All experiments were carried out under the Dutch and European law with approval of the local Institutional Animal Care and Use Committee (IACUC) of the VU University (Amsterdam, Netherlands). Wt and 2b5^ho animals were used. All animals were weaned at P21 and had ad libitum access to food and water. The mice were housed with a 12 h light and dark cycle.

Four-month-old mice were sacrificed by cervical dislocation. Brains were taken out and the olfactory bulbs, cerebella and forebrains were removed. Astrocytes from the remaining structures (gray and white matter structures, including striatum, hippocampi and basal nuclei) were isolated. Brain tissue was minced with a scalpel in Hank’s balanced salt solution (HBSS) without magnesium and calcium (Gibco) at 4°C. The tissue was dissociated with a papain solution containing 20 mM PIPES (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.1 mM EDTA, 5.5 mM L-cysteine-HCl, 40 U/ml DNase and 20 U/ml papain for 30 min at 37°C. Cells were plated in poly-L-ornithine (PLO)-coated flasks and cultured in DMEM/F12 with 15% fetal bovine serum (FBS) (Hyclone), 1% sodium pyruvate (Gibco), 100U penicillin, 100 μg/ml streptomycin (Gibco) and 10 μg/ml gentamicin (Gibco). The cells were passaged twice before they were used for experiments. All chemicals were purchased from Sigma–Aldrich unless otherwise stated. Every experiment was replicated in independent cultures derived from different mice (number of experiments is indicated in figure legends as, e.g., n = 3).

Astrocytes were plated in 6 cm dishes (∼250,000 cells/dish) and cultured until 80–90% confluent. Cells were starved for methionine for 15 min in AHA medium [DMEM/F12 without methionine, lysine, arginine and phenol red (Thermo Fisher Scientific)] supplemented with 91.3 mg/L L-lysine, 147.5 mg/L L-arginine, dialysed FBS and phenol red, after which AHA (Bachem) was added to AHA medium for the indicated time in a final concentration of 2 mM. Cells were harvested in lysis buffer composed of 50 mM Tris pH 7.5, 100 mM NaCl, 0.5 mM EDTA and 0.5% sodium dodecyl sulfate (SDS).

The newly synthesized proteins were labeled using the Click-iT^® Protein buffer kit according to the manufacturer’s protocol (Invitrogen). In short, the AHA molecules in the cell lysate were coupled to biotin. Proteins were precipitated by subsequent addition of six volumes of methanol, 1.5 volume of chloroform and 4 volumes of water. The protein pellet was dissolved in 50 mM Tris-HCl pH 7.5 with 1% SDS before subjection to Western blot analysis. Samples were loaded on 12% Criterion^TM TGX Stain-Free^TM Protein Gels (Bio-Rad), allowing detection of total protein load. Biotinylated AHA-labeled proteins were stained with 400 ng/ml streptavidin (680 nm) and visualized at 700 nm in an Odyssey system (Odyssey^® Fc, LI-COR). The amount of AHA-staining was corrected for the total protein load determined by Gel Doc^TM EZ System (Bio-Rad).

Astrocytes were plated in ½ area 96 well plates (∼5000 cells/well) and cultured for 2 days in AHA-SILAC medium (Thermo Fisher Scientific, custom-made DMEM/F12 without methionine, arginine, lysine and phenol red), with addition of 15% dialyzed serum, 0.005% phenol red, SILAC amino acids (94.2 mg/L [¹³C₆] L-lysine 152 mg/L [¹³C₆] L-arginine or 96 mg/L [¹³C₆, ¹⁵N₄] L-lysine and 154.3 mg/L [¹³C_6, ¹⁵N₄] L-arginine) and 17.2 mg/L L-methionine. The cells were starved for methionine from 15 min and cultured with 2 mM AHA (Bachem) or methionine overnight in half well area plates. Cell viability (ATP levels, CellTiter-Glo) was measured according to manufacturer’s instructions (Promega). In short, cells were kept at room temperature for 30 min. CellTiter-Glo was added in the same volume as the culture medium. The plate was shaken for 2 min and left standing for 10 min before measuring luminescence with a Victor2 plate reader (PerkinElmer Life Sciences).

