Cell Culture, Transient Transfection, and Stress Treatment
HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), high glucose, supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin, at 37 °C in a 95% air/5% CO2 atmosphere. SH-SY5Y cells were cultured in D-MEM/F12 (1:1) GlutaMAX, supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin in a 95% air/5% CO2 atmosphere. Cells were plated on 10 cm Petri dishes (2 × 106 cells/well) for immunoprecipitation studies, on coverslips (1.5 × 105 cells/well) pre-coated with 0.01% poly-l-lysine solution for immunocytochemistry studies, or in 6-well plates (1.5 × 105 cells/well) pre-coated with 0.01% poly-l-lysine solution for Park7 siRNA experiments. Transfection was performed 24 h after plating using the Effectene Transfection Reagent kit (QIAGEN) using procedures supplied by the manufacturer. d-Sorbitol (Sigma) was diluted in standard growth medium to yield a 0.4 or a 0.2 M concentration. For oxidative stress treatment, 24 h after transfection, cells were exposed to 200 μM paraquat for 24 h or to 1 mM hydrogen peroxide for 2 h. Cycloheximide (CHX; Sigma-Aldrich) was used at 50 μg/ml for 30 min.
To identify interaction partners of GFP-tagged DJ-1, we used the GFP-Trap technique, a high-quality GFP binding system based on a single domain antibody against GFP derived from Camelids. Cells were lysed 48 h after transfection. Each confluent 10 cm cell culture dish was washed twice in ice-cold phosphate buffer saline (PBS) and lysed on ice for 5 min in 400 μl lysis buffer (20 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X100 supplemented with Roche EDTA free complete mini protease inhibitors and PhosSTOP phosphatase inhibitors). Lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. Supernatants were collected and GFP-trap beads (20 μl per reaction, Chromotek) were used according to the manufacturer instructions to immunoprecipitate GFP-DJ-1. Lysates from untransfected cells were used as negative controls. To identify endogenous DJ-1 interacting proteins, Dynabeads M-270 Epoxy were coated with a polyclonal goat anti-DJ-1 antibody (ab4150, Abcam) using the Dynabeads Antibody Coupling Kit (Invitrogen). Typically, 5 μg of antibody were used per 1 mg of Dynabeads M-270 Epoxy and 1.5 mg of Ab-coupled beads was used per reaction. Cell lysis was performed as described above and immunoaffinity purification was achieved by mixing at 4 °C for 1 h 30 min. Magnetic beads were then collected using a magnet and washed three times with dilution buffer (lysis buffer with no Triton). The DJ-1 protein complex was eluted from the beads for 10 min at 75 °C in 1× SDS sample buffer. DJ-1 (exogenous and endogenous) complex was separated on a 10% SDS PAGE, stained with Coomassie Blue stain compatible with mass spectrometry (ProtoBlue safe, National Diagnostic), and sent for mass spectrometry analysis at the Cambridge Centre for Proteomics, University of Cambridge, UK. For the validation of DJ-1 interaction partners, the immunocomplex was analyzed by immunoblotting. In the case of RNase A treatment, the enzyme was added to lysates to yield final concentrations of 1 mg/ml, and lysates were left at room temperature for 25 min followed by incubation with the beads as described above.
Mass Spectrometry Analysis
Each gel lane was cut into five equally sized bands and washed, reduced in 2 mM DTT for 1 h at RT, alkylated in 10 mM Iodoacetamide for 30 min at RT and digested in-gel with 2 μg sequencing-grade porcine trypsin (Promega) overnight at 37 °C. Digests were concentrated using a speedvac and resuspended in 0.1% formic acid. All LC-MS/MS experiments were performed using a Dionex Ultimate 3000 RSLC nanoUPLC (Thermo Fisher Scientific Inc., Waltham, MA, USA) system and a QExactive Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Separation of peptides was performed by reverse-phase chromatography at a flow rate of 300 nl/min and a Thermo Scientific reverse-phase nano Easy-spray column (Thermo Scientific PepMap C18, 2 μm particle size, 100 A pore size, 75 μm i.d. × 50 cm length). Peptides were loaded onto a pre-column (Thermo Scientific PepMap 100 C18, 5 μm particle size, 100 A pore size, 300 μm i.d. × 5 mm length) from the Ultimate 3000 autosampler with 0.1% formic acid for 3 min at a flow rate of 10 μl/min. After this period, the column valve was switched to allow elution of peptides from the pre-column onto the analytical column. Solvent A was water + 0.1% formic acid and solvent B was 80% acetonitrile, 20% water + 0.1% formic acid. The linear gradient employed was 2–40% B over 30 min. The LC eluant was sprayed into the mass spectrometer by means of an Easy-spray source (Thermo Fisher Scientific Inc.). All m/z values of eluting ions were measured in an Orbitrap mass analyzer, set at a resolution of 70,000. Data-dependent scans (Top 20) were employed to automatically isolate and generate fragment ions by higher energy collisional dissociation (HCD) in the quadrupole mass analyzer and measurement of the resulting fragment ions was performed in the Orbitrap analyzer, set at a resolution of 17,500. Peptide ions with charge states of 2+ and above were selected for fragmentation. Post-run, the data was processed using Protein Discoverer (version 1.4, ThermoFisher). Briefly, all MS/MS data were converted to mgf files and these files were then submitted to the Mascot search algorithm (Matrix Science, London UK) and searched against the Uniprot human database (UniProt_Human_Oct13 9606, 153,168 sequences; 54,677,058 residues) using a fixed modification of carbamidomethyl (C) and a variable modification of oxidation (M). The peptide mass tolerance was set to 10 ppm, the fragment ion mass tolerance to 0.1 Da, and the maximum number of missed cleavages to 2. Peptide identifications were accepted if they could be established at greater than 95.0% probability. emPAI scores as calculated as part of the MASCOT search algorithm (Matrix Science, London) was used for semi-quantitative analysis.
