DYT1 genotype does not influence performance in the rotarod after the administration of 3-NP.

<p>The latency to fall from the accelerating rotarod was measured 3 times daily for 3 consecutive days at the different time-points indicated. The average value for each time point was used for analysis. Two-way ANOVA for repeated measures showed no interaction between genotype and time point for the saline or 3-NP groups and no effect of genotype. Time influenced performance in both the saline (<i>F</i>[3,66] = 16.72; p<0.0001) and 3-NP (<i>F</i>[3,69] = 8.81; p<0.0001) groups. Post-test Bonferroni comparisons to performance at baseline were done (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).</p

Levels of torsinA in the striatum are slightly increased upon treatment with 3-NP.

<p>(A) Representative western blot showing torsinA expression in striatal lysates of animals receiving 50 mg/kg/day of 3-NP or saline controls. (B) Quantification of torsinA expression as described in the text for animals that received 50 or 20 mg/kg/day of 3-NP (N: 5–7/group). ANOVA showed a non-significant trend in the 50 mg/kg/day group (p = 0.07) and significant differences in the 20 mg/kg/day group (*p = 0.01).</p

The effects of 3-NP on motor behavior are not influenced by the DYT1 genotype.

<p>(A) Distance traveled over a 30 minutes period at baseline, at day 8 of the injections (“crisis”), at the end of the IP injections and 4 and 8 weeks later. Repeated measures two-way ANOVA showed no interaction between genotype and time point for animals receiving saline or 3-NP and no effect of genotype but a significant effect of time in both the saline (<i>F</i>[4,84] = 11.83; p<0.0001) and 3-NP (<i>F</i>[4,92] = 29.42; p<0.0001) groups. Post-hoc Bonferroni analysis was performed comparing each time point to the baseline value, with significance shown in the graph (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) (B) The pattern of the movement was affected by the administration of 3-NP, as mice developed hindlimb dysfunction. Shown are representative plots of locomotion for individual animals at day 16. Black line denotes no movement, green line slow movements and red line fast movements. (C) Analysis of the number of transitions between speeds per distance, indicating erratic movements, indicates a significant worsening of the gait pattern in the immediate post-injection period for the 3-NP group. Repeated measures ANOVA demonstrated no interaction between genotype and performance over time and no effect of genotype in the saline and 3-NP groups. There was a significant effect of time in the saline (<i>F</i>[4,84] = 3.44; p = 0.01) and 3-NP (<i>F</i>[4,92] = 9.38; p<0.0001) groups. Post-hoc Bonferroni analysis comparing each value to baseline demonstrates a significant increment in the number of transitions at days 8 and 16 for the 3-NP but not the saline group (*p<0.05, **p<0.01).</p

DYT1 KI mice are resistant to death caused by 3-NP.

<p>(A) Change in weight during the injection period expressed as a percentage of the initial weight. Two way ANOVA for repeated measures demonstrates a significant interaction between time and experimental group (<i>F</i>[42,630] = 3.34; p<0.0001). Post-test Bonferroni was done using the WT Saline group as a reference (*p<0.05; **p<0.01). (B) Kaplan Meyer survival curve for DYT1 and control mice demonstrates statistically significant differences in mortality upon treatment with 3-NP between both genotypes. The Gehan-Breslow-Wilcoxon test was used for statistical analysis. All animals that received saline survived. There was no mortality beyond the injection period.</p

Model for the differential aggregation properties and processing of 2UIM and 3UIM ataxin-3.

<p>In the absence of polyglutamine expansion, 3UIM ataxin-3 follows a multi-domain aggregation mechanism to generate limited oligomeric species without detectable formation of SDS-insoluble fibrillar aggregates. In contrast, 2UIM ataxin-3 exists in at least two monomeric states: the native conformation, in which the hydrophobic tail remains buried and protected from the aqueous environment, and an aggregation-prone conformation with an exposed hydrophobic tail. The aggregation prone monomer can revert to the native conformation or oligomerize through both the self-association propensity of the Josephin domain (like 3UIM ataxin-3) and hydrophobic interactions of the 2UIM-specific domain. Within 2UIM oligomers, the hydrophobic C-termini will associate, increasing the local polyglutamine concentration beyond that seen in 3UIM oligomers, favoring formation of detergent-insoluble aggregates. Unstable forms of monomer and oligomer are subject to protein quality control mechanisms, including proteasomal degradation for 2UIM ataxin-3. Insoluble fibrils, which are less well handled by protein quality control systems, accumulate as biochemically and microscopically detectable aggregates.</p

Ataxin-3 is alternatively spliced in <i>ATXN3</i> YAC transgenic and human brain.

