Ethical compliance statement
The Ethics Committee of the Capital Region of Denmark approved the study (H-4-2011-157), and written informed consent was obtained from each participant before enrolment.
Human iPS cell culture and differentiation into Neurons & Astrocytes
Human dermal fibroblasts cultures were obtained from skin biopsies from two symptomatic (H150, H151) and one pre-symptomatic (H242) FTD3 individuals (all from the same family). Those were expanded as primary fibroblasts and reprogrammed using non-integrative episomal plasmids . All three participants carry a heterozygous mutation with G-to-C transition in the 5′acceptor splice site of exon 6 in CHMP2B on chromosome 312. Basic characterization and generation of gene-corrected isogenic controls was achieved via the CRISPR-Cas9 system described in our previous study (H150 ISO, H151 ISO and H242 ISO) .
hiPSCs were cultured with ESC medium consisting of Essential 8™ basal medium with supplement (Thermo Fisher Scientific, A1517001) and passaged 1:3–6 with 0.5 mM EDTA on to Vitronectin™-coated (Thermo Fisher Scientific, A14700) dishes.
Neural differentiation of hiPSCs was achieved via a modified dual SMAD protocol and neuronal characterization was performed as described previously . Briefly, iPSC lines were differentiated toward neural-specific progenies employing a modified dual SMAD protocol  . Neural induction was initiated by changing the medium to neural basic medium consisted of 50% DMEM/F-12 medium, 50% Neural basal medium (Thermo Fisher Scientific, 10,888–022), N2 (Thermo Fisher Scientific, 17,502–048), B27 without Vitamin A (Thermo Fisher Scientific, 12,587–010), supplemented with dual SMAD inhibitors SB431542 (Selleck, S1067) and LDN193189 (Selleck, S2618). Subsequently, cells were split onto poly-O-Lysine/laminin (Sigma, L2020) for terminal differentiation applying the following media: Neural basic medium supplemented with 20 ng/ml BDNF (Cell Guidance Systems, GFH1–10), 10 ng/ml GDNF (Cell Guidance Systems, GFH2–10), 50 μM db-cAMP (Sigma, D0627) and 200 μM L-Ascorbic acid 2-phosphate (Sigma, A8960). The whole protocol from hiPSC to matured neurons takes 7 weeks.
The initial steps of astrocyte differentiation followed a previously published protocol  . Astrocyte progenitors were plated at a seeding density of 50.000 cells/cm2 in AMM medium supplemented with 10 ng/ml of Activin A (Thermo Fisher Scientific, PHG9014), 10 ng/ml HRGβ1 (Peprotech, 100–03), 200 ng/ml of IGF1 (Peprotech, 100–11), 1% sodium pyruvate (Thermo Fisher Scientific, 1,136,070), 10% heat-inactivated and toxin-free FBS (Invitrogen) and 200 μM L-Ascorbic acid 2-phosphate (Sigma, A8960). Medium was changed every other day and cells were differentiated for 35+ days. The efficiency of terminal astrocyte differentiation was monitored by assaying for expression of S100β, AQP4 and SOX9 by immunocytochemical staining and RNA seq. In the current study, astrocyte progenitor cells were differentiated for 10 weeks for immunocytochemical analyses and metabolic assays.
Sample collection and protein isolation
Neurons were collected on ice in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 7.3 mM Na2HPO4, 0.9 mM CaCl2 and 0.5 mM MgCl2, pH 7.4) with protease inhibitor (Complete tablets, Roche). Cell pellets were dissolved in lysis buffer consisting of 6 M urea (Sigma), 2 M thiourea (Sigma), 20 mg/ml sodium dodecyl sulfate (SDS, GE Healthcare), 40 mM N-ethylmaleimide (NEM, Sigma) and protease inhibitor followed by sonication on ice and incubation for 30 min at room temperature (RT).
Following methanol-chloroform precipitation, proteins were denatured and reduced in 6 M urea, 2 M thiourea, and 10 mM tris(2-carboxyethyl) phosphine (TCEP, ThermoFisher). After vortexing, the samples were incubated for 2 h with 1 μl endoproteinase Lys-C (Wako) at RT. The samples were diluted 10 times in 20 mM TCEP in 20 mM triethylammonium bicarbonate (TEAB, Sigma) buffer, pH 7.5 and sonicated on ice, followed by digestion with 1 μg trypsin (Sigma) per 50 μg peptide overnight (O/N) at RT. Protein concentration was measured by Qubit® (Thermo Scientific) according to the manufacturer’s instructions. Seventy microgram of each sample was labeled with Tandem Mass Tag (TMT) 10plex Isobaric label Reagents (Thermo Scientific) according to the manufacturer’s instructions. The labeled peptides were mixed 1:1:1:1:1:1 and dried.
