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

Horzinski L, Kantor L, Huyghe A, Schiffmann R, Elroy-Stein O, Boespflug-Tanguy O, Fogli A - BMC Neurol (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculté de Médecine, INSERM U931-CNRS 6247-Génétique, Reproduction et Développement, Clermont-Ferrand, France.

ABSTRACT

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.

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Effect of the lymphocytes EBV-transformation on the expression level of the four mRNA markers of the ER-stress response. Thapsigargin (incubation with Thapsigargin (Tg) at 2 μg/ml for 4 h) was used to induce ER-stress in lymphoblastoid cell lines (EIL, black bars) and primary lymphocytes (PL, white bars) from control (C1, C2 and 1283-1) and eIF2B-mutated (1241-1, 807-1, 648-1 and 648-2) patients. Expression levels of ATF4 (A), GRP78 (B), ASNS (C) and CHOP (D) mRNA were determined by quantitative real-time polymerase chain reaction. The level of each transcript was calculated using the 2^(-ΔΔCt) method, followed by normalization of the primary Ct data to beta2M level. Results are shown as fold-change between ER-stress (Tg incubation) compared to normal conditions.
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Figure 2: Effect of the lymphocytes EBV-transformation on the expression level of the four mRNA markers of the ER-stress response. Thapsigargin (incubation with Thapsigargin (Tg) at 2 μg/ml for 4 h) was used to induce ER-stress in lymphoblastoid cell lines (EIL, black bars) and primary lymphocytes (PL, white bars) from control (C1, C2 and 1283-1) and eIF2B-mutated (1241-1, 807-1, 648-1 and 648-2) patients. Expression levels of ATF4 (A), GRP78 (B), ASNS (C) and CHOP (D) mRNA were determined by quantitative real-time polymerase chain reaction. The level of each transcript was calculated using the 2^(-ΔΔCt) method, followed by normalization of the primary Ct data to beta2M level. Results are shown as fold-change between ER-stress (Tg incubation) compared to normal conditions.

Mentions: As primary eIF2B-mutated fibroblasts exhibit heightened ER-stress response mediated by hyper-induction of ATF4 [6], a possible explanation for the lack of difference between eIF2B-mutated and normal EIL on the ER-stress activation could be the immortalization of the PL by EBV. Therefore, we evaluated the impact of EBV immortalization on ER-stress response by comparing EIL and PL from four eIF2B-mutated patients (Table 1) and three healthy individuals using the same stress induction procedure. We found an expected increase in the level of ATF4, CHOP, ASNS and GRP78 mRNA in all the cells, but it was more profound in PL (Figure 2). No significant differences were found in the activation of ER-stress response between eIF2B-mutated and control PL, as was demonstrated in EIL (Figure 1), and extended here to PL (Figure 2). Therefore, modifications induced by EBV-immortalization of PL are not responsible for the absence of differential ER-stress response between eIF2B-mutated and control cells.


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

Horzinski L, Kantor L, Huyghe A, Schiffmann R, Elroy-Stein O, Boespflug-Tanguy O, Fogli A - BMC Neurol (2010)

Effect of the lymphocytes EBV-transformation on the expression level of the four mRNA markers of the ER-stress response. Thapsigargin (incubation with Thapsigargin (Tg) at 2 μg/ml for 4 h) was used to induce ER-stress in lymphoblastoid cell lines (EIL, black bars) and primary lymphocytes (PL, white bars) from control (C1, C2 and 1283-1) and eIF2B-mutated (1241-1, 807-1, 648-1 and 648-2) patients. Expression levels of ATF4 (A), GRP78 (B), ASNS (C) and CHOP (D) mRNA were determined by quantitative real-time polymerase chain reaction. The level of each transcript was calculated using the 2^(-ΔΔCt) method, followed by normalization of the primary Ct data to beta2M level. Results are shown as fold-change between ER-stress (Tg incubation) compared to normal conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2967530&req=5

Figure 2: Effect of the lymphocytes EBV-transformation on the expression level of the four mRNA markers of the ER-stress response. Thapsigargin (incubation with Thapsigargin (Tg) at 2 μg/ml for 4 h) was used to induce ER-stress in lymphoblastoid cell lines (EIL, black bars) and primary lymphocytes (PL, white bars) from control (C1, C2 and 1283-1) and eIF2B-mutated (1241-1, 807-1, 648-1 and 648-2) patients. Expression levels of ATF4 (A), GRP78 (B), ASNS (C) and CHOP (D) mRNA were determined by quantitative real-time polymerase chain reaction. The level of each transcript was calculated using the 2^(-ΔΔCt) method, followed by normalization of the primary Ct data to beta2M level. Results are shown as fold-change between ER-stress (Tg incubation) compared to normal conditions.
Mentions: As primary eIF2B-mutated fibroblasts exhibit heightened ER-stress response mediated by hyper-induction of ATF4 [6], a possible explanation for the lack of difference between eIF2B-mutated and normal EIL on the ER-stress activation could be the immortalization of the PL by EBV. Therefore, we evaluated the impact of EBV immortalization on ER-stress response by comparing EIL and PL from four eIF2B-mutated patients (Table 1) and three healthy individuals using the same stress induction procedure. We found an expected increase in the level of ATF4, CHOP, ASNS and GRP78 mRNA in all the cells, but it was more profound in PL (Figure 2). No significant differences were found in the activation of ER-stress response between eIF2B-mutated and control PL, as was demonstrated in EIL (Figure 1), and extended here to PL (Figure 2). Therefore, modifications induced by EBV-immortalization of PL are not responsible for the absence of differential ER-stress response between eIF2B-mutated and control cells.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculté de Médecine, INSERM U931-CNRS 6247-Génétique, Reproduction et Développement, Clermont-Ferrand, France.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus