1. Developmental Biology
  2. Neuroscience

TorsinB overexpression prevents abnormal twisting in DYT1 dystonia mouse models

  1. Jay Li
  2. Chun-Chi Liang
  3. Samuel S Pappas  Is a corresponding author
  4. William T Dauer  Is a corresponding author
  1. Medical Scientist Training Program, University of Michigan, United States
  2. Cellular and Molecular Biology Graduate Program, University of Michigan, United States
  3. Department of Neurology, University of Michigan, United States
  4. Peter O’Donnell Jr. Brain Institute, Departments of Neuroscience and Neurology & Neurotherapeutics, University of Texas Southwestern, United States
https://doi.org/10.7554/eLife.54285
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Cite this article
  1. Jay Li
  2. Chun-Chi Liang
  3. Samuel S Pappas
  4. William T Dauer
(2020)
TorsinB overexpression prevents abnormal twisting in DYT1 dystonia mouse models
eLife 9:e54285.
https://doi.org/10.7554/eLife.54285
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5 figures, 3 tables and 1 additional file

Figures

Figure 1 with 5 supplements
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TorsinB deletion worsens torsinA-related motor and neuropathological phenotypes.

(A) Nissl staining of P28 Emx1(A)-CKO and Emx1(A+B)-CKO mice. Emx1(A+B)-CKO mice exhibit significant atrophy of Cre-expressing brain regions including cortex (*) and hippocampus (^). (B) GFAP staining of P28 Emx1(A)-CKO and Emx1(A+B)-CKO brains. Emx1(A+B)-CKO mice exhibit severe reactive gliosis in Cre-expressing regions, including the cerebral cortex and the hippocampus. (C) Cortical thickness of P28 Emx1(A)-CKO and Emx1(A+B)-CKO mice. Cortical thickness is reduced by 10.4% in Emx1(A)-CKO mice (unpaired t-test t16 = 5.834, p<0.0001; control n = 9, Emx1(A)-CKO n = 9). Cortical thickness is reduced by 64.8% in Emx1-dCKO (unpaired t-test t11 = 30.16, p<0.0001; control n = 4, Emx1(A+B)-CKO n = 9). (D) Representative images from CUX1 and CTIP2 stained cerebral cortex of Emx1(A)-CKO and Emx1(A+B)-CKO mice and their respective littermate controls. (E) CUX1 and CTIP2 counts in P28 Emx1(A)-CKO and Emx1(A+B)-CKO mice. CUX1+ neurons are not significantly reduced in Emx1(A)-CKO mice (unpaired t-test t16 = 0.8469, p=0.4095; control n = 9, Emx1(A)-CKO n = 9). CUX1+ cells are significantly reduced in Emx1(A+B)-CKO mice (77.0% reduction; unpaired t-test t8 = 12.86, p<0.0001, control n = 4, Emx1(A+B)-CKO n = 6). CTIP2+ neurons are not significantly reduced in Emx1(A)-CKO sensorimotor cortex (unpaired t-test t16 = 0.02552, p=0.98; control n = 9; Emx1(A)-CKO n = 9). CTIP2+ neurons are significantly reduced in Emx1(A+B)-CKO sensorimotor cortex (98.6%; unpaired t-test t7 = 7.636, p=0.0001, control n = 4, Emx1(A+B)-CKO n = 5). (F) Proportion of Emx1(A)-CKO and Emx1(A+B)-CKO mice exhibiting limb clasping during tail suspension. A significantly greater proportion of Emx1(A+B)-CKO compared to Emx1(A)-CKO mice exhibit limb clasping during tail suspension (Chi square test χ2 = 9.1, p=0.0026; Emx1(A)-CKO n = 12, Emx1(A+B)-CKO n = 14).

Figure 1—source data 1

Behavioral and histological data on Emx1(A)-CKO and Emx1(A+B)-CKO mice.

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Figure 1—figure supplement 1
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TorsinB null mice exhibit no apparent organismal or neuropathological phenotypes.

