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.     

Eukaryotic Initiation Factor 2B (eIF2B) GEF Activity as a Diagnostic Tool for EIF2B-Related Disorders

Background

In recent years, the phenotypes of leukodystrophies linked to mutations in the eukaryotic initiation factor 2B genes have been extended, classically called CACH/VWM (Childhood ataxia with cntral hypomyélination/vanishing white matter disorder). The large clinical spectrum observed from the more severe antenatal forms responsible for fetal death to milder adult forms with an onset after 16 years old and restricted to slow cognitive impairment have lead to the concept of eIF2B-related disorders. The typical MRI pattern with a diffuse CSF-like aspect of the cerebral white matter can lack particularly in the adult forms whereas an increasing number of patients with clinical and MRI criteria for CACH/VWM disease but without eIF2B mutations are found. Then we propose the use of biochemical markers to help in this difficult diagnosis. The biochemical diagnosis of eIF2B-related disorder is difficult as no marker, except the recently described asialotransferrin/transferrin ratio measured in cerebrospinal fluid, has been proposed and validated until now. Decreased eIF2B GEF activity has been previously reported in lymphoblastoid cell lines from 30 eIF2B-mutated patients. Our objective was to evaluate further the utility of this marker and to validate eIF2B GEF activity in a larger cohort as a specific diagnostic test for eIF2B-related disorders.

Methodology/Principal Findings

We performed eIF2B GEF activity assays in cells from 63 patients presenting with different clinical forms and eIF2B mutations in comparison to controls but also to patients with defined leukodystrophies or CACH/VWM-like diseases without eIF2B mutations. We found a significant decrease of GEF activity in cells from eIF2B-mutated patients with 100% specificity and 89% sensitivity when the activity threshold was set at ≤77.5%.

Conclusion

These results validate the measurement of eIF2B GEF activity in patients'' transformed-lymphocytes as an important tool for the diagnosis of eIF2B-related disorders.     

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.     

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 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.     

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.     

Heightened stress response in primary fibroblasts expressing mutant eIF2B genes from CACH/VWM leukodystrophy patients

Childhood ataxia with central nervous system hypomyelination (CACH), also called vanishing white matter (VWM) leukoencephalopathy, is a fatal genetic disease caused by mutations in eukaryotic initiation factor 2B (eIF2B) genes. The five subunits eIF2B factor is critical for translation initiation under normal conditions and regulates protein synthesis in response to cellular stresses. Primary fibroblasts from CACH/VWM patients and normal individuals were used to measure basal eIF2B activity as well as global protein synthesis and ATF4 induction in response to stress in the endoplasmic reticulum. We show that although the cells expressing mutant eIF2B genes respond normally to stress conditions by reduced global translation rates, they exhibit significantly greater increase in ATF4 induction compared to normal controls despite equal levels of stress and activity of the upstream eIF2α kinase. This heightened stress response observed in primary fibroblasts that suffer from minor loss of basal eIF2B activity may be employed as an initial screening tool for CACH/VWM leukodystrophy.     

CSF N-Glycan Profiles to Investigate Biomarkers in Brain Developmental Disorders: Application to Leukodystrophies Related to eIF2B Mutations

Background

Primary or secondary abnormalities of glycosylation have been reported in various brain diseases. Decreased asialotransferrin to sialotransferrin ratio in cerebrospinal fluid (CSF) is a diagnostic marker of leukodystrophies related to mutations of genes encoding translation initiation factor, EIF2B. We investigated the CSF glycome of eIF2B-mutated patients and age-matched normal individuals in order to further characterize the glycosylation defect for possible use as a biomarker.

Methodology/Principal Findings

We conducted a differential N-glycan analysis using MALDI-TOF/MS of permethylated N-glycans in CSF and plasma of controls and eIF2B-mutated patients. We found in control CSF that tri-antennary/bisecting and high mannose structures were highly represented in samples obtained between 1 to 5 years of age, whereas fucosylated, sialylated structures were predominant at later age. In CSF, but not in plasma, of eIF2B-mutated patient samples, we found increased relative intensity of bi-antennary structures and decreased tri-antennary/bisecting structures in N-glycan profiles. Four of these structures appeared to be biomarker candidates of glycomic profiles of eIF2B-related disorders.

