Figure 1.
HDAC3 inhibitors increase both soluble and insoluble Htt-ex1s but prefer long Qs.
A–F: Indicated amounts of HDAC3 inhibitors T247, T326, and T130 were added to C3 or C4 HeLa stable cell lines. The cells were harvested after 48 h of incubation and the fraction soluble in 1% Triton X-100 was subjected to western blot analysis (A–C). The insoluble fraction was subjected to a filter trap assay (D–E). Signals were detected by anti-GFP antibodies and chemiluminescence. Signal intensities were normalized to no inhibitors (DMSO only) = 100. The band from an anti-actin blot is shown as a loading control. Panels A, D: T247, B, E: T326, C, F: T130. *P≤0.05, **P≤0.01, ***P≤0.001 vs. 0×IC50 by ANOVA with multiple comparisons. N = 3. G, H: HDAC3 inhibitors do not increase Htt-ex1 mRNA levels. Effect of HDAC3 inhibitors on Htt-ex1 expression levels were assayed by qPCR. G: internal control = GAPDH, H: internal control = ACTB. Expression level was normalized to no inhibitor = 1.0. There was no statistical significance by ANOVA with multiple comparisons. N = 3.
Figure 2.
Effect of HDAC3 on cytoplasmic and nuclear Htt aggregates.
A: Aspartate at the 166th and 168th amino acid of HDAC3 is crucial for its activity. An empty plasmid (–), FLAG tagged wild-type (wt), or D166A + D168A mutant (DA) of HDAC3 were overexpressed in 293T cells. After immunoprecipitation using anti-FLAG antibodies, pan-histone deacetylase activity was measured by fluorometric analysis. *P≤0.05 vs. empty plasmid by ANOVA and multiple comparisons. N = 3. Anti-FLAG and anti-actin western blots from cell lysates are shown below. B–C: HDAC3 overexpression reduces nuclear Htt-ex1 aggregates. Empty vector (–), FLAG-tagged wild-type or DA mutant HDAC3 were transfected to E3 and N3 cells. Amount of aggregate measured by filter trap assay are shown in B and C. *significant against – and DA by ANOVA and multiple comparisons. N = 3. D–G: Empty vector (–), FLAG-tagged wild-type or DA mutant HDAC3 were transfected to E3 and N3 cells. Cells harboring inclusion bodies are counted and their fraction in total cells was plotted in 2D and E. Representative GFP images of low powered magnification fields are shown in 2F and 2G. *significant against – and DA by ANOVA and multiple comparisons. H: HDAC3 shRNA reduces HDAC3 amount by 70%. Molecular weight markers are shown at the left side. I–J: HDAC3 knockdown increases nuclear aggregates. HDAC3 shRNA was transfected into E3 or N3 cells and the 1% TritonX-100 insoluble fraction was subjected to filter trap assay. *P = 0.0003 by t-test. N = 3.
Figure 3.
HDAC3 inhibitors have differential effects on cytoplasmic and nuclear Htt-ex1 aggregates.
A: HDAC3 inhibitors increase aggregated nuclear Htt-ex1. For filter trap analysis, three independently made insoluble fractions were analyzed on one single membrane; thus, there are error bars shown for 0×IC50s. *P≤0.05, ***P≤0.001 vs. each 0×IC50 by ANOVA and multiple comparisons. N = 3. B: HDAC3 inhibitors reduce cytoplasmic soluble Htt-ex1s. The effect of various HDAC inhibitors on 1% TritonX-100 soluble cytoplasmic Htt-ex1s. Indicated amount of HDAC inhibitors were added to E3 (cytoplasmic) or N3 (nuclear) cells for 48 h. Quantitated band intensity was normalized to each band without HDAC inhibitors (0×IC50); thus, there are no error bars. **P≤0.01, ***P≤0.001 vs. each 0×IC50 by ANOVA with multiple comparisons. N = 3. Anti-actin blots are shown for loading control.
Figure 4.
HDAC3 preferably binds to nuclear Htt with long Qs.
A: GST-HDAC3 binds directly to either cytoplasmic or nuclear Htt-ex1. GST pull-down assay of E1, E2, E3 (cytoplasm), and N1, N2, N3 (nuclear) HeLa cell lysates is shown. Pulled-down fraction was analyzed by anti-GFP or GST antibodies. *Non-specific band. B: HDAC3 immunoprecipitates almost exclusively with nuclear Htt-ex1s. E1, E3 (cytoplasm), N1, and N3 (nuclear) HeLa cells were transfected with FLAG-tagged HDAC3 and those lysates were immunoprecipitated with anti-FLAG antibodies immobilized to protein G agarose beads. The pre-IP fraction and the IPed fraction were analyzed using anti-FLAG or anti-GFP antibodies. Molecular weight markers are shown on the left. C: HDAC3 associates exclusively with nuclear inclusion bodies. E3 or N3 cells were fixed and stained with anti-HDAC3 antibodies and visualized by Alexa 546 conjugated secondary antibodies. Arrowheads: inclusion bodies with no HDAC3 signals associated. Arrows: HDAC3 signal-associated inclusion bodies. Bar = 20 µm.
Figure 5.
HDAC3 inhibitors affect proteasome activity.
A–C: HDAC3 inhibitors inhibit proteasome activity. Three different HDAC3 inhibitors were added to HeLa cell cultures at the indicated concentrations. After 48 h of incubation, the proteasome activity of 5 µg total protein in a PBS lysate was measured using a fluorometric assay. A: T247, B: T326, C: T130. *P≤0.05 vs. each 0×IC50 by ANOVA with multiple comparisons. N = 3. D, E: HDAC3 inhibitors show a differential effect on cytoplasmic and nuclear proteasome activity. After incubating with the indicated amount of HDAC3 inhibitors for 48 h, cells were fractionated and the proteasome activity of 5 µg total protein from each fraction was independently measured. *P<0.05, **P<0.001 0×IC50 vs. 5×IC50 by t-tests N = 3. F: HDAC inhibitors have little or no direct proteasome inhibitory effect. Total protein (5 µg) from a HeLa cell PBS extract was subjected to the proteasome activity assay. During the incubation period for activity measurement, the indicated amount of HDAC inhibitors, or lactacystin as positive control, were added. Relative activity was shown with DMSO = 100%. ***P≤0.001 vs. DMSO by ANOVA with multiple comparisons. N = 3.
Figure 6.
Htt aggregates inhibit HDAC3 activity.
A: HDAC3 activity is suppressed upon either cytoplasmic or nuclear Htt-ex1 expression. After two days of transfection in 293T cells, the HDAC3 activity of cellular lysates was measured using a fluorescence-based assay. B: Htt-ex1 overexpression does not alter pan-HDAC activity. After two days of transfection in 293T cells, the pan-HDAC activity was measured using a fluorescence-based assay.