Disruption of Protein Processing in the Endoplasmic Reticulum of DYT1 Knock-in Mice Implicates Novel Pathways in Dystonia Pathogenesis

Abstract

Dystonia type 1 (DYT1) is a dominantly inherited neurological disease caused by mutations in TOR1A, the gene encoding the endoplasmic reticulum (ER)-resident protein torsinA. Previous work mostly completed in cell-based systems suggests that mutant torsinA alters protein processing in the secretory pathway. We hypothesized that inducing ER stress in the mammalian brain in vivo would trigger or exacerbate mutant torsinA-induced dysfunction. To test this hypothesis, we crossed DYT1 knock-in with p58(IPK)-null mice. The ER co-chaperone p58(IPK) interacts with BiP and assists in protein maturation by helping to fold ER cargo. Its deletion increases the cellular sensitivity to ER stress. We found a lower generation of DYT1 knock-in/p58 knock-out mice than expected from this cross, suggesting a developmental interaction that influences viability. However, surviving animals did not exhibit abnormal motor function. Analysis of brain tissue uncovered dysregulation of eiF2α and Akt/mTOR translational control pathways in the DYT1 brain, a finding confirmed in a second rodent model and in human brain. Finally, an unbiased proteomic analysis identified relevant changes in the neuronal protein landscape suggesting abnormal ER protein metabolism and calcium dysregulation. Functional studies confirmed the interaction between the DYT1 genotype and neuronal calcium dynamics. Overall, these findings advance our knowledge on dystonia, linking translational control pathways and calcium physiology to dystonia pathogenesis and identifying potential new pharmacological targets.

Significance statement: Dystonia type 1 (DYT1) is one of the different forms of inherited dystonia, a neurological disorder characterized by involuntary, disabling movements. DYT1 is caused by mutations in the gene that encodes the endoplasmic reticulum (ER)-resident protein torsinA. How mutant torsinA causes neuronal dysfunction remains unknown. Here, we show the behavioral and molecular consequences of stressing the ER in DYT1 mice by increasing the amount of misfolded proteins. This resulted in the generation of a reduced number of animals, evidence of abnormal ER protein processing and dysregulation of translational control pathways. The work described here proposes a shared mechanism for different forms of dystonia, links for the first time known biological pathways to dystonia pathogenesis, and uncovers potential pharmacological targets for its treatment.

Keywords: dystonia; eif2alpha; endoplasmic reticulum; p58; proteomics; torsina.

Figure 1.

Generation of DYT1 knock-in/p58 knock-out mice. Tor1a^+/ΔE;p58^+/− and Tor1a^+/+;p58^+/− mice were bred to generate six potential genotypes. A, Shown are the expected (dotted line) and obtained number of animals per genotype based on a normal Mendelian distribution. B, Weekly weights are shown for the cohort of animals maintained until age 9 months. Two-way ANOVA for repeated measures showed a positive interaction of genotype and time (p < 0.001). Bonferroni's post test indicated an effect of the p58, but not the DYT1, genotype.

Figure 2.

Behavioral characterization of adult DYT1 knock-in/p58 knock-out and control mice. Mice of the four genotypes indicated were assessed at 6 and 9 months of age for motor function. A, Spontaneous locomotor activity in the open field (shown as total distance traveled in 30 min). B, Beam-crossing task, measured as time to transverse (in seconds) and number of hindlimb slips. C, Performance on the rotarod on 3 consecutive trial days at ages 6 and 9 months. D, Clasping score in the tail-hanging test as described in the Materials and Methods. Statistical differences were assessed by ANOVA with Tukey's post hoc test. *p < 0.05; **p < 0.01.

Figure 3.