Conclusion

Our results suggest a dynamic development of normal CSF N-glycan profiles from high mannose type structures to complex sialylated structures that could be correlated with postnatal brain maturation. CSF N-glycome analysis shows relevant quantitative changes associated with eIF2B related disorders. This approach could be applied to other neurological disorders involving developmental gliogenesis/synaptogenesis abnormalities.     

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.     

Architecture of the eIF2B regulatory subcomplex and its implications for the regulation of guanine nucleotide exchange on eIF2

Eukaryal translation initiation factor 2B (eIF2B) acts as guanine nucleotide exchange factor (GEF) for eIF2 and forms a central target for pathways regulating global protein synthesis. eIF2B consists of five non-identical subunits (α–ϵ), which assemble into a catalytic subcomplex (γ, ϵ) responsible for the GEF activity, and a regulatory subcomplex (α, β, δ) which regulates the GEF activity under stress conditions. Here, we provide new structural and functional insight into the regulatory subcomplex of eIF2B (eIF2B^RSC). We report the crystal structures of eIF2Bβ and eIF2Bδ from Chaetomium thermophilum as well as the crystal structure of their tetrameric eIF2B(βδ)₂ complex. Combined with mutational and biochemical data, we show that eIF2B^RSC exists as a hexamer in solution, consisting of two eIF2Bβδ heterodimers and one eIF2Bα₂ homodimer, which is homologous to homohexameric ribose 1,5-bisphosphate isomerases. This homology is further substantiated by the finding that eIF2Bα specifically binds AMP and GMP as ligands. Based on our data, we propose a model for eIF2B^RSC and its interactions with eIF2 that is consistent with previous biochemical and genetic data and provides a framework to better understand eIF2B function, the molecular basis for Gcn⁻, Gcd⁻ and VWM/CACH mutations and the evolutionary history of the eIF2B complex.     

Visual Search Elicits the Electrophysiological Marker of Visual Working Memory

Background

Although limited in capacity, visual working memory (VWM) plays an important role in many aspects of visually-guided behavior. Recent experiments have demonstrated an electrophysiological marker of VWM encoding and maintenance, the contralateral delay activity (CDA), which has been shown in multiple tasks that have both explicit and implicit memory demands. Here, we investigate whether the CDA is evident during visual search, a thoroughly-researched task that is a hallmark of visual attention but has no explicit memory requirements.

Methodology/Principal Findings

The results demonstrate that the CDA is present during a lateralized search task, and that it is similar in amplitude to the CDA observed in a change-detection task, but peaks slightly later. The changes in CDA amplitude during search were strongly correlated with VWM capacity, as well as with search efficiency. These results were paralleled by behavioral findings showing a strong correlation between VWM capacity and search efficiency.

Conclusions/Significance

We conclude that the activity observed during visual search was generated by the same neural resources that subserve VWM, and that this activity reflects the maintenance of previously searched distractors.     

Decreased GABAB receptor function in the cerebellum and brain stem of hypoxic neonatal rats: Role of glucose,oxygen and epinephrine resuscitation

Background-

Hypoxia during the first week of life can induce neuronal death in vulnerable brain regions usually associated with an impairment of cognitive function that can be detected later in life. The neurobiological changes mediated through neurotransmitters and other signaling molecules associated with neonatal hypoxia are an important aspect in establishing a proper neonatal care.

Methods-

The present study evaluated total GABA, GABA_B receptor alterations, gene expression changes in GABA_B receptor and glutamate decarboxylase in the cerebellum and brain stem of hypoxic neonatal rats and the resuscitation groups with glucose, oxygen and epinephrine. Radiolabelled GABA and baclofen were used for receptor studies of GABA and GABA_B receptors respectively and Real Time PCR analysis using specific probes for GABA_B receptor and GAD mRNA was done for gene expression studies.

