Donor characteristics and human brain collection
Cervical spinal cord tissue was extracted from all donors. Controls (n = 10) and ALS patients were stratified according to disease familial history and by the presence of C9orf72 repeat expansion (n = 10) and TDP43 pathology (n = 10). The summary of donor information can be found in Table 1 and individual donor details in Supplementary Table 1.
Table 1
Summary demographic information of donors. Data presented as mean ± SD
Experimental group | n | Age at death (years) | Post-mortem intervals (hrs) | Gender (% Male) |
Control | 10 | 60 ± 11 | 90 ± 29 | 50 |
Sporadic ALS with TDP43-pathology | 10 | 67 ± 13 | 72 ± 20 | 60 |
C9orf72 repeat expansion -positive | 10 | 62 ± 10 | 55 ± 29 | 50 |
Preparation of synaptoneurosomes from human post-mortem tissue
Post-mortem human cervical spinal cord tissue was homogenized, filtered and centrifuged to yield synaptoneurosome preparations as described [31]. In summary, spinal cord tissue was homogenized using glass homogenizers on ice with homogenization buffer (25mM HEPES (pH 7.5), 120mM NaCl, 5mM KCl, 1mM MgCl2, 2mM CaCl2) with the addition of protease and phosphatase inhibitors (Roche #11,836,153,001, Thermo Scientific #A32959). The homogenized tissue was filtered using 80µm nylon filter, resulting in a total homogenate sample (TH), which was partially aliquoted and stored on dry ice. A 5µm filter (Millipore, SLSV025LS) was then used to filter the remainder of the TH and the sample underwent a 5 min centrifugation at 1000 x g. The pellets generated went through a series of washes in homogenization buffer and disposal of supernatant before the final pellet was weighed and resuspended in protein extraction buffer (100mM Tris–HCL Buffer (pH 7.6) 4% SDS with 1% protease inhibitor cocktail (Roche #11,836,153,001)) in a 1:5 dilution in relation to pellet weight. Further sample homogenisation by hand occurred before final centrifugation at 17,000 x g at 4°C for 20 min, from which the pellet was disposed of and supernatant collected as the extracted synaptoneurosome (SNS) proteome. Micro BCA Protein Assay Kit (Thermo Scientific #23,235) was then used to determine protein concentration of the samples for further experiments.
Extracted SNS protein (25µg) from each individual case was pooled based on stratification of presence of C9orf72 repeat expansion, TDP43 pathology and control. This pooling resulted in 3 representative groups, each consisting of 10 individual cases, for proteomic analysis (pool 1 – control (n = 10), pool 2 - ALS-C9orf72 (n = 10), pool 3 – sporadic ALS with TDP43 pathology (n = 10)). The remaining SNS sample from each individual was kept for any further validation experiments.
Human iPSCs
The hiPSC lines used in this study are listed in Table 2. HiPSCs were cultured at 37°C (5% CO2, 5% O2) on Matrigel®-coated (Corning, 354277) 6-well plates using mTeSR1 medium (Stem Cell Technologies, 83850). At 80% confluence, the colonies were detached using Dispase (Stem Cell Technologies, 07923) and passaged in a 1:3 or 1:6 split ratio. Analysis of mycoplasma contamination was performed using the MycoStrip™ - Mycoplasma Detection Kit (Invivogen, rep-mysnc-50).
