Journal: PLoS Biology

Article Title: HDAC4 Reduction: A Novel Therapeutic Strategy to Target Cytoplasmic Huntingtin and Ameliorate Neurodegeneration

doi: 10.1371/journal.pbio.1001717

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

Article Snippet: The cross between Hdh Q150 and Hdac4 HET mice, both on a C57BL/6 background, was performed at Novartis.

Techniques: Expressing

Journal: PLoS Biology

Article Title: HDAC4 Reduction: A Novel Therapeutic Strategy to Target Cytoplasmic Huntingtin and Ameliorate Neurodegeneration

doi: 10.1371/journal.pbio.1001717

Figure Lengend Snippet: (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 ( n = 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 ( n = 6). (C) Seprion ligand ELISA was used to quantify aggregate load in the striatum, cortex, and cerebellum of Hdh Q150 and Dble:: Hdh Q150 mice at 6 and 10 mo of age. Values for the Dble:: Hdh Q150 mice were plotted as a percentage of aggregate load of Hdh Q150 mice ( n ≥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 . (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) ( n = 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. p values were calculated using Student's t test.

Article Snippet: The cross between Hdh Q150 and Hdac4 HET mice, both on a C57BL/6 background, was performed at Novartis.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Comparison, High Molecular Weight, Isolation, Agarose Gel Electrophoresis

Journal: PLoS Biology

Article Title: HDAC4 Reduction: A Novel Therapeutic Strategy to Target Cytoplasmic Huntingtin and Ameliorate Neurodegeneration

doi: 10.1371/journal.pbio.1001717

Figure Lengend Snippet: (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 35 S-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 Hdh 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, Hdh Q20, and Hdh 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 Hdh Q80 homozygous mice. (E) Representative immunofluorescence images of cortex from 14-wk-old R6/2 and 23-mo-old Hdh 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.

Article Snippet: The cross between Hdh Q150 and Hdac4 HET mice, both on a C57BL/6 background, was performed at Novartis.

Techniques: Mutagenesis, Staining, Recombinant, Western Blot, Immunoprecipitation, Immunofluorescence, Immunodetection