Astrocytes were plated in 10 cm dishes (∼750,000 cells/dish) and cultured until 80% confluent. The culture medium was replaced with AHA-SILAC medium. Cells were grown for 4 days. SILAC labels were reversed in biological duplicates. Cells were subsequently starved for methionine for 15 min and AHA was added as described in the previous section. Astrocytes were further cultured for 2 h or otherwise indicated. The cells were subsequently washed with cold PBS and lysed in urea lysis buffer (supplied with the Click-IT Protein enrichment Kit, Invitrogen).

For the protein analysis of secreted proteins conditioned medium samples from wt and 2b5^ho astrocytes (2 h) were concentrated using Amicon^® Ultra-15 centrifuge filter tubes (3 kDa, Merck). Samples were diluted with urea lysis buffer to a volume of 400 μl per sample.

The AHA-labeled proteins from cell lysates as well as from the conditioned medium were enriched using the Click-iT^® Protein Enrichment Kit according to the manufacturer’s protocol (Invitrogen) with some minor modifications. In short, the AHA-labeled proteins were bound to the resin (16 h), following an iodoacetamide treatment and several washing steps. The AHA-labeled proteins bound to the resin were dissolved in 50 mM ammonium bicarbonate with 3 M urea. Digestion was performed at 37°C by adding 0.1 μg Lys-C for 4 h followed by addition of 1 μg trypsin overnight. The peptides were separated from the resin by briefly centrifuging them through a 0.8 ml spin columns (Thermo Fisher Scientific). The flow through contains the peptides. Peptide samples were stored at -80°C until further use. See also Figure 1 for an overview of the enrichment procedure.

Peptides were analyzed using a Q Exactive^TM Hybrid Quadrupole-Orbitrap^TM Mass Spectrometer (Thermo Fisher Scientific), which was connected to a 1290 Infinity II LC System (Agilent). The trap column was made of C18 (Dr. Maisch Reprosil) material and the analytical column was a 50 cm, 50 μm inner diameter Poroshell C18 (Agilent) column. Both the trap and analytical columns were packed in-house. Solvent A consisted of 0.1% formic acid (Merck) in deionized water (Merck) and Solvent B of 0.1% formic acid in 80% acetonitrile (Biosolve). Peptides were first trapped at 50 μl/min with solvent A and then eluted with solvent B in a 120 min gradient at 100 nl/min: 0–10 min, 100% solvent A; 10.1–105 min, 13–40% solvent B; 105–108 min, 40–100% solvent B; 108–109 min, 100% solvent B; 109–110 min, 0–100% solvent A; 110–120 min, 100% solvent A. The Orbitrap was operated in a data-dependent manner, with the following settings: ESI voltage, 1700 V; inlet capillary temperature 320°C; full-scan automatic gain control (AGC) target, 3 × 10⁶ ions at 35000 resolution; scan range, 350–1500 m/z; Orbitrap full-scan maximum injection time, 250 ms; MS2 scan AGC target, 5 × 10⁴ ions at 17500 resolution; maximum injection, 120 ms; normalized collision energy, 25; dynamic exclusion time, 30; isolation window 1.5 m/z; 10 MS2 scans per full scan.

The LC-MS/MS data were processed with MaxQuant (v1.5.2.8) and MS2 spectra were searched with the Andromeda search engine against the mouse proteome in UniProt (17155 entries, downloaded on 2015-10-27). Enzyme specificity was set to Trypsin/P and two missed cleavages were allowed. SILAC labels (K6/8, R6/10), methionine for AHA substitution and methionine oxidation were set as variable modifications and cysteine carbamidomethylation was set as fixed modification. Minimum peptide length was 7 amino acids. Mass tolerance was set to 20 ppm for peptide masses and 0.6 Da for fragmentation masses. The false discovery rate (FDR) threshold was set to 1% for identifications. Minimal ratio count was set to 2 for protein quantification and the functions “match between runs” and “requantify” were enabled. Data were further analyzed in Perseus (v1.5.0.0). Protein groups were kept for further analysis if they were detected in at least three of the biological replicates. A t-test was performed and protein groups were considered significant if the p-value was <0.05.

