Mutations causing childhood ataxia with central nervous system hypomyelination reduce eukaryotic initiation factor 2B complex formation and activity

Childhood ataxia with central nervous system hypomyelination (CACH), or vanishing white matter leukoencephalopathy (VWM), is a fatal brain disorder caused by mutations in eukaryotic initiation factor 2B (eIF2B). eIF2B is essential for protein synthesis and regulates translation in response to cellular stresses. We performed mutagenesis to introduce changes equivalent to 12 human CACH/VWM mutations in three subunits of the equivalent factor from yeast (Saccharomyces cerevisiae) and analyzed effects on cell growth, translation, and gene expression in response to stresses. None of the mutations is lethal or temperature sensitive, but almost all confer some defect in eIF2B function significant enough to alter growth or gene expression under normal or stress conditions. Biochemical analyses indicate that mutations analyzed in eIF2Balpha and -epsilon reduce the steady-state level of the affected subunit, while the most severe mutant tested, eIF2Bbeta(V341D) (human eIF2B(betaV316D)), forms complexes with reduced stability and lower eIF2B activity. eIF2Bdelta is excluded from eIF2Bbeta(V341D) complexes. eIF2B(betav341D) function can be rescued by overexpression of eIF2Bdelta alone. Our findings imply CACH/VWM mutations do not specifically impair responses to eIF2 phosphorylation, but instead cause protein structure defects that impair eIF2B activity. Altered protein folding is characteristic of other diseases, including cystic fibrosis and neurodegenerative disorders such as Huntington, Alzheimer's, and prion diseases.     

A Point Mutation in Translation Initiation Factor 2B Leads to a Continuous Hyper Stress State in Oligodendroglial-Derived Cells

Background

Mutations in eukaryotic translation initiation factor 2B (eIF2B) cause Childhood Ataxia with CNS Hypomyelination (CACH), also known as Vanishing White Matter disease (VWM). The disease is manifested by loss of brain myelin upon physiological stress. In a previous study, we showed that fibroblasts isolated from CACH/VWM patients are hypersensitive to pharmacologically-induced endoplasmic reticulum (ER) stress. Since brain cells from affected individuals are not available for research, we wished to assess the effect of eIF2B mutation on oligodendroglial-derived cells.

Methodology/Principal Findings

A rat oligodendroglial-derived cell line was used for a stable knock-down of eIF2B5 followed by stable expression of mutated eIF2B5(R195H) cDNA. In response to a pharmacological ER-stress agent, eIF2B5(R195H) expressing cells exhibited heightened ER-stress response demonstrated by hyper induction of ATF4, GADD34, Bip, PDIA1, PDIA3, PDIA4 and PDIA6 proteins. Moreover, even in the absence of a pharmacological stress agent, eIF2B5(R195H)-expressing cells exhibited high basal levels of ATF4, GADD34 and ER-associated Bip, PDIA1 and PDIA3.

Significance

The data provide evidence that oligodendroglial-derived cells expressing a mutated eIF2B constantly use their stress response mechanism as an adaptation mean in order to survive. The current study is the first to demonstrate the effects of eIF2B5 mutation on ER homeostasis in oligodendroglial-derived cells.     

Poor Cerebral Inflammatory Response in eIF2B Knock-In Mice: Implications for the Aetiology of Vanishing White Matter Disease

Background

Mutations in any of the five subunits of eukaryotic translation initiation factor 2B (eIF2B) can lead to an inherited chronic-progressive fatal brain disease of unknown aetiology termed leucoencephalopathy with vanishing white matter (VWM). VWM is one of the most prevalent childhood white matter disorders, which markedly deteriorates after inflammation or exposure to other stressors. eIF2B is a major housekeeping complex that governs the rate of global protein synthesis under normal and stress conditions. A previous study demonstrated that Eif2b5^R132H/R132H mice suffer delayed white matter development and fail to recover from cuprizone-induced demyelination, although eIF2B enzymatic activity in the mutant brain is reduced by merely 20%.

Principal Findings

Poor astrogliosis was observed in Eif2b5^R132H/R132H mice brain in response to systemic stress induced by peripheral injections of lipopolysaccharide (LPS). Even with normal rates of protein synthesis under normal conditions, primary astrocytes and microglia isolated from mutant brains fail to adequately synthesise and secrete cytokines in response to LPS treatment despite proper induction of cytokine mRNAs.

Conclusions

The mild reduction in eIF2B activity prevents the appropriate increase in translation rates upon exposure to the inflammatory stressor LPS. The data underscore the importance of fully-functional translation machinery for efficient cerebral inflammatory response upon insults. It highlights the magnitude of proficient translation rates in restoration of brain homeostasis via microglia-astrocyte crosstalk. This study is the first to suggest the involvement of microglia in the pathology of VWM disease. Importantly, it rationalises the deterioration of clinical symptoms upon exposure of VWM patients to physiological stressors and provides possible explanation for their high phenotypic variability.     