Table 2
Detailed information on the hiPSC lines used in this study
Group | ID | Gene | Mutation | Age | Sex | Origin | Catalog # |
ALS | ALSC9orf72 I | C9orf72 | (G4C2)1.8kb | 60 | ♂ | Ulm University | NA |
ALSC9orf72 II | C9orf72 | (G4C2)6−8kb | 46 | ♂ | Cedars-Sinai | CS29iALS-C9 |
ALSC9orf72 III | C9orf72 | (G4C2)2.7kb | 50 | ♀ | Cedars-Sinai | CS30iALS-C9 |
ALSFUS I | FUS | p.R521C | 57 | ♀ | Ulm University | NA |
ALSFUS II | FUS | c.1484delG | 27 | ♂ | Ulm University | NA |
ALSFUS III | FUS | c.1504delG | 19 | ♂ | Ulm University | NA |
ALSTARDBP I | TARDBP | p.Gly298Ser | 62 | ♂ | Cedars-Sinai | CS47iALS-TDP |
ALSTARDBP II | TARDBP | p.N390D | 26 | ♂ | Cedars-Sinai | CS5ZLDiALS |
ALSSOD1 I | SOD1 | p.A5V | 40 | ♀ | Cedars-Sinai | CS07iALS-SOD1A4 |
ALSSOD1 II | SOD1 | p.G94A | 57 | ♂ | Cedars-Sinai | CS2RJViALS |
ALSTBK1 I | TBK1 | p.Thr77TrpfsX4 | 41 | ♂ | Ulm University | NA |
ALSTBK1−FUS | TBK1 FUS | p.Tyr185X p.R524G | 54 | ♀ | Ulm University | NA |
Control | Healthy I | NA | NA | 45 | ♀ | Ulm University | NA |
Healthy II | NA | NA | 64 | ♂ | BioCat GmbH | SC600A-WT |
Healthy III | NA | NA | 49 | ♂ | Cedars-Sinai | CS0YX7iCTR |
Healthy IV | NA | NA | 52 | ♀ | Cedars-Sinai | CS14iCTR-21 |
CorrectedC9orf72 | C9orf72 | CRISPR-corrected (G4C2)6−8kb | 46 | ♂ | Cedars-Sinai | CS29iALS-C9n1.ISO |
Differentiation of hiPSC-derived motoneurons
Motoneuron differentiation was carried out as previously described [11]. Briefly, hiPSC colonies were detached and cultivated in suspension in ultra-low attachment flasks T75 for 3 days for the formation of embryoid bodies (EBs) in hESC medium (DMEM/F12 + 20% knockout serum replacement + 1% NEAA + 1% β-mercaptoethanol + 1% antibiotic-antimycotic + SB-431542 10 µM + Dorsomorphin 1 µM + CHIR 99021 3 µM + Purmorphamine 1 µM + Ascorbic Acid 200ng/µL + cAMP 10 µM + 1% B27 + 0.5% N2). On the fourth day, medium was switched to MN Medium (DMEM/F12 + 24 nM sodium selenite + 16 nM progesterone + 0.08 mg/mL apotransferrin + 0.02 mg/mL insulin + 7.72 µg/mL putrescine + 1% NEAA, 1% antibiotic-antimycotic + 50 mg/mL heparin + 10 µg/mL of the neurotrophic factors BDNF, GDNF, and IGF-1, SB-431542 10 µM, Dorsomorphin 1 µM, CHIR 99021 3 µM, Purmorphamine 1 µM, Ascorbic Acid 200ng/µL, Retinoic Acid 1 µM, cAMP 1 µM, 1% B27, 0.5% N2). Ultimately, after 5 further days of cultivation EBs were dissociated into single cells with Accutase (Sigma Aldrich) and plated onto µDishes, 24-well µPlates (Ibidi) or 6-well plates (Corning) pre-coated with Growth Factor Reduced Matrigel (Corning).
