Dystonia Type 6 Gene Product Thap1: Identification Of a 50 kDa DNA-binding Species In Neuronal Nuclear Fractions

Mutations in THAP1 result in dystonia type 6, with partial penetrance and variable phenotype. The goal of this study was to examine the nature and expression pattern of the protein product(s) of the Thap1 transcription factor (DYT6 gene) in mouse neurons, and to study the regional and developmental distribution, and subcellular localization of Thap1 protein. The goal was accomplished via overexpression and knock-down of Thap1 in the HEK293T cell line and in mouse striatal primary cultures and western blotting of embryonic Thap1-null tissue. The endogenous and transduced Thap1 isoforms were characterized using three different commercially available anti-Thap1 antibodies and validated by immunoprecipitation and DNA oligonucleotide affinity chromatography. We identified multiple, novel Thap1 species of apparent Mr 32 kDa, 47 kDa, and 50–52 kDa in vitro and in vivo, and verified the previously identified species at 29–30 kDa in neurons. The Thap1 species at the 50 kDa size range was exclusively detected in murine brain and testes and were located in the nuclear compartment. Thus, in addition to the predicted 25 kDa apparent Mr, we identified Thap1 species with greater apparent Mr that we speculate may be a result of posttranslational modifications. The neural localization of the 50 kDa species and its nuclear compartmentalization suggests that these may be key Thap1 species controlling neuronal gene transcription. Dysfunction of the neuronal 50 kDa species may therefore be implicated in the pathogenesis of DYT6

Trichostatin A, valproic acid and HDACi 4b up-regulate markers of the differentiated MSN but do not increase survival of striatal neurons <i>in vitro</i>.

<p>(<b>a</b>) Over 2,000 neurons were counted for each condition, derived from 3 separate platings. Untreated control, BDNF (10 ng/ml), HDACi 4b (5 µM), trichostatin A (10 nM) and valproic acid (3 mM), p = 0.94, one-way ANOVA. (<b>b–d</b>) Primary striatal neurons were treated with (b) TSA (10 nM), (c) VPA (3 mM), or (d) HDACi 4b (5 µM) for 24 h, each of which increased the level of DARPP-32 protein. Representative of 3 platings, each performed in duplicate. (<b>e</b>) Calbindin is increased after 24 h of treatment with VPA, (<b>f</b>) Transcript levels of DARPP-32, ARPP-21, D1 type receptors (D1R), D2 type receptors (D2R), encephalin (ENK) and dynorphin (DYN) were measured by RT-qPCR after 24 h treatment with BDNF or TSA. N = 5–8; +/− SEM (*p<0.05, **p<0.01, ***p<0.001).</p

VPA and TSA do not inhibit induction of Egr-1 by BDNF, but do inhibit induction of Nab2 by BDNF.

<p>(<b>a</b>) Primary striatal neurons were treated with BDNF (10 ng/ml), VPA (3 mM), or BDNF (10 ng/ml) plus VPA (3 mM) for 15, 30, and 60 min. The level of Egr-1 was determined by western blotting. Results representative of at least 2 separate platings, performed in triplicate. Error bars indicate SEM (*p<0.05, **p<0.01, ***p<0.001). (<b>b</b>) Primary striatal neurons were treated with BDNF (10 ng/ml), VPA (3 mM), or BDNF (10 ng/ml) plus VPA (3 mM) for 3 hours and the level of Nab2 was determined by western blotting. Results representative of at least 2 separate platings, performed in triplicate. Error bars indicate SEM (*p<0.05). (<b>c</b>) Primary striatal neurons were treated with BDNF (10 ng/ml), TSA (10 nM), or BDNF (10 ng/ml) plus TSA (10 nM) for 3 hours. Levels of Egr-1 and Nab2 were determined by Western blotting. Results representative of 3 separate platings performed in duplicate. Error bars indicate SEM (*p<0.05, **p<0.01, ***p<0.001). (<b>d</b>) Primary striatal cultures were treated as in (b) followed by gel shift analysis. Anti-Nab2, 2 µg, was added before addition of DNA except in Lane 5, where it was added after the labeled oligonucleotide. Representative of 2 experiments performed in duplicate. The band representing binding of Egr-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076842#pone.0076842-Keilani1" target="_blank">[7]</a> is represented by the filled arrow, and the super-shift band generated by addition of α-Nab2 is indicated by the unfilled arrow. (<b>e</b>) Primary striatal cultures were treated with vehicle (NI:non-infected), Ad-GFP (Adenovirus – GFP), or Ad-Nab2 (Adenovirus – Nab2) with and without BDNF, as described in Methods. Representative of 3 platings, performed in duplicate. Error bars indicate SEM (*p<0.05, **p<0.01, ***p<0.001).</p

AcH3 is increased in striatal neurons by HDAC inhibitors and is enriched downstream of the transcription start site in <i>Ppp1r1b</i> striatal chromatin.

<p>(<b>a</b>) Cells were treated with BDNF (10 ng/mL), (a) trichostatin A (10 nM), (b) valproic acid (3 mM) or (c) HDACi 4b (5 µM) for 3 h. N = 3−5, +/− SEM (*p<0.05, ***p<0.001). (<b>b</b>) Chromatin was purified from adult mouse striatal tissue (STR) (DARPP-32-positive) and NIH-3T3 cells (DARPP-32-negative). Chromatin immunoprecipitation coupled with real-time PCR showed an increased association of acetylated histone H3 within a 1 kb region downstream of the DARPP-32 transcriptional start site (TSS) in striata relative to NIH 3T3 cells. Results are representative of two separate experiments performed in triplicates. Values for IgG are set at 100%. Error bars indicate SEM (*p<0.05).</p

Bcl11b protein level is down-regulated in the presence of HDAC inhibitors.

<p>(<b>a</b>) Ontogeny of Bcl11b in striatum in Swiss-Webster mice. Bcl11b levels decrease markedly post-natally, particularly between the first and second week of life. (<b>b</b>) Primary striatal neurons were treated with VPA (3 mM) or TSA (10 nM) with or without BDNF (10 ng/ml) for 24 h. Similar results were observed after treatment with compound 4 b (not shown). N = 3–5; +/− SEM (*p<0.05, **p<0.01, ***p<0.001).</p