4.1 Cell culture
U2OS-iMLS and U2OS-iMLS-PRKN FlpIn TRex cells [12] were cultured with DMEM with high glucose and glutamine and supplemented with 10% FBS and 1% Pen-Strep at 37 ºC and 5% CO2. Cells were selected with 100 µg/mL hygromycin and 5 µg/mL blasticidin (Thermo Fisher Scientific, R21001). Additional 2 µg/mL puromycin (Sigma Aldrich, P7255) were used for U2OS-iMLS-PRKN cells. To induce the expression of the mitophagy reporter, 500 µg/mL doxycycline (Clontech, 631311) was added for the last 24 h. These cells were obtained from Prof. Anne Simmonsen Lab (Faculty of Medicine, Institute of Basic Medical Sciences and Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, University of Oslo, Norway).
ARPE-19 MitoQC cells were maintained in DMEM:F12 (1:1) supplemented with 15% de FBS, 1% glutamine 2 mM and 1% de pen-strep (0.5 mg/ml) at 37 ºC and 5% CO2. Cells were selected with 800 µg/mL hygromycin. This cell line was obtained from Dr. Ian Ganley Lab (School of Life Sciences, University of Dundee, Scotland) [16].
Peripheral blood samples were obtained from thirteen control subjects, four sporadic (sALS) patients, four ALS patients carrying a mutation in SOD1 (SOD1-ALS) and two ALS patients carrying a mutation in TARDBP (TARDBP-ALS). Blood samples were used to isolate peripheral blood mononuclear (PBMC) on Lymphoprep™ density-gradient centrifugation. Patients were diagnosed by neurologists from the Hospital Universitario 12 de Octubre (Madrid, Spain) applying El Escorial criteria [32]. Samples were obtained after signing an informed consent. All procedures were approved by the Hospital 12 de Octubre and the Spanish Council of Higher Research Institutional Review Board and are in accordance with National and European Union Guidelines.
4.2 Compound preparation
All the compounds were prepared with a stock concentration of 25 or 10 mM in DMSO. The final % of DMSO in cell culture was not higher than 0.1%. BafA1 (Enzo Life Sciences, BCL-CM110-0100) and MRT68921 (Cayman Chemical, 1190379-70-4) were also prepared in DMSO. Deferiprone (DFP) (Pubchem, 379–405) and hydroxychloroquine (HCQ) were dissolved in water. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (Sigma, C2920) was dissolved in ethanol.
4.3 Mitophagy Assay
U2OS-iMLS, U2OS-iMLS-PRKN and ARPE-19 MitoQC cells were seeded at a final concentration of 50,000 cells/mL in a 96-well plate (U2OS cells) and in crystals in a 24-well plate (ARPE-19 cells). The next day, cells were treated as indicated. In the case of U2OS-iMLS, 500 µg/mL doxycycline was added to medium to induce the expression of the reporter. After 24 h, the cells were fixed with 3.7% paraformaldehyde (PFA) 200 mM Hepes and incubated with Hoechst (Invitrogen, H1399) to stain the nuclei. The U2OS cells were maintained in the plates in PBS until images were acquired. The ARPE-19 MitoQC cells were seeded in crystals and incubated with DAPI (Sigma-Aldrich, 2D9542) to stain the nuclei. Then, the crystals were mounted over slides with ProLong Diamond Antifade Mountant (Thermo, P36961). The slides were kept for 24 h, at RT and then at 4°C until images were acquired.
4.4 Immunostaining
The U2OS-iMLS cells were seeded at a final concentration of 50,000 cells/mL in a 96-well plate. The next day, the cells were treated as indicated. After 24 h, the cells were fixed with 3.7% PFA 200 mM Hepes pH 7 and incubated with Hoechst (Invitrogen, H1399) to stain the nuclei. The cells were incubated with 0.2% NP-40 to permeabilize them. Then, the cells were washed with 1% BSA in PBS 1X and incubated with the primary antibody (LC3; 1:500, MBL-PM036) for 1h at 37°C. After washing steps, the cells were incubated with the secondary antibody Rabbit DayLight 649 (1:500) for 30 min at RT. Finally, the cells were kept in PBS until imaged.
