Fig 1: Neuronal activity and persistent inflammatory nociception trigger the nuclear export of HDAC4 and histone 3 acetylation in spinal cord neurons.a Representative images of cultured primary spinal cord neurons (DIV10) immunostained for endogenous HDAC4 (green) and the neuronal marker NeuN (red); Hoechst (blue) marks the nuclei. Scale bar is 20 µm. b Quantification of the relative fluorescence intensity of the nuclear HDACs signal in spinal cord neurons normalized to respective controls. Each point represents the mean value derived from one independent experiment. Ca. 40 cells were analyzed per condition and experiment (HDAC1 n = 4 F(3,8) = 1.120; HDAC3 n = 4 F(3,12) = 0.9239; HDAC4 n = 6 F(3,20) = 8.676; HDAC5 n = 4 F(3,12) = 3.210; HDAC7 n = 6 F(3,20) = 2.726; HDAC9 n = 5 F(3,20) = 0.7916; HDAC10 n = 4 F(3,8) = 2.502; HDAC11 n = 4 F(3,12) = 0.6623). c Quantification of the relative fluorescence intensity of the nuclear signal of acetylated histone H3-Lys9 (AcH3) in spinal cord neurons, treated as indicated. Ca. 30 cells were analyzed per sample (n = 4 F(3,12) = 5.854). d Representative images of primary cultured spinal cord neurons (DIV10) immunostained for AcH3 (green). Cells were treated with Bic (50 µM) for the indicated times. Nuclei were labelled with Hoechst (blue) and NeuN (red) as a neuronal marker. Scale bar is 40 µm. e Representative images of spinal cord dorsal horn sections of lumbar spinal segments L3–5 of mice 24 h after intraplantar CFA or saline injection, immunostained for HDAC4 or acetylated histone H3-Lys9 (AcH3) shown in green; NeuN (red). Nuclei were labeled with Hoechst (blue) and scale bar is 40 µm. Higher magnifications of the upper laminae are shown in the right panels with a scale bar of 40 µm. f Quantifications of the average nuclear fluorescence intensities from immunolabeled HDACs and AcH3 in neurons of the spinal cord dorsal horn, normalized to values obtained from saline-injected mice and integrated over a time course series (n = 4 mice per condition and timepoint). Timepoints used were 0.5, 2, 6, and 24 h. Statistically significant differences were determined by one-way ANOVA followed by Dunnett’s post hoc test (b, c) or two-tailed Student’s t-test (f). ***p < 0.001; **p < 0.01; *p < 0.05. In bar graphs, each point represents a value derived from one independent culture or mouse. Graphs represent mean ± SEM. See also Supplementary Figs. 1 and 2.
Fig 2: SAHA specifically down regulates Hdac7 mRNA levels. Hdac7 expression is decreased in Hdac7+/− mice and not altered by the presence of the R6/2 transgene. (A) Hdac7 mRNA expression levels are shown as relative expression ratios to the geometric mean of the Actb and Grin1 housekeeping genes in 13 week old mouse cerebellum from SAHA-treated WT (grey) and R6/2 (black) mice together with vehicle treated WT (white) and R6/2 (striped) mice (n>8 per genotype). Error bars are S.E.M. (B, C) Hdac7 expression level is shown as a relative expression ratio to the geometric mean of three housekeeping genes in (B) striatum and (C) cerebellum of 14 week old wild-type (Wt, white), Hdac7+/− knockdown (Hdac7) (grey), R6/2 (black) and R6/2 mice with Hdac7+/− knockdown (R6/2-Hdac7, striped) mice (n>8 per genotype). Error bars are S.E.M. (D) Representative western immunoblot of 50 µg of cortical homogenate from 14 week old wild-type (WT), R6/2, Hdac7+/− and R6/2-Hdac7+/− mice. Blots were probed with an antibody that recognizes Hdac7 (120 kDa) and a non-specific band (70 kDa). (E) Quantification of Hdac7 protein expression levels in WT (white) and Hdac7+/− (grey) mice. Quantification was performed on blots containing four samples per genotype using the non-specific band for reference. Blots were additionally probed with an antibody to α-tubulin to confirm equal protein loading (data not shown). Error bars represent standard deviation from the mean (n = 4). * p<0.05, ** p<0.01, *** p<0.001.
Fig 3: HDAC7 is present in the nucleus and cytoplasm of neuronal cells in the mouse striatum.Representative confocal microscopy images of coronal sections of brains from wild-type (WT) and R6/2 transgenic mice at 14 weeks of age immuoprobed with antibodies to NeuN to identify neurons and with anti-HDAC7. Nuclei were visualized using TO-PRO-3 (blue). Scale bar 20 µm.
Fig 4: HDAC7 genetic reduction does not alter R6/2 physiological or behavioural phenotypes.The R6/2 phenotypes (A) weight loss, (B) hypothermia, (C) impaired RotaRod performance and (D) reduced grip strength are not ameliorated by genetic knock-down of Hdac7 expression. Error bars represent S.E.M. Shown to the right of each graph are the P values arising from GLM repeated measures ANOVA analysis of the data.
Fig 5: Comparable changes in striatal and cerebellar gene expression changes between R6/2 and R6/2-HDAC7 mice.Relative expression ratios to the geometric mean of three housekeeping genes [44] for transcripts in striatum (A) and cerebellum (B) of 15 week old WT (white), Hdac7+/− (grey), R6/2 (black) and R6/2-Hdac7 (striped) mice. Error bars correspond to S.E.M. (n>8). * P<0.05. Hdac7 genotype does not modulate R6/2 transgene expression level in striatum (C) or cerebellum (D) of 15 week old mice R6/2 (black) and R6/2-Hdac7+/− (striped). Error bars correspond to S.E.M. (n>8). Cnr1, cannabinoid receptor 1; Darpp32, Dopamine and cAMP regulated neuronal phosphoprotein; Drd2, dopamine D2 receptor; Penk1, proenkephalin; Psme1, proteasome activator subunit 1 (PA28 alpha) Reg alpha; Uchl1, ubiquitin C-terminal hydrolase L1; Igfbp5, insulin-like growth factor binding protein 5.
Supplier Page from MilliporeSigma for Anti-Histone Deacetylase 7 (HDAC7) (KG-17) antibody produced in rabbit