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Originally published In Press as doi:10.1074/jbc.M704097200 on December 31, 2007

J. Biol. Chem., Vol. 283, Issue 12, 7568-7579, March 21, 2008
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The Dystonia-associated Protein TorsinA Modulates Synaptic Vesicle Recycling*Formula

Alessandra Granata{ddagger}§, Rose Watson{ddagger}, Lucy M. Collinson{ddagger}, Giampietro Schiavo{ddagger}1, and Thomas T. Warner§2

From the {ddagger}Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom and the §Department of Clinical Neurosciences, UCL Institute of Neurology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, United Kingdom

Received for publication, May 17, 2007 , and in revised form, December 24, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant {Delta}E-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of {Delta}E-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the {Delta}E-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that {Delta}E-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The majority of cases of early onset, primary torsion dystonia are caused by a dominantly inherited mutation in the DYT1 (TOR1A) gene on chromosome 9q34 (1). DYT1 dystonia manifests in childhood, typically with dystonia in a limb that spreads to the trunk and other limbs, usually sparing cranio-cervical muscles (2, 3). There is no evidence for neurodegeneration in DYT1 dystonia, implying that abnormal movements are caused by a functional neuronal defect (4). All cases of typical DYT1 dystonia are caused by an in-frame GAG deletion ({Delta}GAG302/303; {Delta}E) in DYT1 gene, resulting in the loss of a glutamic acid in the C-terminal region of the encoded protein, torsinA (1).

TorsinA is a member of the AAA ATPase superfamily of chaperone-like proteins (5). In mammalian neuronal cells, torsinA is found throughout the cytoplasm, neurite processes, and growth cones (6, 7). TorsinA has also been found in the lumen of the endoplasmic reticulum (ER)3 and in the space between the inner and the outer membrane of the nuclear envelope (NE) (8-11). In contrast, in cells overexpressing the mutant ({Delta}E-torsinA), the protein is redistributed from ER to NE and accumulates in large perinuclear membranous inclusions, which appeared to arise from the nuclear envelope (7, 12-14). TorsinA-positive inclusions have been found in the midbrain of DYT1 patients, suggesting that they are relevant to the pathogenesis of DYT1 dystonia (15). In SH-SY5Y neuroblastoma cells, {Delta}E-enriched inclusions contain the vesicular monoamine transporter 2 (VMAT2), a membrane-associated protein involved in loading dopamine vesicles (14). Other indirect evidence for abnormal dopaminergic function in DYT1 dystonia comes from cell models showing that torsinA affects the membrane distribution of the dopamine transporter and influences the activation of dopaminergic D2 receptors in a transgenic mouse model (16, 17). Consistent with these observations, torsinA is highly expressed in dopaminergic neurons of the substantia nigra (18, 19). More recent work in a DYT1 transgenic mouse model has suggested that mutant torsinA impaired dopamine release (20).

To investigate the role of torsinA further, we performed a yeast two-hybrid screening using full-length wt-torsinA and {Delta}E-torsinA as bait. Snapin, a SNAP25 (synaptosomal associated protein of 25 kDa)-binding protein (21), was identified and its interaction with both wild type and mutant torsinA confirmed by in vitro and in vivo assays. Snapin is thought to promote the maturation/priming of synaptic vesicles (SVs) by interacting with components of the SNARE complex (21, 22). In view of this observation, we investigated whether overexpression of wild type or mutant torsinA affects SV recycling in SH-SY5Y cells. Additionally, to understand the functional link between torsinA and snapin, we examined the effects of siRNA-based knockdown of both proteins. Our findings suggest that overexpression of wt-torsinA as well as knockdown of the endogenous torsinA negatively affects SV turnover, whereas {Delta}E-torsinA appears to act as a loss of function mutant by enhancing synaptic membrane turnover.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Antibodies—The reagents were from Sigma-Aldrich, unless otherwise specified. The mouse monoclonal anti-hemagglutinin (HA) antibody is from the Cancer Research UK Monoclonal Antibody Service. Rabbit polyclonal Syt-163 antibody was raised against a peptide corresponding to the N-terminal 19 residues of rat synaptotagmin I (SytI). Rabbit polyclonal anti-snapin and anti-green fluorescent protein (GFP) antibodies were kind gifts from Dr. R. Jahn (Max Planck Institute, Göttingen, DE) (23) and Dr. T. Hunt (Cancer Research UK London Research Institute). The monoclonal anti-SytI M48 and anti-SGI LF19 antibodies were kindly provided by Dr. T. H. Söllner (University of Heidelberg, DE) and by Prof. H. Winler, (University of Innsbruck, Innsbruck, Austria), respectively. The monoclonal anti-human torsinA antibody is from Cell Signaling Technology (Danvers, MA), the monoclonal anti-protein-disulfide isomerase (PDI) antibody is from Stressgen (Victoria, Canada), the polyclonal anti-VAMP2 is from Wako Chemical (Osaka, JP), and the rat torsinA is from Abcam (Cambridge Science Park, Cambridge, UK). AlexaFluor® 488-, 555-, and 647-conjugated goat anti-rabbit and anti-mouse secondary antibodies were from Invitrogen. Horseradish peroxidase-conjugated secondary antibodies were from DAKO UK (Ely, UK) and ECL was from GE Healthcare (Little Chalfont, UK).

Cell Culture—SH-SY5Y cell lines stably transfected with pcDNA3.1, containing either wild type DYT1, GAG-deleted DYT1 (DYT1-{Delta}E) or no insert (14) were grown in 1:1 mixture of Eagle's minimal essential medium (Promochem, Middlesex, UK), Ham's F-12 nutrient mixture (Invitrogen) and 10% fetal calf serum at 37 °C and 5% CO2 under selective conditions (0.4 mg/ml G418; Invitrogen).