Analysis of the PERK–eiF2α pathway. A, Schematic representation of the ER and post-ER components of the PERK-eiF2α ER stress pathway. B, Western blot analysis in cerebellar tissue obtained from 3-week-old and 9-month-old mice for expression of the indicated proteins (n = 3 different animals per genotype). C, Quantification of total torsinA levels for each genotype as shown in B. D, Quantification of the Western blot signal for different proteins shown in B. For phosphorylated proteins, the phosphorylated to total protein ratio is shown. E, Lectin blot of cerebellar lysates from three different animals per genotype (top) with quantification (bottom). Equal total protein amounts were loaded. Statistical differences were assessed by ANOVA with Tukey's post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4.

Analysis of the PERK–eiF2α pathway in DYT1 transgenic rats. A, C, Western blot analysis of cerebellar lysates obtained from DYT1 transgenic rats and nontransgenic littermates for expression of the indicated proteins (n = 5 per genotype). B, D, Quantification of the experiments shown in A and C. Statistical differences were assessed by ANOVA with Tukey's post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5.

Proteomic analysis of cerebellar lysates from 3-week-old mice. A, Cerebellar lysates obtained from Tor1a^+/ΔE or Tor1a^+/+ mice on a p58^+/+ or p58^−/− background were processed as described in the experimental procedures. Lysates from Tor1a^+/ΔE or Tor1a^+/+ mice were labeled with Cy3 and Cy5, respectively, mixed, and subjected to 2-DIGE (overlap images shown). A subset of the spots that were differentially expressed between the Tor1a^+/ΔE and Tor1a^+/+ genotypes (circled) were picked for MS-MALDI-TOFF identification (spot IDs correspond to those shown in Table 1). B, Magnification of the top boxed area in A. The two circled proteins were identified as Hspa9, suggesting posttranslational modification by Tor1a^+/ΔE only in the p58^−/− background. C, Magnification of the bottom boxed area in A. The top two circled proteins were identified as Erp29 and the bottom two as Prdx6, also suggesting posttranslational modifications by Tor1a^+/ΔE only in the p58^−/− background.

Figure 6.

Western blot analysis of selected proteins identified by the proteomic analysis. Cerebellar lysates (n = 3 animals per genotype) were used for Western blot analysis in 3-week-old (A) and 9-month-old (B) mice with the indicated antibodies. Statistical differences were assessed by ANOVA with Tukey's post hoc test. *p < 0.05; **p < 0.01.

Figure 7.

Analysis of calcium transients in cerebellar slices of 3- to 4-week-old mice. A, Montage showing a cerebellar slice in an imaging chamber. Three structures are visible in the differential interference contrast (DIC) optics: the molecular layer, the granule cell layer, and the white matter. The slice is anchored with a nylon mesh (∼1 mm intervals). Arrows show the direction of the 90 mm KCl application. A red square shows a representative area for calcium imaging. B, Resting fluorescence image of the cerebellar slice before stimulation (F₀). C, Changes in fluorescence intensity (ΔF/F₀) before KCl application (left) and during the peak response (right). D, Representative ΔF/F₀ trace. The left inset shows a region of interest used for calculating ΔF/F₀. The right inset shows an enlarged trace near the peak, demonstrating the effect of running average on reducing noise for measuring the peak height (yellow trace). E, Running-averaged ΔF/F₀ traces under the control condition (left) and under a treatment with tunicamycin (right). Individual traces (top) and averaged traces (bottom). A single trace is derived from a single slice. F, Peak heights of ΔF/F₀ traces. Statistical significance was assessed using Student's t test, with two-tailed p-values. *p < 0.05; **p < 0.01; n = 7 or 8 slices in each condition.

Figure 8.

Analysis of protein expression in putamen and cerebellum of DYT1 patients and controls. A, Brain tissue from three DYT1 patients (cerebellum and putamen from one and only cerebellum or putamen from the other two) and controls (putamen from five subjects and cerebellum from three subjects) were subject to Western blot analysis to evaluate the PERK-eiF2α pathway and dysregulated calcium-related proteins from the proteomic analysis in mice. B, Quantification of expression levels normalized to the first control subject is shown. Statistical analysis was not performed due to the limited number of samples from each anatomical region.