A new function and complexity for protein translation initiation factor eIF2B

eIF2B is a multisubunit protein that is critical for protein synthesis initiation and its control. It is a guanine nucleotide exchange factor (GEF) for its GTP-binding protein partner eIF2. eIF2 binds initiator tRNA to ribosomes and promotes mRNA AUG codon recognition. eIF2B is critical for regulation of protein synthesis via a conserved mechanism of phosphorylation of eIF2, which converts eIF2 from a substrate to an inhibitor of eIF2B GEF. In addition, inherited mutations affecting eIF2B subunits cause the fatal disorder leukoencephalopathy with Vanishing White Matter (VWM), also called Childhood Ataxia with Central nervous system Hypomyelination (CACH). Here we review findings which reveal that eIF2B is a decameric protein and also define a new function for the eIF2B. Our results demonstrate that the eIF2Bγ subunit is required for eIF2B to gain access to eIF2•GDP. Specifically it displaces a third translation factor eIF5 (a dual function GAP and GDI) from eIF2•GDP/eIF5 complexes. Thus eIF2B is a GDI displacement factor (or GDF) in addition to its role as a GEF, prompting the redrawing of the eIF2 cycling pathway to incorporate the new steps. In structural studies using mass spectrometry and cross-linking it is shown that eIF2B is a dimer of pentamers and so is twice as large as previously thought. A binding site for GTP on eIF2B was also found, raising further questions concerning the mechanism of nucleotide exchange. The implications of these findings for eIF2B function and for VWM/CACH disease are discussed.     

Nck in a complex containing the catalytic subunit of protein phosphatase 1 regulates eukaryotic initiation factor 2alpha signaling and cell survival to endoplasmic reticulum stress

Stress imposed on the endoplasmic reticulum (ER) induces the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2) on Ser51. This results in transient inhibition of general translation initiation while concomitantly activating a signaling pathway that promotes the expression of genes whose products improve ER function. Conversely, dephosphorylation of eIF2alphaSer51 is accomplished by protein phosphatase 1 (PP1c) complexes containing either the protein CReP or GADD34, which target PP1c to eIF2. Here, we demonstrate that the Src homology (SH) domain-containing adaptor Nck is a key component of a molecular complex that controls eIF2alpha phosphorylation and signaling in response to ER stress. We show that overexpression of Nck decreases basal and ER stress-induced eIF2alpha phosphorylation and the attendant induction of ATF4 and CHOP. In contrast, we demonstrate that the mouse embryonic fibroblasts lacking both isoforms of Nck (Nck1-/-Nck2-/-) show higher levels of eIF2alpha phosphorylation and premature induction of ATF4, CHOP, and GADD34 in response to ER stress and finally, are more resistant to cell death induced by prolonged ER stress conditions. We establish that a significant amount of Nck protein localizes at the ER and is in a complex with eIF2 subunits. Further analysis of this complex revealed that it also contains the Ser/Thr phosphatase PP1c, its regulatory subunit CReP, and dephosphorylates eIF2alpha on Ser51 in vitro. Overall, we demonstrate that Nck as a component of the CReP/PP1c holophosphatase complex contributes to maintain eIF2alpha in a hypophosphorylated state. In this manner, Nck modulates translation and eIF2alpha signaling in response to ER stress.     

Mutations linked to leukoencephalopathy with vanishing white matter impair the function of the eukaryotic initiation factor 2B complex in diverse ways

Leukoencephalopathy with vanishing white matter (VWM) is a severe inherited human neurodegenerative disorder that is caused by mutations in the genes for the subunits of eukaryotic initiation factor 2B (eIF2B), a heteropentameric guanine nucleotide exchange factor that regulates both global and mRNA-specific translation. Marked variability is evident in the clinical severity and time course of VWM in patients. Here we have studied the effects of VWM mutations on the function of human eIF2B. All the mutations tested cause partial loss of activity. Frameshift mutations in genes for eIF2Bepsilon or eIF2Bbeta lead to truncated polypeptides that fail to form complexes with the other subunits and are effectively null mutations. Certain point mutations also impair the ability of eIF2Bbeta or -epsilon to form eIF2B holocomplexes and also diminish the intrinsic nucleotide exchange activity of eIF2B. A point mutation in the catalytic domain of eIF2Bepsilon impairs its ability to bind the substrate, while two mutations in eIF2Bbeta actually enhance eIF2 binding. We provide evidence that expression of VWM mutant eIF2B may enhance the translation of specific mRNAs. The variability of the clinical phenotype in VWM may reflect the multiple ways in which VWM mutations affect eIF2B function.     

Paradoxical Sensitivity to an Integrated Stress Response Blocking Mutation in Vanishing White Matter Cells

The eukaryotic translation initiation factor eIF2B promotes mRNA translation as a guanine nucleotide exchange factor (GEF) for translation initiation factor 2 (eIF2). Endoplasmic reticulum (ER) stress-mediated activation of the kinase PERK and the resultant phosphorylation of eIF2’s alpha subunit (eIF2α) attenuates eIF2B GEF activity thereby inducing an integrated stress response (ISR) that defends against protein misfolding in the ER. Mutations in all five subunits of human eIF2B cause an inherited leukoencephalopathy with vanishing white matter (VWM), but the role of the ISR in its pathogenesis remains unclear. Using CRISPR-Cas9 genome editing we introduced the most severe known VWM mutation, EIF2B4^A391D, into CHO cells. Compared to isogenic wildtype cells, GEF activity of cells with the VWM mutation was impaired and the mutant cells experienced modest enhancement of the ISR. However, despite their enhanced ISR, imposed by the intrinsic defect in eIF2B, disrupting the inhibitory effect of phosphorylated eIF2α on GEF by a contravening EIF2S1/eIF2α^S51A mutation that functions upstream of eIF2B, selectively enfeebled both EIF2B4^A391D and the related severe VWM EIF2B4^R483W cells. The basis for paradoxical dependence of cells with the VWM mutations on an intact eIF2α genotype remains unclear, as both translation rates and survival from stressors that normally activate the ISR were not reproducibly affected by the VWM mutations. Nonetheless, our findings support an additional layer of complexity in the development of VWM, beyond a hyperactive ISR.