(A) Tor1b-/- postnatal growth. Tor1b-/- mutants exhibit no significant changes in body weight from age P15 to P56 compared to controls (two-way repeated measures ANOVA genotype x age, interaction F2, 14 = 0.6325, p=0.5458; wild-type n = 4, torsinB KO n = 5). (B) Nissl analysis of P56 Tor1b-/- brains. Tor1b-/- mutants did not exhibit any differences in brain morphology compared to littermate controls. (C) GFAP staining of P56 Tor1b-/- brains. Tor1b-/- mutants did not exhibit gliosis. (D) Cortical thickness measurements of P56 Tor1b-/- mice. Cortical thickness does not differ significantly between WT and Tor1b-/- mice (unpaired t-test t6 = 0.09577, p=0.9268; wild-type n = 4, torsinB KO n = 4). (E) Proportion of P56 Tor1b-/-- mice and WT littermates that exhibit limb clasping during tail suspension. No mice of either genotype exhibited limb clasping.

Figure 1—figure supplement 1—source data 1

Data on histological analysis of torsinB KO mice.

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Figure 1—figure supplement 2
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Emx1(A+B)-CKO mice do not exhibit neuropathological abnormalities at birth.

(A) Illustration of examined genotypes. Each row of boxes within the circles denote the presence or absence of Tor1a (top row) or Tor1b (bottom row) alleles following Cre recombination. The presence of a letter (‘A’ or ‘B’) indicates an intact allele, whereas the absence of a letter indicates a deleted allele. (B) CTIP2 neuron counts in P0 Emx1(A+B)-CKO sensorimotor cortex. CTIP2+ neuronal counts in Emx1(A+B)-CKO mice do not differ significantly from littermate controls at P0 (non-parametric Mann-Whitney test U = 38.5, p=0.1911; control n = 13, Emx1(A+B)-CKO n = 9). (C) Cortical thickness measurements at P0 in Emx1(A+B)-CKO mice. Cortical thickness does differ significantly at P0 in Emx1(A+B)-CKO mutants (unpaired t-test t20 = 0.2663, p=0.7927; control n = 13, Emx1(A+B)-CKO n = 9).

Figure 1—figure supplement 2—source data 1

Data on P0 characterization of Emx1(A+B)-CKO mouse brains.

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Figure 1—figure supplement 3
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Emx1(A+B)-CKO neuropathological abnormalities that emerge postnatally are restricted to the forebrain.

Gross morphology of Emx1(A+B)-CKO and control brains. Emx1(A+B)-CKO causes grossly apparent atrophy in the forebrain Cre field while hindbrain morphology appears unchanged.

Figure 1—figure supplement 4
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Further assessment of cortical neurons in Emx1(A+B)-CKO mice.

(A) Cortical neuron soma size in Emx1-CKO. Neuronal soma size is unchanged in Emx1(A)-CKO cortical neurons of layer I-III or layer IV-VI (two-way ANOVA main effect of genotype F1, 396 = 0.04729, p=0.8280, main effect of layers F1, 396 = 0.3768 p=0.5397, interaction F1, 396 = 0.01256, p=0.9108, control n = 100, Emx1(A)-CKO n = 100). (B) CTIP2 neuron counts in layer Vb of Emx1-CKO sensorimotor cortex at P28. Emx1-CKO mice undergo a 10.6% reduction of CTIP2 neurons in layer Vb (unpaired t-test t16 = 2.383, p=0.0299, control n = 9, Emx1(A)-CKO n = 9). Source data for Figure 1—figure supplement 4 can be found in Figure 1—figure supplement 4—source data 1. Each tab of the spreadsheet is titled with a letter matching the corresponding figure panel.

Figure 1—figure supplement 4—source data 1

Information pertaining to further characterization of cortical neurons in Emx1(A)-CKO mice.

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Figure 1—figure supplement 5
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Emx1(A+B)-CKO growth and survival curves.

(A) Growth curves of Cre control and Emx1(A+B)-CKO mice. Emx1(A+B)-CKO mice exhibit reduced postnatal growth (two-way repeated measures ANOVA main effect of genotype F1, 19 = 27.14, p<0.0001, main effect of age F28, 532 = 240.3 p<0.0001, interaction F28, 532 = 46.89, p<0.0001; asterisks denote the following p-values from Sidak’s multiple comparisons tests: *=p < 0.05, **=p < 0.01, **=p < 0.001, ****=p < 0.0001; Cre control n = 9, Emx1(A+B)-CKO n = 12). (B) Survival curves of Cre control and Emx1(A+B)-CKO mice. Emx1(A+B)-CKO mice exhibit lethality starting the 3rd postnatal week, and the humane endpoint for survival and behavioral analysis is P28 (Gehan-Breslow-Wilcoxon method χ2 = 5.256, p=0.0219; Cre control n = 41, Emx1(A+B)-CKO n = 41). Source data for Figure 1—figure supplement 5 can be found in Figure 1—figure supplement 5—source data 1. Each tab of the spreadsheet is titled with a letter matching the corresponding figure panel.