Yeast Two-hybrid Screening—The Matchmaker yeast two-hybrid system 3 (Clontech, Mountain View, CA) was used according to the manufacturer's instructions. To generate the baits, human full-length DYT1 and DYT1-{Delta}E cDNAs were inserted in-frame into the HindIII and BamHI sites of the pGBKT7 vector (Clontech). The bait was transformed into Saccharomyces cerevisiae Y187 strain (MATa), which was then mated with AH109 yeast strain (MATa) pretransformed with an adult human brain cDNA library. Positive clones were selected for growth on Ade-/His-/Trp-/Leu-/{alpha}-galactosidase plates. Plasmid DNA was isolated and transformed in Escherichia coli using pGEM®T Easy Vector System (Promega, Madison, WI), and DNA sequencing was performed using automated methods. To confirm the specificity of the interactions, cDNA from positive colonies was rescued, retransformed in fresh yeast cells, and tested for β-galactosidase activity, using the yeast β-galactosidase assay kit (Pierce) according to the manufacturer's instructions.

Expression and Purification of Recombinant Proteins—Full-length DYT1 and DYT1-{Delta}E and their six deletion mutants (tors1-3) were amplified by PCR from pGBKT7 and the C-terminal deletion mutant of human snapin from pSFV1-PV-IRES-GFP vector (a kind gift from Dr. J. Rett (Physiologisches Institut, Homburg, Germany)) (22) and subcloned into EcoRI and SalI of PGEX-T4-1 (GE Healthcare) as glutathione S-transferase (GST) fusion protein. A vector encoding full-length snapin fused to GST was kindly provided by Dr. R. Jahn (23). Protein expression was induced in E. coli BL21 by the addition of 400 µM isopropyl-β-D-thiogalactopyranoside for 4 h at 30 °C (50). Upon lysis of the bacteria in 20 mM Tris-HCl, pH 7.5, 0.5% Tween, 2 mM EDTA, 0.1% 2-mercaptoethanol, 4 µg/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride, and EDTA-free protein inhibitors (Roche, Mannheim, DE), fusion proteins were purified on glutathione-Sepharose beads for 1 h at 4 °C, washed three times with PBS containing 0.05% Tween, 0.5 M NaCl and eluted in 20 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0. Purified torsinA and its mutant were then dialyzed against 20 mM Hepes-KOH, pH 7.0, 200 mM KCl, 2 mM 2-mercaptoethanol, and 0.5 mM ATP, whereas snapin and its fragment were dialyzed against PBS.

In Vitro Transcription-Translation and Pulldown Assays—For pulldown assays, GST fusion proteins containing torsinA, {Delta}E-torsinA, snapin, and its deletion mutants were bound to glutathione-Sepharose in Hank's buffer (20 mM Hepes-NaOH, pH 7.4, 0.44 mM KH2PO4, 0.42 mM NaH2PO4, 5.36 mM KCl, 136 mM NaCl, 0.81 mM MgSO4, 1.26 mM CaCl2, 6.1 mM glucose) containing 0.1% BSA (Hank's-BSA) for 1 h at 4 °C. The beads were then blocked with 2% BSA in Hank's buffer for 1 h at 4 °C and washed three times with Hank's. 35S-Labeled proteins were generated using TNT Quick coupled transcription/translation system (Promega), precleared on glutathione-Sepharose beads for 1 h at 4 °C, and then incubated with either prebound GST fusion proteins or GST alone for 1 h at 4 °C in Hank's-BSA. Glutathione-Sepharose beads were then washed with ice-cold Hank's-BSA containing 250 mM NaCl, 1% Triton X-100 and resuspended in loading buffer. Eluted proteins were then analyzed by autoradiography. The gel was stained with Coomassie Blue to visualize the GST fusion proteins. SH-SY5Y cell lines expressing wild type HA-tagged fusion torsinA and {Delta}E-torsinA were washed in PBS, scraped, and then lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.4, 0.1 mM EDTA, and protease inhibitors) containing 0.5% CHAPS (Calbiochem, Darmstadt, Germany) for 30 min at 4 °C under constant agitation. Precleared cell extracts were incubated with immobilized GST-snapin or GST overnight at 4 °C. After six washes with lysis buffer, the bound proteins were eluted in loading buffer and analyzed by Western blot.

Immunoprecipitation and Western Blot—Cell extracts from SH-SY5Y expressing HA-tagged wild type DYT1, DYT1-{Delta}E (20 µg protein/lane), prepared as above, were incubated with anti-snapin antibodies (30 µg of antibody/sample; 1 mg of antibody for 1 ml of resin) overnight at 4 °C. As a negative control, the cell lysates were mixed with an irrelevant antibody (anti-GFP). Protein A-Sepharose beads (GE Healthcare) were then added to each samples and incubated for 1 h at 4 °C under constant stirring. After extensive washes with lysis buffer containing 0.5% CHAPS, the beads were resuspended in loading buffer, boiled, and analyzed in SDS-PAGE followed by Western blot. Immunoprecipitates were blotted with anti-HA (1:1000) and anti-snapin (1:250) antibodies. Lysates of SH-SY5Y cells expressing wild type DYT1, DYT1-{Delta}E, or the control vector pcDNA3.1 were resuspended in loading buffer, boiled, and analyzed by Western blot (10 µg/lane). Nitrocellulose membranes were incubated with anti-VAMP2 (1:1000), anti-synaptotagmin I (M48, 1:200), or anti-actin (1:1000) antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were revealed by ECL and quantified (n = 3) using National Institutes of Health Image 1.61 software.

Hippocampal Neuron Culture—E18 mouse embryos were dissected in PBS, pH 7.4, containing 0.6% glucose (PBS-G). Once removed, hippocampi were kept on ice in 1 ml of PBS-G buffer. Trypsin was added to 0.025% final concentration, and the tissues were incubated at 37 °C for 15 min with constant stirring. The cells were centrifuged at 1,000 rpm for 5 min at room temperature and then resuspended in growth medium (Dulbecco's modified Eagle's medium, 10% horse serum, 2 mM glutamine, 4.5 g glucose, 1 mM sodium pyruvate, gentamicin, penicillin, and streptomycin) prior to plating on coverslips pretreated with poly-L-lysine (0.1 mg/ml) for 1 h at room temperature. The following day, the growth medium was replaced with differentiating medium (Neurobasal, 2% B27, 2 mM glutamine, penicillin, and streptomycin). The cells were analyzed for immunofluorescence after 4 days.

Immunofluorescence—SH-SY5Y cell lines were plated onto glass coverslips and allowed to grow overnight. PC12 cells were grown on poly-L-lysine-coated coverslips and differentiated with 100 ng/ml nerve grow factor in Dulbecco's modified Eagle's medium for 72 h (24).