Results-

The adaptive response of the body to hypoxic stress resulted in a reduction in total GABA and GABA_B receptors along with decreased GABA_B receptor and GAD gene expression in the cerebellum and brain stem. Hypoxic rats supplemented with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GAD. Resuscitation with oxygen alone and epinephrine was less effective in reversing the receptor alterations.

Conclusions-

Being a source of immediate energy, glucose can reduce the ATP-depletion-induced changes in GABA and oxygenation, which helps in encountering hypoxia. The present study suggests that reduction in the GABA_B receptors functional regulation during hypoxia plays an important role in central nervous system damage. Resuscitation with glucose alone and glucose and oxygen to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

The Prognostic Value of Altered eIF3a and Its Association with p27 in Non-Small Cell Lung Cancers

Background

Over-expressed eukaryotic initiation factor 3a (eIF3a) in non-small cell lung cancer (NSCLC) contributed to cisplatin sensitivity. However, the role of eIF3a in oncogenesis was still controversial. This study was designed to investigate the prognostic impact of eIF3a and p27 in radically resected NSCLC patients.

Methods

The expression levels of subcellular eIF3a and p27 were evaluated immunohistochemically in 537 radically resected NSCLC samples, and another cohort of 210 stage II NSCLC patients. Disease specific survival (DSS) and disease free survival (DFS) were analyzed by Kaplan-Meier method and Cox regression model.

Results

The subcellular expression of eIF3a was strongly correlated with status of p27 (Spearman rank coefficient correlation for cytoplasmic eIF3a and p27 = 0.653, for nuclear staining = 0.716). Moreover, survival analysis revealed favorable prognostic impact of nuclear eIF3a, p27, and the combination high nuclear staining on NSCLC (Hazards Ratio = 0.360, 95%CI = 0.109–0.782, P = 0.028). In addition, interaction research between biomarkers and chemotherapy status disclosed cisplatin-based regimen trend to prolong DSS of stage II NSCLC patients with high eIF3a-C (P = 0.036)and low p27-N (P = 0.031).

Conclusions

Our findings suggested altered eIF3a expression closely correlated with p27 status, and the association was of prognostic value for resected NSCLC. Altered expression of eIF3a and p27 predicted prognosis of NSCLC independently.     

MicroRNAs Show Mutually Exclusive Expression Patterns in the Brain of Adult Male Rats

Background

The brain is a major site of microRNA (miRNA) gene expression, but the spatial expression patterns of miRNAs within the brain have not yet been fully covered.

Methodology/Principal Findings

We have characterized the regional expression profiles of miRNAs in five distinct regions of the adult rat brain: amygdala, cerebellum, hippocampus, hypothalamus and substantia nigra. Microarray profiling uncovered 48 miRNAs displaying more than three-fold enrichment between two or more brain regions. Notably, we found reciprocal expression profiles for a subset of the miRNAs predominantly found (> ten times) in either the cerebellum (miR-206 and miR-497) or the forebrain regions (miR-132, miR-212, miR-221 and miR-222).

Conclusions/Significance

The results indicate that some miRNAs could be important for area-specific functions in the brain. Our data, combined with previous studies in mice, provides additional guidance for future investigations of miRNA functions in the brain.     

Gene Expression of ANP,BNP and ET-1 in the Heart of Rats during Pulmonary Embolism

Aims

Atrial natriuretic petide (ANP), brain natriuretic peptide (BNP) and endothelin-1 (ET-1) may reflect the severity of right ventricular dysfunction (RVD) in patients with pulmonary embolism (PE). The exact nature and source of BNP, ANP and ET-1 expression and secretion following PE has not previously been studied.