Figure 1—figure supplement 5—source data 1

Growth and survival data on Emx1(A+B)-CKO mice.

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Figure 2 with 1 supplement
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TorsinB dose-dependently worsens a DYT1 model.

(A) Illustration of examined genotypes. Each row of boxes within the circles denote the presence or absence of Tor1a (top row) or Tor1b (bottom row) alleles following Cre recombination. The presence of a letter (‘A’ ‘ΔE’ or ‘B’) indicates an intact allele, whereas the absence of a letter indicates a deleted allele. (B) Cortical thickness of P28 Emx1-SKI;Tor1b+/+, Emx1-SKI;Tor1b+/-, and Emx1-SKI;Tor1b-/- mice. TorsinB loss reduces cortical thickness in Emx1-SKI mice in a dose-dependent manner (2% reduction in Emx1-SKI;Tor1b+/+, 25.2% reduction in Emx1-SKI;Tor1b+/-, and 54.5% decrease in Emx1-SKI;Tor1b-/-; two way ANOVA main effect of background genotype F1,13 = 128.3, p<0.0001, age F2,13 = 62.65, p<0.0001, and interaction F2,13 = 36.98, p<0.0001; Sidak’s multiple comparisons test p=0.9323 for Tor1b+/+, p<0.0001 for Tor1b+/-, p<0.0001 for Tor1b-/-; Tor1b+/+ control n = 3; Emx1-SKI n = 5, Tor1b+/- control n=3, Emx1-SKI n = 3, Tor1b-/- control n=2, Emx1-SKI n = 3). (C) CUX1+ cell counts in sensorimotor cortex of P28 Emx1-SKI;Tor1b+/+ and Emx1-SKI;Tor1b-/- mice. There is no significant reduction in the number of CUX1+ cells in Emx1-SKI;Tor1b+/+ sensorimotor cortex (unpaired t-test t16 = 0.8469, p=0.4095; control n = 3, SKI n = 5). Simultaneous deletion of two torsinB alleles reduces CUX1+ cell counts by 44.0% (unpaired t-test t3 = 2.655, p=0.0766; control n = 2, SKI n = 3) though this reduction does not reach statistical significance. (D) CTIP2+ cell counts in sensorimotor cortex of P28 Emx1-SKI;Tor1b+/+ and Emx1-SKI;Tor1b-/- mice. CTIP2+ neuronal cell counts are not reduced in Emx1-SKI;Tor1b+/+ mice (unpaired t-test t6 = 0.8844, p=0.4105; control n = 3, SKI n = 5). CTIP2+ cell counts in Emx1-SKI;Tor1b-/- are significantly reduced by 96.6% (unpaired t-test t3 = 24.44, p<0.0001; control n = 2, SKI n = 3). (E) Proportion of Emx1-SKI;Tor1b+/+ and Emx1-SKI;Tor1b+/- mice exhibiting limb clasping during tail suspension. A significantly greater proportion of Emx1-SKI;Tor1b+/- mice exhibit limb clasping during tail suspension compared to Emx1-SKI;Tor1b+/+ (Chi square test χ2 = 7.441, p=0.0064; Emx1-SKI;Tor1b+/+n = 12, Emx1-SKI;Tor1b+/- n = 11).

Figure 2—source data 1

Raw data on histological and behavioral characterization of Emx1-SKI mice with two, one, and zero intact torsinB alleles.

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Figure 2—figure supplement 1
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Novel allele design and histological analysis of Emx1-SKI;Tor1b-/- mice and Emx1-SKI;Tor1b+/- mice.

