Diagnosis of WFS was confirmed in patients by direct sequencing of the WFS1 gene and/or multiplex ligation-dependent probe amplification (MLPA; SALSA MLPA P163 GJB-WFS1 probemix, MRC-Holland, The Netherlands), as described previously . Skin biopsies in WFS patients (n = 2 with nonsense mutations) and healthy volunteers (n = 2) were performed.
Next step included reprogramming process of skin fibroblasts to induced pluripotent stem (iPS) cells and then to trans-differentiated neural stem cells (NSC) with following induction of ER stress. This allowed us to create an experimental human cell model of WFS with enhanced ER stress effect.
Fibroblasts collected from skin biopsy were transformed into iPS and NSC in cooperation with Celther Company, Poland. Next, iPS cells were cultured in Essential 8 (Life Technologies, CA, USA) on protein-coated culture vessels of the extracellular matrix (Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix Geltrex, Life Technologies, CA, USA; 1:100). After reaching the appropriate confluence, iPS cell colonies were passed using 0.5 mM EDTA (Sigma-Aldrich, Germany) on new culture vessels and also extracellular matrix protein-coated, until the number of cells required for the analysis was reached.
The NSC were cultured in PSC Neural Induction Medium (Life Technologies, CA, USA) on protein-coated culture vessels of the extracellular matrix (Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix Geltrex, Life Technologies, CA, US; 1:100). After reaching appropriate confluence, the cells passed using StemPro Accutase (Life Technologies, CA, USA) on new culture vessels and also extracellular matrix protein-coated, until the number of cells required for the analysis was reached.
ER stress induction
The cells were treated with ER stress inductor tunicamycin (TM, Sigma-Aldrich, Germany; 5 µg/ml) dissolved in DMSO (Sigma-Aldrich, Germany). The cells were incubated with TM and under control conditions (culture medium). After 8 h of incubation, the medium was collected from both culture vessels and centrifuged (200 x g, 2 min). After removing the supernatant, the remaining cells were rinsed with PBS solution and then peeled off from the vessel surface. The cells were centrifuged (200 x g, 5 min) and counted and then aliquoted for further analyses.
The NSC were then prepared for proteomic analysis, i.e. dry pellet of 10 million cells. For this purpose, an appropriate number of cells were washed twice with cold buffered saline solution without ions (PBS w/o Ca2+, Mg2+; vWR, PA, USA), inundated with a volume of cold PBS appropriate for the surface of the culture vessel, scraped and centrifuged (330 x g, 5 min, 4°C). After supernatant removal, the cells were quickly frozen in liquid nitrogen and then stored at − 80°C. The whole procedure was performed on ice.
Proteomics - materials
Isobaric TMT reagents and the BCA protein concentration assay kit were from ThermoFisher Scientific (Rockford, IL, USA). Empore-C18 material for in-house made Stage Tips was from 3 M (Saint Paul, MN, USA). Sep-Pak cartridges (100 mg size) were purchased from Waters (Milford, MA, USA). All solvents used for Liquid chromatography (LC) were purchased from J.T. Baker (Central Valley, PA, USA). Mass spectrometry (MS)-grade trypsin and Lys-C protease were purchased from ThermoFisher Scientific and Wako (Boston, MA, USA), respectively. Complete protease inhibitors were from Millipore Sigma (Saint Louis, MO, USA). Unless otherwise noted, all other chemicals were purchased from ThermoFisher Scientific.
