Sensitivity to 3-NP of striatal cells expressing hMTH1 and wild-type or mutant murine <i>htt</i>.

<p>Striatal cells derived from wild-type Hdh<i>^Q7/Q7</i> and mutant <i>Hdh^Q111/Q111</i> mice were transfected with hMTH1. (A) Proteins were separated and probed with an antibody against hMTH1. (B) Intracellular localization of hMTH1 (green fluorescence) in <i>Hdh^Q111/Q111</i> and <i>Hdh^Q111/Q111</i>+hMTH1. Nuclei were counterstained by DAPI. (C) LDH release. LDH release from striatal cells into culture medium was measured 24 hr after continuous exposure to 20 mM 3-NP. Hdh<i>^Q7/Q7</i> (grey bar) and Hdh<i>^Q7/Q7</i>+hMTH1 (dashed bar); <i>Hdh^Q111/Q111</i> (black bars) and <i>Hdh^Q111/Q111</i>+hMTH1 (open bar). Mean±SE, n = 4. (D) Clonal survival. Cloning efficiency was determined at 33°C after 24 hr continuous exposure to the indicated 3-NP concentrations. Mean±SD, n = 2. (E) Coulter counter assay. Survival of non-proliferating cells measured in a Coulter Counter. Mean±SD, n = 3. The asterisks indicate significant differences by Student's <i>t</i>-test (p values = 0.02) between <i>Hdh^Q111/Q111</i> and <i>Hdh^Q111/Q111</i>+hMTH1.</p

Oxidative DNA damage in the brain.

<p>Brains from wild-type mice that had been treated twice per day for 5 days with 60 mg/kg 3-NP were examined for DNA 8-oxodG by immunohistochemistry. Nuclei of striatum, parietal and frontal cortex were counterstained by DAPI.</p

Muscle specific expression of Vp16-Jazz in transgenic mice.

<p>A: Western Blot analysis of total proteins extracted from the skeletal muscle of wild type (wt) and transgenic mice derived from two different founders (tg9 and tg41). The expression of Vp16-Jazz transgene was monitored by the anti-myc monoclonal 9E10 antibody. Detection of α−tubulin was used to normalize the amount of proteins. B: Western Blot analysis of total proteins extracted from the skeletal muscle, heart and brain of transgenic mice from families tg9 and tg41.</p

3-NP-induced toxicity in wild-type and hMTH1-Tg^+/− and hMTH1-Tg^+/+ transgenic mice.

<p>Groups of wild-type (n = 20), <i>hMTH1-Tg^+/−</i> (n = 16) and <i>hMTH1-Tg^+/+</i> (n = 16) mice were injected i.p. twice daily for 5 days with 60 mg/kg 3-NP. Wild-type (black bars), <i>hMTH1-Tg^+/−</i> (grey bars) and <i>hMTH1-Tg^+/+</i> (white bars). (A) Weight loss. Body weight, measured immediately before the first injection on the indicated days, is expressed as a percentage of the pretreated body weight. (B) Motor impairment. Mice were monitored twice a day for dystonia and/or gait abnormalities. Neurological score was as follows: intermittent dystonia of one hindlimb: 1; intermittent dystonia of two hindlimbs: 2; permanent dystonia of hindimbs: 3; uncoordinated and wobbling gait or recumbency: 3; near death recumbency: 4. For each animal, the highest neurological score reached at any time of the observation period was considered. Values are mean±standard errors. (C) Cumulative mortality. The non-surviving fraction at the end of 5-day treatment is expressed as a percentage of starting total. (D) Striatal lesion formation. The percentage of animals with detectable post-mortem striatal lesions is shown. (E) Size of striatal lesions. Postmortem measurements of striatal lesions along the rostrocaudal axis. The asterisks indicate a P<0.05 <i>vs</i> wild-type according to One-way Anova and Tukey multiple comparison post-hoc test for panels A, B and E and to χ² test for panels C and D.</p

hMTH1 protein in <i>hMTH1-Tg^+/+</i> MEFs and protection against oxidative stress.

<p>(A) Expression of hMTH1 in wild-type and homozygous <i>hMTH1-Tg^+/+</i> MEFs. Total cell extracts were separated by 12%-SDS polyacrylamide electrophoresis, blotted and probed with an antibody against hMTH1 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000266#pgen.1000266-Kang1" target="_blank">[23]</a>. The human SHSY5S neuroblastoma and SV40-transformed MRC5 cell lines are shown for comparison (left panel). Western blotting of subcellular fractions of <i>hMTH1-Tg^+/+</i> MEFs with anti-hMTH1 (right panel). Proteins were separated on 18%-SDS polyacrylamide electrophoresis and cytocrome <i>c</i> was used to quantify mitocondrial cell extracts. (B) Steady-state levels of oxidized guanine. DNA and RNA from wild-type (filled bars) and <i>hMTH1-Tg^+/+</i> (open bars) MEFs were digested to nucleosides and 8-oxodG and 8-oxoG were separated and quantified by HPLC-EC. Values are expressed as ratio to DNA and RNA guanine, respectively. Values are the mean±SE of 3 independent determinations. Asterisks indicate statistically significant differences (p-value = 0.005 and 0.015 for DNA and RNA, respectively; <i>t</i>-test). (C) Levels of oxidized guanine following oxidant treatment. 8-oxodG and 8-oxoG were measured by HPLC-EC in DNA and RNA extracted from MEFs exposed to 40 mM KBrO₃ for 30 min (time 0) and after the indicated times of repair incubation in drug-free medium (30, 60, 120 min). Wild type (filled bars) and <i>hMTH1-Tg^+/+</i> (open bars). Values are the mean±SE of 3 independent determinations. Asterisks indicate a p-value≤0.05; <i>t</i>-test.</p

Vp16-Jazz and utrophin up-regulation.