(A) Design of the Tor1aΔE floxed Tor1b allele. (B) Cortical thickness of P0 Emx1-SKI-B0 mice. The thickness of the Emx1-SKI-B0 cortical tissue is unchanged at P0 (unpaired t-test t3 = 0.5425, p=0.6252; control n = 3, Emx1-SKI;Tor1b-/- n = 2). (C) CUX1+ and CTIP2+ counts in the sensorimotor cortex of P28 Emx1-SKI;Tor1b+/- mice. There is a decrease of CUX1+ cells by 8.0% (unpaired t-test t4 = 2.705, p=0.0538; control n = 3, Emx1-SKI;Tor1b+/- n = 5) that does not reach statistical significance in Emx1-SKI;Tor1b+/- mice compared to Cre controls. There is no reduction of total CTIP2+ neurons in the Emx1-SKI;Tor1b+/- mice compared to Cre controls (unpaired t-test t4 = 0.9128, p=0.4130; control n = 3, Emx1-SKI;Tor1b+/- n = 5).

Figure 2—figure supplement 1—source data 1

Further histological characterization of Emx1-SKI mice with varying amounts of torsinB.

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Figure 3 with 3 supplements
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TorsinB augmentation eliminated all phenotypes in CNS conditional torsinA null mice.

(A) Cartoon illustration of ROSA26 locus engineered to express torsinB. Top: In absence of Cre, torsinB expression is prevented by a floxed ‘STOP’ cassette. Bottom: TorsinB is expressed following Cre deletion of the floxed ‘STOP’ cassette. (B) Western blot analysis of whole brain lysates probed with an anti-torsinB antibody. Mice expressing both the Nestin-Cre and B-OE alleles exhibit Cre-dependent overexpression of torsinB. (C) Quantification of western blots of whole brain lysates from Nestin-Cre and Nestin-Cre;B-OE littermates probed with anti-torsinB antibody. The B-OE allele causes significant torsinB overexpression (unpaired t-test t4 = 2.947, p=0.0421; Nestin-Cre n = 3, Nestin-Cre;B-OE n = 3). Quantification was performed by comparing torsinB expression in Cre control samples to that of Nestin-Cre;B-OE samples diluted 1:50. (D) Image of Cre control, Nes(A)-CKO, and Nes(A)-CKO;B-OE littermates at P7 (in order left to right). TorsinB overexpression restores postnatal growth. (E) Growth curves of Cre control, Cre control;B-OE, N(esA)-CKO, and Nes(A)-CKO;B-OE mice. TorsinB overexpression almost entirely restores growth in N-CKO mice (two-way repeated measures ANOVA main effect of genotype F2, 17 = 14.16, p=0.0002, main effect of age F9, 153 = 775.7 p<0.0001, interaction F18, 153 = 6.727, p<0.0001; asterisks denote the following p-values from Tukey’s multiple comparisons tests: *=p < 0.05, **=p < 0.01, **=p < 0.001, ****=p < 0.0001; Cre control n = 7, Cre control;B-OE n = 6, N(A)-CKO;B-OE n = 7). (F) Survival curves of Cre control, Cre control;B-OE, Nes(A)-CKO, and Nes(A)-CKO;B-OE mice. TorsinB overexpression eliminates lethality in N-CKO mice (Gehan-Breslow-Wilcoxon method χ2 = 36.16, p<0.0001; Cre control n = 7, Cre control;B-OE n = 7, Nes(A)-CKO n = 10, Nes(A)-CKO;B-OE n = 7). (G) Nissl staining of P8 brains from Cre control, Cre control;B-OE, Nes(A)-CKO, and Nes(A)-CKO;B-OE mice. TorsinB overexpression eliminates the morphological defects characteristic of Nes(A)-CKO mice. (H) GFAP staining of P8 brains from Cre control, Cre control;B-OE, Nes(A)-CKO, and Nes(A)-CKO;B-OE mice. GFAP immunostaining illustrates the characteristic gliotic changes in Nes(A)-CKO cortex (arrows), thalamus, deep cerebellar nuclei, and hindbrain (circles). TorsinB overexpression eliminates gliotic changes in Nes(A)-CKO mice.

Figure 3—source data 1

Biochemical and organismal characterization of Nes(A)-CKO;B-OE mice.

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Figure 3—figure supplement 1
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Quantification of torsinB in Nestin-Cre;B-OE liver tissue.

