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

Laetitia Horzinski,Raphael Schiffmann,Odile Boespflug-Tanguy,Orna Elroy-Stein,Anne Fogli,Aurélia Huyghe,Liraz Kantor

doi:10.1186/1471-2377-10-94

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.

Highlights

Eukaryotic translation initiation factor 2B, 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
We previously reported a decrease Eukaryotic translation initiation factor 2B (eIF2B) GEF activity in Epstein-Barr virus (EBV)-immortalised lymphocytes (EIL, or lymphoblasts) from patients with eIF2B-related disorder that seemed to correlate with disease severity [7]. eIF2B GEF activity is subjected to diverse modes of regulation
One of the inhibitory mechanisms is mediated by phosphorylation of Ser 51 of eIF2a subunit by four specific kinases (PERK, GCN2, PKR and HRI), each of which is activated by specific stress conditions including endoplasmic reticulum (ER)stress [8]. eIF2 phosphorylation mediates the inhibition of eIF2B, leading to reduction of global translation initiation which is accompanied by translational activation of specific sub-class of mRNAs encoding rescue proteins

Summary

Introduction

Eukaryotic 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. It has been shown that eIF2B mutations induce a decrease of eIF2B GEF activity, leading to the translation activation of GCN4 mRNA, the yeast homolog of ATF4 involved in stress regulation [10,11]. Later on, heightened ER-stress response mediated by hyper-induction of ATF4 was observed in primary fibroblasts from eIF2B-mutated patients [9], and in rat oligodendroglialderived cells expressing mutated human EIF2B5 gene [12]. A single study reported stressrelated differences between control and eIF2B-mutated patient’s EIL: mutated EIL lost their ability to respond to heat stress by eIF2a phosphorylation, suggesting a lack of inhibition of eIF2B GEF activity under stress conditions [17] leading to a potentially disturbed cellular response

Methods

Results

Discussion

Conclusion