recommendations. Activity of the V-ATPase was inhibited by addition of 5 mM Bafilomycin A1 (Sigma-Aldrich) to each microsomal fraction during the reaction. In parallel, replicate microsomal samples without ATP were used to determine non- specific background absorbance. Microsomal fractions from non- transfected cells were used to determine non-specific ATPase activity. Assay samples were incubated with 0.5 mM ATP for 30 min at room temperature, reactions were terminated, and absorbance at 650 nm was measured. Absorbance from replicate samples without ATP was removed from the equivalent sample containing ATP, and the absorbance from non-transfected microsomes was also subtracted from each sample. Microsomal samples were subjected to Western blotting with anti-V5 antibodies and ATP13A2-V5 protein levels were determined by densitometry and used to normalize ATPase activity in each sample. Data represent Pi release from ATP expressed as a percent of WT ATP13A2 activity. Immunocytochemistry and confocal microscopy Immunocytochemical staining was conducted as previously described [36]. Briefly, cells grown on glass coverslips were fixed with 4% paraformaldehyde (PFA) and incubated with primary antibodies for 1–2 h and anti-rabbit or mouse IgG-AlexaFluor- 488, -546 or -633 secondary antibodies (Invitrogen) for 1 h. Nuclei were stained with 49,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Peterborough, UK). Coverslips were mounted with Mowiol 4–88 mounting medium (Sigma-Aldrich). Transiently transfected primary cortical neurons or SH-SY5Y cells were immunostained with combinations of rabbit polyclonal anti-bIII- tubulin antibody (Sigma-Aldrich), mouse monoclonal anti-V5 antibody (Invitrogen), or rabbit monoclonal anti-calreticulin antibody (EMD Millipore). Confocal images were acquired on a Zeiss LSM 700 confocal microscope using Zeiss ZEN confocal software (2009; Carl Zeiss AG, Feldbach, Switzerland) and a Plan- Apochromat 63x/1.40 oil objective in x, y and z planes. Image analysis was performed using Fiji software (Image Processing and Analysis in Java) or Imaris software (Bitplane AG, Zurich, Switzerland). Images were subjected to deconvolution using HuygensPro software (Scientific Volume Imaging, Hilversum, Netherlands). Pearson’s correlation coefficients (Rcoloc) were calculated in Fiji between the indicated fluorescent channels. Representative images are taken from a single z-plane at a thickness of 0.15 mm. Cell viability assays SH-SY5Y cells were seeded in growth medium in 96-well plates (25,000 cells/well) and transiently transfected with plasmid DNA. After 48 h, cellular viability was assessed by MTS reduction assay (CellTiter 96 Cell Proliferation Assay; Promega, Madison, WI, USA) according to manufacturer’s instructions. Absorbance was measured on an Apollo-1 LB 911 microplate photometer (Berthold Technologies GmbH, Regensdorf, Switzerland) and background absorbance from media alone was subtracted. Viability was expressed as a percent of control (empty plasmid). Neurite outgrowth assays Primary cortical cultures at DIV 3 were co-transfected with V5- ATP13A2 or empty vector and EGFP plasmids at a 10:1 DNA molar ratio. At DIV 6, cultures were fixed with 4% PFA and References Pathogenic Effect of Missense Mutations in ATP13A2 subjected to immunocytochemistry with mouse anti-MAP2 antibody (Sigma-Aldrich) and anti-mouse IgG-AlexaFluor-633 antibody (Invitrogen). Where indicated primary cortical neurons were treated with cadmium (30 mM) or nickel (50 mM) for 24 h prior to fixation. Fluorescent images were acquired using an EVOS inverted fluorescence digital microscope (Advanced Microscopy Group, Bothell, WA, USA) with a 10x objective. EGFP images were pseudo-colored using ICA1 in NIH ImageJ software to improve the contrast of neuritic processes, and used for neurite length measurements. The length of EGFP+ neurites from MAP2+ cortical neurons were measured using the line tool function of NIH ImageJ software by an investigator blinded to each condition. Only neurons that had extended neurites were measured whereas neurons without processes were excluded from the analysis. The longest EGFP+ neurite (i.e. axon) was measured and used for comparison amongst groups. In each experiment, cortical neuronal processes from EGFP+/MAP+ neurons (n = 67– 88) randomly sampled across five coverslips from at least three independent experiments were measured. For neurons treated with heavy metals, EGFP+/MAP+ neurites from control (n = 73– 156), cadmium (n = 39–119) or nickel (n = 32–72) treatment groups were measured. Statistics Data were analyzed by one-way ANOVA with Newman-Keuls post-hoc analysis as indicated. P,0.05 was considered significant. Supporting Information Figure S1 Analysis of human ATP13A2 variant mRNA expression levels. A, Agarose gel electrophoresis indicates similar DNA quantity and integrity for each ATP13A2 expression plasmid (500 ng DNA). Equivalent quantities of each plasmid were employed for transient transfection of HEK-293T cells. B, Quantitative RT-PCR was conducted on mRNA-derived cDNAs from HEK-293T cells transiently expressing V5-tagged human ATP13A2 variants. PCR primers specific for plasmid-derived human ATP13A2 were employed that amplify the 39 end of ATP13A2 incorporating the V5 epitope tag sequence. Bars represent the relative levels of plasmid-derived human ATP13A2 mRNA normalized to endogenous b-actin mRNA levels expressed in arbitrary units (mean6SEM, n = 4 independent experiments). Non-significant (ns) compared to WT ATP13A2 by one-way ANOVA with Newman-Keuls post-hoc analysis. (TIF) Acknowledgments The authors would like to thank Dr. Christian Kubisch (University of Cologne, Germany) for kindly providing expression plasmids for WT and KRS mutant ATP13A2-V5, Dr. Adam Linstedt (Carnegie Mellon University, Pittsburgh, PA) for providing the HA-SPCA1 plasmid, and members of the EPFL SV BioImaging and Optics platform for assistance with confocal microscopy. Author Contributions Conceived and designed the experiments: AP AM AT DJM. Performed the experiments: AP AM AT KS RM SS LG. Analyzed the data: AP AM AT KS RM SS LG DJM. Contributed reagents/materials/analysis tools: AP DJM. Wrote the paper: DJM. 1. Gasser T (2009) Mendelian forms of Parkinson’s disease. Biochim Biophys Acta 1792: 587–596. 2. Hardy J, Cai H, Cookson MR, Gwinn-Hardy K, Singleton A (2006) Genetics of Parkinson’s disease and parkinsonism. Ann Neurol 60: 389–398. PLoS ONE | www.plosone.org 15 June 2012 | Volume 7 | Issue 6 | e39942