(A) Western blot of whole liver lysates from Nestin-Cre and Nestin-Cre;B-OE littermates probed with anti-torsinB antibody. (B) Quantification of western blots of whole brain lysates from Nestin-Cre and Nestin-Cre;B-OE littermates probed with anti-torsinB antibody. The B-OE allele does not alter torsinB overexpression in liver, which is outside of the Cre field (unpaired t-test t4 = 4560, p=0.6720; Nestin-Cre n = 3, Nestin-Cre;B-OE n = 3).

Figure 3—figure supplement 1—source data 1

Information pertaining to torsinA expression in torsinB overexpression brain tissue.

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Figure 3—figure supplement 2
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Analysis of torsinA expression in Nestin-Cre;B-OE brain tissue.

(A) Western blot of whole brain lysates from Nestin-Cre and Nestin-Cre;B-OE littermates probed with anti-torsinA antibody. (B) Quantification of western blots of whole brain lysates from Nestin-Cre and Nestin-Cre;B-OE littermates probed with anti-torsinA antibody. TorsinA expression is not altered in mice overexpressing torsinB (unpaired t-test t4 = 0.5566, p=0.6075; Nestin-Cre n = 3, Nestin-Cre;B-OE n = 3)).

Figure 3—figure supplement 2—source data 1

Information on liver torsinB expression levels.

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Figure 3—figure supplement 3
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TorsinB overexpression prevents overtly abnormal postures and gross disruption of brain morphology in N-CKO mice.

(A) Table of overtly abnormal postural phenotypes displayed by N(A)-CKO and N(A)-CKO;B-OE mice at P10. TorsinB overexpression eliminates these phenotypes that are prevalent in N(A)-CKO mice (stiff hindlimb: Chi square test χ2 = 11.48, p=0.0007; twisted hindpaw: Chi square test χ2 = 15, p=0.0001). (B) Brain area measurements of P8 Nestin-Cre control, Cre control;B-OE, N(A)-CKO, and N(A)-CKO;B-OE. TorsinB overexpression prevents the reduction in brain size characteristic of N(A)-CKO mice (two-way ANOVA main effect of genotype F1, 8 = 13.54, p=0.0062, main effect of torsinB level F1, 8 = 3.103, p=0.1162, interaction F1,8 = 8.916, p=0.0174; asterisks denote the following p-values from Tukey’s multiple comparisons tests: *=p < 0.05; Cre control n = 3, Cre control;B-OE n = 3; N(A)-CKO n = 3, N(A)-CKO;B-OE n = 3).

Figure 3—figure supplement 3—source data 1

Behavioral and brain morphological characterization of Nes(A)-CKO;B-OE mice.

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Figure 4
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TorsinB overexpression rescues ΔE torsinA phenotypes.

TorsinB overexpression prevents striatal cholinergic interneuron degeneration and dystonic-like movements. (A) Growth curves of Cre control, Cre control;B-OE, Nes-SKI, and Nes-SKI;B-OE mice. Nes-SKI mice exhibited reduced postnatal growth, which was partially rescued by torsinB overexpression (two-way repeated measures ANOVA main effect of genotype F3, 26 = 60.65, p<0.0001, main effect of age F1.828, 47.53 = 794.8 p<0.0001, interaction F12, 104 = 9.831, p<0.0001; Cre control n = 7, Cre control;B-OE n = 8, Nes-SKI n = 7, Nes-SKI;B-OE n = 8). (B) Table of overtly abnormal postural and developmental phenotypes displayed by Nes-SKI and Nes-SKI;B-OE mice at P15. TorsinB overexpression reduces phenotypes that are prevalent in Nes-SKI mice (squinty eyes: Chi square test χ2 = 5.529, p=0.0187; twisted hindpaw: Chi square test χ2 = 6.234, p=0.0125). (C) Nissl staining of P10 brains from Cre control, Cre control;B-OE, Nes-SKI, and Nes-SKI;B-OE mice. TorsinB overexpression eliminates the morphological defects characteristic of Nes-SKI mice. (D) GFAP staining of P10 brains from Cre control, Cre control;B-OE, Nes-SKI, and Nes-SKI;B-OE mice. GFAP immunostaining illustrates the characteristic gliotic changes in Nes-SKI, including in cortex (arrows) and thalamus (circle). TorsinB overexpression eliminates gliotic changes in Nes-SKI mice.

Figure 4—source data 1

Information characterizing Nes-SKI;B-OE mice.

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Figure 5 with 2 supplements
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TorsinB overexpression prevents striatal cholinergic interneuron degeneration and dystonic-like movements.