<p>A: Vp16-Jazz chromatin immunoprecipitation, performed in skeletal muscle derived from wt mice and transgenic mice (family tg9) using myc monoclonal antibody/protein G-agarose beads or protein G-agarose beads as a control (no-Ab). Immunoprecipitates from each sample were analyzed for the presence of utrophin promoter by PCR. A sample representing linear amplification of the total input chromatin (input) was included (lane 1). As control, samples from transgenic mice were also tested for the presence of dystrophin promoter sequence. B: Real-time PCR analysis of the utrophin gene expression rate in Vp16-Jazz transgenic mice (tg9 and tg41) and control wt mice. The gene expression ratio between utrophin and β-glucoronidase (GUS) and β2-microglobulin (β2M) is shown as means±S.D. from three independent experiments performed in triplicate. C: Western blot of total protein extracts derived from skeletal muscle and heart from wt mice and Vp16-Jazz transgenic mice (tg9 and tg41) incubated with monoclonal antibody against utrophin. The same membrane was incubated with anti-α-tubulin monoclonal antibody for loading normalization. D: Relative utrophin expression of wt and transgenic mice (tg9 and tg41) was determined by densitometric analysis. E: Total protein extracts from skeletal muscle of wt and transgenic mice (tg9 and tg41) were subjected to immunoblotting to detect the expression levels of the dystrophin and α-sarcoglycan proteins. The anti-α-tubulin and anti-myc antibodies were used to normalize the protein content and to test the Vp16-Jazz transgene expression respectively.</p

Effects of Vp16-Jazz in transgenic mice.

<p>Immunohistochemistry of Tibialis Anterior (TA) muscle derived from wt (panel A) and transgenic mice tg9 (panel B) stained with anti-utrophin antibody. Nuclei are counterstained with Hoechst 33258. C: TA from wt and transgenic mice tg9 were co-stained with anti-utrophin antibody and the α-bungarotoxin-Alexa Fluor to visualize the acetylcholine receptor (AChR) at the neuromuscular junctions. The anti-utrophin monoclonal antibody reveals an extra-synaptic distribution of utrophin only in transgenic mice. D: Relationship between contractile response (g) and stimulation frequency (Volts) in diaphragm and EDL muscle preparations obtained from wt, tg9 and tg41 mice. E: Scatter plots of the DEG with natural log transformed expression values averaged over the 4 replicates. The x–axis reports the mean of the replicates of transgenic mice, while the y-axis is the mean across the replicates of the control wt mice. Expression values are colour coded with red representing up-regulated genes in tg9/tg41 and green down-regulated genes compared to wild-type and lines on the graph set at 2-folds differential expression.</p

Vp16-Jazz and its DNA target sequence.

<p>A: Schematic representation of the Vp16-Jazz gene in the pMex-vector, used to generate transgenic mice. The 9 base pair long Vp16-Jazz DNA target sequence is indicated. B: The nucleotide sequence of the mouse utrophin promoter A. The Vp16-Jazz DNA target sequence is indicated in bold characters and underlined. The main transcription factor binding sites present in this promoter region are indicated.</p

Construction and characterization of a transgenic mouse expressing the <i>hMTH1</i> cDNA.

<p>(A) A BamH1-EcoRV fragment (509 bp) derived from pcDEBΔ <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000266#pgen.1000266-Kang1" target="_blank">[23]</a> encoding the <i>hMTH1</i> cDNA was subcloned into the gWIZ vector under the control of the CMV promoter. This vector transfected into wild-type MEFs expressed the hMTH1 protein (data not shown). The MscI-KpnI fragment (2481 bp) was used in the construction of the transgenic mouse. (B) Determination of transgene copy number. Genomic DNA from mouse tails was analysed by Southern blot analysis (left panel). In the right panel 2, 5, 10, 20, 40 copies of the MscI-KpnI fragment were analysed. Both blots were probed with the same probe. Copy number was determined by comparison. The arrow indicates the DNA from the mouse that was used as a founder for the colony. (C) Analysis by FISH of the number of hMTH1 integrations in hemizygous (<i>hMTH1-Tg^+/−</i>) and homozygous (<i>hMTH1-Tg^+/+</i>) strains. (D) Expression of <i>hMTH1</i> mRNA. RT-PCR was performed using total RNA from the indicated organs and human specific primers. RT-PCR for the <i>GADPH</i> gene is used as an internal control. <i>hMTH1</i> and <i>GAPDH</i> fragments are respectively 200 bp and 330 bp. (E) Western blot analysis of transgene expression. Total proteins (20–40 µg) from a range of tissues were separated by SDS polyacrylamide electrophoresis, blotted and probed with an antibody against hMTH1. β-tubulin was used as a loading control.</p

KBrO₃ sensitivity of <i>hMTH1-Tg^+/+</i> and wild-type MEFs.

<p>Wild-type (closed symbols) and <i>hMTH1-Tg^+/+</i> (open symbols) MEFs were treated with KBrO₃ for 30 min at the indicated concentrations. Viability was measured by MTT assay 48 hr later. The graphs are the mean±SD of 3 independent experiments.</p