The cells were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature. SH-SY5Y were washed with PBS and incubated with 50 mM NH4Cl for 5 min. The cells were rinsed and PBS containing 10% goat serum was added for 1 h at room temperature. PC12 cells were incubated in blocking solution buffer (10% goat serum, 2% BSA, and 0.25% gelatin) for 1 h. Primary antibodies were diluted (anti-HA, 1:1000; anti-torsinA, 1:200; anti-snapin, 1:250; anti-SytI M48, 1:250; anti-VAMP2, 1:500; anti-SGI, 1:1000; and anti-PDI, 1:500) in PBS containing 0.05% saponin and 10% goat serum and incubated for 1 h at room temperature. After rinsing with PBS three times for 10 min, AlexaFluor® 488-, 555-, and 647-conjugated secondary antibodies diluted in PBS (1:500) were applied for 1 h at room temperature. The coverslips were then washed and mounted with Mowiol 4-88 (EMD Bioscience, La Jolla, CA). Treatment with cycloheximide (10 µg/ml; Calbiochem) on control cells and cells expressing wt-torsinA was carried for 15 min, before fixing with 4% PFA and processing as described above. The images were acquired by confocal microscopy (Zeiss LSM510; Carl Zeiss, Jena, Germany) with a 63x Plan-Apochromat oil immersion objective.

Electron Microscopy—SH-SY5 cells expressing {Delta}E-torsinA were fixed with 8% PFA, 0.1 M phosphate buffer, pH 7.4, by adding directly to the cell medium at 37 °C for 10 min, followed by fixation in 4% PFA/phosphate buffer for 30 min at room temperature. The cells were embedded in gelatin, cryoprotected in 2.3 M sucrose, and frozen in liquid nitrogen, and ultrathin cryosections were cut using an FC6 cryo ultramicrotome (Leica Microsystems UK) (25). Cryosections were immunolabeled with polyclonal rabbit anti-snapin (1:500) and 10-nm protein A gold (Cell Microscopy Centre, UMC, Utrecht, The Netherlands). Differentiated PC12 cells were grown in 3-cm plastic dishes and fixed as above. The samples were processed for flat embedding and ultrathin cryosectioning (26). Endogenous snapin was detected using polyclonal rabbit anti-snapin (1:500) and 10-nm protein A gold. The cryosections were viewed in a 1010 transmission electron microscope (Jeol UK), and the images were captured with an Ultrascan 1000 digital camera and Digital Micrograph software (Gatan UK).

SV Recycling Assays—To monitor SV recycling, two independent exo-endocytic assays were performed. The first (27) is based on a polyclonal antibody (Syt-163) against the intraluminal domain of rat SytI. The antibody was diluted (3 µg/ml) in KRH medium (25 mM Hepes-NaOH, pH 7.4, 125 mM NaCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2 mM CaCl2, 6 mM glucose) containing 5 mM KCl or KRH medium with high potassium (25 mM Hepes-NaOH, pH 7.4, 100 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2 mM CaCl2, 6 mM glucose) and added directly to the cells. After 5 min at 37 °C, the cells were washed five times with KRH, fixed (4% PFA, 0.12 M sucrose in PBS), and permeabilized with PBS containing 0.05% Triton X-100. The cells were stained with AlexaFluor® 488-conjugated secondary goat anti-rat antibody, washed, and mounted. The images were acquired by confocal microscopy using a 63x Plan-Apochromat oil immersion objective with the pinhole fully open. The experiment was repeated three times, and ten random pictures for each sample were taken for quantification purposes. Mean fluorescence intensity of the cells was measured using ImageJ 1.36b.

The second strategy is based on the uptake and unloading of the styril dye FM1-43 in SH-SY5Y cells stably expressing HA-tagged wild type DYT1, DYT1-{Delta}E, or control vector. SH-SY5Y plated on MatTek dishes (MatTek, Ashland, MA) were incubated with 10 µM FM1-43 dye (Invitrogen) in high potassium buffer (5 mM Hepes-NaOH, pH 7.4, 37 mM NaCl, 100 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 10 mM glucose) for 1 min, followed by 1 min of incubation with Advasep-7 (1 mM, CyDex, Lenexa, KS) and by two washes with low potassium buffer (5 mM Hepes-NaOH, pH 7.4, 132 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 10 mM glucose) to remove the surface-bound dye. As negative control, FM1-43 was added to the cells in absence of calcium (5 mM Hepes-NaOH, pH 7.4, 37 mM NaCl, 100 mM KCl, 3.5 mM MgCl2, 250 mM EGTA, 10 mM glucose) and in the presence of 50 µM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (Invitrogen). Images were taken every 2 s with a Zeiss LSM 510 microscope equipped with a Nikon x63, 1.4 NA Plan Ph3 oil-immersion objective. The excitation was provided by a 488-nm argon laser, and emitted light was collected using 560 filter set (Omega Optical, Brattleboro, VT). The mean fluorescence intensity of five individual cells for each sample in three independent experiments was measured using Zeiss LSM 510 software version 3.2.

RNA Interference—On-target plus Smartpool of siRNAs for human torsinA (60 nM) and human snapin (20 nM; Dharmacom, Chicago, IL) were used to knockdown gene expression. siRNAs were transfected into SHSY-5Y cells using 1-2 µl of Lipofectamin 2000 (Invitrogen) in OptiMEM (Invitrogen). 5 h after transfection, the medium was replaced with Eagle's minimal essential medium: Ham's F-12 nutrient mixture, 10% fetal calf serum, and the cells were used at 72 h after transfections. The cells transfected with 20-60 nM scrambled oligonucleotides were used as control. RNA interference-mediated knockdown of torsinA and/or snapin was verified by immunoblot and immunofluorescence analysis using monoclonal anti-torsinA and rabbit anti-snapin antibodies as previously described.