Cells were washed twice with sterile PBS and then lysed on ice for 10 min in lysis buffer . Lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. Supernatants were collected and protein concentration was determined by the Bradford method. Samples were stored at − 80 °C until used. Proteins were separated on a 10% SDS polyacrylamide gel (10 μg of total proteins per well) and transferred to a polyvinylidene difluoride membrane. Membranes were incubated for 1 h in TBST 5% dried milk to saturate all non-specific binding sites. Incubation with primary antibodies was overnight at 4 °C, using mouse anti-DJ-1 antibody (1:1000; sc-55572, Santa Cruz Biotechnology), rabbit anti-β-tubulin (1:1000; #2128, Cell Signaling Technology), rabbit anti-eIF4A3 (1:1000; ab32485, Abcam), or goat anti-TIA1 (1:200; sc-1751, Santa Cruz Biotechnology). Blots were developed using horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000; Vector Laboratories) and the ECL chemiluminescence system (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific).
siRNA Knockdown of DJ-1
ON-TARGETplus human PARK7 (11315) siRNA, SMARTpool (catalog no L-005984-00-0005) was purchased from Dharmacon siRNA Technologies (GE Healthcare) and dissolved in 1X siRNA Buffer to obtain a 20 μM stock stored in aliquots at − 20 °C before use. ON-TARGETplus non-targeting pool siRNA (catalog no D-001810-10-05) was used as a negative control, siGLO Red (catalog no D-001630-02-05) was used as transfection control, and ON-TARGETplus GAPD Control Pool (catalog no D-001830-10-05) was used as a positive control. HEK 293T cells were transfected according to the manufacturer’s specifications using DharmaFECT 1 Transfection Reagent and treated with sorbitol or lysed 72 h after transfection.
Cells were fixed in 4% paraformaldehyde in PBS for 20 min at 37 °C and then incubated in 1% bovine serum albumin (BSA) in PBS 0.2% Triton for 30 min at room temperature. Primary antibodies were diluted 1:100 (anti-DJ-1, #5933, Cell Signaling Technology), 1:100 (anti-DJ-1, sc-55572, Santa Cruz Biotechnology), 1:500 (anti-DJ-1, NBP1-92715, Novus Biologicals), 1:200 (anti-G3BP, #611126, BD transduction Laboratories), 1:200 (anti-eIF3η (N-20), sc-16377, Santa Cruz Biotechnology), 1:100 (anti-TIA1 (C-20), sc-1751, Santa Cruz Biotechnology), 1:1000 (anti-p54-RCK, A300-416, Bethyl Laboratories), 1:1000 (anti-p70 S6 kinase α/Hedls, sc-8418 Santa Cruz Biotechnology), 1:500 (anti-Tau 5, Calbiochem #577801), 1:200 (anti-eIF4A3, ab32485, Abcam) in blocking solution and incubated overnight at 4 °C. After washing in PBS, cells were incubated for 2 min in 1:2000 Hoechst 33342 trihydrochloride, 10 mg/ml solution (Invitrogen), in PBS. Secondary antibodies conjugated to Alexa 488, Alexa 546, Alexa 594, Alexa 647 (Invitrogen) were diluted 1:500 in PBS 0.2% Triton + 1% BSA and incubated at room temperature for 1 h. Finally, cells were rinsed in PBS, and coverslips were mounted in Mowiol.
