mid #12605; [33]), human GFP-Rab9A (plasmid #12663; [33]) and human GFP-Rab11A (plasmid #12674; [33]) were obtained from Addgene. The following antibodies were employed: mouse monoclonal anti-V5 and anti-V5-peroxidase (Invitrogen); mouse monoclonal anti-MAP2 (clone HM-2) and anti-b-tubulin (clone TUB 2.1), and rabbit polyclonal anti-bIII-tubulin (Sigma-Aldrich, Buchs, Switzerland); mouse monoclonal anti-HA-peroxidase (Roche Applied Science, Basel, Switzerland); mouse monoclonal anti-LAMP1 (clone LY1C6) and rabbit monoclonal anti-calreti- culin (clone EPR3924) (EMD Millipore, Billerica, MA, USA); rabbit polyclonal anti-Giantin (ab24586; Abcam, Cambridge, UK); peroxidase-coupled anti-mouse and anti-rabbit IgG, light chain-specific secondary antibodies (Jackson ImmunoResearch, Inc., West Grove, PA, USA); anti-rabbit IgG and anti-mouse IgG coupled to AlexaFluor-488, -546 and -633 (Invitrogen). Cell culture and transient transfection Human SH-SY5Y neuroblastoma cells (CRL-2266; ATCC, Manassas, VA, USA [34,35]) or HEK-293T cells (Invitrogen) were maintained in Dulbecco` s modified Eagle’s Medium (Invitrogen) supplemented with 10% foetal bovine serum (FBS) and 1X penicillin/streptomycin at 37uC in a 5% CO2 atmosphere. Cells were transfected with plasmid DNAs using FuGENE HD reagent (Roche Applied Science) according to manufacturer’s recommen- dations. For biochemical assays cells were routinely harvested at 48–72 h post-transfection. Primary neuronal cultures Primary cortical neurons were prepared from rats as previously described [17,36]. Whole brains were dissected from Sprague- Dawley P0 rats and the cerebral cortices were isolated stereo- scopically and dissociated by digestion in media containing papain (20U/ml, Sigma-Aldrich) and mechanical trituration. Cells were plated in 35 mm dishes on glass coverslips coated with poly-D- lysine (20 ng/ml; BD Biosciences, Allschwil, Switzerland) and mouse laminin (33 mg/ml; Invitrogen) and cultured in Neurobasal media containing B27 supplement (2% w/v), L-glutamine (500 mM) and penicillin/streptomycin (100 U/ml) (Invitrogen). At days-in-vitro (DIV) 3, cortical cultures were treated with cytosine b-D-arabinofuranoside (AraC, 10 mM) to inhibit glial cell division. Primary cortical neurons were transfected with plasmid DNAs using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer’s instructions to a maximum of 5 mg total DNA per 35 mm dish of cells. Cell fractionation and western blotting For western blotting, HEK-293T or SH-SY5Y cells maintained in growth medium in 6 well plates (250,000 cells/well) were transiently transfected with 2 mg of each plasmid DNA. After 48 h, cells were harvested in media, resuspended in lysis buffer (1X PBS pH 7.4, 1% Triton-X-100, Complete Mini protease inhibitor cocktail [Roche Applied Sciences]) and rotated at 4uC for 1 h. Lysates were centrifuged at 17,500 g for 10 min at 4uC, and the resulting pellet and supernatant (Triton-soluble) fractions were collected. The pellet was further solubilized by sonication in RIPA buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton-X-100, 2.5% sodium deoxycholate, 1% SDS, Com- plete Mini protease inhibitor cocktail [Roche Applied Sciences]) to produce the Triton-insoluble fraction (RIPA-soluble). Triton- soluble and Triton-insoluble fractions were combined 1:1 with 2X Laemmli sample buffer (Bio-Rad AG, Reinach, Switzerland) containing 5% 2-mercaptoethanol and resolved by SDS-PAGE (7.5%), transferred to Protran nitrocellulose (0.2 mm; Perkin Elmer, Schwerzenbach, Switzerland) and subjected to Western Pathogenic Effect of