Allele-specific silencing of mutant ataxin-3 reduced the levels of mutant protein and mRNA.

<p>(A) Western blot analysis of cerebellar lysates stained with 1H9 antibody for ataxin-3. (B–D) Differences in the levels of high molecular weight protein species were detected between animals co-injected with MUT+shAtx3 (<i>n</i> = 3) and animals control (co-injected with MUT+shGFP, <i>n</i> = 3), whereas no differences were found in ataxin-3 endogenous levels. Normalization of protein levels was made with β-tubulin protein endogenous levels. (E) Quantitative real-time PCR analysis of cerebelar lysates shows a reduction in the ataxin-3 mRNA levels in the animals co-injected with MUR+shAtx3 compared to controls. Endogenous <i>hprt</i> mRNA was used as an internal control for the normalization and quantitative analysis of the ataxin-3 mRNA levels. Values are represented as mean ± SEM. *Statistical significance (<i>n</i> = 3; **<i>P</i><0.01; ***<i>P</i><0.001; Unpaired Student's <i>t</i>-test).</p

Allele specific silencing of mutant ataxin-3 prevents the appearance of balance and motor coordination abnormalities.

<p>(a) Time course of mice behavior tests: rotarod performance test, footprints patterns analysis and activity box monitoring. (b) Rotarod test. Mice were placed in a rotarod accelerating from 0 to 40 r.p.m. in 5 min. Mice co-injected with Atx3 MUT and shAtx3 (closed circles, <i>n</i> = 8) showed an improvement performance relative to mice co-injected with Atx3 MUT and shGFP (control) (open circles, <i>n</i> = 7), especially at 8 and 10 weeks post-injection. Wild-type non-injected mice are represented as WT (<i>n</i> = 8). Values are expressed as mean ± SEM. *<i>P</i><0.05 (2-way ANOVA Bonferroni <i>post</i>-test). (c–f) Footprint analysis of shAtx3 and shGFP-treated Atx3 MUT mice. (c) Stride length. Mice injected with RNA interference against mutant ataxin-3 (MUT+shAtx3) have an increased stride length relative to control (MUT+shGFP) starting at 6 weeks post-injection. Values are expressed as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (2-way ANOVA Bonferroni <i>post</i>-test). (d) Footprint overlap. The distance between the front and hind footprint placement or overlap is reduced in shAtx3-injected mice relative to control, starting at 6 weeks post-injection. Values are expressed as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 (2-way ANOVA Bonferroni <i>post</i>-test). (e) Hind base width. The shAtx3-injected mice have a narrow hind base width relative to control, starting at 6 weeks post-injection. Values are expressed as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 (2-way ANOVA Bonferroni <i>post</i>-test). (f) Front base width. The shAtx3-injected mice have a narrow front base width relative to control, starting at 8 weeks post-injection. Values are expressed as mean ± SEM. *<i>P</i><0.05 (2-way ANOVA Bonferroni <i>post</i>-test). In general the patterns clearly differ, showing that shAtx3-injected mice display longer strides, evenly spaced and accurately positioned footprints when compared to control-treated MJD mice.</p

Allele-specific silencing of mutant ataxin-3 leads to diminution of the number of intranuclear aggregates.

<p>Confocal microscopy of ataxin-3, β-galactosidase, with nuclei counterstained in blue (DAPI). Mice injected with mutant ataxin-3 and short-hairpin against GFP (upper panel, MUT+ shGFP) exhibit aggregates of mutant ataxin-3 ((b,f), red, arrows) co-localizing with the short-hairpin RNA ((c,g), green, arrows); whereas aggregates from mice injected with mutant ataxin-3 and short-hairpin against mutant ataxin-3 (lower panel, MUT+ shAtx3) practically do not co-localize(l,p). This indicates that cells expressing the shRNA against mutant ataxin-3 do not display aggregates. Scale bar: 10 µm. (Q) Quantification of the mean number of aggregates per section. Silencing of mutant ataxin-3 significantly decreased the presence of mutant ataxin-3 aggregates (MUT+shAtx3) as compared to control (MUT+shGFP). Values are represented as mean ± SEM. *Statistical significance (<i>n</i> = 6; ***<i>P</i><0.001; Unpaired Student's <i>t</i>-test).</p

Allele-specific silencing prevents Purkinje cells pathology.

