PMC

The myelination process in hippocampal slice cultures. A: MBP immunostaining showed that myelin was undetectable in hippocampal slice cultures at 7 days of culture. B: MBP immunostaining showed abundant myelin in hippocampal slice cultures at 21 days of culture. N = 3 cultured slices. Scale bar = 50 μm.

Sal treatment decreased hypomyelination and oligodendrocyte loss elicited by IFN-γ. A–D: MBP immunostaining revealed that 100 U/ml of IFN-γ markedly inhibited myelination in cultured hippocampal slices and that 40 μmol/L sal treatment reduced the inhibition (A: untreated; B: sal control; C: IFN-γ; D: IFN-γ + sal). N = 3 cultured slices. E–I: Rip immunostaining showed that oligodendrocytes in cultured hippocampal slices exposed to IFN-γ were significantly reduced compared to the untreated slices (I: *P < 0.05) and that 40 μmol/L sal treatment significantly attenuated the severity of oligodendrocyte loss (I: *P < 0.05) (E: untreated; F: sal control; G: IFN-γ; H: IFN-γ + sal). N = 3 cultured slices, error bars represent SD. Scale bars: 150 μm (D); 25 μm (H).

Sal treatment increased the levels of p-eIF2α in the cultured hippocampal slices exposed to IFN-γ. A: Western blot analysis showed that IFN-γ slightly elevated the levels of p-eIF2α and ATF4 in cultured hippocampal slices and that sal treatment further increased the levels of p-eIF2α and ATF4. B: Densitometry analysis of Western blot results showed that 100 U/ml of IFN-γ treatment increased the levels of p-eIF2α and ATF4 in cultured hippocampal slices compared to untreated slices or sal-treated slices (*P < 0.05), and that 40 μmol/L sal combined with 100 U/ml of IFN-γ treatment further elevated the levels of p-eIF2α and ATF4 in cultured hippocampal slices compared to 100 U/ml of IFN-γ-treated slices (*P < 0.05). The relative protein levels are relative to actin. The experiments have been repeated three times, error bars represent SD. C: Western blot analysis revealed that IFN-γ dramatically reduced the levels of MBP in cultured hippocampal slices and that sal treatment ameliorated the reduction. D: Densitometry analysis of Western blot results showed that 100 U/ml of IFN-γ significantly reduced the levels of MBP in cultured hippocampal slices (*P < 0.05) and that 40 μmol/L sal treatment significantly ameliorated the reduction (*P < 0.05). The relative protein levels are relative to actin. The experiments have been repeated three times, error bars represent SD.

GADD34 inactivation increased the levels of p-eIF2α in oligodendrocytes in mice expressing IFN-γ in the CNS. A: Real-time PCR analysis revealed increased levels of GADD34 mRNA in the brain of mice expressing IFN-γ in the CNS. GADD34 mRNA levels are relative to the housekeeping gene GAPDH. N = 3 animals, error bars represent SD, *P < 0.05. B and C: CC1 and GADD34 double labeling showed elevated levels of GADD34 in oligodendrocytes (arrows, double-positive cells) in the corpus callosum of 21-day-old mice expressing IFN-γ in the CNS. Inset: High-magnification image showed the distribution of GADD34 in oligodendrocytes. D: CC1 and p-eIF2α double labeling showed modest activation of eIF2α in oligodendrocytes (arrows, double-positive cells) in the corpus callosum of 21-day-old mice expressing IFN-γ in the CNS, and GADD34 inactivation further increased the level of p-eIF2α in oligodendrocytes (arrows, double-positive cells). B, C, and D: n = 3 animals. E: Quantitative analysis showed that the presence of IFN-γ significantly increased the percentage of CC1/p-eIF2α double-positive cells in the corpus callosum of 21-day-old mice, and GADD34 inactivation further increased the percentage. N = 3 animals, error bars represent SD, *P < 0.05. Scale bars: 4 μm (C, inset); 10 μm (C, D).

GADD34 inactivation did not significantly affect immune response induced by IFN-γ. A: CD3 immunostaining showed few infiltrated T cells (arrow, CD3-positive T cells) in the corpus callosum of 21-day-old IFN-γ^CNS+; GADD34 WT mice compared to control IFN-γ^CNS−; GADD34 WT mice or IFN-γ^CNS−; GADD34 mutant mice. Moreover, the numbers of infiltrated T cells (arrow, CD3-positive T cells) in IFN-γ^CNS+; GADD34 mutant mice were comparable to IFN-γ^CNS+; GADD34 WT mice. N = 3 animals. B: CD11b immunostaining showed that the presence of IFN-γ dramatically activated microglia/macrophages in the corpus callosum of 21-day-old IFN-γ^CNS+; GADD34 WT mice compared to control IFN-γ^CNS−; GADD34 WT mice or IFN-γ^CNS−; GADD34 mutant mice. Nevertheless, the activation of microglia/macrophages in IFN-γ^CNS+; GADD34 mutant mice was comparable to IFN-γ^CNS+; GADD34 WT mice. N = 3 animals. C: Real-time PCR analysis revealed significantly increased levels of IFN-γ, TNF-α, and iNOS mRNA in the brain of 21-day-old IFN-γ^CNS+; GADD34 WT mice compared to control IFN-γ^CNS−; GADD34 WT mice, or IFN-γ^CNS−; GADD34 mutant mice (*P < 0.05). Importantly, GADD34 inactivation did not change the levels of IFN-γ, TNF-α, and iNOS mRNA in the brain mice expressing IFN-γ in the CNS. N = 3 animals, error bars represent SD. Scale bars: 10 μm (A); 25 μm (B).

GADD34 inactivation ameliorated oligodendrocyte loss and hypomyelination elicited by IFN-γ. A and B: CC1 immunostaining showed that oligodendrocytes in the corpus callosum of 21-day-old IFN-γ^CNS+; GADD34 WT mice were significantly reduced compared to control IFN-γ^CNS−; GADD34 WT mice (B, *P < 0.05). In contrast, oligodendrocyte numbers in IFN-γ^CNS+; GADD34 mutant mice were comparable to control IFN-γ^CNS−; GADD34 WT mice or IFN-γ^CNS−; GADD34 mutant mice. N = 3 animals. C: MBP immunostaining showed severe hypomyelination in the corpus callosum of 21-day-old IFN-γ^CNS+; GADD34 WT mice, and GADD34 inactivation protected against hypomyelination elicited by IFN-γ. N = 3 animals. D: Electron microscope analysis revealed that the presence of IFN-γ reduced the number of myelinated axons in the corpus callosum of 21-day-old mice on a GADD34 WT background and that GADD34 inactivation ameliorated the reduction in myelinated axons in mice expressing IFN-γ in the CNS. N = 3 animals. E: Electron microscope analysis revealed that the percentage of myelinated axons in the corpus callosum of 21-day-old IFN-γ^CNS+; GADD34 WT mice is significantly reduced compared to IFN-γ^CNS−; GADD34 WT mice or IFN-γ^CNS−; GADD34 mutant mice (*P < 0.05). Nevertheless, the percentage of myelinated axons in the corpus callosum of IFN-γ^CNS+; GADD34 mutant mice are comparable to IFN-γ^CNS−; GADD34 mutant mice. N = 3 animals. B and E: Error bars represent SD. Scale bars: 25 μm (A); 50 μm (C); 200 nm (D).