To assess the presence of an N-terminal signal peptide in the proteins, we subjected the proteins from the proteomic screen to the SignalP 4.1 server (Bendtsen et al., 2004; Petersen et al., 2011). For all UniProt IDs the FASTA files were downloaded from the UniProt website and all Fasta files are automatically subjected to the SignalP 4.1 server. The presence or absence of a signal peptide for each UniProt ID was predicted with the SignalP 4.1 algorithms and the results were saved to Excel. The UniProt IDs linked back to the list of all identified protein names. Biomart was used to identify proteins containing a transmembrane domain. The number of proteins with a signal peptide was calculated for the proteins, the synthesis of which was affected by eIF2B𝜀^Arg191His as well as for the other proteins that are not regulated by the same mutation. Next the number of regulated proteins with a signal peptide were subdivided in ‘upregulated’ and ‘downregulated’.

The sequences of the mRNA variants encoding the proteins identified in the AHA-SILAC proteomes were downloaded from the NCBI database including the start and stop codon location. The 5′-UTR features “uORFs, %GC and thermodynamic stability (ΔG in kcal/mol)” were determined for all mRNAs (Babendure et al., 2006). We compared each mRNA characteristic for all proteins found in the AHA-SILAC proteomic screen and compared the characteristics for the proteins regulated by eIF2B𝜀^Arg191His and non-regulated proteins. The 5′-UTR length, %GC and ΔG from mRNAs encoding the proteins found in the AHA-SILAC proteomes were analyzed per protein. When a protein is potentially translated from more than 1 mRNA variant (taken from the NCBI database), we counted and analyzed all features of all mRNA variants per protein and plotted all as a proportional representation per protein so that the analysis was not skewed by proteins with multiple variants. 5′-UTR lengths of less than 13 nucleotides were omitted from analyses as AUG codons less than 13 nt from the cap are inefficient to initiate translation (Hinnebusch, 2011). The % of GC in the 5′-UTRs was calculated. To calculate the thermodynamic stability, the free energy (ΔG) of 5′-UTRs was determined using UNAFold¹, which predicts thermodynamic stability. The uORF was defined as upstream AUG/CUG/GUG/UUG/ACG with a purine (A/G) at the -3 position and a guanine at the +4 position (Ingolia et al., 2011; Ferreira et al., 2013; Noderer et al., 2014). Scripts used for the analyses are found at: https://github.com/LisanneWisse/UORF or https://github.com/LisanneWisse/RefSeq.

Overrepresentation of specific pathways was analyzed with DAVID [based on gene ontology (GO)-terms] between the proteins that are affected by the eIF2B𝜀^Arg191His mutation and all the proteins found in the AHA-SILAC proteome (Huang et al., 2009a,b).

Astrocytes were plated in 10 cm dishes (∼750,000 cells/dish) and cultured until 80% confluent. Cells were washed with cold PBS, collected by scraping in PBS and pelleted by centrifugation (5 min, 1000 × g, 4°C). The method is based on the protocol described². Cells were lysed in harvesting buffer containing 10 mM HEPES pH 7.9, 50 mM NaCl, 0.5 M sucrose, 0.1 mM EDTA, 0.5% Triton, 1 mM DTT, phosphatase inhibitors (0.5 mM activated NaVO₃, 25 mM β-glycerophosphate, 50 mM NaF) and protease inhibitors (Roche). Lysates were centrifuged (10 min 500 × g, 4°C) and the supernatant was used for Western blot analyses.

Mice were sacrificed by cervical dislocation at 4 months of age. Brains were removed, snap-frozen in liquid nitrogen and stored at -80°C until further use. Lysates were prepared by grinding the brain samples with a pestle and mortar under liquid nitrogen. The powder was lysed in cytoplasmic lysis buffer (20 mM Tris pH 7.4, 100 mM KAc, 3 mM MgAc₂, 2 mM DTT, 1.5% IGEPAL, 1.5% sodium deoxycholate (SODC), 1x HALT (protease and phosphatase inhibitory cocktail, Thermo Fisher Scientific]. The samples were homogenized with a pestle followed by trituration through a 23G needle. The samples were centrifuged (10 min, 10.000 × g, 4°C) and the supernatant was aliquoted into pre-chilled microfuge tubes. Protein concentrations were determined using a Quick Start^TM Bradford Protein Assay (Bio-Rad). For RNA isolation, TRIzol^TM Reagent (Invitrogen) was added to 50 μl of the supernatant and total RNA was isolated as described under RNA isolation and cDNA synthesis.