Hydrophobic interaction liquid chromatography (HILIC) and high pH fractionation
To reduce sample complexity, peptides were fractionated using HILIC as previously described . Samples were diluted in 0.1% TFA and approximately 50 μg peptide was fractioned. Samples were then dissolved in 90% ACN, 0.1% TFA (solvent B) and loaded onto a packed TSKgel Amide-80 (Tosoh Bioscience) micro-capillary column (450 m OD × 320 m ID × 17 cm) using an Agilent 1200 Series HPLC (Agilent). Peptides were separated using a gradient from 100 to 60% solvent B (A = 0.1% TFA) running for 30 min at a flow-rate of 6 μl/min. Fractions were collected every 1 min and combined into 12–15 final fractions based on the UV chromatogram, and subsequently dried by vacuum centrifugation.
To increase the coverage of the peptides, high pH fractionation was also performed using approximately 50 μg peptide. Briefly, the sample was dissolved in 1% ammonium hydroxide (NH3, Sigma), pH 11, and loaded on a R2/R3 column equilibrated with 0.1% NH3. The peptides were eluted in a stepwise fashion using a gradient of 5–60% ACN/0.1% NH3. All fractions were dried by vacuum centrifugation and stored at − 20 °C.
Samples were resuspended in 0.1% formic acid (FA) and loaded onto a two-column EASY-nLC system (Thermo Scientific). The pre-column was a 3 cm-long fused silica capillary (100 μM inner diameter) with a fitted end and packed with ReproSil - Pur C18 AQ 5 μm whereas the analytical column was a 17 cm-long fused silica capillary (75 μm inner diameter) and packed with ReproSil-Pur C18 AQ 3 μm reversed-phase material (both resins from Dr. Maisch Ammerbuch-Entringen).
Peptides were eluted with an organic solvent gradient from 100% phase A (0.1% FA) to 34% phase B (95% ACN, 0.1% FA) at a constant flow-rate of 250 nL/min. Depending on the samples based on the HILIC, the gradient was from 1 to 30% solvent B in 60 min or 90 min, 30 to 50% solvent B in 10 min, 50–100% solvent B in 5 min and 8 min at 100% solvent B.
The nLC was connected online to a QExactive HF Mass Spectrometer (Thermo Scientific) operated at positive ion mode with data-dependent acquisition. The Orbitrap acquired the full MS scan with an automatic gain control (AGC) target value of 3 × 106 ions and a maximum fill time of 100 ms. Each MS scan was acquired at high-resolution (120,000 full-width half-maximum (FWHM)) at m/z 200 in the Orbitrap with a mass range of 400–1400 Da. The 12 most abundant peptide ions were selected from the MS for higher energy collision-induced dissociation (HCD) fragmentation (collision energy: 34 V). Fragmentation was performed at high resolution (60,000 FWHM) for a target of 1 × 105 and a maximum injection time of 60 ms using an isolation window of 1.2 m/z and a dynamic exclusion. All raw data were viewed in Thermo Xcalibur v3.0.
Proteomic data analysis
Raw data were processed using Proteome Discoverer (v2.1, ThermoFisher) and searched against the Swissprot human database using an in-house Mascot server (v2.3, Matrix Science Ltd.) and the Sequest HT search engine. Database searches were performed with the following parameters: precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.03 Da (HCD fragmentation) and a maximum of 2 missed cleavages for trypsin. All identified peptides were filtered against a Decoy database using Percolator resulting in a false discovery rate (FDR) of 0.01 (FDR < 0.01). Only peptides with Mascot rank 1 and cut-off value of Mascot score > 15 and a SEQUEST HT ∆Cn of 0.1 were considered for further analysis.
Quantitation of identified peptides was performed on Log2-values of measured signal to noise (S/N) values and the data were normalized based on the median. Quantification of proteins was obtained by merging unique non-modified peptides using the R Rollup function (using the DanteR package) including at least two unique peptides per protein and using the mean of the S/N. The intensity factors for the experimental conditions for each peptide or protein were subtracted.
Significantly-regulated proteins were defined via a combination of limma testing and rank products  using the crosstalk.bmb.sdu.dk publicly-accessible app, with a q-value of 0.05 for significantly-regulated proteins.