MS sample processing
All proteomic analyses for each patient and control samples were performed in triplicates. Cell pellets were lysed using 8 M urea, 200 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) at pH 8.5 with protease inhibitors (one tablet per 10 mL of lysis buffer. Samples were further homogenized and DNA was sheared via sonication using a probe sonicator (20x 0.5 sec pulses; level 3). Total protein was determined using a BCA assay and proteins were stored at -80oC until future use. A total of 25 µg of protein was aliquoted for each condition and TMT channel for further downstream processing. Protein extracts were reduced using 5 mM tris-(2-carboxyethyl) phosphine (TCEP) for 15 min at room temperature. Next, samples were alkylated with 10 mM iodoacetamide for 30 min in the dark at room temperature. To facilitate the removal of incompatible reagents, proteins were precipitated using chloroform and methanol. Briefly, to 100 µL of each sample, 400 µL of methanol was added, followed by 100 µL of chloroform with thorough vortexing. Next, 300 µL of HPLC grade water was added and samples were vortexed thoroughly. Each sample was centrifuged at 14 000 x g for 5 min at room temperature. The upper aqueous layer was removed and the protein pellet was washed twice with methanol and centrifuged at 14 000 x g for 5 min at room temperature. Protein pellets were re-solubilized in 200 mM EPPS buffer and digested overnight with Lys-C (1:100, enzyme:protein ratio) at room temperature. The next day, trypsin (1:100 ratio) was added and incubated at 37 oC for an additional 6 hrs in a ThermoMixer set to 1000 RPM.
To each digested sample, 30% anhydrous acetonitrile was added and 25 µg of peptides were labeled using ~ 55 µg of TMTPro reagents (TMT1-TMT16) for 1 hour at room temperature with constant agitation. Following labeling, 5% hydroxylamine was added to quench excess TMT reagent. To equalize protein loading a ratio check was performed by pooling ~ 2 µg of each TMT-labeled sample. Samples were pooled and desalted using an in-house packed C18 Stage Tip and analyzed by liquid chromatography (LC) tandem mass spectrometry (MS/MS). Normalization factors derived from the ratio check were used to pool samples 1:1 across all TMT channels and the combined sample was desalted using a 100 mg Sep-Pak solid phase extraction cartridge. Eluted peptides were further fractionated using basic-pH reversed-phase (bRP) on an Agilent 300 extend C18 column and were collected into a 96 deep-well plate. Samples were consolidated into 24 fractions as previously described, and 12 nonadjacent fractions were desalted using Stage Tips prior to analyses using LC-MS/MS [16–18].
Mass spectrometry and data acquisition
All mass spectrometry data were acquired using an Orbitrap Fusion Lumos mass spectrometer in-line with a Proxeon nanoLC-1200 Ultra performance LC (UPLC) system. TMT labeled peptides were separated using an in-house packed 100 µm capillary column with 35 cm of Accucore 150 resin (2.6 µm, 150 Å) (ThermoFisher Scientific) using either a 120 min LC gradient from 4 to 24% acetonitrile in 0.125% formic acid per run. Eluted peptides were acquired using synchronous precursor selection (SPS-MS3) method for TMT quantification. Briefly, MS1 spectra were acquired at 120K resolving power with a maximum of 50 ms ion injection in the Orbitrap. MS2 spectra were acquired by selecting the top 10 most abundant features via collisional induced dissociation (CID) in the ion trap using an automatic gain control (AGC) of 15K, quadrupole isolation width of 0.5 m/z and a maximum ion time of 50 ms. These spectra were passed in real time to the external computer for database searching. Intelligent data acquisition (IDA) using real-time searching (RTS) was performed using Orbiter as previously described [19, 20]. Peptide spectral matches were analyzed using the Comet search algorithm designed for spectral acquisition speed [21, 22]. Real-time access to spectral data was enabled by the ThermoFisher Scientific Fusion API. Briefly, peptides were filtered using simple filters that included the following: not a match to a reversed sequence, maximum PPM error 50, minimum XCorr of 0.5, minimum deltaCorr of 0.10 and minimum peptide length of 7. If peptide spectra matched to above criteria, an SPS-MS3 scan was performed using up to 10 b- and y-type fragment ions as precursors with an AGC of 200K for a maximum of 200 ms with a normalized collision energy setting of 45.
