Dystonia-associated mutations cause premature degradation of torsinA protein and cell-type-specific mislocalization to the nuclear envelope

Abstract

An in-frame 3 bp deletion in the torsinA gene resulting in the loss of a glutamate residue at position 302 or 303 (torsinA DeltaE) is the major cause for early-onset torsion dystonia (DYT1). In addition, an 18 bp deletion in the torsinA gene resulting in the loss of residues 323-328 (torsinA Delta323-8) has also been associated with dystonia. Here we report that torsinA DeltaE and torsinA Delta323-8 mutations cause neuronal cell-type-specific mislocalization of torsinA protein to the nuclear envelope without affecting torsinA oligomerization. Furthermore, both dystonia-associated mutations destabilize torsinA protein in dopaminergic cells. We find that wild-type torsinA protein is degraded primarily through the macroautophagy-lysosome pathway. In contrast, torsinA DeltaE and torsinA Delta323-8 mutant proteins are degraded by both the proteasome and macroautophagy-lysosome pathways. Our findings suggest that torsinA mutation-induced premature degradation may contribute to the pathogenesis of dystonia via a loss-of-function mechanism and underscore the importance of both the proteasome and macroautophagy in the clearance of dystonia-associated torsinA mutant proteins.

Figure 1.

TorsinA is enriched in the NE in SH-SY5Y cells but not in HeLa cells. (A) HeLa or SH-SY5Y cells expressing C-terminally HA-tagged torsinA WT were stained with primary antibodies against HA and ER marker KDEL, followed by detection with secondary antibodies conjugated to TR (KDEL, red) or FITC (torsinA, green). Hoechst stain was used to visualize the nucleus. (B) Quantification shows the relative distribution of torsinA and KDEL in the NE versus the ER. Data represent mean ± SE from at least three independent experiments. *Significantly different from the NE/ER ratio of KDEL in SH-SY5Y cells (P < 0.05). #Significantly different from the NE/ER ratio of KDEL in HeLa cells (P < 0.05). (C) NE preference of torsinA was determined by normalizing the NE/ER ratio of torsinA in HeLa or SH-SY5Y cells to the corresponding NE/ER ratio of KDEL in the same cells. Data represent mean ± SE from at least three independent experiments. *Significantly different from HeLa cells (P < 0.05). Scale bar, 10 µm.

Figure 2.

Endogenous torsinA shows preferential NE localization in primary cortical neurons compared with fibroblasts. (A) Mouse embryonic fibroblasts or primary cortical neurons were stained with primary antibodies against torsinA and ER marker KDEL, followed by detection with secondary antibodies conjugated to TR (KDEL, red) or FITC (torsinA, green). Hoechst stain was used to visualize the nucleus. Neuronal cell body shows an enlarged view of the cell body area from the same neuron. (B) Quantification shows the relative distribution of torsinA and KDEL in the NE versus the ER. Data represent mean ± SE from at least three independent experiments. *Significantly different from the NE/ER ratio of KDEL in cortical neurons (P < 0.05). (C) NE preference of torsinA was determined by normalizing the NE/ER ratio of torsinA in fibroblasts or neurons to the corresponding NE/ER ratio of KDEL in the same cells. Data represent mean ± SE from at least three independent experiments. *Significantly different from fibroblasts (P < 0.05). Scale bar, 10 µm.

Figure 3.

Dystonia-associated mutations cause torsinA translocation to the NE in SH-SY5Y cells but not in HeLa cells. (A) HeLa or (B) SH-SY5Y cells expressing C-terminally HA-tagged torsinA WT, ΔE or Δ323–8 were stained with primary antibodies against HA and ER marker KDEL, followed by detection with secondary antibodies conjugated to TR (KDEL, red) or FITC (torsinA, green). Hoechst stain was used to visualize the nucleus. (C) Quantification shows the relative distribution of torsinA and KDEL in the NE versus the ER. Data represent mean ± SE from at least three independent experiments. *Significantly different from the NE/ER ratio of torsinA WT in SH-SY5Y cells (P < 0.05). (D) NE preference of torsinA was determined by normalizing the NE/ER ratio of torsinA in HeLa or SH-SY5Y cells to the corresponding NE/ER ratio of KDEL in the same cells. Data represent mean ± SE from at least three independent experiments. *Significantly different from the NE preference of torsinA WT in SH-SY5Y cells (P < 0.05). Scale bar, 10 µm.

