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


METHODS
We 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).


RESULTS
Despite 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.


CONCLUSIONS
Taken 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 discriminatory markers in eIF2B-related disorders.
Faculté de Médecine, INSERM U931-CNRS 6247-Génétique, Reproduction et Développement, Clermont-Ferrand, France.
Laetitia
L
Horzinski
Liraz
L
Kantor
Aurélia
A
Huyghe
Raphael
R
Schiffmann
Orna
O
Elroy-Stein
Odile
O
Boespflug-Tanguy
Anne
A
Fogli
D051701:Activating Transcription Factor 4 / Q000235:genetics / Q000378:metabolism*; D000293:Adolescent; D015153:Blotting, Western; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors / Q000633:toxicity; D020717:Eukaryotic Initiation Factor-2B / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D007223:Infant; D056784:Leukoencephalopathies / Q000235:genetics / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D009154:Mutation; D014176:Protein Biosynthesis / Q000187:drug effects / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013312:Stress, Physiological / Q000502:physiology*; D019284:Thapsigargin / Q000633:toxicity
C497109:ATF4 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; D020717:Eukaryotic Initiation Factor-2B; C000717247:HSPA5 protein, human; D051701:Activating Transcription Factor 4; D019284:Thapsigargin