Mass spectrometry data analysis
All acquired data were searched using the open-source Comet algorithm using a previously described informatics pipeline [23–25]. We acknowledge Dr. Steven Gygi for use of a custom CORE data analysis software as part of the pipeline. Briefly, peptide spectral libraries were first filtered to a peptide false discovery rate (FDR) of less than 1% using linear discriminant analysis employing a target-decoy strategy. Spectral searches were done using a custom fasta formatted database which included common contaminants, reversed sequences with the following parameters: 50 PPM precursor tolerance, fully tryptic peptides, fragment ion tolerance of 0.9 Da and a static modification by TMT (+ 304.2071 Da) on lysine and peptide N termini. Carbamidomethylation of cysteine residues (+ 57.021 Da) was set as a static modification while oxidation of methionine residues (+ 15.995 Da) was set as a variable modification. Resulting peptides were further filtered to obtain a 1% protein FDR and proteins were collapsed into groups. Reporter ion intensities were adjusted to correct for impurities during synthesis of different TMT reagents according to the manufacturers’ specifications. Lastly, protein quantitative values were column normalized so that the sum of the signal for all protein in each channel was equal to account for sample loading differences and a total sum signal-to-noise of all report ion ions of 100 was required for analysis.
RNA isolation and microarrays gene expression study – transcriptomics analysis
Dry pellets containing 1 million iPS cells each were suspended in 200 µl of RNA-Later Solution (Life Technologies, Carlsbad, CA, USA) for microarray expression analysis. Cells were stored at -80°C. Total RNA was extracted by using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. The contamination of DNA was removed by DNA digestion using RNase-Free DNase Set (Qiagen, Hilden, Germany). RNA concentration was determined by spectrophotometrical measurement using NanoDrop 8000 Spectrophotometer (Life Technologies, Carlsbad, CA, USA). The quality of extracted RNA samples was assessed with Agilent 2200 TapeStation Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) using RNA Screen Tape Kits or High Sensitivity RNA Screen Tape Kits (Agilent Technologies, Santa Clara, CA, USA). Samples were stored at − 80°C and selected for further analysis if they had a RNA Integrity Number (RIN) > 8.
Next, a next generation transcriptome-wide gene-level expression profiling was performed using Clariom™ S Assay (Applied Biosystems, Thermo Fisher Scientific, MA, US). In this study, the Affymetric GeneChip® System 3000Dx v.2 platform (Thermo Fisher Scientific, MA, US) consisting of GeneChip® Hybridization Oven, GeneChip® Fluidics Station 450Dx and GeneChip® Scanner 3000Dx with AutoLoader was used.
Assessment of mitochondrial metabolic activity
In order to validate the results obtained from the proteomic analysis an additional assessment of mitochondrial respiratory chain activity in NSC WFS cells and NSC control cells has been evaluated with the use of Resazurin, as described previously . An aqueous stock solution of Resazurin (Sigma R7017) with a final concentration of 0.5mM (500µM) was used. After the removal of the culture medium, the cells were rinsed twice with PBS containing Ca2+ and Mg2+ ions. Next, 0.5ml of PBS (Ca2+/Mg2+) containing 5mM glucose and 0.5mM Resazurin was added to each well of the 24 – well plate where the desired cells number was previously seeded. Fluorescence measurement was carried out using the Infinite M200Pro reader (Tecan Trading AG, Switzerland) with the Magellan operating software.
Quantification of protein level by sulforhodamine B (SRB) assay
The protein concentration in each well was determined after the measurements of mitochondrial metabolic activity to standardize this parameter to the protein level in each well of the multi well plate using an SRB assay . After the measurements of mitochondrial metabolic activity, cells were fixed in 1% acetic acid in ice-cold methanol for at least 24 h and the standard SRB procedure was followed. Colorimetric SRB measurement was performed using Infinite M200 plate reader (Tecan Trading AG, Switzerland) with the 595nm wavelength.