Synaptosome isolation from hiPSC-derived motor neurons
Synaptosomes were isolated from DIV42 hiPSC-derived motor neurons (MNs) as previously described [48]. hiPSC-MNs, plated on 6-well tissue culture plates at a seeding density of 300,000 cells/well, were washed quickly with DPBS twice, and gently scraped and collected in 1 ml of Buffer A (10 mM HEPES pH 7.4, 2 mM EDTA, 5 mM sodium orthovanadate, 30 mM sodium fluoride, 20 mM β-glycerol phosphate, protease inhibitor cocktail). The cells were homogenized for 30 strokes using a glass Dounce tissue grinder (Kimble, 885302-0002 and 885303-0002) and the homogenate (Ho) was centrifuged at 500 x g for 5 mins at 4°C. The supernatant containing the nuclei- and cell debris-free total lysate (S1) was further centrifuged at 10,000 x g for 15 mins at 4°C to yield a cytosolic supernatant (S2) and the crude synaptosomal pellet (P2). Synaptosomes were obtained by resuspending P2 in 200 µl Buffer B (10 mM HEPES pH 7.4, 2 mM EDTA, 2 mM EGTA, 5 mM sodium orthovanadate, 30 mM sodium fluoride, 20 mM β-glycerol phosphate, 1% TritonX, protease inhibitor cocktail). Further centrifugation at 20000 x g for 80 mins at 4°C led to the separation of the soluble, synaptic cytosol (S3) fraction in the supernatant and the insoluble, enriched postsynaptic density (P3) fraction as the pellet. The P3 fraction was then resuspended in 70 µl of Buffer C (50 mM Tris pH 9.0, 5 mM sodium orthovanadate, 30 mM sodium fluoride, 20 mM β-glycerol phosphate, 1% sodium deoxycholate, protease inhibitor cocktail). Protein concentration was estimated using the BCA method according to manufacturer’s instructions (ThermoFisher Scientific, 23227). Finally, 20 µg each of S1 and P2 fractions were flash frozen in liquid nitrogen and shipped with dry ice for proteomic analysis. S1, S3 and P3 fractions were used for Western blot experiments.
Liquid chromatography–mass spectrometry
100 µg of total protein per group, obtained from post-mortem spinal cord samples, was processed using S-trap mini protocol (Protifi) as described [31]. Processing of 3 sample repeats yielded: pool 1–90µg of protein, pool 2–155µg of protein and pool 3–96µg of protein) and the equivalent of 40µg peptides was used for each TMT channel. Samples were digested using trypsin at 1:50 dilution at 37°C overnight in 150µl TEAB (final concentration 100nM). Centrifugation at 4000g for 30s in 160µl of 50mM TEAB eluted peptides from S-trap mini spin column. Further centrifugations were performed in 160µl of 2% aqueous formic acid and finally 160µl of 50% acetonitrile/0.2% formic acid. Tryptic peptides from each sample eluate produced were pooled, dried and then quantified with Pierce Quantitative fluorometric Peptide Assay (Thermo Scientific). A TMTsixplex™ Isobaric Label Reagent Set (90061) Pierce High pH Reversed-Phase Peptide Fractionation kit (Thermo Scientific, #84,868) was used to label samples with TMT following manufacturer’s protocol. Desalted tryptic peptides for each sample were dissolved in 100µl of 100mM TEAB, then the 6 TMT labels were dissolved in anhydrous acetonitrile, added to the different samples and incubated for 1 hour at room temperature. Labeling reaction was stopped with 8µl of 5% hydroxylamine per sample. Following TMT labeling, samples were mixed, desalted and dried in speed-vac at 30°C. Samples were then re-dissolved with 200µl of ammonium formate (NH4HCO2) (10mM, pH 9.5) and peptides were fractionated using High pH RP Chromatography. Two technical replicates were generated using the 3 pooled samples giving the 6-plex set up for TMT labelling. Full method as described [31]. Mass spectrometry analysis of the post-mortem samples was performed in the The ‘FingerPrints’ Proteomics Facility at the School of Life Sciences, University of Dundee carried out as described [31]. In summary, peptide analysis was conducted using a Q-Exactive-HF (Thermo Scientific) mass spectrometer coupled with a Dionex Ultimate 3000 RSLC Nano (Thermo Scientific). Using the following buffers: buffer A (0.1% formic acid in Milli-Q water (v/v)) and buffer B (80% acetonitrile and 0.1% formic acid in Milli-Q water (v/v). Sample aliquots were loaded onto a trap column 10 µL/min (100 µm×2 cm, PepMap nanoViper C18 column, 5 µm, 100 Å, Thermo Scientific) equilibrated in 0.1% formic acid and the column was washed for 5 min at the same flow rate. Column was then switched in-line with a Thermo Scientific, resolving C18 column (75µm×50cm, PepMap RSLC C18 column, 2µm, 100 Å). At a constant rate of 300 nl/min peptides were eluted from the column. A constant column temperature of 50°C was maintained. Q-Exactive HF was run in data dependent positive ionization mode. Voltage for the source was set to 2.4kV with a capillary temperature of 250°C. Mass accuracy wasis checked prior to the before the start of sample analysis.