For cell lysates approximately 10 μg and for brain lysates 50–60 μg of protein was loaded on a 12% SDS-polyacrylamide gel with 2,2,2-trichloroethanol (TCE) which allow detection of total protein load (Ladner et al., 2004). Proteins were transferred onto a PVDF membrane (Bio-Rad). Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C. Membranes were washed with TBS-Tween20 (0.1%) and incubated with an HRP-conjugated secondary goat anti-rabbit IgG antibody (Dako, P0448) for 2 h at room temperature. The membranes were washed three times with TBS-Tween (0.1%) and once with TBS, incubated with SuperSignal^TM West Femto (Thermo Fisher Scientific) and imaged (Odyssey^® Fc, LI-COR). Protein expression was corrected for total amount of protein determined by Gel Doc^TM EZ System (Bio-Rad).

Astrocytes were washed twice with PBS and collected in TRIzol^TM Reagent (Invitrogen). RNA was isolated according to the manufacturer’s protocol. In short, 1/5 volume chloroform was added to the TRIzol^TM Reagent. The samples were centrifuged (10 min, 12000 × g, 4°C) and the water layer was transferred to a fresh tube. Half a volume of isopropanol and 1/250 volume of linear acrylamide (Ambion) was added and the samples were centrifuged for 20 min at 4°C and 12000 × g. The pellet was washed twice with 70% ethanol and resuspended in non-DEPC treated water (Ambion). RNA was precipitated with 175 mM sodium acetate (pH 5.2) and 70% ethanol and incubated for 30 min at -20°C. The samples were centrifuged (30 min, 4°C, 12000 × g) and the pellets were washed twice with 70% ethanol and resuspended in non-DEPC-treated water.

RNA quality and quantity were determined by measuring the A260 and A280 (NanoDrop 2000, Thermo Fisher Scientific). cDNA was synthesized in a 20 μl reverse transcription reaction: 1x first strand buffer (Invitrogen), random hexamers (0.02 μg/μl; Qiagen), oligoDT (0.02 μg/μl; Qiagen), dNTPs (1 mM each; Roche), DTT (1 mM; Invitrogen), RNaseOUT (0.25 U/μl; Invitrogen), Superscript III (5 U/μl; Invitrogen) and 1.5 μg total RNA were incubated for 2 h at 50°C. RNAseH (62.5 U/μl; Invitrogen) was added and incubated for 30 min at 37°C followed by 15 min at 70°C.

mRNA levels were determined with qPCR using a LightCycler^® 480 II Instrument (Roche). For each 10 μl sample a mixture of LightCycler^® 480 SYBR Green I Master (Roche), primers (1 pmol/μl) and cDNA (0.1 μl) was used. Used primers are listed in Table 1. Gapdh mRNA was used as reference.

Astrocytes were plated in half-area 96 well plates (∼3000 cells/dish, CELLSTRAR) to 80% confluency. pNL1.3-Gapdh (80 ng) was transfected into the cells using 0.24 μl FuGENE^® 6 according to the manufacturer’s protocol (Promega). Luciferase activity in cells and culture medium was measured using the protocol of Promega and a Wallac 1420 Victor2 Microplate Reader (Perkin Elmer). The ratio of extracellular/intracellular luciferase activity determines the relative secretion.

Astrocytes were plated in half-area 96 well plates (∼3000 cells/dish, CELLSTAR). Each pNL1.1 promoter/5′-UTR construct (40 ng) was co-transfected with internal control pGL3-Gapdh (20 ng) using FuGENE^® 6 Transfection Reagent (0.24 μl) according to the manufacturer’s protocol. Luciferase activity was measured using the protocol of Promega and a GloMax^® Discover System (Promega). The ratio Nluc:Fluc determines the expression of Nluc corrected for well-by-well differences.