Figure 1
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  		FIGURE 1.
A, snapin interacts specifically with torsinA and its {Delta}E mutant. The strength of interaction of wt-torsinA and {Delta}E-torsinA with snapin was quantified by a β-galactosidase assay on single yeast colonies as shown in supplemental Fig. S1. The β-galactosidase activity of wt-torsinA and {Delta}E-torsinA co-transformed with snapin was comparable with the positive control (wt-torsinA and KLC1), which was set to 100%. No colonies were observed for single transformation with wt-torsinA, {Delta}E-torsinA, and snapin alone. n = 3 colonies were measured for each sample. The error bars represent S.E. B, schematic representation of the full-length and truncated versions of wild type and mutant torsinA. SP, sequence peptide; CC, coiled-coil domain. The filled region (residues 91-181) denotes the ATPase domain. {Delta}GAG302/303 indicates the position of the 3-bp deletion. Wild type and mutant GST-torsinA bound 35S-labeled snapin in an in vitro pulldown assay. The corresponding Coomassie Blue staining of the SDS-PAGE gel is shown in supplemental Fig. S2A. C, schematic representation of full-length and truncated C-terminal snapin (CC-snapin). HS, hydrophobic sequence; CC, coiled-coil domain. Both snapin and CC-snapin interact with 35S-labeled wt- and {Delta}E-torsinA. The Coomassie Blue staining corresponding to this experiment is shown in supplemental Fig. S2B. Lanes Ts, Twt, and T{Delta}E show 1/10 of the starting material for snapin, wild type, and mutant torsinA, respectively. D, SH-SY5Y cells lines expressing HA-wt-torsinA and HA-{Delta}E-torsinA were incubated with GST-snapin and GST prebound to glutathione beads. The bound material was analyzed by Western blot using an anti-HA antibody. wt and mutant torsinA bind snapin with equal efficiency. The Ponceau staining of the nitrocellulose membranes corresponding to this experiment is shown in supplemental Fig. S2C. E, endogenous snapin forms a complex with wt and mutant torsinA in SH-SY5Y extracts. The lysates were incubated with either anti-snapin or anti-GFP antibodies, and the immunoprecipitated material was analyzed by Western blot with an anti-HA antibody. wt- and {Delta}E-torsinA bind endogenous snapin with equal strength. Lanes Twt and T{Delta}E show 1/10 of the starting material. Snapin bound to the beads was detected by Western blot as shown in supplemental Fig. S2D. IB, immunoblot.

 
Statistical Analysis—Student's t test analysis was assessed using Kaleidagraph version 4 (Synergy Software).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Wild type and Mutant TorsinA-interacting Proteins—We undertook a yeast two-hybrid screening to identify proteins that interact with both wild type torsinA (wt-torsinA) and mutant torsinA ({Delta}E-torsinA). Human full-length DYT1 (DYT1-wt) and its GAG-deleted mutant (DYT1-{Delta}E) were used as baits to screen an adult human brain cDNA library under high stringency conditions. As a result, eight independent clones were isolated using DYT1-{Delta}E and five using DYT1-wt. Three clones obtained using {Delta}E-torsinA as a bait encoded for the C-terminal region (residues 112-300) of snapin, a synaptic protein previously involved in the regulation of neurotransmitter release at central synapses (21).

The interaction of wt-torsinA and {Delta}E-torsinA with snapin was first verified by an independent yeast two-hybrid analysis (supplemental Fig. S1). The β-galactosidase activity of single colonies was measured within 2 h and compared with that of a yeast strain co-transformed with wt-torsinA and its previously identified binding partner kinesin light chain 1 (KLC1) (6). As shown in Fig. 1A, the strength of the interaction of wt-torsinA with full-length snapin and KCL1 was comparable under our experimental conditions. No significant difference was found between the binding of snapin with wt-torsinA and {Delta}E-torsinA.

These interactions were confirmed in GST binding assays: wt-torsinA and {Delta}E-torsinA expressed as GST recombinant fusion proteins were able to bind in vitro translated snapin (Fig. 1B). These results confirmed the specificity of the binding, because no interaction of snapin with GST alone was observed. To identify the specific domains involved in this interaction, a series of truncated mutants of wt-torsinA, {Delta}E-torsinA, and snapin were generated and tested in pull down assays (Fig. 1B). Deletion clones for wt-torsinA and {Delta}E-torsinA, corresponding to its first 181 residues and including the ATP-binding domain (tors-1) or only spanning the C-terminal coiled-coil region (residues 251-332; tors-3), were unable to bind snapin. In contrast, a fragment containing both ATP-binding and coiled-coil domains (residues 91-332; tors-2) showed the same intensity of binding of the full-length proteins (Fig. 1B). This finding indicates that the binding site is situated in this region, and the N terminus is not required for the interaction with snapin. In agreement with the results shown in Fig. 1A, no significant difference was observed in snapin binding between wt-torsinA and its mutant (Fig. 1B).

In a parallel experiment, full-length snapin and its C-terminal fragment (residues 83-136; CC-snapin), expressed as GST recombinant proteins, were equally able to bind both wt-torsinA and {Delta}E-torsinA (Fig. 1C). In contrast, no binding to immobilized GST was detected. This result indicates that the coiled-coil region of snapin alone is sufficient to mediate the interaction with both wild type and mutant torsinA.

A pulldown experiment was also performed using detergent extracts derived from stably transfected SH-SY5Y cells expressing wt and {Delta}E-torsinA tagged with an HA epitope. Specific interaction of GST-snapin with both wild type and mutant torsinA was revealed using an anti-HA antibody (Fig. 1D). Snapin appeared to bind equally {Delta}E-torsinA and wt-torsinA, whereas no binding was detected with GST alone. The ability of the endogenous snapin to interact with wt-torsinA and {Delta}E-torsin was also analyzed by coimmunoprecipitation from extracts derived from SH-SY5Y cells expressing HA-wt-torsinA and HA-{Delta}E-torsinA (Fig. 1E). Consistent with the pulldown results shown in Fig. 1D, snapin was able to bind both wt and mutant torsinA. In contrast, no specific binding was detected using a polyclonal anti-GFP antibody (Fig. 1E).

Snapin Partially Co-localizes with Mutant TorsinA in SH-SY5Y Cells—To gain further insights into the interaction between torsinA and snapin in living cells, we performed an immunofluorescence analysis in SH-SY5Y cells overexpressing torsinA using antibodies against endogenous snapin and the HA tag of torsinA. At the cell periphery, snapin co-localizes with synaptotagmin I (SytI), a specific marker of SVs and secretory granules, and this co-distribution is not grossly altered by the overexpression of {Delta}E-torsinA (supplemental Fig. S3). However, in these cells, endogenous snapin also accumulates in the typical torsinA-positive inclusions (Fig. 2), a finding further confirmed by cryoimmuno electron microscopy (Fig. 2G). Snapin showed little co-localization with torsinA on the NE (Fig. 2, A-C), a previously described site of accumulation of {Delta}E-torsinA (28, 29).