Fluorescent immunocytochemistry and confocal microscopy
For immunofluorescence staining, cells were fixed with 4% formaldehyde in PBS solution for 15 min, permeabilized with 1% Triton X-100 in PBS solution for 15 min and blocked with 2% bovine serum albumin in PBS solution for 1 h. Thereafter, cells were incubated with the following primary antibodies overnight at 4 °C: anti-MAP 2 (1:500; Sigma, M1406), anti-TUJ1 (1:500; Covance, MRB-435P), anti-TAU (1:200; Dako, A0024) and anti-CTIP2 (1:200; Abcam, ab28448). Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1:500; Molecular Probes) and Alexa Fluor 594 (1:500; Molecular Probes). Cells were washed in PBS and nuclei counterstained with DAPI (Sigma-Aldrich, D9542). Finally, coverslips of growing cells were transferred onto glass slides with mounting medium (Dako, S3023) and imaged immediately using sequential line scanning with a Leica TCS SP5 II inverted confocal microscope.
Incubation experiments using 13C-labeled substrates
Stable isotope 13C and gas chromatography coupled to mass spectrometry can be used to obtain information regarding metabolic processes as the natural abundance of 13C is scarce and, accordingly, the sensitivity for detection of endogenous metabolites is limited. This low natural abundance allows for exogenous employment of 13C-labeled substrates to study the significance of diverse metabolic pathways. Therefore, in order to investigate metabolism in cultured neurons and astrocytes, these cells can be incubated in the presence of 13C-labelled substrates and the percent 13C enrichment determined in various metabolites, thereby mapping the pathways. To achieve this, culture medium was removed and cells were washed twice with 1 ml of PBS. Cell cultures were subsequently incubated for 90 min at 37 °C in 2 ml serum-free culture medium containing 2.5 mM [U-13C] glucose (neurons) or in the presence of 250 μM [U-13C] glutamate plus 2.5 mM unlabeled glucose (neurons and astrocytes) or 0.5 mM [U-13C] glutamine (neurons) plus unlabeled glucose. In order to avoid excitotoxic effects of glutamate in neurons during the incubation period, two glutamate receptor antagonists CNQX (25 μM), a selective antagonist of the α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) and kainate receptor subtypes, and D-AP5 (100 μM), an N-methyl-D-aspartate (NMDA) antagonist, were also present in the medium . Following incubation, medium was collected and cells were washed with PBS, lysed and extracted with 70% ethanol. Cell extracts were centrifuged at 20,000 g for 20 min (4 °C) to separate the soluble extract (supernatant) from the insoluble components (pellet). Supernatants were lyophilized and reconstituted in water for subsequent biochemical analyses.
Metabolic mapping using gas chromatography coupled to mass spectrometry (GC-MS)
Extract and media samples were adjusted to pH 1–2 with HCl and evaporated to dryness under nitrogen flow. Analytes were extracted into an organic phase (96% ethanol/benzene) followed by derivatization with 14% DMF/86% MTBSTFA with a modified procedure . Standards containing unlabeled metabolites of interest and cell extracts were separated and analysed in a gas chromatograph (Agilent Technologies 7820A chromatograph, J&W GC column HP-5MS, parts no. 19091S-433) coupled to a mass spectrometer (Agilent Technologies, 5977E). Isotopic enrichment of the metabolites of interest was corrected for natural abundance of 13C using the unlabeled standards and calculated according to . Data are presented as labelling (%) of M + X, where M is the mass of the unlabelled molecule and X is the number of labelled C-atoms in a given metabolite.
Seahorse XFe96 glycolysis assay
Both principal cellular bioenergetic processes - glycolysis and mitochondrial respiration - can be assessed simultaneously with the Seahorse platform (Seahorse Biosciences-Agilent Technologies, USA) in different brain-derived preparations. We have previously demonstrated alterations in mitochondrial function in FTD3 neurons using this approach . Here, glycolytic activity was evaluated by measuring the extracellular acidification rate (ECAR) using a Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Biosciences-Agilent Technologies, USA). Human neuronal progenitor cells cultured with neuronal maturation factors for 1 week were seeded in a Seahorse 96-well cell culture microplate at a density of 6.5 × 103 cells/well. The cells were then allowed to adhere and further mature for 3 to 4 weeks. On the day of the assay, the culture medium was changed for unbuffered DMEM (pH 7.4) supplemented with 2.5 mM glucose and the cells were equilibrated for 10 min at 37 °C in a CO2-free incubator. The pH of the reagents used to test mitochondrial function was adjusted to 7.4. The ECAR measurement cycle consisted of 3 min mix and 3 min measurement of the oxygen level. Testing of glycolytic function was evaluated by the sequential injection of the ATP synthesis inhibitor, oligomycin (2 μM final concentration) and a mixture of rotenone (0.5 μM) and antimycin A (0.5 μM) with one ECAR measurement after the first injection and two final measurement cycles. ECAR was simultaneously recorded and calculated by the Seahorse XFe96 software, Wave. Subsequent to the Seahorse analysis, protein content was measured for each well using the Pierce BCA assay with BSA as standard. Based on the ECAR measurements, mitochondrial respiration was calculated by subtracting non-mitochondrial respiration (minimum measurement after rotenone/antimycin injection) from the last measurement obtained before oligomycin injection. Furthermore, we recorded the oxygen consumption rate (OCR) . With these data we were able to calculate the coupling efficiency (in %) as the mitochondrial ATP production rate/ the basal respiration rate × 100 (Seahorse bioscience, Agilent Technologies) . Results are expressed as mean values ± standard error of the mean (S.E.M.), (n = 3). The mean values are based on 12 replicates from the three batches of neurons from the three different FTD patient cell lines (H150, H151 and H242).