Astrocytes were cultured in 6 cm dishes (∼250,000 cells/dish) until 70% confluent. Medium was replaced 72 and 24 h before harvesting. At the time of harvesting culture medium was removed from the cells and stored at -80°C until analysis. Cells were washed with ice-cold PBS and metabolites were extracted from cells in 0.5 ml lysis buffer containing methanol/acetonitrile/dH₂O (2:2:1). Samples were spun at 16.000 × g for 15 min at 4°C. Supernatants were collected for LC-MS analysis. 10 μl of conditioned medium was added to 1 mL of lysis buffer containing methanol/acetonitrile/H₂O (2:2:1) and prepared as above.

LC-MS analysis was performed on an Exactive mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex Ultimate 3000 autosampler and pump (Thermo Fisher Scientific). The MS operated in polarity-switching mode with spray voltages of 4.5 and -3.5 kV. Metabolites were separated using a SeQuant^® ZIC^®-pHILIC HPLC Columns (2.1 mm × 150 mm, 5 μm, guard column 2.1 mm × 20 mm, 5 μm; Merck) using a linear gradient of acetonitrile and eluent A [20 mM (NH₄)₂CO₃, 0.1% NH₄OH in ULC/MS grade water (Biosolve)]. Flow rate was set at 150 μl/min. Metabolites were identified and quantified using LCQUAN^TM Quantitative Software (Thermo Fisher Scientific) on the basis of exact mass within 5 ppm and further validated by concordance with retention times of standards. Metabolites were quantified using LCQUAN^TM Quantitative Software (Thermo Fisher Scientific). Peak intensities were normalized based on median peak intensity.

The program Factor was used to correct for differences between experiments (qPCR, Western blot, cell viability) but not between other conditions (genotype, treatments) (Ruijter et al., 2006). Statistical analysis of qPCR, Western blot and cell viability experiments were performed with a two way ANOVA followed by a Sidaks multiple comparison test, using GraphPad Prism software. Statistical analysis of the transfection data was performed using a T-test per construct.

A Chi-square analysis was used to measure significant differences in the presence of signal peptides, transmembrane regions and SP-targeting proteins as well as for the analysis on the 5′-UTR features using GraphPad Prism software.

The secretion assay was statistically tested with a multilevel analysis in SPSS. Differences were significant when p < 0.05.

L-azidohomoalanine incorporation was measured in wt and 2b5^ho astrocytes to investigate if the general protein synthesis rate is affected by the homozygous Arg191His mutation in eIF2B𝜀 (Figure 1). Label incorporation increased linearly for a period of 4 h (Figure 1B). The incorporation occurred with similar kinetics in 2b5^ho and wt astrocytes (Figure 1B), indicating that the eIF2B𝜀 Arg191His mutation did not significantly affect protein synthesis rate. The viability of both wt and 2b5^ho astrocytes was not influenced by AHA or SILAC treatment (Figure 1C). Expression of stress-related mRNAs was unaffected by the AHA-labeling protocol (Supplementary Figure 1). These results allow further proteomic analyses with an AHA incorporation pulse of 2 h.

The AHA proteomic labeling approach was performed to identify and quantify proteins that are differentially translated in 2b5^ho astrocytes. SILAC-labeled wt and 2b5^ho astrocytes were subjected to AHA labeling for 2 h. After AHA labeling cells were harvested and subjected to bead-based enrichment using a Click-iT chemistry approach. Bound proteins were digested and resulting peptides were analyzed by LC-MS/MS. In four biological replicates, we identified a total of 2888 proteins across both wt and 2b5^ho astrocytes. 1240 proteins were detected in at least 3 out of 4 biological replicates for both wt and 2b5^ho astrocytes and used for further analysis. Accumulation of 72 proteins was increased and of eight proteins decreased in the 2b5^ho AHA-proteome (p < 0.05, student’s T-test) (Figure 2A and Supplementary File 1).