Figure 2
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  		FIGURE 2.
{Delta}E-torsinA partially co-localizes with endogenous snapin in SH-SY5Y cells. Cells overexpressing {Delta}E-torsinA (A and D, green) were stained with a monoclonal anti-HA antibody. Endogenous snapin was revealed with a rabbit polyclonal anti-snapin antibody (B and E, red) and analyzed by confocal microscopy. Merged pictures are shown in C and F. Scale bars, 5 µm (A-C) and 2 µm (D-F). G, cryosections of {Delta}E-torsinA expressing SH-SY5Y cells were immunolabeled with a polyclonal anti-snapin antibody followed by 10-nm protein A gold and imaged by transmission electron microscopy. Gold particles were concentrated in membrane whorls. Scale bar, 200 nm.

 
We also analyzed the distribution of snapin in SH-SY5Y overexpressing wt-torsinA. The diffuse cellular distribution of both snapin and wt-torsinA prevented the identification of any specific site of co-localization (data not shown). Therefore, to assess whether the overlapping between these proteins could occur in the ER where a pool torsinA resides (8-10), we co-stained with the ER marker PDI (supplemental Fig. S4, B and F). This approach revealed a level of co-localization of torsinA and snapin in this subcellular compartment (supplemental Fig. S4, D and H, in white). However, the inhibition of protein synthesis by cycloheximide caused the loss of co-localization between snapin and PDI without altering the overlap between snapin and wt-torsinA (supplemental Fig. S4, L and P, in cyan) or the overall distribution of PDI (supplemental Fig. S4, J and N, in red). These observations suggest that in SH-SY5Y cells, wt-torsinA and snapin are co-localized both in the secretory pathway and in a peripheral compartment depleted of ER proteins and containing exocytic markers, such as SytI.

Snapin and Endogenous TorsinA Overlap on Secretory Organelles in PC12—To identify this compartment, we analyzed the distribution of endogenous torsinA and snapin using specific antibodies in PC12 cells differentiated for 72 h with nerve grow factor (Fig. 3). Both torsinA and snapin showed a punctate pattern in the cytosol (Fig. 3, A, C, and E) with accumulation at the neurite tips (Fig. 3, B and D), where extensive co-localization was detected (Fig. 3F). Similarly, the two endogenous proteins showed similar distribution in primary hippocampal neurons with extensive overlap in both the cell bodies and the neural processes (supplemental Fig. S5). Cryosections of differentiated PC12 cells obtained using a flat-embedding technique allowed us to preserve the in situ orientation of the neurites (26) and localize endogenous snapin by immunolabeling with anti-snapin antibody decorated with 10-nm protein A gold. Numerous gold particles were associated to a subset of dense core granules at the neurite tips (Fig. 3, G and H). No gold label was detected on dense core granules incubated only with 10-nm protein A gold on the control sections (data not shown). Although the anti-torsinA antibody was able to detect endogenous torsinA by immunofluorescence (Fig. 3, A and B), the antibody was not compatible with the processing required for cryo-immunoelectron microscopy (data not shown).


Figure 3
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  		FIGURE 3.
Endogenous torsinA co-localizes with snapin in PC12 cells. Differentiated PC12 cells were stained for endogenous torsinA (A and B, red) and for endogenous snapin (C and D, green). The merged images (E and F, in yellow) show high levels of co-localization in the neurite tips. Scale bars, 5 µm (A, C, and E) and 2 µm (B, D, and F). G, an overview of the architecture of differentiated PC12 cells by electron microscopy of cryosections using the flat-embedding technique. A neurite tip packed with dense core granules lies next to the body of another cell. Areas of fluorescence co-localization in neurite tips (E and F) are likely to correspond to the dense core granules, which are labeled with snapin immunogold (10 nm). H shows a higher magnification of the boxed area in G. Scale bars, 2.5 µm (G) and 250 nm (H).

 
To verify that the co-localization of torsinA and snapin was restricted to these organelles, PC12 cells were co-stained for SGI, a secretory granule marker. As shown in Fig. 4, several SGI-positive organelles contained torsinA and snapin (Fig. 4, G and H, in white); however, other SGI-containing vesicles were positive only for snapin or torsinA, suggesting that neurite tips hold a heterogeneous population of granules, which may contain either one or both proteins.

TorsinA Affects SV Recycling—In the view of previous studies suggesting the involvement of torsinA in dopamine transport (16) and the putative role of snapin in neurotransmitter release (21, 31), we tested whether torsinA could play a role in SV trafficking. To this end, we performed an exo-endocytosis assay in SH-SY5Y cells, using an antibody against the intravesicular domain of SytI (Syt-163). This antibody is internalized upon fusion of the SV with the presynaptic membrane, which determines the exposure of the luminal domain of SytI on the cell surface (27, 32). Therefore, the signal detected with this antibody in resting conditions and upon stimulation reflects the level of SytI exposed on the plasma membrane at steady state or upon SV exocytosis and recycling, respectively. SH-SY5Y cells stably transfected with wt and {Delta}E-torsinA were incubated with the Syt-163 antibody in resting (5 mM KCl; Fig. 5, A, C, and E) or depolarizing conditions (100 mM KCl; Fig. 5, B, D, and F). The cells were then washed, fixed, permeabilized, and finally stained with a fluorescently conjugated secondary antibody, prior to immunofluorescence analysis. Mean fluorescence intensity of random fields was quantified (Fig. 5G). As expected, the average level of SytI detected on the surface of control cells under depolarizing conditions was significantly higher (Fig. 5B) than in resting cells (Fig. 5A). In SH-SY5Y cells overexpressing wt-torsinA, SytI labeling on the plasma membrane on stimulation was reduced compared with the control cells (Fig. 5D), suggesting that overexpression of torsinA severely impairs SV turnover. In contrast, cells overexpressing {Delta}E-torsinA showed a significant accumulation of Syt-163 signal on the plasma membrane in resting conditions (Fig. 5E), which did not increase upon depolarization (Fig. 5F). Therefore, overexpression of {Delta}E-torsinA appears to affect the rate of vesicle endocytosis in SH-SY5Y cells, resulting in an accumulation of SytI on the plasma membrane and alteration of membrane identity.