(A) Duration of abnormal movements during one minute of tail suspension in P70 Cre control, Cre control;B-OE, Dlx(A)-CKO, and Dlx(A)-CKO;B-OE mice. TorsinB overexpression significantly reduces severity of limb clasping (two-way ANOVA main effect of genotype F1,44 = 47.45, p<0.0001, torsinB level F1,44 = 36.86, p<0.0001, and interaction F1,44 = 49.96, p<0.0001; Cre control n = 10, Cre control;B-OE n = 12, Dlx(A)-CKO n = 11, Dlx(A)-CKO;B-OE n = 15). (B) Prevalence of trunk twisting in Dlx(A)-CKO and Dlx(A)-CKO;B-OE mice. TorsinB overexpression significantly reduces prevalence of trunk twisting (Chi square test χ2 = 18.77, p<0.0001; Dlx(A)-CKO n = 11, Dlx(A)-CKO; B-OE n = 15). (C) Stereologic counts of striatal cholinergic interneurons in P70 Cre control, Cre control;B-OE, Dlx(A)-CKO, and Dlx(A)-CKO;B-OE brains. TorsinB overexpression prevents ChI degeneration characteristic of Dlx(A)-CKO mice (two-way ANOVA main effect of genotype F1,22 = 52.45, p<0.0001, ROSA-Tor1b allele F1,22 = 45.54, p<0.0001, and interaction F1,22 = 41.90, p<0.0001; Cre control n = 6, Cre control;B-OE n = 6, Dlx(A)-CKO n = 7, Dlx(A)-CKO;B-OE n = 7). (D) Representative images of P70 striatum immunostained with antibody to ChAT. ChAT+ cell density is reduced in Dlx(A)-CKO striatum while it appears normal in Dlx(A)-CKO;B-OE striatum.

Figure 5—source data 1

Information on behavioral and histological characterization of Dlx(A)-CKO;B-OE mice.

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Dlx-Cre;B-OE mice exhibit no apparent motor or organismal phenotype.

(A) Western blots of striatal and cerebellar lysates probed with anti-torsinB antibody. Dlx5/6-Cre selectively activates the B-OE allele in forebrain structures (striatum); torsinB expression in hindbrain structures (e.g., cerebellum) is unaffected. (B) Postnatal weight gain of Dlx5/6-Cre;B-OE mice. Weight gain is normal in Dlx5/6-Cre;B-OE mice compared to littermate controls (two-way repeated measures ANOVA genotype x age interaction F3, 30 = 9439, p=0.4318; Cre control n = 6, Cre; B-OE n = 6). (C) Development of neonatal reflexes in Dlx5/6-Cre; B-OE mice. Neonatal reflexes including righting (two-way repeated measures ANOVA genotype x age interaction F2, 20 = 1.858, p=0.1819; Cre control n = 6, Cre;B-OE n = 6), negative geotaxis (two-way repeated measures ANOVA genotype x age interaction F2, 20 = 2.031, p=0.1574; Cre control n = 6, Cre;B-OE n = 6), and forelimb hang (unpaired t-test t1 = 2317, p=0.8214; Cre control n = 6, Cre;B-OE n = 6) are normal in Dlx5/6-Cre;B-OE mice, indicating normal gross and motor development.

Figure 5—figure supplement 1—source data 1

Infomation pertaining to growth and preweaning reflexes of Dlx-Cre;B-OE mice.

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Dlx-Cre;B-OE mice exhibit no apparent neuropathological phenotype.

(A) Cortical thickness of P70 Cre control, Cre control;B-OE, Dlx(A)-CKO, and Dlx(A)-CKO;B-OE mice. Cortical thickness is unchanged (two-way ANOVA main genotype x torsinB level interaction F1,12 = 0.0002485, p=0.9877; n = 4 for all genotypes). (B) Striatal volume of P70 Cre control, Cre control;B-OE, Dlx(A)-CKO, and Dlx(A)-CKO;B-OE mice. Striatal volume is unchanged (two-way ANOVA genotype x torsinB level interaction F1,12 = 0.03657, p=0.9515; n = 4 for all genotypes). (C) Striatal medium and small neuron counts in P70 Cre control, Cre control;B-OE, Dlx(A)-CKO, and Dlx(A)-CKO;B-OE mice. The overall number of striatal neurons is unchanged two-way ANOVA genotype x torsinB level interaction F1,12 = 0.01144, p=0.9166; n = 4 for all genotypes). (D) GFAP staining from Cre control, Cre control;B-OE, Dlx(A)-CKO, and Dlx(A)-CKO;B-OE mice. Labels show striatum (Str) and corpus callosum (CC). GFAP staining shows no reactive gliosis within the striatum of any mice.