For the 6plex TMT analysis the following Q-Exactive HF parameters were used. Full MS were acquired in a scan range of 335–1600 m/z at resolution 120k and target values of 3x106 charges with maximum IT set to 50 ms. In each scan cycle, the 15 most intense precursor ions were picked up at target values of 1x105 charges and maximum IT set to 200 ms and fragmented using higher energy collision-induced dissociation (HCD) of 32. MS/MS spectra were recorded with resolution 60k. Sequenced precursor masses were excluded from further selection for 45 seconds.
For experiments with hiPSC-derived MNs, protein extracts were precipitated with ice-cold acetone-methanol at -20°C overnight. The proteins were pelleted by centrifugation (2000 x g, 20 min, 4°C) and washed three times with 80% ice-cold acetone. Dried proteins were resolved in the digestion buffer (6 M urea, 2 M thiourea, 10 mM Tris, pH 8.0). For proteome analyses 10 micrograms of proteins per sample were digested in solution with trypsin as described previously [77], and resulting peptides were desalted with C18 StageTips [49]. For the phosphoproteome, in each case one milligram of protein was digested as described above. Peptides were further purified on Sep-Pak 18 cartridges (Waters) and subjected to phosphopeptide enrichment by MagReSyn Ti-IMAC (ReSyn Bioscience) as described previously [26]. In brief, 100 µl of magnetic bead suspension per sample and enrichment round was washed with 70% ethanol, followed by washing with 1% NH4OH. Before peptide loading, beads were equilibrated with a loading buffer containing 1 M glycolic acid and 5% TFA in 80% ACN. Elution from the beads was performed three times with 1% NH4OH. The pooled eluates were further purified by C18 StageTips and peptides were subjected to two consecutive rounds of enrichment. LC-MS analyses of desalted peptides [49] were performed on an EASY-nLC 1200 UHPLC coupled to a Q Exactive HF, or quadrupole Orbitrap Exploris 480 mass spectrometer (all Thermo Scientific).
Separations of the peptides and enriched phosphopeptides were done as described previously [26, 77] with slight modifications: peptides were injected onto the column in HPLC solvent A (0.1% formic acid) at a flow rate of 500 nl/min and subsequently eluted with an 127 minute (proteome) or 57 minute (phosphoproteome) segmented gradient of 10-33-50-90% of HPLC solvent B (80% acetonitrile in 0.1% formic acid) at a flow rate of 200 nl/min. Both mass spectrometers were operated in a positive ion and data-dependent acquisition mode.
For the Q Exactive HF mass spectrometer, full MS were acquired in a scan range of 300–1650 m/z at resolution 60k and target values of 3x106 charges with maximum IT set to 25 ms. In each scan cycle, the 12 (proteome) or 7 (phosphoproteome) most intense precursor ions were picked up at target values of 105 charges and maximum IT set to 45 ms and 220 ms, respectively, and fragmented using higher energy collision-induced dissociation (HCD) of 27. MS/MS spectra were recorded with resolution 30k (proteome) and 60k (phosphoproteome), respectively. In all proteome and phosphoproteome measurements, sequenced precursor masses were excluded from further selection for 30 seconds.
For the quadrupole Orbitrap Exploris 480 mass spectrometer, full MS were acquired in a scan range of 300–1750 m/z range at resolution 60k with an automatic gain control (AGC) set to standard and a maximum ion injection time set to automatic. The 20 most intense precursor ions were sequentially fragmented with a normalized collision energy of 28 in each scan cycle using HCD fragmentation. MS/MS spectra were recorded with a resolution of 15,000, whereby fill time was set to automatic. In all measurements, sequenced precursor masses were excluded from further selection for 30 seconds.