We performed a DAVID pathway analysis to investigate if the eIF2B𝜀^Arg191His-regulated proteins share common functions, localizations or other features. This analysis showed enrichment for N-terminally glycosylated proteins, membrane proteins, proteins with disulfide bonds as well as proteins that contain a signal peptide among the set of eIF2B𝜀^Arg191His-regulated proteins (Figure 2B). This outcome suggests that many regulated proteins follow the secretory pathway. Since signal peptides and transmembrane domains are associated with targeting to the secretory pathway (von Heijne, 1990; Petersen et al., 2011; Borgese, 2016), we tested all 1240 identified proteins for signal peptides or transmembrane domains. Signal peptides were found in 237 proteins and transmembrane domains in 177. The number of proteins predicted to pass through the secretory pathway was significantly enriched in the 2b5^ho proteome (Table 3). 27 out of the 80 regulated proteins harbor a signal peptide and 24 have at least one transmembrane domain; 13 of the proteins harbor both a signal peptide and a transmembrane domain (in total 38 proteins out of 80). Intriguingly, the proteins annotated as migrating through the secretory pathway were all upregulated in 2b5^ho astrocytes. It is possible that these proteins accumulate more in 2b5^ho astrocytes during the 2-h labeling pulse due to a secretory pathway flux difference and not to an actual increase in translation rate. To investigate this, we analyzed the AHA-enriched secretome from wt and 2b5^ho cultures after a 2-h AHA labeling. We detected 22 labeled proteins in the secretome, of which three (APOE, CST3 and POSTN) were significantly reduced in the 2b5^ho astrocyte-conditioned medium. Intracellularly, APOE and POSTN were not changed by eIF2B𝜀^Arg191His. CST3 was significantly increased intracellularly in 2b5^ho astrocytes. No other protein from the original 27 eIF2B𝜀^Arg191His-regulated proteins with a signal peptide was detected in the secretomes of wt and 2b5^ho astrocytes. To further address the hypothesis that 2b5^ho astrocytes have an altered secretory pathway flux, we evaluated the flux of proteins migrating through the secretory pathway with a reporter assay. The assay showed robust secretion of the Secluc reporter (Supplementary Figure 2) and secretion differences between wt and 2b5^ho astrocytes were not detected (Figure 3).

To investigate if the proteins that were found in the screen are indeed differentially expressed at the translational level, we selected eleven candidates that are either increased or decreased in wt vs. 2b5^ho cultures. We quantified their mRNA levels to discriminate whether differences in protein amounts are regulated at the transcriptional or the translational level. Only one of the selected candidates differed in mRNA expression between wt and 2b5^ho astrocytes (Figure 4A), suggesting that the increased accumulation of the other 10 proteins was not due to increased transcription or mRNA stability. Of the 11 candidates two were further investigated at the protein level by Western blot. We found that PROS1 and SLC3A2 were indeed significantly increased at the total protein level (Figure 4B). This result confirms the data from the proteomic screen and also demonstrates increased accumulation of these candidate proteins.

We next investigated several targets in mouse brain. At the mRNA level seven out of nine targets were similar between wt and 2b5^ho brains (Supplementary Figure 3A). Two proteins were further assessed using Western blot (Supplementary Figure 3B). SLC3A2 protein abundance was also increased in brain lysates; however, this correlated with increased Slc3a2 mRNA levels, suggesting transcriptional regulation in brain. PROS1 protein abundance was also increased in brain lysates, but Pros1 mRNA abundance was not. This finding suggests translational upregulation of PROS1 both in cell culture and brain.