4.5 Image Acquisition
Images of the U2OS-iMLS cells in 96-well plates were obtained under an ImageXpress Micro Confocal microscope (Molecular Devices), placed in the Advanced Light Microscopy Facility at Gaustad (University of Oslo, Norway). Six images per well were automatically taken at 20X in order to quantify around 1000 cells per condition.
Images of the ARPE-19 MitoQC cells in 24-well plates were obtained with an AF6000 LX widefield multidimensional microscopy system and CLSMLEICA TCS SP8 STED 3X placed in the confocal laser and multidimensional microscopy in vivo facility at CIB Margarita Salas (Madrid, Spain). Five to six images were manually taken at 40X and to have around 100 cells per coverslip.
Image analysis was done with CellProfiler [33]. In order to identify red-only structures per cell segmentations of nuclei, cells and mitochondrial network were performed. Mitochondrial structures were further filtered as “yellow” or “red-only” based on the ratio between their EGFP and mCherry integrated intensities. The final number of red-only structures per cell was used as a mitophagy rate readout. Later, this pipeline was modified in order to analyse mitophagy in cells seeded in 24-well plates.
4.6 Western Blotting
ARPE-19 MitoQC cells were seeded at a final concentration of 200,000 cells/mL in a 6-well plate. After 24 h, the cells were treated as indicated. Proteins were extracted with lysis buffer (50 mM Tris-HCl pH 6.8, 10% glycerol (v/v) and 2% sodium dodecyl sulfate (w/v), in distilled water with protease inhibitors 1X (Sigma, P8783) and phosphatase inhibitors (1 mM sodium orthovanadate [Sigma, S6508], 1 mM sodium fluoride [Sigma, 201154], and 5 mM sodium pyrophosphate decahydrate [Sigma, 221368]). The proteins were scrapped and transferred to a tube. The samples were heated at 95°C for 15 min and spin and stored at − 20°C until used.
Human lymphoblasts were seeded at a final concentration of 106 cells/mL in a 24-well plate and treated as indicated. After washing steps, pellets were stored at -80°C until used or treated with the lysis buffer to extract the proteins. The samples were heated at 95°C for 15 min and spin and stored at − 20°C until used.
To quantify protein, a bicinchoninic acid protein assay kit (Pierce, 23227) was used, and 12 µg of proteins was loaded with 10 mM dithiothreitol and 0.005% bromophenol blue in Criterion TGX Precast Midi Protein gels (BioRad, 5671124) and transferred to polyvinylidene fluoride membranes (BioRad, 170 − 4157) activated with 100% methanol (Panreac, 131091.1214) for 2 min. Transfer was done with Trans-Blot Turbo Transfer (BioRad) for 14 min (two waves of 7 min each) at 25 V. After the transfers, protein bands were detected with Ponceau Red (Sigma, 78376). Membranes were washed with PBS 1X-Tween20 (Bio-Rad, 170–6531) (PBS-T) and blocked with 5% (w/v) milk in PBS-T for 1h, shaking at RT. After blocking, membranes were washed with PBS-T and incubated with primary antibodies anti-LC3 (Sigma, L7543), anti-p62 (Abcam, 56416), anti-TOMM20 (Santa Cruz Biotechnology, sc-11415), anti-TIMM23 (BD Bio, 611222), anti-vinculin (Abcam, ab129002) and anti-GAPDH (Abcam, ab8245) in 3% (w/v) BSA, 0.01% azide in PBS overnight, shaking at 4°C. Membranes were washed 3× 10 min with PBS-T before secondary antibody incubation (in blocking buffer for 1 h). The membranes were also washed 3× 10 min with PBS-T before membrane imaging with the Chemiluminescent system of ChemiDoc™ MP Imaging System from Bio-Rad.