Figure 4.

Dystonia-associated mutations have no effect on torsinA oligomerization. (A) Interaction of torsinA WT with itself. SH-SY5Y cells expressing C-terminally Myc-tagged torsinA WT and C-terminally HA-tagged torsinA WT or HA vector were lysed and subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with anti-HA and anti-Myc antibodies. Asterisks indicate torsinA degradation products. (B) Interaction of torsinA WT with dystonia-associated mutant torsinA. SH-SY5Y cells expressing C-terminally HA-tagged torsinA WT and C-terminally Myc-tagged torsinA WT, ΔE, Δ323–8 or Myc vector were lysed and subjected to immunoprecipitation with anti-Myc antibody followed by immunoblotting with anti-HA and anti-Myc antibodies.

Figure 5.

The NT region of torsinA is sufficient for torsinA oligomerization. (A) Domain structure of torsinA and its deletion construct. S, signal sequence; H, hydrophobic domain; A, Walker A motif; B, Walker B motif. (B) Interaction of torsinA WT with torsinA NT region. SH-SY5Y cells expressing C-terminally Myc-tagged torsinA WT or NT and C-terminally HA-tagged torsinA WT or HA vector were lysed and subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with anti-HA and anti-Myc antibodies. Asterisks indicate torsinA degradation products. (C) Interaction of dystonia-associated mutants with torsinA NT region. SH-SY5Y cells expressing C-terminally HA-tagged torsinA NT and C-terminally Myc-tagged torsinA WT, ΔE, Δ323–8 or Myc vector were lysed and subjected to immunoprecipitation with anti-Myc antibody followed by immunoblotting with anti-HA and anti-Myc antibodies.

Figure 6.

Dystonia-associated mutations cause premature degradation of torsinA protein. SH-SY5Y cells expressing HA-tagged torsinA WT (open square), ΔE (closed circle) or Δ323–8 (closed triangle) were pulse-labeled for 1 h in [³⁵S]Met/Cys-containing medium and chased with non-radioactive Met/Cys for the indicated time. Time points were taken every 24 h over 5 days (A) and every 6 h over the first 24 h (B). ³⁵S-labeled WT or mutant torsinA proteins were immunoprecipitated from lysates with anti-HA antibodies and detected by SDS–PAGE and autoradiography. The levels of WT or mutant torsinA proteins were quantified and plotted relative to the corresponding torsinA levels at 0 h. Data represent mean ± SE of the results from at least three independent experiments.

Figure 7.

Effects of proteasome, autophagy and lysosome inhibition on WT and mutant torsinA levels. (A) SH-SY5Y cells expressing HA-tagged torsinA WT, ΔE or Δ323–8 were treated with the indicated proteolysis inhibitors or DMSO control. Lysates were analyzed by SDS–PAGE and immunoblotting with anti-HA and anti-actin antibodies. (B) The relative level of WT or mutant torsinA was measured by quantification of the intensity of the WT or mutant torsinA band and normalized to the actin level in the corresponding cell lysate. Results are shown as mean ± SE from at least three independent experiments. *Significantly different from the corresponding vehicle (DMSO)-treated control cells expressing the same type of torsinA (P < 0.05). #Significantly different from the vehicle-treated, torsinA WT-expressing cells (P < 0.05).

Figure 8.

Degradation of torsinA mutants by both the proteasome and lysosome pathways. SH-SY5Y cells expressing HA-tagged torsinA WT (A), ΔE (B) or Δ323–8 (C) were pulse-labeled for 1 h with medium containing [³⁵S]Met/Cys and chased with non-radioactive Met/Cys containing MG132 (closed circle), E64 (closed triangle) or vehicle control (open square) for the indicated time. Lysates were immunoprecipitated with anti-HA antibodies and detected by SDS–PAGE and autoradiography. Proteins levels were quantified using a PhosphorImager and plotted relative to the corresponding torsinA levels at 0 h. Data are shown as mean ± SE of the results from at least three independent experiments. The asterisks indicate a statistically significant (P < 0.05) increase in the level of torsinA in MG132-treated cells versus vehicle-treated controls. The plus sign indicates an increase approaching significance (P < 0.065) in the level of torsinA in MG132-treated cells versus vehicle-treated controls. The pound signs indicate a statistically significant (P < 0.05) increase in the level of torsinA in E64-treated cells versus vehicle-treated controls.