MS data processing
MS data were processed using default parameters of the MaxQuant software (v1.5.2.8, 1.6.2.10 or v1.6.14.0; a final analysis with the version 1.6.14.0 was performed to ensure reproducibility of the coverage across the analysis) [12]. Extracted peak lists were submitted to database search using the Andromeda search engine [13] to query a target-decoy (Elias, Gygi 2007) database of Homo sapiens (96,817 entries, downloaded on 11th of December 2019) and 285 commonly observed contaminants.
In database search, full tryptic specificity was required and up to two missed cleavages were allowed. Carbamidomethylation of cysteine was set as fixed modification, whereas protein N-terminal acetylation, and oxidation of methionine were set as variable modifications. In addition, phosphorylation of serine, threonine, and tyrosine were set as variable modification for phosphoproteome analysis. Initial precursor mass tolerance was set to 4.5 ppm, and 20 ppm at the fragment ion level. Peptide, protein and modification site identifications were filtered at a false discovery rate (FDR) of 0.01. The iBAQ (Intensity Based Absolute Quantification) and LFQ (Label-Free Quantification) algorithms were enabled, as was the “match between runs” option [35, 55].
The MS proteomics data obtained with post-mortem samples have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [45] with the dataset identifier PXD000000000".
Western Blot
Western blots using human post-mortem samples were carried out as described [31]. Briefly, 10µg of sample with 4X Laemmli sample buffer (Bio-Rad, #1,610,747) and beta-mercaptoethanol was denatured for 5 min at 95°C. Using 4–20% Tris-Glycine 1.0mm polyacrylamide pre-cast gels (Termo Fisher, #WXP42020BOX), 5 µl protein ladder (Li-Cor #928–70,000) was loaded along with the 10µg samples. Following electrophoresis, the transfer step was conducted using the iBlot™ 2 Gel Transfer Device (Invitrogen, #IB21001) and nitrocellulose membrane using precast transfer stacks (Invitrogen, #IB23001). Membrane was stained for total protein with REVERT™ 700 Total Protein stain (Li-Cor, REVERT™ 700 Total Protein Stain Kits, #926– 11,010) before being imaged with the Li-Cor Odyssey system. Membrane was destained post-imaging as instructed in the manufacturer's protocol and blocked with 5% milk/TBST or 5% BSA/TBST solutions for 1 hour. After which, primary antibody solutions diluted in the appropriate block solution were added for 24 hours. Following which, the membrane was thoroughly washed, and the secondary antibody diluted in block was applied for 1 hour. After further washes, the membrane was imaged using the Li-Cor Odyssey system.
Western blots with samples from hiPSC-derived MNs were performed by resolving equal concentrations of protein (determined by Bradford Assay) on 10% acrylamide SDS-PAGE gels, which were then transferred to a nitrocellulose membrane using a Trans-Blot Turbo device (BioRad, USA). To block non-specific binding sites, the membranes were incubated with a 5% BSA solution (diluted in TBS pH 7.5 + 0.2% TWEEN) for two hours and incubated with the primary antibody overnight at 4°C. Afterwards, blots were washed 3 times with TBS + 0.2% TWEEN, incubated with HRP-conjugated secondary Ab for 1 hour, and again washed 3 times. Chemiluminescent signal was detected using the ECL detection kit (ThermoFisher Scientific, 32106) and a MicroChemi 4.2 device (DNR Bio Imaging System). For quantification, Gel-analyzer Software 2010a was used. The values of the proteins of interest were normalized against the loading control, β-actin.
Immunocytochemistry
Immunostainings were performed as previously described [11]. Following fixation with 4% paraformaldehyde (containing 10% sucrose), cells were first incubated with blocking solution (PBS + 10% Goat Serum + 0.2% Triton-x100) for two hours at room temperature and subsequently with primary antibodies diluted in the same blocking solution overnight at 4°C. After three washes with PBS (15 minutes each), the cells were incubated with secondary antibodies (diluted 1:1000 in PBS) for two hours at room temperature. Then, cells were washed again 3 times and mounted with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36935) and Ibidi Mounting Medium (Ibidi, 5001).