<p>Fluorescence microscopy analysis for the DARPP-32 (a,b) and calbindin (c,d) proteins highlighting the Purkinje cells. An increased expression of DARPP-32 neuronal marker was observed for the shAtx3-treated mice, relative to control-treated. Note the improved Purkinje cell morphology in shAtx3-treated mice (arrows) as compared to shrunken-sized Purkinje cells in control-treated (arrow heads). Scale bar: 40 µm. (E) Quantification of optical densitometry of DARPP-32 immunoreactivity. Silencing of mutant ataxin-3 significantly increased DARPP-32 expression (MUT+shAtx3) as compared to control (MUT+ shGFP). *Statistical significance (<i>n</i> = 5; ***<i>P</i><0.001; Unpaired Student's <i>t</i>-test). (f–i) Fluorescence microscopy analysis for the calbindin protein highlighting the Purkinje cells and the molecular layer (c,d). An increased expression of calbindin neuronal marker was observed for the shAtx3-treated mice (d), relative to control-treated (c). Note the improved Purkinje cell morphology in shAtx3-treated mice and increased expression of calbindin in the molecular layer as compared to control. Scale bar: 40 µm. (F) Quantification of optical densitometry of calbindin immunoreactivity. *Statistical significance (<i>n</i> = 5; **<i>P</i><0.01; Unpaired Student's <i>t</i>-test).</p

Allele specific silencing of mutant ataxin-3 reduces hyperactivity.

<p>(a, b) Analysis of the activity in the last 30 minutes (one zone). (a) Distance travelled. Mice injected with shAtx3 travelled less than control-injected mice starting at 8 weeks post-injection. Values are expressed as mean ± SEM. *<i>P</i><0.05, ** <i>P</i><0.001 (2-way ANOVA Bonferroni <i>post</i>-test). (b) Time spent on resting. Mice injected with shAtx3 spent more time resting than control-injected mice starting at 8 weeks post-injection. Values are expressed as mean ± SEM. *<i>P</i><0.05 (2-way ANOVA Bonferroni <i>post</i>-test). (c) Plots of the moved track in the arena during the 40 minutes in the activity box at 10 weeks post-injection showed a clear difference between animals injected with shAtx3 and control mice. (d–f) Analysis minute-by-minute of distance travelled in the last time-point (10 weeks post-injection). (d) Distance travelled in the entire arena (one zone) during the first 10 minutes of the test. Values are expressed as mean ± SEM. *<i>P</i><0.05, ***<i>P</i><0.001 (2-way ANOVA Bonferroni <i>post</i>-test). (e) Analysis of the periphery of the arena (zone 1) during the first 10 minutes. This zone is associated with comfort and shelter. (f) Analysis of the center of the arena (zone 2) during the first 10 minutes. This zone is associated with reduced anxiety and fear. Mice injected with shAtx3 displayed a reduced hyperactivity relative to control, preferring the comfort zone (zone 1) relative to the center (zone 2). Taken together the significant differences were evident almost in all time points. Values are expressed as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (2-way ANOVA Bonferroni <i>post</i>-test).</p

Silencing of mutant ataxin-3 reduces neurodegeneration.

<p>Cresyl violet staining. Note the increased cell number and improved cell morphology in shAtx3-treated mice (a–d). Scale bar: 20 µm. (E) Quantification of Purkinje cell number per µm. Silencing of mutant ataxin-3 significantly increased the cell number as compared to control. *Statistical significance (<i>n</i> = 4; **<i>P</i><0.01; Unpaired Student's <i>t</i>-test). Moreover, the quantification of molecular (F) and granular layer (DG thickness also revealed a cellular preservation in mice injected with shAtx3 (<i>n</i> = 5; *<i>P</i><0.05; **<i>P</i><0.01; Unpaired Student's <i>t</i>-test, respectively). Apoptotic effect was detected in neurons (white arrows) in the control animals detected by TUNEL assay (red channel) (h–j) compared to mice co-injected with MUT+shAtx3 where no apoptotic effect was detected (k–m). ML, molecular layer; PCL, Purkinje cell layer; GL, granular layer.</p

Allele-specific silencing of mutant ataxin-3 reduces the number of intranuclear inclusions.