<p>(A) Schematic representation of the <i>ATXN3</i> gene showing exons that encode specific functional domains. Untranslated regions (U) are not drawn to scale. The splicing pattern of the originally identified 2UIM ataxin-3 transcript is shown below, while above is shown the alternative splicing that links exon 10 to exon 11, generating 3UIM ataxin-3. Asterisks indicate exons that encode amino acids comprising the catalytic triad, polyQ denotes the polyglutamine domain, and the arrowhead indicates a polymorphic Tyr/Stop-encoding residue within the hydrophobic domain (Φ) of the C-terminus of 2UIM ataxin-3. C-terminal amino acid sequences are shown below the diagram, beginning with shared sequence in both isoforms extending from the polyQ domain, followed by the divergent sequences for the 2UIM and 3UIM isoforms; residues omitted in some SNP variants of the 2UIM isoform are shown in grey. (B) Diagram showing 5′ ataxin-3 splice variants identified and confirmed by sequencing. Multiple variants are detectable in mature mRNA from adult murine brain (and fetal brain, data not shown) by RT-PCR, using primers targeting the 5′UTR/exon 1 junction and exon 9 (arrows). All identified splice variants that maintain the open reading frame eliminate at least one catalytic triad residue, and thus are not likely to encode an active DUB. Darkly shaded areas are downstream of a frameshift-induced stop codon. (C–D) Endogenous <i>Atxn3</i> and transgenic <i>ATXN3</i> “full length” splice variants were amplified by RT-PCR using species-specific (human, hum; murine, ms) and sequence-specific (10-exon 2UIM-encoding, 10; 11-exon 3UIM-encoding, 11) primers. 10-exon and 11-exon variants are both detectable in mature mRNA: (C) endogenous <i>Atxn3</i> from all murine samples and unexpanded <i>ATXN3</i> from MJD15.4(+/−) brain; and (D) expanded <i>ATXN3</i> from MJD84.2(+/−) brain, and unexpanded <i>ATXN3</i> from pooled human brain tissue (hum). Perinatal day 1–3 (P_Q84), adult (A), or fetal (F) sources were used, as indicated. Note: Primers are not drawn to scale; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013695#s2" target="_blank">Materials and Methods</a> for exact sequences and locations.</p

2UIM and 3UIM ataxin-3 display similar DUB activity against defined ubiquitin chains <i>in vitro</i>.

<p>(A–C) Recombinant GST-ATXN3(Q22) (3UIM or 2UIM) can cleave K48-linked hexaubiquitin (A), K63-linked tetraubiquitin (B), and mixed linkage K48-K63-K48 tetraubiquitin (C) chains. Results with catalytically inactive GST-ataxin-3 (C14A mutant) are also shown. (D–E) Recombinant 2UIM and 3UIM GST-ATXN3(Q22) cleave Ub-AMC at a similar rate. (D) Ub-AMC reaction curves. Both 3UIM and 2UIM ataxin-3 area able to cleave Ub-AMC, while reactions with either an unrelated control protein (the non-DUB F-box protein FBXO2) or buffer only show no cleavage. Error bars show standard deviations. (E) There is no significant difference between the initial reaction velocity of 2UIM and 3UIM ataxin-3(Q22) (p>0.4 by a 2 tailed heteroscedastic Student's t-test).</p

2UIM ataxin-3 is more prone to aggregate than 3UIM ataxin-3.

