was 10 nM. Cells were seeded on poly-L lysine (PLL) coated plates (35,000 cells/cm2) and then transfected 48 hours before experimentation. Primary cortical rat neurons were prepared from E18 rat embryos, following previously published procedures [65]. Neurons were seeded on poly-ornithin-coated 24-well plates at a density of 2 125,000 cells/cm . Cells were maintained in Neurobasal medium (Gibco/Invitrogen), supplemented with 5 mg/ml transferrin, 1% PSN, 0.5 mM L-Glutamine, 2% B27 supplement. Primary neurons were infected equimolarly with lentiviruses one day after isolation and then cultured for 6 days before experimental use. SH-SY5Y cells (DSMZ number ACC 209) were cultured in DMEM F-12 with glutamine (Lonza) supplemented with 15% (v/ v) fetal calf serum, non-essential amino acids (Invitrogen) and penicillin/streptomycin. Transfections were performed using Lipofectamine Plus (Invitrogen) according to the manufacturer’s instructions. The following plasmids were described earlier: [A53T]a-Synuclein and mito-DsRed [51]. For downregulation of TRAP1, SH-SY5Y cells were reversely transfected with the indicated siRNA and co-transfected with mito-DsRed and empty vector or [A53T]a-Synuclein using Lipofectamin RNAiMax (Invitrogen) according to the manufacturers instruction. 24 hours after transfection, fresh medium was added. Cells were analyzed 48 h after transfection. Fluorescent staining of mitochondria. SH-SY5Y cells were plated on 15 mm glass coverslips and co-transfected with mito-DsRed and the indicated DNA constructs. At 24 h after transfection, cells were washed twice with ice-cold PBS, fixed with 3.7% paraformaldehyde for 10 minutes at room temperature and washed twice with PBS before mounting the coverslips. Transfected cells were identified by co-expression of mito- DsRed. Cells were categorized in two classes according to their mitochondrial morphology [75]. Cells displaying an intact network of tubular mitochondria were classified as tubular. When this network was disrupted and mitochondria appeared predominantly spherical or rod-like, they were classified as fragmented. The mitochondrial morphology of at least 300 cells per coverslip was determined in a blinded manner using a Leica DMRB microscope. Quantifications were based on triplicates of at least three independent experiments. Confocal images of representative cells were obtained using a Zeiss LSM 510 microscope. a- Synuclein was detected using a monoclonal anti-rat antibody described previously [51]. b-Actin was detected using a monoclonal antibody from Sigma. Cell Culture Oxidative Stress Testing and Measurement of Cell Viability Cells were incubated for 16 hours in the presence of either hydrogen peroxide (100 mM) or rotenone in DMSO (HEK293: 200 mM, rat cortical: 1 mM rotenone). Rotenone control cells were treated with equivalent amount of DMSO alone. After overnight oxidative stress treatment, cells were fixed in 4% PFA for 10 min, before permeabilization in PBT for 10 min. Cells were incubated with Hoechst nuclear stain for 30 min before imaging on a fluorescent microscope (Leica DMI6000B). Using a macro within the Leica Qwin V3 quantification software, cell number remaining in each well was assessed by counting total fluorescent nuclei (6 images per well at 106, with minimum 6 wells per genotype in a 24-well culture dish). ATP Synthesis Assay Method was adapted from [86]. Cells were trypsinized and washed three times in cold PBS. Cells were then resuspended in incubation medium (26105 cells/ml, 25 mM Tris, 125 mM KCl, TRAP1 Mitigates a-Synuclein Toxicity 2 mM K+EDTA, 10 mM KH2PO4, pH 7.4) and permeabilized with digitonin (40 mg/ml). To measure ATP produced via complexes I, III, IV, 26104 cells (minimum of 8 replicates per experiment) were resuspended in a complex incubation medium supplemented with 1 mg/ml BSA, 2 mM ADP, 10 mM gluta- mate, 2 mM malate. After incubation for 20 min at 37uC, the reaction was stopped by addition of 6% perchloric acid, and samples were neutralized with 3 M K2CO3. ATP in each sample was measured in a plate reader using the CellTiter-Glo Luminescent reagent (Promega) following manufacturer-provided instructions. Total ATP levels were obtained using an ATP standard curve (ATP from Sigma), with final calculation expressing ATP as pmoles ATP per minute per 1 million cells, and plotted as percentage of control values. Measurement of Steady State Cellular ATP 26104 HEK293 cells/well were seeded (12 replicates per experiment) in an opaque white 96-well plate. Total ATP levels were measured as stated above. Assessment of Mitochondrial Membrane Potential Following manufacturer-provided instructions, cells (6 replicates per experiment) in a 96-well plate (black sided, clear bottom) were incubated with the mitochondrial probe JC-1 (3 mg/ml; Invitro- gen) in full medium for 30 min at 37uC. After washing in PBS, JC- 1 mitochondrial aggregates were measured in a plate reader (excitation: 530 nm, emission: 590 nm). As a control, JC-1 fluorescence was measured in the presence of the mitochondrial membrane potential inhibitor CCCP (Carbonyl cyanide m- chlorophenylhydrazone, 50 mM). Protein content per well was quantified using the Bradford assay. Fluorescence in relation to total protein was displayed as percentage of control values. Immunocytochemistry Cells were plated on PLL-coated glass slips. 48 hours after transfection, cells were fixed in 4% PFA for 10 min, permeabilized in PBT for 10 min, followed by blocking in 1% BSA and overnight incubation with the primary antibody at 4uC. Antibodies used: monoclonal rat anti-a-Synuclein (1:500, Alexis Biochemicals/ Enzo Life Sciences); monoclonal mouse anti-NeuN (1:500, Chemicon); monoclonal mouse anti-TRAP1 (1:300, Alexis Biochemicals). For visualization of mitochondria cells pretreated for 4 hours with 1 mM rotenone, the cells were incubated with Mitotracker Orange CMTMRos (300 nM, following manufactur- er’s instructions, Invitrogen) for 30 min at 37uC prior to fixation. Cells were incubated with respective secondary antibodies for one hour (all secondary antibodies 1:1000, anti-mouse or rat Alexa- Fluor-488, 543, 633, Invitrogen), and mounted using Mowiol (Calbiochem), with or without the anti-bleaching agent DABCO or nuclear stain Hoechst (Sigma-Aldrich). Mitochondrial Isolation Mitochondria were isolated from HEK293 cells transfected with [A53T]a-Synuclein using the following protocol: Cells were suspended in MB buffer (70 mM sucrose, 10 mM HEPES, 1 mM EDTA, 210 nM Mannitol (pH 7.5) and protease inhibi- tors), homogenized with an injection needle (27G L0 19 mm, 5–6 strokes) and centrifuged at 7506g for 7 min. After centrifugation the pellet was resuspended in MB buffer, homogenized using the same injection needle and centrifuged. This procedure was repeated four times. The resulting supernatants were pooled and centrifuged at 100006g for 30 min. This mitochondria-containing pellet was resuspended in MB buffer and further centrifugated at PLoS Genetics | www.plosgenetics.org 12 February 2012 | Volume 8 | Issue 2 | e1002488