A point mutation in translation initiation factor eIF2B leads to function--and time-specific changes in brain gene expression.

BACKGROUND: Mutations in eukaryotic translation initiation factor 2B (eIF2B) cause Childhood Ataxia with CNS Hypomyelination (CACH), also known as Vanishing White Matter disease (VWM), which is associated with a clinical pathology of brain myelin loss upon physiological stress. eIF2B is the guanine nucleotide exchange factor (GEF) of eIF2, which delivers the initiator tRNA(Met) to the ribosome. We recently reported that a R132H mutation in the catalytic subunit of this GEF, causing a 20% reduction in its activity, leads under normal conditions to delayed brain development in a mouse model for CACH/VWM. To further explore the effect of the mutation on global gene expression in the brain, we conducted a wide-scale transcriptome analysis of the first three critical postnatal weeks. METHODOLOGY/PRINCIPAL FINDINGS: Genome-wide mRNA expression of wild-type and mutant mice was profiled at postnatal (P) days 1, 18 and 21 to reflect the early proliferative stage prior to white matter establishment (P1) and the peak of oligodendrocye differentiation and myelin synthesis (P18 and P21). At each developmental stage, between 441 and 818 genes were differentially expressed in the mutant brain with minimal overlap, generating unique time point-specific gene expression signatures. CONCLUSIONS: The current study demonstrates that a point mutation in eIF2B, a key translation initiation factor, has a massive effect on global gene expression in the brain. The overall changes in expression patterns reflect multiple layers of indirect effects that accumulate as the brain develops and matures. The differentially expressed genes seem to reflect delayed waves of gene expression as well as an adaptation process to cope with hypersensitivity to cellular stress

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

Activated primary astrocytes-microglia cultures from Eif2b5^R132H/R132H mice do not exhibit increased global translation and display impaired production of cytokines.

<p>Primary mixed glia cultures were isolated from Eif2b5^R132H/R132H (Mut) and wild-type (WT) mice. (A) Representative analysis of PE-labelled anti-CD11b staining followed by flow cytometry analysis. Both Mut and WT cultures contained 93–95% CD11b-negative (gray area) cells. (B) Immunoblot analysis of 3 independent experiments using anti-TLR4 and anti-β-actin antibodies. No statistically significant difference between WT and Mut cells was observed. (C) WT and Mut cells were treated with 2 µg/ml LPS for 48 h and metabolically labelled with [³⁵S]L-methionine and [³⁵S]L-cysteine for 30 min followed by determination of [³⁵S]-Met/Cys incorporation following trichloroacetic acid precipitation. The incorporation level (cpm/µg protein) in untreated WT cells was set at 100%. Bars represent the means ± SEM of 3 independent experiments, *p<0.02. (D, E) WT and Mut cultures were incubated with 2 µg/ml LPS for the indicated times followed by cell harvest and media collection. A representative immunoblot analysis of intracellular IL-6 and IL-1β protein levels is shown and the average (of 3 independent experiments) of IL-6/β-actin and IL-1β/β-actin in Mut relative to WT is provided in (D); protein concentrations of IL-6, TNF-α and MCP-1 in the media was measured by ELISA and a representative of 3 independent experiments is shown in (E).</p

Induction of IL-6 and IL-1β in response to demand is impaired in Eif2b5^R132H/R132H mice brain.

<p>Intraperitoneal injections of 5 mg/kg LPS were administered to 4–6 four-weeks-old Eif2b5^R132H/R132H (Mut) and wild-type (WT) mice. At 6 h post-injection, mice were sacrificed and brains were removed. (A) Representative immunoblot analysis using anti-TLR4 and anti-β-actin antibodies. Data represent the average of TLR4/β-actin level of 3 mice per group ± SEM. (B) Total RNA was extracted and subjected to qRT-PCR analysis of IL-6 and IL-1β mRNA levels. Bars represent the average of 5 mice per group, normalised to untreated WT±SEM. No significant (NS) differences between Mut and WT were observed. (C) Representative immunoblot analyses using anti-IL-6, anti-IL-1β and anti-β-actin. Data represent the average of IL-6/β-actin and IL-1β/β-actin of 3-6 mice per group ±SEM relative to WT.</p

Activated Eif2b5^R132H/R132H primary microglial cells exhibit impaired production of cytokines.

<p>Primary microglial cells were isolated from Eif2b5^R132H/R132H (Mut) and wild-type (WT) mice. (A) Representative analysis of PE-labelled anti-CD11b staining followed by flow cytometry analysis. Both Mut and WT cultures were 92–95% CD11b-positive (white area). (B,C,D) Both cultures were treated with 0.1 µg/ml LPS for the indicated times followed by media collection and cell harvest for RNA and protein extraction. qRT-PCR of IL-6, TNF-α and IL-1β mRNA levels is shown in (B), bars represent the average of 3 independent assays, normalised to WT cells at 3 h of LPS treatment ± SEM. Intracellular IL-6 and IL-1β protein levels were determined by immunoblot analyses with β-actin as loading control, representative blots of 3 independent experiments are shown in (C); protein concentrations of IL-6 and TNF-α in the media was measured by ELISA and a representative of 3 independent experiments is shown in (D).</p

Activated Eif2b5^R132H/R132H primary astrocytes exhibit impaired production of cytokines.

<p>Primary astrocytes were isolated from Eif2b5^R132H/R132H (Mut) and wild-type (WT) mice. (A) Representative analysis of PE-labelled anti-CD11b staining followed by flow cytometry analysis. Both Mut and WT cultures were 99.4–99.7% CD11b-negative (gray area). (B,C,D) Both cultures were treated with 2 µg/ml LPS for the indicated times followed by media collection and cell harvest for RNA and protein extraction. qRT-PCR of IL-6 and TNF-α mRNA levels is shown in (B), bars represent the average of 3 independent assays, normalised to 48 h±SEM. No significant (NS) differences between Mut and WT were observed; intracellular IL-6 protein level was determined by immunoblot analysis with β-actin as loading control. A representative blot of 3 independent experiments is shown and the average of IL-6/β-actin in Mut relative to WT is provided in (C); protein concentrations of IL-6 and TNF-α in the media was measured by ELISA and a representative of 3 independent experiments is shown in (D).</p

eIF2B5^R132H/R132H mice exhibit impaired astrogliosis.

<p>Intraperitoneal injections of 5 mg/kg LPS or PBS were administered to 7 Eif2b5^R132H/R132H (Mut) and 7 wild-type (WT) mice at P14 and again at P15. At P21 mice were sacrificed and brains were removed. (A) Representative immunoblot analysis of whole brain using anti-GFAP and anti-GAPDH antibodies. Bars represent the average of GFAP/GAPDH from 4 mice per group ± SEM (*p<0.05). (B) Brain slices were analyzed by immunohistochemistry for reactive astrocytes using anti-GFAP antibodies. The total stained area of the thalamus region was quantified and normalised to that of PBS-injected wild-type mice. Representative GFAP positive cells are shown (photographed with ×40 objective). Bars represent the average of 3 mice per group ± SEM (*p<0.0001).</p