10 research outputs found

    Quantitative Proteomic Analysis Identifies Targets and Pathways of a 2‑Aminobenzamide HDAC Inhibitor in Friedreich’s Ataxia Patient iPSC-Derived Neural Stem Cells

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    Members of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors show promise as therapeutics for the neurodegenerative diseases Friedreich’s ataxia (FRDA) and Huntington’s disease (HD). While it is clear that HDAC3 is one of the important targets of the 2-aminobenzamide HDAC inhibitors, inhibition of other class I HDACs (HDACs 1 and 2) may also be involved in the beneficial effects of these compounds in FRDA and HD, and other HDAC interacting proteins may be impacted by the compound. To this end, we synthesized activity-based profiling probe (ABPP) versions of one of our HDAC inhibitors (compound 106), and in the present study we used a quantitative proteomic method coupled with multidimensional protein identification technology (MudPIT) to identify the proteins captured by the ABPP 106 probe. Nuclear proteins were extracted from FRDA patient iPSC-derived neural stem cells, and then were reacted with control and ABPP 106 probe. After reaction, the bound proteins were digested on the beads, and the peptides were modified using stable isotope-labeled formaldehyde to form dimethyl amine. The selectively bound proteins determined by mass spectrometry were subjected to functional and pathway analysis. Our findings suggest that the targets of compound 106 are involved not only in transcriptional regulation but also in posttranscriptional processing of mRNA

    The Effects of Pharmacological Inhibition of Histone Deacetylase 3 (HDAC3) in Huntington’s Disease Mice

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    <div><p>An important epigenetic modification in Huntington’s disease (HD) research is histone acetylation, which is regulated by histone acetyltransferase and histone deacetylase (HDAC) enzymes. HDAC inhibitors have proven effective in HD model systems, and recent work is now focused on functional dissection of the individual HDAC enzymes in these effects. Histone deacetylase 3 (HDAC3), a member of the class I subfamily of HDACs, has previously been implicated in neuronal toxicity and huntingtin-induced cell death. Hence, we tested the effects of RGFP966 ((<i>E</i>)-N-(2-amino-4-fluorophenyl)-3-(1-cinnamyl-1<i>H</i>-pyrazol-4-yl)acrylamide), a benzamide-type HDAC inhibitor that selectively targets HDAC3, in the N171-82Q transgenic mouse model of HD. We found that RGFP966 at doses of 10 and 25 mg/kg improves motor deficits on rotarod and in open field exploration, accompanied by neuroprotective effects on striatal volume. In light of previous studies implicating HDAC3 in immune function, we measured gene expression changes for 84 immune-related genes elicited by RGFP966 using quantitative PCR arrays. RGFP966 treatment did not cause widespread changes in cytokine/chemokine gene expression patterns, but did significantly alter the striatal expression of macrophage migration inhibitory factor (<i>Mif)</i>, a hormone immune modulator associated with glial cell activation, in N171-82Q transgenic mice, but not WT mice. Accordingly, RGFP966-treated mice showed decreased glial fibrillary acidic protein (GFAP) immunoreactivity, a marker of astrocyte activation, in the striatum of N171-82Q transgenic mice compared to vehicle-treated mice. These findings suggest that the beneficial actions of HDAC3 inhibition could be related, in part, with lowered <i>Mif</i> levels and its associated downstream effects.</p></div

    Summary of cytokines array gene expression changes in striatum due to RGFP966 treatment.

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    <p>A. Heatmap of expression values for 84 cytokine/chemokine genes showing two-way clustering of expression levels and treatment groups. Red denotes increased relative gene expression levels for the indicated groups, with green denoting decreased expression levels. B. Volcano plots of expression changes due to RGFP966 treatment showing three different comparisons, as indicated. Dotted line on y-axis denotes the significance cut-off of p-value<0.05, using one-way ANOVA. Dotted lines on x-axis denote a fold-change cut-off of > +/- 2.</p

    Real-time qPCR results showing altered expression of <i>Mif</i> and <i>Il1b</i> in striatum and cortex of RGFP966 treated WT and N171-82Q mice.