Pharmacological treatment
The effect of Docosahexaenoic acid (Sigma-Aldrich D2534) treatment was tested on hiPSC-derived MN cultured in 24-well plates differentiated from the ALS cell lines. Treatment was carried out form DIV 21 until DIV 42, by replacing half of the medium every second day, using a final concentration of 100 µM. The effect of DHA treatment was determined by measuring the levels of the phenotypic rescue using immunofluorescence.
The Tetanus neurotoxin (Sigma-Aldrich T3194) was used at a final concentration of 15 nM. The treatment was performed in hiPSC-derived MN differentiated from the Healthy I cell line, starting from DIV 42 and carried out for 24 h.
Primary rat cortical neurons
Primary cultures of rat cortical neurons were prepared from rat embryos (Sprague-Dawley rats, Janvier Laboratories) at embryonic day 18 as previously described [9]. Briefly, cerebral cortices were manually dissected under stereomicroscopic guidance. After 10 minutes of incubation with 0.25% trypsin-EDTA (Gibco), the tissues were washed three times with DMEM (Gibco) (containing 10% foetal bovine serum, 1% penicillin/streptomycin, and 1% GlutaMAX, henceforth referred to as DMEM+) and thus mechanically dissociated. Following a filtration through a 100µm mesh filter, the dissociated cells were plated on poly-L-lysine-coated (Sigma-Aldrich) glass coverslips or plastic dishes and cultured in Neurobasal Medium (Gibco) (containing 1% P/S, 1% GlutaMAX and 2% B27 – henceforth NB+).
Poly-(GA)175-EGFP aggregates were over-expressed in primary cortical neurons using an AAV9 vector under the control of the human Synapsin 1 promoter to ensure selective neuronal expression. Viruses were produced by the Penn Vector Core (University of Pennsylvania, Philadelphia, USA). Transduction of neurons was performed at DIV 3 with either AAV9-hSyn-poly(GA)175-EGFP or AAV9-hSyn-EGFP (a gift from Bryan Roth; Addgene viral prep # 50465-AAV9) as a control.
Microscopy
Fluorescence microscopy was performed with a Thunder imaging system (Leica) equipped with a DFC9000 sCMOS camera, a HC PL Fluotar 20X air (N.A. 0.4) objective and using the LasX software (Leica).
Confocal microscopy was performed with a laser-scanning microscope (Leica DMi8) equipped with an ACS APO 63X oil DIC immersion objective. Images were acquired using the LasX software (Leica), with a resolution of 1024x1024 pixels and a number of Z-stacks (step size of 0.3 µm) encompassing the entire cell soma.
Image analysis
To analyze the intensity levels of nuclear phospho-c-JunSer63 in immunostaining, the Z-stack was collapsed with the maximum intensity projection tool of ImageJ and the phospho-c-JunSer63 signal was measured within a region of interest (ROI) drawn using as a reference the neuronal nucleus (identified in the DAPI channel).
To analyze the intensity of SYP puncta, four ROIs (130x130 µm) were randomly drawn in each picture on the MAP2 channel to cover neuronal dendrites, then synaptic clusters were traced with the FindFoci plugin of ImageJ, using the Max Entropy algorithm.
The neuroprotective effect of the DHA treatment was assessed by assessing the number of neurons in the entire field of view acquired, using the MAP2 channel.
The same computational parameters and post-acquisition adjustments were used to analyze images from the same tests and to display figures.
Antibody list
The following primary antibodies were used for Western Blot experiments with post-mortem samples: anti-Lamin (Proteintech, 10298-1-AP; diluted 1:1000), anti-Synaptophysin (Abcam, ab8049; diluted 1:1000), anti-PSD95 (Cell Signaling, 3450; diluted 1:1000), anti-RTN3 (Proteintech, 12055-2-AP; diluted 1:500), anti-TDP43 (Abcam, ab133547; diluted 1:500) and anti-SerpinA3 (Proteintech, 12192-1-AP; diluted 1:500). The secondary antibodies used were Donkey anti-Mouse IgG IRDye® 800CW (Li-Cor, 923-32212) or the Donkey anti-Rabbit IgG IRDye® 800CW (Li-Cor, 926-32213).