Figure 4
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  		FIGURE 4.
Endogenous torsinA and snapin overlap with SGI in PC12 cells. PC12 after 72 h of differentiation in presence of nerve grow factor, were fixed and stained for SGI with a specific monoclonal mouse antibody (A and B, green), endogenous torsinA (C and D, red), and endogenous snapin (E and F, blue). The merged images (G and H, in white) show a population of granules triple-positive for SGI, snapin and torsinA. Scale bars, 5 µm (A, C, E, and G) and 2 µm (B, D, F, and H).

 
The different efficiency of membrane recycling seen in wt-torsinA- and {Delta}E-torsinA-expressing cells may be dependent on an altered rate of SV biogenesis, which could be down-regulated by overexpression of wt-torsinA and increased by {Delta}E-torsinA. We therefore quantified the total amount of SytI and VAMP2, another marker of SVs and secretory granules, in control SH-SY5Y cells and in cells expressing wt and {Delta}E-torsinA (Fig. 6). Whereas the concentration of SytI remained constant in the three cell lines both in immunofluorescence and immunoblotting (Fig. 6, A-C and G), VAMP2 expression was higher in {Delta}E-torsinA-positive cells (Fig. 6F) than control cells and was further reduced in wt-torsinA-expressing cells (Fig. 6E). These findings suggest that torsinA is unlikely to control the biogenesis of the vesicles; however, it seems to tightly regulate the expression of specific vesicle proteins, such as VAMP2, or a subpopulation of secretory vesicles expressing VAMP2.


Figure 5
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  		FIGURE 5.
SytI is accumulated on the surface of SH-SY5Y cells expressing mutant torsinA. SH-SY5Y cells stably transfected with control vector (A and B), wt-torsinA (C and D), and {Delta}E-torsinA (E and F) were incubated in resting (5 mM K+; A, C, and E) and depolarizing conditions (100 mM K+; B, D, and F). In control cells, the intensity of SytI labeling increases in response to 100 mM K+ (B) compared with resting cells (A). The cells expressing wt-torsinA show a strongly reduced response to depolarization (D), whereas {Delta}E-torsinA cells display an increased surface accumulation of SytI in resting conditions (E), which is not significantly changed upon stimulation (F). Scale bar, 10 µm. G, quantification of the anti-SytI antibody uptake. The empty columns refer to resting conditions (5 mM K+), whereas the filled columns show the extent of SytI staining in depolarizing conditions (100 mM K+) (n = 3). The error bars represent S.E. The asterisks indicate p < 0.05 in Student's t test.

 


Figure 6
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  		FIGURE 6.
TorsinA regulates selectively the expression of VAMP2 in SH-SY5Y cells. Control (A and D), wt-torsinA (B and E), and {Delta}E-torsinA (C and F) SH-SY5Y expressing cells were stained for SytI and VAMP2 in immunofluorescence (A-F) and Western blot (G). {Delta}E-torsinA expressing cells display an increase of VAMP2 expression (150% ± 3.5 S.D., n = 3; F) as compared with control cells (set to 100%; D). In contrast, cells expressing wt-torsinA (E) show a reduction of VAMP2 levels to 50% ± 4.0 S.D. of the control. Actin was used as loading control in G. The level of expression of SytI remains constant in all cell lines. The intensity of the bands was quantified as described under "Experimental Procedures."

 
In the light of these results, we sought to test SV exo-endocytosis using FM1-43 uptake (33, 34). This styril dye selectively labels organelles undergoing exocytosis and endocytosis in living cells, providing a means of monitoring these processes in real time. In our experimental protocol, SH-SY5Y cells bearing a control plasmid or expressing either wt-torsinA or {Delta}E-torsinA were exposed for 1 min to the dye in high potassium conditions (100 mM KCl) prior to rapid washing in control medium and imaging (5 mM KCl; supplemental Fig. S6, A, D, and G; see "Experimental Procedures"). The experiment was repeated three times, and the quantitative analysis of the uptake is shown in Fig. 7A. We found that FM1-43 uptake is higher in {Delta}E-torsinA-expressing cells (140 ± 10; p < 0.01) and lower in wt-torsinA cells (77 ± 3; p < 0.05) compared with control SH-SY5Y cells (105 ± 6). To monitor the rate of SV exocytosis, FM1-43-loaded cells were treated after 30 s with 100 mM KCl, resulting in a strong depolarization and fluorescence loss (supplemental Fig. S6, B, E, and H). Unstimulated cells were used as control (base line). After stimulation, the residual fluorescence was higher in the mutant and lower in the wt-torsinA SH-SY5Y cells than the control (Fig. 7B). However, once the signal was normalized for the different loading, the amount and overall kinetics of FM1-43 release was equal in control, wt, and mutant torsinA-expressing cells (Fig. 7C), suggesting that exocytosis is not affected by wt- and {Delta}E-torsinA overexpression. To confirm that FM1-43 release was due to bona fide Ca2+-dependent exocytosis, we repeated the experiment in the presence of extracellular and intracellular Ca2+ chelators. This treatment completely blocked FM1-43 release, demonstrating that exo-endocytosis in SH-SY5Y cells is strictly Ca2+-dependent (Fig. 7D).