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    <p>Groups of mice were treated with RGFP966 (25 mg/kg) for 12 weeks beginning at 8 weeks of age. Bar graphs shown the mean +/- S.E.M. expression value from n = 5–6 mice per group normalized to the expression of <i>Hprt</i>. Significant differences of p<0.05 were measured by a two-tailed, unpaired Student’s <i>t</i> test and are indicated by an asterisk (*).</p

    The effects of RGFP966 (25 mg/kg) on striatal volume in WT and N171-82Q transgenic mice.

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    <p>Representative photomicrographs of the striatum at ~0.62 Bregma under each condition are shown on the left. Scale bar = 40 μm. Bar graphs show quantitation of striatal volume. The total area of the striatum was assessed using sections 125 μm apart spanning the striatum from ~bregma, 1.18 to 0.38 mm. One-way ANOVA (Dunnett’s post-test) revealed significant differences between vehicle-treated wild type and N171-82Q transgenic mice and also a significant difference between vehicle-treated and RGFP966-treated N171-82Q mice (*, P<0.05). Bars represent mean score ± SEM (n = 6 to 7 per group).</p

    Cytotoxicity (MTS) assays on resting murine BM cells and human MNCs.

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    <p>(A) Ficoll-Hypaque-separated murine BM cells were plated in triplicate with 1R-Chl (10, 100, 500, and 1000 nM) without any growth factors or mitogens. After 72 and 96 hours, 20 µL of Celltiter 96 Aqueous One solution (Promega, WI) was added. The absorbances of the MTS metabolites were read corresponding to the numbers of metabolically active cells. (B) Ficoll-Hypaque-separated human MNCs were treated as above, and the viabilities of the cells were assessed after 72 and 96 hours.</p

    Additive effects of 1R-Chl and imatinib treatments.

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    <p>(A) Murine BM cells transduced with unmutated BCR-ABL were treated with 500 nM imatinib, 500 nM 1R-Chl, and 500 nM 1S-Chl individually, or in combination of either 500 nM 1R-Chl or 500 nM 1S-Chl with 500 nM imatinib. (B) Same treatment routines were used on CML patient MNCs. The CML patient MNCs were treated 500 nM imatinib, 500 nM 1R-Chl, and 500 nM 1S-Chl individually, or 500 nM 1R(S)-Chl in combination with 500 nM imatinib. (C) Same experiments were done on normal blood donor MNCs.</p

    Chemical structure of 1R-Chl, imatinib, dasatinib, and their effects on BCR-ABL unmutated and mutated genes transduced into murine BM cells.

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    <p>(A) 1R-Chl (left) targets the DNA sequences 5′-WGGWGW-3′. Bold, imidazole rings. imatinib (middle) targets Bcr-Abl kinase, and dasatinib (right) targets 14 out of 15 Bcr-Abl mutants. (B) Murine BM cells transduced with unmutated BCR-ABL and single point mutation Y253H, E255K, and T315I genes were tested for the effectiveness of 1R-Chl (125 nM to 1000 nM), 1S-Chl (500 nM and 1000 nM), imatinib (500 nM and 5000 nM; IC<sub>50</sub> = 260 nM for the native BCR-ABL transduced cells) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003593#pone.0003593-OHare1" target="_blank">[6]</a>, and dasatinib (10 nM and 100 nM; IC<sub>50</sub> = 0.8 nM for the native BCR-ABL transduced cells) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003593#pone.0003593-OHare1" target="_blank">[6]</a>. Triplicate experiments were done, and the numbers of colonies were quantified 7 days after initial plating.</p

    Effects of 1R-Chl on colony formation of CML patient MNCs and normal human MNCs.

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    <p>(A) MNCs from CML patients were cultured with rh stem cell factor, rh IL-3, rh GM-CSF with or without erythropoietin which lead to CFU-GM or BFU-E cell lineages. The experiments were done in duplicate with 1R-Chl ranging from 125 to 1000 nM, 1S-Chl at 500 and 1000 nM, and imatinib at 500 and 5000 nM. The colonies were counted and results were calculated as the percentage of the control plates (without treatment) after 2 weeks. (B) MNCs from normal donors were cultured and plated under the same condition as CML patient cells. The cells were exposed to 500 and 1000 nM 1R-Chl, 500 and 1000 nM 1S-Chl, and 500 and 5000 nM imatinib. The colonies were counted and percent growth inhibition was calculated after 2 weeks.</p
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