Enhanced integrated stress response promotes myelinating oligodendrocyte survival in response to interferon-gamma

Abstract

The T-cell-derived, pleiotropic cytokine interferon (IFN)-gamma is believed to play a key regulatory role in immune-mediated demyelinating disorders of the central nervous system, including multiple sclerosis and experimental autoimmune encephalomyelitis. Our previous work has demonstrated that the endoplasmic reticulum (ER) stress response modulates the response of oligodendrocytes to this cytokine. The ER stress response activates the pancreatic ER kinase, which coordinates an adaptive program known as the integrated stress response by phosphorylating translation initiation factor 2alpha (eIF2alpha). In this study, we found that growth arrest and DNA damage 34 (GADD34), a stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2alpha, was selectively up-regulated in myelinating oligodendrocytes in mice that ectopically expressed IFN-gamma in the central nervous system. We also found that a GADD34 mutant strain of mice displayed increased levels of phosphorylated eIF2alpha (p-eIF2alpha) in myelinating oligodendrocytes when exposure to IFN-gamma, as well as diminished oligodendrocyte loss and hypomyelination. Furthermore, treatment with salubrinal, a small chemical compound that specifically inhibits protein phosphatase 1(PP1)-GADD34 phosphatase activity, increased the levels of p-eIF2alpha and ameliorated hypomyelination and oligodendrocyte loss in cultured hippocampal slices exposed to IFN-gamma. Thus, our data provide evidence that an enhanced integrated stress response could promote oligodendrocyte survival in immune-mediated demyelination diseases.

Figures

Figure 1

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).

Figure 2

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-γCNS+; GADD34 WT mice were significantly reduced compared to control IFN-γCNS−; GADD34 WT mice (B, *P < 0.05). In contrast, oligodendrocyte numbers in IFN-γCNS+; GADD34 mutant mice were comparable to control IFN-γCNS−; GADD34 WT mice or IFN-γCNS−; GADD34 mutant mice. N = 3 animals. C: MBP immunostaining showed severe hypomyelination in the corpus callosum of 21-day-old IFN-γCNS+; 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-γCNS+; GADD34 WT mice is significantly reduced compared to IFN-γCNS−; GADD34 WT mice or IFN-γCNS−; GADD34 mutant mice (*P < 0.05). Nevertheless, the percentage of myelinated axons in the corpus callosum of IFN-γCNS+; GADD34 mutant mice are comparable to IFN-γCNS−; GADD34 mutant mice. N = 3 animals. B and E: Error bars represent SD. Scale bars: 25 μm (A); 50 μm (C); 200 nm (D).

Figure 3

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-γCNS+; GADD34 WT mice compared to control IFN-γCNS−; GADD34 WT mice or IFN-γCNS−; GADD34 mutant mice. Moreover, the numbers of infiltrated T cells (arrow, CD3-positive T cells) in IFN-γCNS+; GADD34 mutant mice were comparable to IFN-γCNS+; 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-γCNS+; GADD34 WT mice compared to control IFN-γCNS−; GADD34 WT mice or IFN-γCNS−; GADD34 mutant mice. Nevertheless, the activation of microglia/macrophages in IFN-γCNS+; GADD34 mutant mice was comparable to IFN-γCNS+; 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-γCNS+; GADD34 WT mice compared to control IFN-γCNS−; GADD34 WT mice, or IFN-γCNS−; 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).

Figure 4

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.

Figure 5

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.

Figure 6

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).