Evaluation of mitochondrial morphology - transmission electron microscopy (TEM)
In order to perform morphological analysis of mitochondria in NSC WFS cells and control cells, transmission electron microscopy (TEM) was used. Cells were fixed by 2.5% glutaraldehyde and 2% paraformaldehyde (Electron Microscopy Sciences, PA, USA) solution for 1 h in 4°C. After washing three times for 10 min in 0.1 M cacodylate buffer (BDH Chemicals, VWR, PA, USA). cells were postfixed in 2% osmium tetroxide (Agar Scientific, Sigma-Aldrich, Germany) for 1 h at room temperature and rinsed three times for 10 min in deionized water. Dehydration was performed by incubating the sample in increasing ethanol concentrations and next in pure propylene oxide. During dehydration cells were stained with 1% uranyl acetate (Serva, Heidelberg, Germany) in 70% ethanol. Finally, cells were embedded in the mixture of propylene oxide (Electron Microscopy Sciences, PA, USA) and Epon resin (Serva, Heidelberg, Germany) then in pure Epon resin. After polymerization in 60°C, seventy nanometer thick sections were cut using Ultramicrotome (Leica, Vienna, Austria) and collected on TEM copper grids (Ted Pella, CA, USA). Electron micrographs were obtained with Morada camera on a JEM 1400 transmission electron microscope at 80 kV (JEOL Co., Tokio, Japan) in the Laboratory of Electron Microscopy Core Facility, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
Validation in publicly available datasets
Dataset for the in silico hypothesis validation - RNA-sequencing data from hippocampi and hypothalami of Wfs1-deficient mice - was obtained from the publicly accessible database – Gene Expression Omnibus (GEO) – under accession number GSE102625. RNA-seq data analysis was performed using Galaxy platform (https://usegalaxy.org). Reads were preprocessed with Trim Galore! (Galaxy Version 0.6.3) on default settings and mapped to the mm10 genome by using TopHat (Galaxy Version 2.1.1). The read coverage was computed with featureCounts (Galaxy Version 1.6.4 + galaxy1). Finally, the expression values were normalized with TPM normalization method (Galaxy Version 0.4.0) for further analyses. Pathway enrichment analysis was performed with Gene Set Enrichment Analysis (GSEA 4.0.0) platform using REACTOME gene sets (REACTOME_MITOCHONDRIAL_PROTEIN_IMPORT and REACTOME_RESPIRATORY_ELECTRON_TRANSPORT).
Initially, wolframin expression was compared between WFS and healthy samples, both before and after administration of tunicamycin, using two-sided unpaired student’s t-test and Bonferroni’s correction for multiple hypothesis testing.
Global differences between study groups were investigated with Principal Component Analysis (PCA) and hierarchical clustering (HCL) performed with Multiple Experiment Viewer (MeV 4.8). In two-dimensional PCA plot, groups were manually clustered and annotated. Parameters of HCL included Euclidean distance as distance measure and average linkage algorithm. Based on these analyses, outlier samples were identified and excluded.
For quantitative proteomics data analysis, fold change for each protein in every comparison was calculated and statistical significance was obtained using two-sided unpaired student’s t-test, followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR). Results were visualised in using the volcano plots.
Gene set enrichment analysis (GSEA) was performed for each comparison of investigated groups with Broad GSEA software (4.0.3) using Reactome 7.0 gene sets collection. Analyses were performed with 1000 permutations (gene set) and default ranking parameters.
GSEA of WFS vs Healthy comparison was visualised as enrichment map in Cytoscape 3.8.0 using the EnrichmentMap 3.3.0 plugin, with overlap coefficient 0.1 and FDR 0.05 as parameters. Network was clustered with AutoAnnotate 1.3.3, manually trimmed and annotated.
Graphical summary of pathways significantly expressed in WFS was created with BioRender.
The ANOVA Kruskal-Wallis test was used for analysis of mitochondrial morphology.