Confocal Laser Scanner Microscopy Analysis
Confocal laser scanner microscopy analysis (CLSM) analysis was performed using an Olympus FV1000 confocal laser scanning microscope. Cells were imaged in sequential mode using a 60X UPlanSAPO Olympus objective, and Kalman filter of 4. The following settings were used for: Hoechst—excitation 405 nm laser line, emission detected between 425 and 475 nm; Alexa 488—excitation 488 nm laser line, emission detected between 500 and 545 nm; Alexa 546—excitation 559 nm laser line, emission 575–675 nm; Alexa 594—excitation 559 nm laser line, emission 575–675; Alexa 647, emission—excitation 635 nm laser line, emission 655–755 nm. The number of SGs/PBs per cell and their average size were counted using the Stress Granule Counter plug-in (Ann Sablina, Lomonosov Moscow State University, Russia) for ImageJ software (Schneider et al. 2012) with the following parameters: number of smoothes: 10, number of smoothes after subtraction: 2, threshold: 3000, Min part size: 2, Max part size: 10, circularity: 0.2. Ten confocal z-slices taken in ten separated fields were counted in each experiment. At least 350 cells were counted per condition for each independent experiment. Cell nuclei were counted manually using the Cell Counter plug-in for ImageJ. For co-localization studies, the ImageJ co-localization plug-in written by Pierre Bourdoncle was employed. Three independent experiments were performed for all conditions.
Precipitation of DJ-1 RNA Complexes
Immunoprecipitation reactions were as described in . HEK 293T cells were allowed to reach confluence in 15 cm petri dishes (n = 4) and harvested in polysome lysis buffer (PLB) (10 mM Hepes, pH 7.0, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT, 100 units/ml RNase OUT and protease inhibitors). Each plate yielded approximately 200 μl of lysate, 100 μl of which was used in each DJ-1 and IgG control IP. DJ-1 (Abcam, ab4150) or IgG isotype control (Abcam, ab37373) antibodies were conjugated to Protein G Dynabeads (Invitrogen) for 10 min at RT with rotation. The conjugated beads were incubated with cell lysate in NET2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40, 20 mM EDTA, 1 mM DTT, 100 units/ml RNase OUT) for 1 h at RT and then washed six times with cold NT2 buffer.
QPCR Analysis of Transcripts
RNA was released from the protein-bead complex by treatment with Proteinase K (beads were resuspended in NT-2 buffer supplemented with 1% SDS, 1.2 mg/ml Proteinase K) and was purified using acid phenol-chloroform followed by precipitation in 100% ethanol containing 0.27 M ammonium acetate, 0.12 M lithium chloride, and 5 mg/ml glycogen (Ambion) at − 80 °C for 16 h. Precipitated RNA was pelleted and washed with 80% ethanol before resuspension in RNase-free H2O. Then, 40 ng of RNA from each sample was used to synthesize cDNA with the Sensiscript® Reverse Transcription kit (QIAGEN) according to the manufacturer’s protocol.
Further, 1 μl of cDNA was used per technical replicate in a 10 μl reaction QPCR reaction with Maxima SYBR Green master mix (Thermo Scientific) and primers at a final concentration of 330 nM. QPCR reactions were performed on a LightCycler 480 system (Roche). Amplification specificity was confirmed by melt curve analysis of QPCR products and –RT controls were included for each sample. Crossing points (Cp) were calculated using the second derivative method. The ratio of mRNA levels in DJ-1 to IgG control samples was calculated using the qpcR package in R Studio —amplification efficiencies were calculated using non-linear regression of sigmoidal curves and incorporated into the ratio calculation. Statistical significance of relative expression levels was tested using a pairwise-reallocation test based upon that used by REST software , where Cp and efficiency values were permutated within control and treatment groups. Ratios were calculated for each permutation and compared to ratios obtained from the original data. The proportion of ratios higher or lower than that obtained from the original data was used to generate the P value of the test.