The primary antibodies used in experiments with hiPSC-derived MNs were: anti-MAP2 (Encor, CPCA-MAP2; diluted 1:1000), Proteostat® aggresome detection kit (Enzo, ENZ-51035-0025; diluted 1:5000), anti-Synaptophysin (Abcam, ab14692; diluted 1:1000), anti-Synaptophysin (Synaptic Systems, 101 004; diluted 1:1000), anti-phospho-c-Jun (Ser63) (Cell Signaling, 91952; diluted 1:1000), anti-phospho SQSTM1/p62 (Ser349) (Cell Signaling, 16177; diluted 1:300). For Western blot experiments, the secondary HRP-conjugated anti-Mouse (1:3000 dilution) and anti-Rabbit (1:1000 dilution) antibodies from DAKO were used. For immunostainings, the following secondary antibodies from Thermo Fisher Scientific were used at 1:1000 dilution: goat anti-Rabbit Alexa Fluor® 488 (A-11034), goat anti-Chicken Alexa Fluor® 568 (A-11041), and goat anti-Guinea Pig Alexa Fluor® 647 (A-21450).
Lactate-dehydrogenase Cytotoxicity Assay
The CyQUANT™ LDH Cytotoxicity Assay (Thermo Fisher Scientific, Waltham, MA, United States; C20300) was used following manufacturer's instructions to measure the amount of cell stress and death within the cultures, by measuring the leaked LDH released from damaged cells in the culture medium. At DIV42, 50 µl of medium were taken from each culture and transferred to a 96-well plate kept at room temperature and protected from light. The medium was then mixed with 50 µl of the reaction mixture given with the kit. Additionally, 50 µl of Stop Solution (included in the kit) was added to each well after 30 minutes of incubation. Gen5 microplate reader (BioTek Instruments, Winooski, VT, United States) was used to measure the absorbance of the Formazan-dye produced by the reaction at 490 nm. To further detect and minimize background signals, the absorbance at 680 nm was measured and subtracted.
Data and statistical analysis
Proteins identified in post-mortem mass spectrometry with less than two unique peptides were removed from the dataset (565 IDs). Similarly, proteins that were not identified in all samples were also excluded from the dataset (30 IDs). Following these data filtering steps, the dataset was analysis ready containing 5497 protein IDs. Reporter intensity value of each protein was log2 transformed and group median corrected to account for minor loading variables. An average of technical replicates per protein were counted for each group, then sALS and C9 + ve group were normalized to the control group providing the fold change value for each protein [1]. To visualize protein distribution by volcano plot, p value for each protein was generated by using multiple t-test with the original FDR method of Benjamini and Hochberg, then p values were -log10 transformed. Ratiometric values were generated by normalizing the average reporter intensity value of each protein of sALS and C9 + ve data to control than 1/group median corrected. Up – or downregulated proteins were identified as a threshold of 20%-fold change (≥ 1.2 or ≤ 0.8 ratio change) [31]. This fold change threshold was chosen as post-hoc validation of protein expression with western blotting can be conducted with these levels of protein change.
Downstream statistical analysis of hiPSC-based proteomics and phosphoproteomics was performed in R (version 3.6.0). The R package proteus (version 0.2.14) was used to analyze MaxQuant’s Proteomics output file “proteinGroups.txt” and the phosphoproteomics output file “Phospho (STY)Sites.txt”. Differential expression (DE) analysis was performed with the R package Limma (version 3.42.2) outside of the package Proteus. As a cut-off for statistical significance, a nominal p value < 0.05 or adjusted p values (adj. P. Val.) < 0.05 were chosen. All data was quantile normalized to account for variation of intensity between samples followed by log2 transformation.