Quantitative determination of intracellular amino acids
Amino acids in cell extracts were separated and quantified by reverse-phase high performance liquid chromatography (HPLC) using an Agilent ZORBAX Eclipse plus C18 column (4.6 × 150 mm, particle size 3.5 μm; 959,963–902, Agilent Technologies, Santa Clara, CA, USA), pre-column online o-phthaldialdehyde derivatization and fluorescence detection (338 nm, 10 nm bandwidth, and reference wavelength 390 nm, 20 nm bandwidth). An Agilent 1260 Infinity system coupled to a 1260 Infinity fluorescence detector (Agilent Technologies) was employed. To elute the amino acids, a mobile phase gradient consisting of a mixture of mobile phase A (10 mM Na2HPO4: 10 mM Na2B4O7, pH 8.2; 5 mM NaN3) and mobile phase B (acetonitrile 45%: methanol 45%: water 10%, v:v:v) was used at a flow of 1.5 ml/min with a total run time of 35 min. The amount of amino acids in the samples was quantified based on standards containing a mixture of the amino acids of interest at increasing known concentrations and injected in parallel with the samples .
Western blot analyses
Cell pellets were lysed in lysate buffer (mM: 150 NaCl, 10 Tris base, 1 EDTA and 1% Triton, pH 8) supplied with cOmplete Protease Inhibitor (Roche, Germany) for 1 h on ice. Proteins were denatured by boiling the sample (5 min) in NuPAGE® LDS Sample Buffer (ThermoFisher Scientific, Carlsbad, CA, USA) with 50 mM DTT. Equal amounts of protein per lane were resolved on a 4–12% Bis–Tris pre-cast gel using the XCell SureLockTM mini-electrophoresis system (ThermoFisher). Proteins were transferred onto a PVDF membrane with constant 30 V for 2 h in NuPAGE® Transfer Buffer (ThermoFisher) with 20% methanol. The membranes were blocked for 1.5 h at room temperature (RT) in 2% (w/v) non-fat skim milk protein and 0.1% Tween-20 in wash buffer (mM: 50 Tris base, 100 NaCl, pH 7.2) and were incubated overnight at 4 °C with antibodies against PAG (1:1000; Abcam, ab93434), GDH2 (1:1000; made and kindly gifted by Ioannis Zaganas ,, GS (1:1000, Abcam, ab73593), GAPDH (1:5000, Santa Cruz biotech, sc-25,778) or β-actin (1:2000; Abcam, ab8227). Following three 5-min washes, the membranes were incubated (1.5 h, RT) with appropriate HRP-conjugated secondary antibodies (Dako, Glostrup, Denmark). All antibodies were diluted in washing buffer with 0.2% (w/v) non-fat skim milk and 0.05% Tween-20. The bands were visualized using ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).
Data and statistical analysis
Data are presented as mean ± S.E.M. of hiPSC-derived neurons or astrocytes from three different FTD3 patient cell lines (H150, H151 and H242) and their respective isogenic controls. In mass spectrometry and western blot analyses, three to four technical replicates per cell line were used for each condition tested. For the 13C-labeling studies, data are depicted as labeling (%) of M + X, where M is the mass of the unlabeled metabolite and X is the number of 13C-labeled carbon atoms. Statistical evaluations comprised unpaired multiple t test followed by Bonferroni-Dunn post hoc test to correct for multiple comparisons or two-way ANOVA correcting for multiple comparisons (Sidak post hoc test) as indicated in figure legends or throughout the text. Data are assumed to be sampled from populations with the same scatter. Analyses were performed using GraphPad Prism 7.0. P values below 0.05 were considered statistically significant.