Figure 5—figure supplement 2—source data 1

Information on neuropathological characterization of Dlx(A)-CKO;B-OE mice.

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Tables

Table 1
Features of mouse models used in this study.

Characteristics of DYT1 dystonia mouse models including extent of Cre field, behavioral phenotype, and neuropathological phenotype.

ModelGenotypeCre fieldOrganismal/behavioral phenotypeHistologic findingsRationale for useImpact of torsinB modulation
Emx1(A)-CKOEmx1-Cre; Tor1aKO/flxForebrain excitatory, prominent in cortex and hippocampusNormal appearing
Limb clasping in a subset of mice		Forebrain-selective neurodegenerationForebrain motor loop involvement
Mild behavioral and neuropathology findings to assess combined torsinA and B LOF		Reduction:
Worsened neuropathology and behavior
Emx1-SKIEmx1-Cre; Tor1aΔE/flxForebrain excitatory, prominent in cortex and hippocampusNormal appearing
Limb clasping in a subset of mice		Forebrain-selective neurodegeneration milder than that seen in Emx1-CKOForebrain motor loop involvement
Presence of disease mutant torsinA		Reduction:
Dose-dependent worsening of neuropathology and behavior
Nes(A)-CKONestin-Cre; Tor1aKO/flxEntire nervous systemLack of postnatal weight gain
Early lethality by 3rd postnatal week
Overtly abnormal postures at rest		Degeneration in multiple sensorimotor regionsClear and robust phenotypes
Widespread involvement of nervous system		Overexpression:
Prevention of lethality, restored weight gain
Prevention of degeneration and gliosis
Nes-SKINestin-Cre; Tor1aΔE/flxEntire nervous systemReduced postnatal weight gain
Overt postural and developmental phenotypes at rest		Degeneration in multiple sensorimotor regionsWidespread involvement of nervous system
Presence of disease mutant torsinA		Overexpression:
Restored weight gain
Prevention of degeneration and gliosis
Dlx(A)-CKODlx5/6-Cre; Tor1aKO/flxForebrain GABAergic and cholinergic, including all striatal neuronsLimb clasping and trunk twisting; symptoms respond to drugs used in human patientsSelective degeneration of dorsal striatal cholinergic interneuronsPredictive validity
Time course of motor abnormalities mimics that of human patients		Overexpression:
Prevention of abnormal limb clasping and twisting movements
Prevention of cholinergic interneuron degeneration
Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional
information
AntibodyRabbit polyclonal α-GFAPDakoCat#Z0334
RRID:AB_10013382		IHC(1:2000)
AntibodyRabbit polyclonal α-CUX1Santa Cruz BiotechnologyCat#sc-13024
RRID:AB_2261231		IHC(1:200)
AntibodyRat monoclonal α-CTIP2AbcamCat#ab18465 RRID:AB_2064130IHC(1:500)
AntibodyGoat polyclonal α-ChATMilliporeCat#AB144P RRID:AB_90560IHC(1:200)
AntibodyDonkey α-rabbit IgG,
Alexa Fluor 555 conjugated		InvitrogenCat#A-31572 RRID:AB_162543IHC(1:800)
AntibodyDonkey α-rat IgG,
Alexa Fluor 488 conjugated		InvitrogenCat# A-21208 RRID:AB_141709IHC(1:800)
AntibodyDonkey α-goat IgG, biotin-SP conjugatedJackson ImmunoresearchCat#705-065-147 RRID:AB_2340397IHC(1:800)
AntibodyRabbit polyclonal α-torsinAAbcam#ab34540 RRID:AB_2240792WB(1:10,000)
AntibodyRabbit polyclonal α-torsinBKind gift of
Schlieker lab		N/AWB(1:1,000)
