Abstract
eIF2alpha is part of a multimeric complex that regulates cap-dependent translation. Phosphorylation of eIF2alpha (phospho-eIF2alpha) is induced by various forms of cell stress, resulting in changes to the proteome of the cell with two diametrically opposed consequences, adaptation to stress or initiation of programmed cell death. In contrast to the robust eIF2alpha phosphorylation seen in response to acute insults, less is known about the functional role of basal levels of eIF2alpha phosphorylation. Here we show that mouse embryonic fibroblasts expressing a nonphosphorylatable eIF2alpha have enhanced sensitivity to diverse toxic insults, including amyloid beta-(1-42) peptide (Abeta), a key factor in the pathogenesis of Alzheimer disease. This correlates with impaired glutathione metabolism because of down-regulation of the light chain, xCT, of the cystine/glutamate antiporter system X(-)(c). The mechanistic link between the absence of phospho-eIF2alpha and xCT expression is nuclear factor ATF4. Consistent with these findings, long term activation of the phospho-eIF2alpha/ATF4/xCT signaling module by the specific eIF2alpha phosphatase inhibitor, salubrinal, induces resistance against oxidative glutamate toxicity in the hippocampal cell line HT22 and primary cortical neurons. Furthermore, in PC12 cells selected for resistance against Abeta, increased activity of the phospho-eIF2alpha/ATF4/xCT module contributes to the resistant phenotype. In wild-type PC12 cells, activation of this module by salubrinal ameliorates the response to Abeta. Furthermore, in human brains, ATF4 and phospho-eIF2alpha levels are tightly correlated and up-regulated in Alzheimer disease, most probably representing an adaptive response against disease-related cellular stress rather than a correlate of neurodegeneration.
Highlights
Is no longer active, and protein synthesis is not initiated
Even in cell culture models, it can be difficult to determine whether a specific biochemical change is associated with the promotion of cell survival or cell death, if there is a graded effect leading from adaptation to death
Several studies have suggested that increased levels of eIF2␣ phosphorylation observed in nerve cells in response to acute toxic insults such as 6-hydroxydopamine [49] and A peptide [50] as well as chronic increases detected in Alzheimer disease (AD) [23,24,25] reflect a contribution of eIF2␣ phosphorylation to nerve cell death
Summary
Cell Culture and Viability Assays—Mouse embryonic fibroblasts derived from embryos genetically engineered to homozygously express the nonphosphorylatable S51A mutation (A/A MEFs) and wild-type controls (S/S MEFs) [10] were a kind gift from Randal J. Mouse cortical neurons were prepared as described previously [33] and seeded at a density of 1 ϫ 105 cells per well onto poly-L-lysine- and laminin-coated 96-well plates using high glucose DMEM supplemented with 10% fetal calf serum with 30 M salubrinal dissolved in DMSO or DMSO alone. 24 h later, the cells were re-plated for the different assays performed in parallel as follows: 8.8 ϫ 105 in 100-mm dishes for protein preparation; 1.65 ϫ 105 in 60-mm dishes for GSH measurement; 3 ϫ 104/well in 24-well plates for [35S]cystine uptake; and 2.5 ϫ 103/well in 96-well plates for oxidative glutamate toxicity, respectively. Because [35S]cystine became unavailable during the course of the experiments, system XcϪ activity in HT22 cells treated with salubrinal was measured as sodium-insensitive, HCA-inhibitable uptake of [3H]glutamate (PerkinElmer Life Sciences).
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