The Dystonia-associated Protein TorsinA Modulates Synaptic Vesicle Recycling
2008; Elsevier BV; Volume: 283; Issue: 12 Linguagem: Inglês
10.1074/jbc.m704097200
ISSN1083-351X
AutoresAlessandra Granata, Rose Watson, Lucy Collinson, Giampietro Schiavo, Thomas T. Warner,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoThe 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 ΔE-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 ΔE-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 ΔE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that ΔE-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. 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 ΔE-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 ΔE-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 ΔE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that ΔE-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. 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 (1Ozelius L.J. Hewett J.W. Page C.E. Bressman S.B. Kramer P.L. Shalish C. de Leon D. Brin M.F. Raymond D. Corey D.P. Fahn S. Risch N.J. Buckler A.J. Gusella J.F. Breakefield X.O. Nat. Genet. 1997; 17: 40-48Crossref PubMed Scopus (900) Google Scholar). DYT1 dystonia manifests in childhood, typically with dystonia in a limb that spreads to the trunk and other limbs, usually sparing cranio-cervical muscles (2Fahn S. Bressman S.B. Marsden C.D. Adv. Neurol. 1998; 78: 1-10Crossref PubMed Google Scholar, 3Bressman S.B. de Leon D. Kramer P.L. Ozelius L.J. Brin M.F. Greene P.E. Fahn S. Breakefield X.O. Risch N.J. Ann. Neurol. 1994; 36: 771-777Crossref PubMed Scopus (155) Google Scholar). There is no evidence for neurodegeneration in DYT1 dystonia, implying that abnormal movements are caused by a functional neuronal defect (4Rostasy K. Augood S.J. Hewett J.W. Leung J.C. Sasaki H. Ozelius L.J. Ramesh V. Standaert D.G. Breakefield X.O. Hedreen J.C. Neurobiol. Dis. 2003; 12: 11-24Crossref PubMed Scopus (138) Google Scholar). All cases of typical DYT1 dystonia are caused by an in-frame GAG deletion (ΔGAG302/303; ΔE) in DYT1 gene, resulting in the loss of a glutamic acid in the C-terminal region of the encoded protein, torsinA (1Ozelius L.J. Hewett J.W. Page C.E. Bressman S.B. Kramer P.L. Shalish C. de Leon D. Brin M.F. Raymond D. Corey D.P. Fahn S. Risch N.J. Buckler A.J. Gusella J.F. Breakefield X.O. Nat. Genet. 1997; 17: 40-48Crossref PubMed Scopus (900) Google Scholar). TorsinA is a member of the AAA ATPase superfamily of chaperone-like proteins (5Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar). In mammalian neuronal cells, torsinA is found throughout the cytoplasm, neurite processes, and growth cones (6Kamm C. Boston H. Hewett J. Wilbur J. Corey D.P. Hanson P.I. Ramesh V. Breakefield X.O. J. Biol. Chem. 2004; 279: 19882-19892Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 7Hewett J. Gonzalez-Agosti C. Slater D. Ziefer P. Li S. Bergeron D. Jacoby D.J. Ozelius L.J. Ramesh V. Breakefield X.O. Hum. Mol. Genet. 2000; 9: 1403-1413Crossref PubMed Scopus (176) Google Scholar). TorsinA has also been found in the lumen of the endoplasmic reticulum (ER) 3The abbreviations used are: ERendoplasmic reticulumSNAREsoluble N-ethylmaleimide-sensitive factor attachment protein receptorwtwild typeNEnuclear envelopeSVsynaptic vesicleHAhemagglutininGFPgreen fluorescent proteinPDIprotein-disulfide isomeraseGSTglutathione S-transferasePBSphosphate-buffered salineBSAbovine serum albuminCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPFAparaformaldehydesiRNAsmall interfering RNASytIsynaptotagmin ISGIsecretogranin I. and in the space between the inner and the outer membrane of the nuclear envelope (NE) (8Hewett J. Ziefer P. Bergeron D. Naismith T. Boston H. Slater D. Wilbur J. Schuback D. Kamm C. Smith N. Camp S. Ozelius L.J. Ramesh V. Hanson P.I. Breakefield X.O. J. Neurosci. Res. 2003; 72: 158-168Crossref PubMed Scopus (99) Google Scholar, 9Liu Z. Zolkiewska A. Zolkiewski M. Biochem. 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A. 2004; 101: 7612-7617Crossref PubMed Scopus (191) Google Scholar, 14Misbahuddin A. Placzek M.R. Taanman J.W. Gschmeissner S. Schiavo G. Cooper J.M. Warner T.T. Mov. Disord. 2005; 20: 432-440Crossref PubMed Scopus (44) Google Scholar). TorsinA-positive inclusions have been found in the midbrain of DYT1 patients, suggesting that they are relevant to the pathogenesis of DYT1 dystonia (15McNaught K.S. Kapustin A. Jackson T. Jengelley T.A. Jnobaptiste R. Shashidharan P. Perl D.P. Pasik P. Olanow C.W. Ann. Neurol. 2004; 56: 540-547Crossref PubMed Scopus (136) Google Scholar). In SH-SY5Y neuroblastoma cells, ΔE-enriched inclusions contain the vesicular monoamine transporter 2 (VMAT2), a membrane-associated protein involved in loading dopamine vesicles (14Misbahuddin A. Placzek M.R. Taanman J.W. Gschmeissner S. Schiavo G. Cooper J.M. Warner T.T. Mov. Disord. 2005; 20: 432-440Crossref PubMed Scopus (44) Google Scholar). 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 (16Torres G.E. Sweeney A.L. Beaulieu J.M. Shashidharan P. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15650-15655Crossref PubMed Scopus (147) Google Scholar, 17Pisani A. Martella G. Tscherter A. Bonsi P. Sharma N. Bernardi G. Standaert D.G. Neurobiol. Dis. 2006; 24: 318-325Crossref PubMed Scopus (132) Google Scholar). Consistent with these observations, torsinA is highly expressed in dopaminergic neurons of the substantia nigra (18Augood S.J. Martin D.M. Ozelius L.J. Breakefield X.O. Penney Jr., J.B. Standaert D.G. Ann. Neurol. 1999; 46: 761-769Crossref PubMed Scopus (125) Google Scholar, 19Shashidharan P. Kramer B.C. Walker R.H. Olanow C.W. Brin M.F. Brain Res. 2000; 853: 197-206Crossref PubMed Scopus (93) Google Scholar). More recent work in a DYT1 transgenic mouse model has suggested that mutant torsinA impaired dopamine release (20Balcioglu A. Kim M.O. Sharma N. Cha J.H. Breakefield X.O. Standaert D.G. J. Neurochem. 2007; 102: 783-788Crossref PubMed Scopus (102) Google Scholar). endoplasmic reticulum soluble N-ethylmaleimide-sensitive factor attachment protein receptor wild type nuclear envelope synaptic vesicle hemagglutinin green fluorescent protein protein-disulfide isomerase glutathione S-transferase phosphate-buffered saline bovine serum albumin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid paraformaldehyde small interfering RNA synaptotagmin I secretogranin I. To investigate the role of torsinA further, we performed a yeast two-hybrid screening using full-length wt-torsinA and ΔE-torsinA as bait. Snapin, a SNAP25 (synaptosomal associated protein of 25 kDa)-binding protein (21Ilardi J.M. Mochida S. Sheng Z.H. Nat. Neurosci. 1999; 2: 119-124Crossref PubMed Scopus (195) Google Scholar), 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 (21Ilardi J.M. Mochida S. Sheng Z.H. Nat. Neurosci. 1999; 2: 119-124Crossref PubMed Scopus (195) Google Scholar, 22Chheda M.G. Ashery U. Thakur P. Rettig J. Sheng Z.H. Nat. Cell Biol. 2001; 3: 331-338Crossref PubMed Scopus (144) Google Scholar). 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 ΔE-torsinA appears to act as a loss of function mutant by enhancing synaptic membrane turnover. 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) (23Vites O. Rhee J.S. Schwarz M. Rosenmund C. Jahn R. J. Biol. Chem. 2004; 279: 26251-26256Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) 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-ΔE) or no insert (14Misbahuddin A. Placzek M.R. Taanman J.W. Gschmeissner S. Schiavo G. Cooper J.M. Warner T.T. Mov. Disord. 2005; 20: 432-440Crossref PubMed Scopus (44) Google Scholar) 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-ΔE 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-/α-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-ΔE 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)) (22Chheda M.G. Ashery U. Thakur P. Rettig J. Sheng Z.H. Nat. Cell Biol. 2001; 3: 331-338Crossref PubMed Scopus (144) Google Scholar) 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 (23Vites O. Rhee J.S. Schwarz M. Rosenmund C. Jahn R. J. Biol. Chem. 2004; 279: 26251-26256Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Protein expression was induced in E. coli BL21 by the addition of 400 μm isopropyl-β-d-thiogalactopyranoside for 4 h at 30 °C (50Lalli G. Herreros J. Osborne S.L. Monteccucco C. Ressetto O. Shiavo G. J. Cell Sci. 1999; 112: 2715-2724Crossref PubMed Google Scholar). 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, ΔE-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 ΔE-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-ΔE (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-ΔE, 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 (24Herreros J. Ng T. Schiavo G. Mol. Biol. Cell. 2001; 12: 2947-2960Crossref PubMed Scopus (156) Google Scholar). 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 63× Plan-Apochromat oil immersion objective. Electron Microscopy—SH-SY5 cells expressing ΔE-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) (25Roposo G. Kleijmeer M.J. Posthuma G. Slot J.W. Geuze H.J. Immunoglod Labeling of Ultrathin Cryosections: Application in Immunology. Blackwell Science, Cambridge, MA1997: 78-85Google Scholar). 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 (26Oorschot V. de Wit H. Annaert W.G. Klumperman J. J. Histochem. Cytochem. 2002; 50: 1067-1080Crossref PubMed Scopus (46) Google Scholar). 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 (27Matteoli M. Takei K. Perin M.S. Sudhof T.C. De Camilli P. J. Cell Biol. 1992; 117: 849-861Crossref PubMed Scopus (283) Google Scholar) 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 63× 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-ΔE, 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 ×63, 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. Statistical Analysis—Student's t test analysis was assessed using Kaleidagraph version 4 (Synergy Software). 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 (ΔE-torsinA). Human full-length DYT1 (DYT1-wt) and its GAG-deleted mutant (DYT1-ΔE) 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-ΔE and five using DYT1-wt. Three clones obtained using ΔE-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 (21Ilardi J.M. Mochida S. Sheng Z.H. Nat. Neurosci. 1999; 2: 119-124Crossref PubMed Scopus (195) Google Scholar). The interaction of wt-torsinA and ΔE-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) (6Kamm C. Boston H. Hewett J. Wilbur J. Corey D.P. Hanson P.I. Ramesh V. Breakefield X.O. J. Biol. Chem. 2004; 279: 19882-19892Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). 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 ΔE-torsinA. These interactions were confirmed in GST binding assays: wt-torsinA and ΔE-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, ΔE-torsinA, and snapin were generated and tested in pull down assays (Fig. 1B). Deletion clones for wt-torsinA and ΔE-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 ΔE-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 ΔE-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 ΔE-torsinA and wt-torsinA, whereas no binding was detected
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