AntibodyGoat α-rabbit IgG,
HRP-linked		Cell SignalingCat#7074 RRID:AB_2099233WB(1:20,000)
AntibodyRabbit polyclonal α-CalnexinEnzo Life SciencesCat#ADI-SPA-860-F RRID:AB_11178981WB(1:20,000)
Sequence-based reagentLox-gtFIntegrated DNA TechnologiesPCR primersCTG ACA CAG TGA GTG AAG GTG C
Sequenced-based reagentLox-gtRIntegrated DNA TechnologiesPCR primersGGT GCT GAG GAA GTG CTG TG
Sequenced-based reagentFrt-gtFIntegrated DNA
Technologies		PCR primersAGG GGC CAT AGA GTG GTT AGG
Sequenced-based reagentFrt-gtRIntegrated DNA
Technologies		PCR primersCTT AGC CGC
TTT GTG CTG
Sequenced-based reagentRosa-gtFIntegrated DNA TechnologiesPCR primersAGT CGC TCT GAG TTG TTA TCA G
Sequenced-based reagentRosa-gtRIntegrated DNA TechnologiesPCR primersCTG ACA CAG TGA GTG AAG GTG C
Commercial assay or kitABC KitVectastainCat#PK6100 RRID:AB_2336819Use kit directions
Commercial assay or kitProlong Gold AntifadeThermo
Scientific		Cat#P36930Use kit directions
Software, algorithmStereoinvestigatorMBF Biosciencehttps://www.mbfbioscience
.com/stereo-investigator _		N/A
Software, algorithmImageJNIHhttps://imagej.nih.gov/ij/;
RRID:SCR_003070		N/A
Software, algorithmPrismGraphpadhttp://www.graphpad.com; RRID:SCR_002798N/A
Strain, strain
background (M. musculus, male and female)		Tor1b+/-University of Connecticut Center for Mouse Genome ModificationN/AB6;129 hybrid
Strain, strain background (M. musculus, male and female)Tor1atm1Wtd/JThe Jackson
Laboratory		RRID:IMSR_JAX:006251B6;129 hybrid
Strain, strain
background
(M. musculus, male and female)		Tor1atm3.1Wtd/JThe Jackson
Laboratory		RRID:IMSR_JAX:025832B6;129 hybrid
Strain, strain background (M. musculus, male and female)STOCK Tor1atm2Wtd/JThe Jackson LaboratoryRRID:IMSR_JAX:025637B6;129 hybrid
Strain, strain background (M. musculus, male and female)Tor1abΔE floxed Tor1b/+This paper (University of Connecticut
Center for Mouse Genome
Modification)		N/AB6;129 hybrid
Mouse line will be
submitted and
available from The Jackson Laboratory
Strain, strain background (M. musculus, male and female)Emx1tm1(cre)Krj/JThe Jackson LaboratoryRRID:IMSR_JAX:005628B6;129 hybrid
Strain, strain
background (M. musculus,
male and female)		Tg(dlx5a-cre)1Mekk/JThe Jackson LaboratoryRRID:IMSR_JAX:008199B6;129 hybrid
Strain, strain background (M. musculus, male and female)Tg(Nes-cre)1kln/JThe Jackson LaboratoryRRID:IMSR_JAX:003771B6;129 hybrid
Strain, strain background
(M. musculus, male and female)		B-OEThis paper (from Biocytogen)N/AB6;129 hybrid
Mouse line will be submitted and available from The Jackson Laboratory
Table 2
Genotyping PCR programs and band sizes.
GenePrimer nameCycleBand sizes
Tor1a ΔE floxed Tor1bLox-gtF94°C, 3 min; 94°C, 30 s; 61.5°C, 30 s; 72°C, 30 s, 34 cycles; 72°C, 5 minWT – 245 bp
Floxed– 336 bp
Lox-gtR
Frt-gtF
Frt-gtR
B-OERosa-gtF94°C, 3 min; 94°C, 30 s; 61°C, 30 s; 72°C, 30 s, 30 cycles; 72°C, 5 minWT – 469 bp
Mutant – 188 bp
Rosa-gtR
Mut-R

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  1. Jay Li
  2. Chun-Chi Liang
  3. Samuel S Pappas
  4. William T Dauer
(2020)
TorsinB overexpression prevents abnormal twisting in DYT1 dystonia mouse models
eLife 9:e54285.
https://doi.org/10.7554/eLife.54285