Figure 1.
eIF2B5R132H/R132H mice exhibit impaired astrogliosis.
Intraperitoneal injections of 5 mg/kg LPS or PBS were administered to 7 Eif2b5R132H/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).
Figure 2.
Eif2b5R132H/R132H primary astrocytes exhibit abnormal morphology and overexpress GFAP-δ.
(A) Primary astrocytes isolated from Eif2b5R132H/R132H (Mut) and wild-type (WT) mice were incubated with 2 µg/ml LPS for 48 h followed by immunostaining for GFAP. Typical “flat” and “star” morphology is shown (photographed with ×63 objective). All cells within random fields (photographed with ×10 objective) were counted and assigned “star” or “flat” morphology (N = number of cells). Bars represent percent of “star” (white) or “flat” (black) morphology (*p<0.001). (B) WT and Mut primary astrocytes were treated with 2 µg/ml LPS for the indicated times, followed by immunoblot analysis of GFAP-δ and β-actin. GFAP-δ/β-actin levels were normalised to untreated WT cells. Bars represent the average of GFAP-δ/β-actin levels of 3 independent experiments ± SEM. (WT, white; Mut, black; *, p<0.005).
Figure 3.
Induction of IL-6 and IL-1β in response to demand is impaired in Eif2b5R132H/R132H mice brain.
Intraperitoneal injections of 5 mg/kg LPS were administered to 4–6 four-weeks-old Eif2b5R132H/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.
Figure 4.
Activated primary astrocytes-microglia cultures from Eif2b5R132H/R132H mice do not exhibit increased global translation and display impaired production of cytokines.
Primary mixed glia cultures were isolated from Eif2b5R132H/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).
Figure 5.
Activated Eif2b5R132H/R132H primary astrocytes exhibit impaired production of cytokines.
Primary astrocytes were isolated from Eif2b5R132H/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).
Figure 6.
Activated Eif2b5R132H/R132H primary microglial cells exhibit impaired production of cytokines.
Primary microglial cells were isolated from Eif2b5R132H/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).