<p>(A) Representative immunofluorescence of Cos7 cells transiently expressing Flag-tagged ataxin-3(Q22) splice isoforms or the UIM3(SA/DG) mutant. Cells were gated by fluorescence intensity into populations of moderate and high expressors. (B) Quantification of puncta per cell in (A). Error bars represent the standard deviation within each bin. Frequency distributions differ significantly between ATXN3(Q22)2UIM and ATXN3(Q22)3UIM and between ATXN3(Q22)2UIM and ATXN3(Q22)UIM3(SA/DG) mutant ataxin-3 (*p<0.0001, ** p<1×10^-11), but not between ATXN3(Q22)3UIM and ATXN3(Q22)UIM3(SA/DG) mutant ataxin-3 by a χ² test for independence, df  = 3. (C) Supernatant (sup) and pellet (pel) fractions of non-denaturing RIPA brain lysates from aged MJD84.2 (ATXN3(Q84)3UIM) and Q71B (ATXN3(Q71)2UIM) hemizygous transgenic mice were analyzed by Western blot with 1H9 anti-ataxin-3 antibody. Insoluble microaggregates are detected at the base of lane wells, whereas soluble transprotein and endogenous ataxin-3 are visualized within the resolving gel. (D) Quantification of the ratio of insoluble to soluble ataxin-3 transprotein seen in (C). 3UIM-predominant MJD84.2 mice show a significantly lower ratio of insoluble:soluble transprotein than 2UIM-only Q71B mice (*p<0.0005 by a 1 tailed heteroscedastic Student's t-test).</p

Mutations in THAP1/DYT6 reveal that diverse dystonia genes disrupt similar neuronal pathways and functions

<div><p>Dystonia is characterized by involuntary muscle contractions. Its many forms are genetically, phenotypically and etiologically diverse and it is unknown whether their pathogenesis converges on shared pathways. Mutations in <i>THAP1</i> [THAP (Thanatos-associated protein) domain containing, apoptosis associated protein 1], a ubiquitously expressed transcription factor with DNA binding and protein-interaction domains, cause dystonia, DYT6. There is a unique, neuronal 50-kDa Thap1-like immunoreactive species, and Thap1 levels are auto-regulated on the mRNA level. However, <i>THAP1</i> downstream targets in neurons, and the mechanism via which it causes dystonia are largely unknown. We used RNA-Seq to assay the <i>in vivo</i> effect of a heterozygote <i>Thap1</i> C54Y or ΔExon2 allele on the gene transcription signatures in neonatal mouse striatum and cerebellum. Enriched pathways and gene ontology terms include eIF2α Signaling, Mitochondrial Dysfunction, Neuron Projection Development, Axonal Guidance Signaling, and Synaptic LongTerm Depression, which are dysregulated in a genotype and tissue-dependent manner. Electrophysiological and neurite outgrowth assays were consistent with those enrichments, and the plasticity defects were partially corrected by salubrinal. Notably, several of these pathways were recently implicated in other forms of inherited dystonia, including DYT1. We conclude that dysfunction of these pathways may represent a point of convergence in the pathophysiology of several forms of inherited dystonia.</p></div

Cortico-striatal synaptic plasticity is altered in <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} derived slices.

<p><b>(A)</b><i>Thap1</i>^<i>+/-</i> mice are deficient in synaptically-induced LTD in dorsolateral striatum compared to wildtype controls (A₁; <i>p</i> < .05), while LTP in the dorsomedial region is intact (A₂). Representative excitatory postsynaptic potential (EPSP) traces in this and panel B were averaged over the baseline period (thin line) and over the final 5 min of recording (thick line), color coded to the graph. Calibration for these and all other traces: 1 mV / 5 ms. <b>(B)</b> In <i>Thap1</i>^{<i>C54Y/+</i>} mice, LTD was not significantly reduced (B₁), but LTP was deficient (B₂; p < .05). Note that wildtype data are the same as for panel A, and that all genotypes were analyzed together. <b>(C)</b> Paired-pulse ratio was not altered in <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} mice. The traces show averaged EPSPs recorded at inter-stimulus interval = 50 ms (thin and thick lines show responses to first and second stimuli, respectively). All graphs show group means ± SEM, and the number of slices/mice for each group are shown in parentheses. Data were analyzed by ANOVAs performed over the final 5 minutes of recording (panels A and B) or on averaged paired-pulse data for each interval (panel C), followed where appropriate by Newman-Keuls <i>post-hoc</i> tests. *p<0.05 See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007169#pgen.1007169.s012" target="_blank">S9 Table</a>.</p