Effect of the Knockdown of TorsinA and Snapin on Membrane Turnover and SV Recycling—To further understand the role of torsinA and snapin in the regulation of exo-endocytosis, an RNA interference study was performed. Control SH-SY5Y cells were transfected with a pool of siRNA for torsinA or snapin or a combination of both, and the efficiency of the knockdown was checked after 3 days by immunofluorescence and immunoblotting (supplemental Fig. S7). Both torsinA and snapin expression was reduced by 90% upon introduction of the siRNA (supplemental Fig. S7, D and H). In the double knockdown, the expression of both snapin and torsinA was reduced by 70% (supplemental Fig. S7, J and K). As a control, cells were transfected with scrambled RNA, and no effect on either torsinA or snapin expression was observed (supplemental Fig. S7, A and B). Exo-endocytosis in cells treated with siRNAs was analyzed according to the two methods described previously. As shown in Fig. 8, SytI labeling of cells transfected with snapin siRNA showed a low signal (78 ± 2, p < 0.05) in response to depolarization compared with the control (set to 100%; Fig. 8, C and D). In contrast, cells treated with torsinA siRNA showed a higher labeling for SytI on the surface prior to stimulation (110 ± 3), which did not increase on depolarization (107 ± 4, p < 0.05; Fig. 8, E and F). Interestingly, this phenotype resembled SHSY5Y cells overexpressing mutant torsinA (Fig. 5, E and F), strongly suggesting that {Delta}E-torsinA is a loss of function mutant. Treatment with both siRNAs had an inhibitory effect, resulting in a decreased exposure of SytI on the membrane upon stimulation (85 ± 3, p < 0.05), similar to the snapin knockdown (Fig. 8, G and H). This suggests that snapin is likely to act upstream from torsinA in the pathway of regulation of the SV turnover.

Cells treated with one or both siRNA for torsinA and snapin were also analyzed for FM1-43 uptake in resting (supplemental Fig. S6, J, M, and P) and depolarizing conditions (supplemental Fig. S6, K, N, and Q). The results shown in Fig. 8J indicate that treatment with both single torsinA or snapin siRNA and double (torsinA + snapin siRNA) negatively affected the uptake of FM1-43 with similar efficiency. This suggests that torsinA and snapin act on the same pathway, and their presence is required to ensure a normal level of endocytosis. Furthermore, knocking down torsinA, snapin, or both proteins resulted in a reduced level of FM1-43 release compared with control cells, after the signal was normalized for the different loading (Fig. 8K), indicating that endogenous levels of torsinA and snapin are also required for regulated secretion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we used yeast two-hybrid analysis to identify a new binding partner of torsinA, the SNARE-associated protein snapin. We have reported that snapin shows a robust interaction with wild type and mutant torsinA. Previously, it has been shown that the ATP-binding domain is essential for torsinA function and cellular distribution (6, 13). Here, we have demonstrated that this portion of torsinA and/or the adjacent linker region has the additional role of recruiting snapin. This mode of binding is different from that described for KLC1, which only requires the coiled-coil region of torsinA for maximal interaction (6).


Figure 7
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  		FIGURE 7.
Effects of wt-torsinA and {Delta}E-torsinA on FM1-43 uptake and release in SH-SY5Y cells. A, FM1-43 uptake in control, wt-torsinA, and {Delta}E-torsinA expressing cells after 1 min of incubation under depolarizing conditions. FM1-43 uptake in {Delta}E-torsinA expressing cells is higher than control cells and is further decreased in SH-SY5Y expressing wt-torsinA (see also supplemental Fig. S6, A, D, and G). B, FM1-43 release upon stimulation with 100 mM K+ in control and wt-torsinA- and {Delta}E-torsinA-expressing cells. The amount of FM1-43 in {Delta}E-torsinA cells is indicated as base line. The arrowhead indicates the addition of 100 mM KCl. C, percentage of FM1-43 dye released in control, wt-torsinA, and {Delta}E-torsinA cells after normalization for the different loading. D, the release of FM1-43 is blocked by the Ca2+ chelators EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester. The error bars represent S.E. *, p < 0.05; **, p < 0.01 in Student's t test.

 
Other binding partners have been described for torsinA, including lamina-associated polypeptide 1 in the NE and a novel transmembrane protein (Lull1) in the ER (35). These multiple interactions suggest that torsinA may have distinct roles in different cellular compartments. Our immunofluorescence studies showed that endogenous torsinA co-localizes with snapin on secretory granules at neurites tips. In cells overexpressing {Delta}E-torsinA, accumulation of snapin was also seen in {Delta}E-positive perinuclear membrane whorls.

Snapin is a ubiquitously expressed protein, which was initially found to be associated with SVs, where it binds the SNARE complex by a direct interaction with SNAP25. The recruitment of snapin to the SNARE complex is thought to be required to enhance the interaction between the SNAREs and SytI (21). This association is a crucial step in the mechanism of exocytosis, which leads to the fusion of SVs with the plasma membrane triggered by Ca2+ influx (36). Protein kinase A modulates neurotransmitter release by targeting snapin for phosphorylation in neurons (22, 37). Despite conflicting evidence about the role of snapin in neurotransmitter release (23), recent analysis of a snapin knockout mouse model supports a role for this protein in Ca2+-dependent neurosecretion (31). In the same study, snapin was also shown to be associated with SVs along with other markers, such as VAMP2, SytI, and VMAT2. Several other proteins have been identified as interacting partners of snapin in both neuronal and non-neuronal cells (38). Interestingly, some are involved in membrane or cargo sorting; BLOC-1 is involved in the biogenesis of endosomal-lysosomal organelles; EBAG9 acts as inhibitor of large dense core vesicles exocytosis in PC12 cells; and the vanilloid receptor interacts with snapin during its transfer from carrier vesicles to the plasma membrane (39-41). Consistent with these findings, our data indicate that snapin knockdown has a negative effect on the exo-endocytic pathway with a consequent reduction in vesicle turnover (SytI labeling) and a decrease in the uptake and release of FM1-43. The interaction reported here of torsinA with snapin supports the hypothesis that torsinA may be involved in or influence SV dynamics in neurons.


Figure 8
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  		FIGURE 8.
Knockdown of torsinA and snapin affect SytI exposure on the plasma membrane and reduce FM1-43 uptake and release. SH-SY5Y cells treated with control (scramble; A and B), snapin (C and D), torsinA (E and F), and double (snapin + torsinA; G and H) siRNAs were analyzed for SytI labeling on the plasma membrane in resting condition (A, C, E, and G) and upon stimulation with 100 mM KCl (B, D, F, and H). Knockdown of snapin and snapin + torsinA decrease the intensity of SytI signal (D, H, and I). In contrast, higher levels of SytI were observed on the plasma membrane in torsinA knockdown cells in resting and stimulated condition (E, F, and I) (in I, empty columns, 5 mM KCl; filled columns, 100 mM KCl). J, FM1-43 uptake in snapin (59% ± 3), torsinA (62% ± 2), and snapin + torsinA (57% ± 4.6) knockdown cells is reduced comparing with the control cells (scramble siRNA; set to 100% ± 5). K, FM1-43 release is reduced in knockdown cells for snapin (30% ± 3), torsinA (26% ± 2), and snapin + torsinA (24% ± 3) compared with control cells after normalization for the different loading (54% ± 8) (n = 3). The error bars represent S.E. Asterisks indicate p < 0.05 in Student's t test.