Methods and Results

Polystyrene microparticles were injected to induce PE in rats. Gene expression of BNP, ANP and ET-1 were determined in the 4 cardiac chambers by quantitative real time polymerase chain reaction (QPCR). Plasma levels of ANP, BNP, ET-1 and cardiac troponin I (TNI) were measured in plasma. PE dose-dependently increased gene expression of ANP and BNP in the right ventricle (RV) and increased gene expression of ANP in the right atrium (RA). In contrast PE dose-dependently decreased BNP gene expression in both the left ventricle (LV) and the left atrium (LA). Plasma levels of BNP, TNI and ET-1 levels dose-dependently increased with the degree of PE.

Conclusion

We found a close correlation between PE degree and gene-expression of ANP, and BNP in the cardiac chambers with a selective increase in the right chambers of the heart.The present data supports the idea of natriuretic peptides as valuable biomarkers of RVD in PE.     

Visual working memory load-related changes in neural activity and functional connectivity

Background

Visual working memory (VWM) helps us store visual information to prepare for subsequent behavior. The neuronal mechanisms for sustaining coherent visual information and the mechanisms for limited VWM capacity have remained uncharacterized. Although numerous studies have utilized behavioral accuracy, neural activity, and connectivity to explore the mechanism of VWM retention, little is known about the load-related changes in functional connectivity for hemi-field VWM retention.

Methodology/Principal Findings

In this study, we recorded electroencephalography (EEG) from 14 normal young adults while they performed a bilateral visual field memory task. Subjects had more rapid and accurate responses to the left visual field (LVF) memory condition. The difference in mean amplitude between the ipsilateral and contralateral event-related potential (ERP) at parietal-occipital electrodes in retention interval period was obtained with six different memory loads. Functional connectivity between 128 scalp regions was measured by EEG phase synchronization in the theta- (4–8 Hz), alpha- (8–12 Hz), beta- (12–32 Hz), and gamma- (32–40 Hz) frequency bands. The resulting matrices were converted to graphs, and mean degree, clustering coefficient and shortest path length was computed as a function of memory load. The results showed that brain networks of theta-, alpha-, beta-, and gamma- frequency bands were load-dependent and visual-field dependent. The networks of theta- and alpha- bands phase synchrony were most predominant in retention period for right visual field (RVF) WM than for LVF WM. Furthermore, only for RVF memory condition, brain network density of theta-band during the retention interval were linked to the delay of behavior reaction time, and the topological property of alpha-band network was negative correlation with behavior accuracy.

Conclusions/Significance

We suggest that the differences in theta- and alpha- bands between LVF and RVF conditions in functional connectivity and topological properties during retention period may result in the decline of behavioral performance in RVF task.     

Differences in gene expression between first and third trimester human placenta: a microarray study

Background

The human placenta is a rapidly developing organ that undergoes structural and functional changes throughout the pregnancy. Our objectives were to investigate the differences in global gene expression profile, the expression of imprinted genes and the effect of smoking in first and third trimester normal human placentas.

Materials and Methods

Placental samples were collected from 21 women with uncomplicated pregnancies delivered at term and 16 healthy women undergoing termination of pregnancy at 9–12 weeks gestation. Placental gene expression profile was evaluated by Human Genome Survey Microarray v.2.0 (Applied Biosystems) and real-time polymerase chain reaction.

Results

Almost 25% of the genes spotted on the array (n = 7519) were differentially expressed between first and third trimester placentas. Genes regulating biological processes involved in cell proliferation, cell differentiation and angiogenesis were up-regulated in the first trimester; whereas cell surface receptor mediated signal transduction, G-protein mediated signalling, ion transport, neuronal activities and chemosensory perception were up-regulated in the third trimester. Pathway analysis showed that brain and placenta might share common developmental routes. Principal component analysis based on the expression of 17 imprinted genes showed a clear separation of first and third trimester placentas, indicating that epigenetic modifications occur throughout pregnancy. In smokers, a set of genes encoding oxidoreductases were differentially expressed in both trimesters.

Conclusions

Differences in global gene expression profile between first and third trimester human placenta reflect temporal changes in placental structure and function. Epigenetic rearrangements in the human placenta seem to occur across gestation, indicating the importance of environmental influence in the developing feto-placental unit.