eIF2B plays an essential role in the regulation of protein synthesis. For this reason, we investigated if the mRNAs of the eIF2B𝜀^Arg191His-regulated proteins share specific features that could explain the translational regulation by eIF2B. We investigated the 5′-UTRs for length, structural stability (%GC and ΔG) and number of uORFs with a Kozak sequence. The 5′-UTR length was significantly increased for mRNAs encoding eIF2B𝜀^Arg191His-regulated proteins (Figure 5). Of note, the mRNAs for the eight proteins decreased in the eIF2B𝜀^Arg191His-regulated proteome have relatively short 5′-UTRs (29-343 bases, median 133). This analysis showed that the eIF2B𝜀^Arg191His mutation differentially influences expression of proteins translated from mRNAs with short 5′-UTRs (<150 bases), which were underrepresented, compared to those with lengthy 5′-UTRs (>550 nucleotides), which were overrepresented; those translated from mRNAs with short 5′-UTRs, if regulated, tend to go down in expression in the mutant cells. The %GC in the 5′-UTRs did not significantly differ between eIF2B𝜀^Arg191His-regulated proteins and other proteins (Figure 5). The thermodynamic stability (ΔG in kcal/mole) of the 5′-UTR seemed to be higher for mRNAs encoding the eIF2B𝜀^Arg191His-regulated proteins, although not significantly (Figure 5). The overall number of uORFs did not significantly differ for mRNA encoding the eIF2B𝜀^Arg191His-regulated proteins compared to the non-regulated proteins (Figure 5). However, mRNAs with high numbers of uORFs (8 or more) were clearly overrepresented in the group of significantly altered proteins.

We investigated the potential translational regulation of some eIF2B𝜀^Arg191His-regulated proteins using reporter constructs encoding nanoluciferase driven by promoter sequences (including the 5′-UTR) of the candidates GAS6, PROS1, SCRN1 and FGFR1. A Gapdh promoter construct was included as non-regulated control. Transfection of each construct yielded similar luciferase expression in wt and 2b5^ho cultures (Figure 6). Transfection of the pNL1.1-Fgfr1 promoter construct did not yield a reliable level of nanoluciferase (approximately two–threefold over the background signal in non-transfected cells) and was therefore omitted from analysis. The levels of nanoluciferase activity expressed with the other constructs were still low in comparison to the Gapdh-promoter-driven expression. Because expression could be increased by replacing the candidate promoter with the Gapdh promoter, we therefore constructed Gapdh-candidate chimeras. With these chimeras we tested the 5′-UTR efficiency of candidates GAS6, SORCS1, PROS1, FGFR1, NRXN1, and DDR1 in wt and 2b5^ho cultures. Neither the pNL1.1-Fgfr1-5′-UTR nor the pNL1.1-Nrxn1-5′-UTR constructs yielded quantifiable nanoluciferase expression. These constructs were omitted from further analyses. The expression from the chimeric promoter-5′-UTR constructs of Gas6 and Pros1 was increased by the Gapdh core promoter in both wt and 2b5^ho cells. Still, none of these constructs yielded an increased nanoluciferase expression in 2b5^ho compared to wt astrocytes (Figure 6).

Because the proteomic data only gave small differences, the VWM phenotype remained difficult to explain. Tiny differences in protein levels and post-translational modifications occasionally lead to metabolic shifts. Thus we checked whether they affected cellular metabolism, using a mass spectrometry-based metabolic screen. We measured intra- and extracellular metabolites 24 and 72 h after replacing the culture medium. We looked at general energy consumption by measuring the uptake and secretion of metabolites from the culture medium (Figure 7A). As expected, both cultures had taken up glutamate (Martin-Jimenez et al., 2017) and aspartate (which use the same transporters) (Bender et al., 1997), reflecting specific astrocyte function. The uptake of glutamate and aspartate did not influence their intracellular levels (Supplementary File 2). Also, both wt and 2b5^ho cultures showed uptake of pyruvate. Astrocytes convert pyruvate into lactate and glutamate/aspartate into glutamine, cis-aconitate and α-ketoglutarate (Martin-Jimenez et al., 2017). Indeed, the wt as well as the 2b5^ho cultures secreted lactate, glutamine, cis-aconitate and α-ketoglutarate (Figure 7A). In addition, we investigated intracellular metabolites (Figure 7B). 6P-gluconate, an intermediate of the pentose phosphate pathway (PPP), was detected in 2b5^ho but not in wt cells at both time points (Figure 7B). A fluctuation over time in NADPH levels was observed. The remaining metabolites tested did not show consistent differences over time between wt and 2b5^ho astrocytes (Figure 7B and Supplementary File 2).