 
Analysis of the effects of torsinA on neurosecretion in SH-SY5Y stably transfected with wild type DYT1 and DYT1-{Delta}E showed that overexpressing wt-torsinA negatively affects SV recycling, as demonstrated by the impairment of FM1-43 uptake following depolarization. In contrast, {Delta}E-torsinA appeared to enhance membrane recycling at the expense of the accuracy of the retrieval of secretory vesicle proteins, such as SytI, from the plasma membrane.

Knockdown of torsinA via RNA interference shows a similar phenotype to snapin, resulting in a general inhibitory effect on the exo-endocytic pathway. Moreover, the ablation of both proteins mirrors the effect of single knockdowns, consistent with the idea that torsinA and snapin play a role in the same pathway. Interestingly, the effect of torsinA knockdown resembles the phenotype caused by {Delta}E-torsinA overexpression. To our knowledge, this is the first direct demonstration that {Delta}E-torsinA is a loss of function mutant, thereby providing evidence of how the {Delta}E mutation may affect torsinA function and cause its pathological effects. Our findings indicate that torsinA plays a role in the recycling of SV membrane, which occurs upon release of neurotransmitters. This strengthens the link between torsinA and abnormal neurotransmission, which may affect the dopamine pathway. Interestingly, Torres et al. (16) proposed that wt-torsinA is able to regulate the distribution of membrane-associated proteins, such as the dopamine transporter receptor, affecting the re-uptake of dopamine. Moreover, Misbahuddin et al. (14) showed that {Delta}E-torsinA enriched inclusions contain VMAT2, which is crucial for maintaining physiological levels of dopamine release.

Dopaminergic pathways have been implicated in the pathogenesis of dystonic movements from study of other forms of dystonias, including L-DOPA-responsive dystonia with diurnal fluctuations (Segawa's dystonia) (42) and hereditary juvenile dystonia-parkinsonism (43). In addition, studies in a transgenic mouse model of DYT1 dystonia found an abnormal balance between the dopaminergic and the cholinergic signaling (17), and abnormal dopamine release has been reported in another DYT1 transgenic mouse (20). Finally, it is tantalizing that, in human brain, torsinA is highly expressed in dopaminergic neurons of the substantia nigra (18).

Although our data suggest that torsinA and snapin play a role in neurosecretion, the molecular mechanism responsible for torsinA-dependent regulation of neurotransmitter release is not completely understood. One hypothesis is that torsinA may have a chaperone-like function (44, 45), regulating the folding of proteins involved in SV trafficking, such us snapin. Interestingly, torsinA displays sequence homology with N-ethylmaleimide-sensitive fusion protein (5), an AAA-ATPase essential for neurosecretion that is responsible for SNARE recycling (46). Alternatively, torsinA may be able to modulate snapin phosphorylation, which is critical for its function (22). Finally, snapin may mediate the association of torsinA with SV and secretory granules. This is consistent with results obtained in human brain showing association of torsinA with SVs in the striatum (18) and with our findings that torsinA and snapin co-distribute with the secretory granules maker SGI in PC12 cells. In addition to these possibilities, which are not mutually exclusive, the effects of torsinA on SytI localization suggest that it may also influence events of membrane sorting not directly dependent on snapin function.

The molecular pathogenesis of {Delta}E-torsinA remains unclear. The formation of membranous whorls caused by expression of {Delta}E-torsinA is evident in E-cadherin-positive and SH-SY5Y transfected cells (7, 14) but has been also observed in other non-neuronal cell lines overexpressing {Delta}E-torsinA (data not shown). These inclusions appear to originate from the NE (47) and have been detected in DYT1 brain, supporting a role in the pathogenesis of dystonia (15). Perinuclear inclusions have also been detected in mice expressing {Delta}E-torsinA (48, 49). In addition, the fact that the phenotype of torsinA knockout is similar to the DYT1-{Delta}E knockin argues that the loss of torsinA function plays an important part in the pathology (30). This is supported by our observation that overexpression of {Delta}E-torsinA and knockdown of endogenous torsinA give rise to the same altered distribution of SytI on the plasma membrane.

In conclusion, our data suggest that torsinA has a regulatory role in vesicle recycling. The {Delta}E mutation impairs torsinA function, uncoupling endocytosis from membrane depolarization, and causing the mislocalization of SV proteins on the cell surface. This in turn may disrupt the neuronal pathways involved in the control of movement. The mechanism by which {Delta}E-torsinA exerts its dominant-negative effect could involve mistargeting of proteins involved in SV trafficking, such us snapin and VMAT-2, into the membranous inclusion bodies. Alternatively, {Delta}E-torsinA might lead to the sequestration of torsinA from other cellular compartments, interfering with its normal functions. One of these functions, highlighted in this paper, may be the maintenance of a functional secretory pathway in neurons.


   FOOTNOTES
 
* This work was supported by a grant from the Wellcome Trust (to A. G.), Cancer Research UK (to L. M. C. and G. S), and the Bachmann-Strauss Dystonia and Parkinson Foundation (to T.T.W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbcorg) contains supplemental Figs. S1-S7. Back

1 To whom correspondence may be addressed. Tel.: 44-20-7269-3300; Fax: 44-20-7269-3417; E-mail: giampietro.schiavo{at}cancer.org.uk. 2 To whom correspondence may be addressed: Tel.: 44-20-7830-2951; Fax: 44-20-7472-6829; E-mail: t.warner{at}medsch.ucl.ac.uk.

3 The abbreviations used are: ER, endoplasmic reticulum; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; wt, wild type; NE, nuclear envelope; SV, synaptic vesicle; HA, hemagglutinin; GFP, green fluorescent protein; PDI, protein-disulfide isomerase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PFA, paraformaldehyde; siRNA, small interfering RNA; SytI, synaptotagmin I; SGI, secretogranin I. Back


   ACKNOWLEDGMENTS
 
We thank A. Weston for preliminary electron microscopy analysis and S. Tabrizi and S. Salinas for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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