Missense Mutations in ATP13A2 blot analysis. Nitrocellulose membranes were probed with mouse monoclonal anti-V5-peroxidase antibody (Invitrogen), or with mouse monoclonal anti-b-tubulin (Sigma-Aldrich) and peroxidase- coupled anti-mouse IgG (Jackson ImmunoResearch) antibodies to control for protein loading. Ponceau S stain was used to assess protein loading of the Triton-insoluble fraction. Proteins were visualized by enhanced chemiluminescence (ECL; GE Healthcare, Glattbrugg, Switzerland) on a FujiFilm LAS-4000 Luminescent Image Analysis system. Quantitation of protein levels by densitometry was conducted on acquired non-saturated images using LabImage 1D software (Kapelan Bio-Imaging Solutions, Leipzig, Germany). Where indicated, HEK-293T cells were treated with 5 mM MG132 (Enzo Life Sciences AG, Lausen, Switzerland) or 50 mM ammonium chloride (AppliChem GmbH, Darmstadt, Germany) for 24 h prior to harvesting. For cycloheximide (CHX) assays, CHX (200 mg/ml; Sigma-Aldrich) was added to transfected cells at 24 h post-transfection, and cells were harvested at 0, 1, 3, 6 and 8 h post-treatment. Quantitative RT-PCR Total RNA was purified from HEK-293T cells transiently expressing human ATP13A2 variants using the RNeasy Plus kit (Qiagen, Valencia, CA, USA) and 200 ng of total RNA was subjected to cDNA synthesis using the High Capacity cDNA RT kit (Applied Biosystems, Carlsbad, CA, USA). Quantitative real- time PCR was performed on a 7900HT Real-Time PCR System with SDS 2.3 software (Applied Biosystems) using Power SYBR Green PCR Mastermix (Applied Biosystems). Relative expression was calculated by normalization of plasmid-derived human ATP13A2 expression to b-actin expression. RT minus control samples run in parallel demonstrated negligible contamination from ATP13A2 plasmid DNA. All Q-PCR reactions were performed in triplicate. Primer sequences were as follows: human b-actin, forward 59-ACCGCGAGAAGATGACCCAGA-39 and reverse 59CAGGGATAGCACAGCCTGGATAGCA-39; plas- mid-derived human ATP13A2, forward 59-GCAGATATCCAG- CACAGTGG-39 and reverse 59-AGACCGAGGAGAGGGT- TAGG-39. ATP hydrolysis assay ATP hydrolysis activity was measured on microsomal fractions by monitoring the release of free c-phosphate (Pi) from ATP. Microsomal fractions were prepared from HEK-293T cells transiently expressing V5-tagged ATP13A2 variants or HA-tagged SPCA1 as previously described [23]. Briefly, HEK-293T cells in 6-well plates were harvested in 600 ml hypotonic buffer (10 mM Tris-HCl pH 7.5, 0.5 mM MgCl2, 1 mM EDTA, 1X Complete protease inhibitor cocktail [Roche Applied Science]) and kept on ice for 10 min. Cells were homogenized by 40 strokes in a glass Dounce homogenizer, followed by addition of 600 ml solution M (10 mM Tris-HCl pH 7.5, 0.5 M Sucrose, 0.3 M KCl, 6 mM b- mercaptoethanol, 40 mM CaCl2) and a further 20 Dounce strokes. Lysates were centrifuged at 8,000 g for 20 min at 4uC and the resulting supernatant fraction was centrifuged at 100,000 g for 45 min. The resulting microsomal pellet was resuspended in 100 ml solution M1 (10 mM Tris-HCl pH 7.5, 0.25 M Sucrose, 0.15 M KCl, 3 mM b-mercaptoethanol, 20 mM CaCl2). Micro- somal fractions were quantified by BCA assay (Pierce Biotechnol- ogy, Rockford, IL) and 4 mg of microsomal proteins were used for each ATPase assay. ATP hydrolysis activity was measured by monitoring the release of free c-phosphate (Pi) from ATP in 96- well plates using a high-sensitivity colorimetric ATPase Assay kit (Innova Biosciences, Cambridge, UK) as per manufacturer’s PLoS ONE | www.plosone.org 14 June 2012 | Volume 7 | Issue 6 | e39942