Evaluation of the endoplasmic reticulum-stress response in eIF2B-mutated lymphocytes and lymphoblasts from CACH/VWM patients

Abstract

BackgroundEukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) and a key regulator of translation initiation under normal and stress conditions, causes an autosomal recessive leukodystrophy of a wide clinical spectrum. EBV-immortalised lymphocytes (EIL) from eIF2B-mutated patients exhibit a decrease in eIF2B GEF activity. eIF2B-mutated primary fibroblasts have a hyper-induction of activating transcription factor 4 (ATF4) which is involved in the protective unfolded protein response (UPR), also known as the ER-stress response. We tested the hypothesis that EIL from eIF2B-mutated patients also exhibit a heightened ER-stress response.MethodsWe used thapsigargin as an ER-stress agent and looked at polysomal profiles, rate of protein synthesis, translational activation of ATF4, and transcriptional induction of stress-specific mRNAs (ATF4, CHOP, ASNS, GRP78) in normal and eIF2B-mutated EIL. We also compared the level of stress-specific mRNAs between EIL and primary lymphocytes (PL).ResultsDespite the low eIF2B GEF activity in the 12 eIF2B-mutated EIL cell lines tested (range 40-70% of normal), these cell lines did not differ from normal EIL in their ATF4-mediated ER-stress response. The absence of hyper-induction of ATF4-mediated ER-stress response in eIF2B-mutated EIL in contrast to primary fibroblasts is not related to their transformation by EBV. Indeed, PL exhibited a higher induction of the stress-specific mRNAs in comparison to EIL, but no hyper-induction of the UPR was noticed in the eIF2B-mutated cell lines in comparison to controls.ConclusionsTaken together with work of others, our results demonstrate the absence of a major difference in ER-stress response between controls and eIF2B-mutated cells. Therefore, components of the ER-stress response cannot be used as discriminantory markers in eIF2B-related disorders.

EIF2B2 mutations in vanishing white matter disease hypersuppress translation and delay recovery during the integrated stress response

“…Similar results have been seen with other VWMD alleles, and in different cellular contexts (Kantor et al 2005;van Kollenburg et al 2006b;Sekine et al 2016;Wong et al 2018). The normal levels of bulk translation in VWMD cells occur despite clear data that many VWMD mutations reduce the exchange rate of GDP for GTP on eIF2α (van Kollenburg et al 2006b;Horzinski et al 2010;Liu et al 2011;Wortham and Proud 2015;Wong et al 2018). This implies that under normal growth conditions the GDP-GTP exchange rate for eIF2α is not rate-limiting for bulk translation.…”

Translational Control during Endoplasmic Reticulum Stress beyond Phosphorylation of the Translation Initiation Factor eIF2α

“…3 and 6A). Efficient translation of the ATF4 mRNA during prolonged stress could not be explained by the current mechanism of upstream ORF-mediated leaky scanning within the ATF4 5Ј-UTR due to the decreased eIF2B activity in stressed cells (19,20,63,64). We have shown previously that eIF2B activity is fully restored during chronic stress in Tg-treated MIN6 cells (26).…”

New Insights into Translational Regulation in the Endoplasmic Reticulum Unfolded Protein Response

“…Heightened signaling in the eIF2(aP)-dependent branch of the UPR has been observed in oligodendroglial cell lines (Kantor et al 2008) and brain samples of VWM/CACH (van Kollenburg et al 2006). eIF2B GEF activity is also impaired in immortalized lymphocytes from patients, but a parallel branch of the UPR, XBP1 splicing, is unaffected (Horzinski et al 2009(Horzinski et al , 2010. In contrast, a heightened UPR but apparently normal GEF activity was found with assays of primary fibroblasts (Kantor et al 2005).…”