mRNA In Situ Hybridization
Cells were plated on NuncLab-Tek II CC2 chamber slides (ThermoFisher Scientific) and fixed with 10% NBF before being processed for RNA ISH using the RNAscope Technology, Multiplex fluorescent assay, Advanced Cell Diagnostics, Hayward, CA, USA. RNAscope probes were designed and provided by Advanced Cell Diagnostics, Hayward, CA, USA: Hs-GPX4 (NM_001039847.2, region 9-943), Hs-EIF4B (NM_001300821.1, region 472-1419), Hs-EIF4EBP1 (NM_004095.3, region 20-863). Positive and negative control probes were respectively Hs-PPIB (NM_000942.4, region 139-989) and DapB (EF191515, region 414-862). All probes were designed as C1 target probes. Staining steps were in accordance with RNAscope protocols with one modification: protease III was incubated for 5 min, as 10 min resulted in a weaker and less clear IF staining. AMP4-AltA-FL was used for the fluorescent labeling (channel 1 in green). For sorbitol-treated cells (1 h, 0.4 M sorbitol), immunofluorescence was performed following RNA ISH: after the last washes at the end of the RNAscope assay protocol, slides were washed in PBS and incubated in 1% bovine serum albumin (BSA) in PBS 0.2% Triton for 30 min at room temperature. Primary antibodies concentration was increased by 100%, as the ISH involves proteolytic treatment which might destroy the antigen of interest. Secondary antibodies were conjugated to Alexa 594 (Invitrogen) for eIF3 and TIA1, or Alexa 647 (Invitrogen) for G3BP and used as previously described. The number of ISH positive dots per cell was counted using the Stress Granule Counter plug-in as previously described for ImageJ software . Ten confocal z-slices taken in ten separated fields were counted in each experiment and about 100 cells were counted per condition for each independent experiment. Cell nuclei were counted manually using the Cell Counter plug-in for ImageJ. For co-localization studies, the co-localization plug-in for ImageJ was used.
Cortical Neuronal Culture
Primary neuronal cultures were obtained from the cortex of 2-day-old rat pups, incubated with 200 U of papain for 30 min at 34 °C and after trypsin inhibitor treatment (T-9253, Sigma Aldrich, St Louis, USA; 10 μg, 45 min at 34 °C) were mechanically dissociated. For immunocytochemistry, neurons were plated at densities of 70,000 cell/dish on chamber slides (80826, IBIDI, München, Germany) precoated with 25 μg/ml poly-d-lysine (Sigma P6407). The plating medium consisted of B27/Neurobasal-A supplemented with 0.5 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen). Experiments were performed after 12 days in culture at which time neurons had formed synapses. Cortical neurons were exposed to N-methyl-d-aspartate (NMDA) 100 μM for 5 or 24 h.
Lactate Dehydrogenase Assay
Twelve DIV primary cortical neurons were treated with NMDA 100 μM for 5 or 24 h. At the end of the treatment, the medium was collected and the release of lactate dehydrogenase (LDH) in the medium was quantified to assess cell viability using the Cytotoxic 96 non-radioactive cytotoxicity assay kit (Promega, WI, USA).
Maintenance and Differentiation of mES Cells
The NesE-Lmx1a ES cell line was propagated on MEF cells as described [22, 23]. The mES cells were differentiated following the 5-stage protocol  with some minor modifications. For the initiation of EB formation (stage 2), cells were dissociated with TryplE Express (Gibco) and purified on gelatinized tissue culture dishes for 45 min. Cells were subsequently plated on non-adherent bacterial dishes for 3 days in EB medium containing FBS (10%; Gibco) to allow EB formation taking place. EBs were subsequently plated on tissue culture dishes and allowed to attach. After attachment, EB medium was changed next day for DMEM/F12 (Gibco) medium containing insulin (Gibco), apo-transferrin, sodium-selenite, and fibronectin (all Sigma) (ITSF medium; stage 3). After 6 days in ITSF medium, neural precursor cells were further expanded and patterned by splitting the cells with TryplE Express and plating them on poly-ornithine and laminin (Sigma) coated 24-well plates containing N3 medium plus 10 ng/ml bFGF (R&D), 100 ng/ml FGF8 (R&D), and 100 nM Hedgehog agonist Hh-Ag1.3 (Curis Inc., USA) (stage 4). After 4 days, neuronal differentiation was initiated by removal of the growth factors and the cells were subsequently kept in N3 medium containing ascorbic acid for 12 days (stage 5). At this stage, the neuronal cultures were treated with either MPP+ (10 and 20 μM), rotenone (50 nM), or DMSO as control for 3, 6, and 12 h. Cell were fixed with 2% PFA for 20 min and analyzed by immunohistochemistry. The following antibodies were used: 1:500 rabbit Nurr1 (E20; Santa Cruz Biotechnology), 1:1000 mouse TH (Millipore), and 1:200 goat eIF3η (N-20, Santa Cruz Biotechnology).
Most data were analyzed with Prism 5 (GraphPad), using one-way ANOVA followed by post-hoc analysis with Tukey’s test. For two independent samples, an unpaired T test was used.
Two-way ANOVA was used for the analysis of CHX effect on SG formation followed by post-hoc analysis with Tukey’s test. P values of less than 0.05 were considered significant for any set of data. In all experiments, results are expressed as means ± SEM.