<p>A–L) Confocal analysis of mutant ataxin-3 aggregates in the Purkinje cells at 10 weeks post-injection. Transgenic MJD mice injected at 21–25 days of age with shGFP (control) display intranuclear inclusions of mutant ataxin-3 (revealed by immunohistochemistry with an HA antibody) in Purkinje cells (revealed by calbindin immunohistochemistry) versus a diffuse expression of mutant ataxin-3 in mice injected with the silencing shAtx3. M) The number of intranuclear inclusions is significantly reduced in transgenic mice injected with shAtx3 compared to control animals injected with shGFP (<i>n</i> = 6, ***<i>P</i><0.001; Unpaired Student's <i>t</i>-test).</p

Allele specific silencing of mutant ataxin-3 improves exploratory and locomotor activity and reduces anxiety in MJD transgenic mice.

<p>A) Representative plots of moved track at 10 weeks post-injection during 40 minutes in the activity box. Animals injected with shAtx3 (<i>n</i> = 8) traveled more that shGFP-injected mice (<i>n</i> = 8). The figure shows representative images that were reproducible among the different groups of animals. B–C) Locomotor horizontal activity of mice was tracked for 30 minutes (after a 10 minutes habituation period), and revealed a significantly better locomotor activity of mice injected with shAtx3 (<i>n</i> = 8) as compared to control mice (shGFP, <i>n</i> = 8), by traveling longer distances with a higher medium velocity. D–E) Two zones analysis of exploratory behavior, revealed that shAtx3-injected mice (<i>n</i> = 8) had increased exploratory behavior and were significantly less anxious than controls (shGFP; <i>n</i> = 8). F–G) This reduced anxiety of shAtx3-injected mice was confirmed through analyzis of the distance in zone two normalized with the overall distance in the arena, and also the number of entries in zone 2 normalized with the overall distance in the arena. *Statistical significance (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; 2-way ANOVA, Bonferonni post-hoc test).</p

Allele-specific silencing of mutant ataxin-3 reduces the levels of mutant aggregated protein.

<p>A) Western blot analysis of cerebellar lysates stained with 1C2 antibody (for the polyglutamine expansion). Differences in levels of high molecular weight protein species were detected between transgenic mice injected with shAtx3 (<i>n</i> = 3) and animals injected with shGFP (<i>n</i> = 3). Normalization of protein levels was made with β-actin protein endogenous levels, and with β-gal marker (reporter gene of shRNAs lentiviral vector). B, D). The quantification of aggregated protein levels (in the stacking gel) revealed significant differences between transgenic mice injected with shAtx3 and control animals injected with shGFP (<i>n</i> = 3; *<i>P</i><0.05; Unpaired Student's <i>t</i>-test). C,E). Oligomerized species were also significantly reduced in animals injected with shAtx3 compared to control animals (<i>n</i> = 3; *<i>P</i><0.05; Unpaired Student's <i>t</i>-test).</p

High resolution image analysis of EM48-positive aggregates indicates major age-dependent differences.

<p>Young (3 week) and old (15 month) rats received a stereotaxic injection of lentiviral vectors encoding Htt171-82Q (2 µl, 200 ng/µl of p24). Histological evaluation was carried out 4 weeks after infection using EM48 immunohistochemistry and image analysis software. The number of EM48 positive objects (aggregates) was determined from images obtained using a 50× objective at different focal depths (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004637#s4" target="_blank">Materials and Methods</a>) so that very small EM48 positive objects could be detected. Typical images acquired at 50× objective in a young rat (<i>A</i>) and an old rat (<i>C</i>). <i>B</i> and <i>D</i> correspond to zoomed images of the rectangles in A and B. Note the high resolution of the images allows reliable detection of even small aggregates. <i>E</i>, Histograms showing the distribution of small objects as a function of size (apparent cross-sectional area) in two animals, indicating that the proportion of very small aggregates in old rats is approximately ten fold higher than in young animals.</p