Phosphosites analysis in total MNs lysate: In order to identify differentially expressed proteins/phosphosite a linear model was then fitted to each protein as follows: exp = ~ condition with “exp” representing expression of a protein and “condition” representing the factor of the experiment with the two levels of patient and control. The same procedure was done for phosphosites and afterwards the phosphosite table was compared against the protein table to exclude phosphosites that were different due to differential expression at the protein level. For graphical visualization we used a volcano plot to show statistical significance -log10(p-value versus log2 fold change).
Proteomics analysis in hiPSC-derived MNs synaptosomal fraction: In order to identify differentially expressed proteins a linear model was then fitted to each protein as follows: exp = ~ X with “exp” representing expression of a protein and “X” representing a combination of the main experimental factor health status with the levels patient and control, and specimen with the levels total lysate and synaptic fraction. For graphical visualization we used a volcano plot to show statistical significance -log10(p-value) versus log2 FC.
For the identification of KEGG pathways and Gene Ontologies (GO) that may be altered we used the g:Profiler (https://biit.cs.ut.ee/gprofiler/gost) or ShinyGo v0.75 online tools with the specification of Homo Sapiens datasets. The protein IDs corresponding to the DE phosphosites and proteins with nominal p-val < 0.05 were copied into the g:Profiler tool. Homo sapiens was selected as the species and for the advanced options the following parameters were considered: only annotated genes, g:SCS threshold, 0.05 threshold and ENTREZGENE_ACC; before clicking on Run query.
Enriched terms were downloaded and plotted using the ggplot2 package for R version 4.1.2 or GraphPad Prism (version 9.5.0). Venn diagram calculation and plotting was generated using the free online software of the Van de Peer Lab (https://bioinformatics.psb.ugent.be/webtools/Venn/). Heatmaps were plotted using hierarchical clustering with Euclidean distance by the heatmap.2 module of the gpltots package of R or the Partek Flow softwares. Z-score was calculated as described before [31]. Protein-protein interaction networks were generated with the OmicsNet 2.0 software [75]. Kinome analysis was performed using the SELPHI tool [46] and the representation of the kinome tree was made using the KinMap tool (http://www.kinhub.org/kinmap/index.html).
To compare two independent groups (genotypes) in western blot, immunocytochemistry and LDH assay, we used unpaired t-test with Welch correction in case of normally distributed data and nonparametric Mann–Whitney test in case of non-normal distribution. Here, experiments were performed in n = 3 replicates and normalized to the average value of the control group to obtain one single value for each cell line (representing a single patient). To analyze the effect of treatments performed in hiPSC-derived neuronal cultures, values obtained by analyzing cultures exposed to vehicle or treatment were compared using a paired t-test. Statistical significance was set at p < 0.05.
Ethics approval
Patients were recruited through the Scottish Motor Neurone Disease Register, ethical approval was obtained from Scotland A Research Ethics Committee 10/MRE00/78 and 15/SS/0216. Use of patient samples for genetic profiling has been approved by the Chief Scientist Office Scotland; MREC/98/0/56 1989–2010, 10/MRE00/77 2011–2013, 13/ES/0126 2013–2015, 15/ES/0094 2015-present. Post-mortem human tissue was requested from the Edinburgh Brain Bank and approved by its ethics committee (21/ES/0087) and the ACCORD medical research ethics committee, AMREC (ACCORD is the Academic and Clinical Central Office for Research and Development, a joint office of the University of Edinburgh and NHS Lothian, approval number 15-HV-016).
All procedures with hiPSC material were in accordance with the ethical committee of the Ulm University (Nr.0148/2009 and 265/12) and in compliance with the guidelines of the Federal Government of Germany. All participants gave informed consent for the study. The use of human material was approved by the Declaration of Helsinki concerning Ethical Principles for Medical Research Involving Human Subjects.
Preparation of rat primary cells was allowed by the Permit Nr. O.103 of Land Baden-Württemberg (Germany), and performed in respect of the guidelines for the welfare of experimental animals issued by the German Federal Government and the Max Planck Society, and the ARRIVE guidelines.