4.7 Biochemical kinase assay
The inhibition experiments were performed in the MRC Phosphorylation Unit (University of Dundee). ULK1 (5–20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/mL BSA, 0.1% mercaptoethanol) was assayed against MBP in a final volume of 25.5 µL containing 50 mM Tris pH 7.5, 0.1 mM EDTA, 10 mM DTT, 0.33 mg/mL substrate, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmol) and incubated for 30 min at room temperature. Assays were stopped by addition of 5 µL of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.
4.8 Animal samples analysis
mCherry-GFP-FIS1101 − 152 (MitoQC) male mice with C57BL/6 genetic background [34] were obtained from Dr. Ian Ganley lab and stablished in the CIB animal facility. Two animals per group were treated intraperitoneally at P90 (postnatal day 90) with IGS2.7 1 mg/Kg for 8 and 24 h and then sacrificed. Vehicle to treat control animals and to dissolve the drug was composed by 5% Tween-80 and 5% DMSO in NaCl 0.9%.
Prp-hTDP-43(A315T) transgenic male mice (TDP-43A135T) and their wild-type (WT) littermate, both with C57BL/6 genetic background, were obtained from Dr. Eva de Lago (Complutense University, Madrid). Four animals per group were treated intraperitoneally with IGS2.7 0.5 and 1 mg/Kg daily for 30 days from P65 until P95. Vehicle to treat control animals and to dissolve the drug was composed by 6.25% Tween-20 and 0.9% DMSO in PBS (Sigma-Aldrich). Animals were sacrificed 24 h after the last drug administration.
B6.Cg-Tg(SOD1-G93A)1Gur/J transgenic male mice (SOD1G93A mice) carrying the G93A mutant form of the human SOD1 transgene (n = 9), and their wild-type (WT) littermate (n = 4), both with C57BL/6 genetic background, were obtained from Dr. Silvia Corrochano (Hospital Clínico). Animals were sacrificed between P112 and P115.
MitoQC spinal cords were extracted and fixed with 3.7% PFA 200mM Hepes. The rest of the spinal cords were fixed with 4% PFA for 24 h, cryoprotected in 30% sucrose (Merck Millipore) and stored at -80 ºC. Sections of 20 µm at the lumbar level of the spinal cords were obtained with a cryostat and collected on gelatin-coated slides.
4.9 Histological procedures
Sections of the spinal cords were used for immunofluorescence. Samples were washed with PBS 1X and permeabilized with 0.3% Triton X-100 (Sigma) in PBS 1X for 15 min in a wet chamber. Samples were again washed with PBS 1X and blocked with the blocking solution BGT (3 mg/mL Bovine Serum Albumine (Nzytech) 10 mM glycine (VWR Chemicals) and 0.25% Triton X-100 in PBS 1X) for 1h at RT in a wet chamber. Next, sections were incubated at 4 ºC overnight with primary antibodies anti-p26 (Progen) and Anti-TOMM20 (Santa Cruz Biotechnology) in blocking solution in a wet chamber. Sections were washed and incubated with secondary antibodies in blocking solution for 1h at RT in a wet chamber. In addition, 1 µg/mL DAPI was used to stain the nuclei. Slides were washed with PBS 1X and mounted with Vectashield antifade mounting medium (Vector Laboratories, H-1000-10).
4.10 Image acquisition and analysis
Spinal cords were imaged in the ventral horn area, where the MN are. Images were obtained under CLSMLEICA TCS SP8 STED 3X microscope placed in the confocal laser and multidimensional microscopy in vivo facility at CIB Margarita Salas (Madrid, Spain) with 63x objective. Z-stacks of four to five sections per animal were obtained.
Image analysis was done with CellProfiler [33]. p62 and TOMM20 area were measured by MN. Ten to fifteen MN were quantified per stack from different Z. First, the mean of the Z-stack (one animal) was calculated. Then, the mean of four animals was obtained and represented.
4.11 Statistics
Statistics was done with the software GraphPad Prism 7, which includes the analysis of the data to normal distribution via the Shapiro-Wilk test. The statistical differences between groups were obtained with the unpaired two tailed t-test to compare two groups or one-way ANOVA followed by Dunnett’s multiple comparison test if there are more than two groups. A p-value lower than 0.05 was considered statistically significant.