce a significant increase of the body weight in R6/2 (B) RotaRod performance is represented as the average latency to fall in each group at 4, 8, 10, 12 and 14 weeks. <i>Hdac3</i> genetic reduction did not ameliorate the impairment in RotaRod performance in R6/2 (C) Average grip strength in each group is represented at 4, 11, 12, 13 and 14 weeks. <i>Hdac3</i> genetic reduction did not induce a significant improvement in the grip strength in R6/2 mice (D) Average activity for each genotype is shown at 5 (upper panel) and 13 (lower panel) weeks of age. <i>Hdac3</i> genetic reduction did not reverse the hypoactivity observed in R6/2 mice (E) Average brain weight for each group was measured at 15 weeks of age. <i>Hdac3</i> genetic reduction did not modify the brain weight loss in R6/2 but a slight increase in brain weight was observed in WT animals **p<0.01. Error bars correspond to SEM (n>12). The same color code (blue = WT; red = Hdac3; green = R6/2 and purple = Dbl) was used for all measured parameters.</p

<i>Hdac3</i> mRNA and protein expression in <i>Hdac3+/−</i> heterozygous mouse brain.

<p>(A) <i>Hdac3</i> mRNA expression levels in 15 week old mouse cortex, cerebellum and striatum are shown as a relative expression ratio to the WT level. <i>Hdac3^+/−</i> (red) and Dbl (purple) mice express the mRNA at 50% of the WT (blue) and R6/2 (green) levels in all brain regions. Error bars correspond to S.E.M. (n = 8) ***p<0.001. (B) Western blot showing the expression of HDAC3 protein in the cytoplasmic (C) and the nuclear (N) fraction of WT mouse whole brain. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used to control for the purity of the fractions. (C) Representative western blot and (D) quantification of cytoplasmic and nuclear fraction prepared from WT (blue), <i>Hdac3^+/−</i> (red), R6/2 (green) and Dbl (purple) 15 week old-mouse whole brains. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used as both purity and loading controls. Cytoplasmic HDAC3 was not affected by <i>Hdac3</i> deletion whereas nuclear HDAC3 was reduced to 60% of the WT level. Error bars correspond S.E.M. (n = 3) *p<0.05.</p

MicroRNA signatures of endogenous Huntingtin CAG repeat expansion in mice

<div><p>In Huntington's disease (HD) patients and in model organisms, messenger RNA transcriptome has been extensively studied; in contrast, comparatively little is known about expression and potential role of microRNAs. Using RNA-sequencing, we have quantified microRNA expression in four brain regions and liver, at three different ages, from an allelic series of HD model mice with increasing CAG length in the endogenous Huntingtin gene. Our analyses reveal CAG length-dependent microRNA expression changes in brain, with 159 microRNAs selectively altered in striatum, 102 in cerebellum, 51 in hippocampus, and 45 in cortex. In contrast, a progressive CAG length-dependent microRNA dysregulation was not observed in liver. We further identify microRNAs whose transcriptomic response to CAG length expansion differs significantly among the brain regions and validate our findings in data from a second, independent cohort of mice. Using existing mRNA expression data from the same animals, we assess the possible relationships between microRNA and mRNA expression and highlight candidate microRNAs that are negatively correlated with, and whose predicted targets are enriched in, CAG-length dependent mRNA modules. Several of our top microRNAs (<i>Mir212</i>/<i>Mir132</i>, <i>Mir218</i>, <i>Mir128</i> and others) have been previously associated with aspects of neuronal development and survival. This study provides an extensive resource for CAG length-dependent changes in microRNA expression in disease-vulnerable and -resistant brain regions in HD mice, and provides new insights for further investigation of microRNAs in HD pathogenesis and therapeutics.</p></div

MicroRNA whose DE validates in Series 1 and Series 2.

<p>The heatmap represents differential expression Z statistics in all binary genotype comparisons for those microRNAs whose association with Q (as a numeric variable) passes the threshold FDR<0.05 in both Series 1 and Series 2. Top left and right panels show validated down- and up-regulated striatum microRNAs, respectively; bottom left and right panels show all validated microRNAs in cortex and cerebellum, respectively.</p

MicroRNA-module pairs with strongest negative correlations and significant (FDR<0.05) enrichment of the module in predicted microRNA targets.

<p>MicroRNA-module pairs with strongest negative correlations and significant (FDR<0.05) enrichment of the module in predicted microRNA targets.</p

Differential expression analysis with respect to CAG length in Series 2.

<p>Bars show numbers of significantly (FDR<0.05) differentially expressed microRNAs in the 4 tissues studied in Series 2; the numbers are also shown next to each bar. Numbers in parentheses represent the counts of distinct microRNA clusters in which the significant microRNAs fall into. Blue and red bars represent microRNAs significantly down- and up-regulated, respectively, with increasing CAG length (Q).</p

Network plots of top hub genes in CAG length-dependent modules and their putative regulator microRNAs.

<p>Each panel shows the top hub genes in one of the CAG length-dependent modules and microRNAs that are negatively correlated with the module eigengene and whose predicted targets are significantly enriched in the module. Predicted microRNA-target relationships are indicated by turquoise lines while the gene-gene co-expression relationships are indicated by red lines (thicker and wider lines indicate higher Topological Overlap). Only modules and microRNAs from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190550#pone.0190550.t001" target="_blank">Table 1</a> are shown whose correlations are less than -0.4; correlations of all mRNA-microRNA pairs shown in this figure are negative.</p