Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma - PubMed (original) (raw)

Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma

Wensheng Lin et al. J Cell Biol. 2005.

Abstract

Interferon-gamma (IFN-gamma) is believed to contribute to immune-mediated demyelinating disorders by targeting the myelin-producing oligodendrocyte, a cell known to be highly sensitive to the disruption of protein synthesis and to the perturbation of the secretory pathway. We found that apoptosis induced by IFN-gamma in cultured rat oligodendrocytes was associated with endoplasmic reticulum (ER) stress. ER stress also accompanied oligodendrocyte apoptosis and hypomyelination in transgenic mice that inappropriately expressed IFN-gamma in the central nervous system (CNS). Compared with a wild-type genetic background, the enforced expression of IFN-gamma in mice that were heterozygous for a loss of function mutation in pancreatic ER kinase (PERK) dramatically reduced animal survival, promoted CNS hypomyelination, and enhanced oligodendrocyte loss. PERK encodes an ER stress-inducible kinase that phosphorylates eukaryotic translation initiation factor 2alpha and specifically maintains client protein homeostasis in the stressed ER. Therefore, the hypersensitivity of PERK+/- mice to IFN-gamma implicates ER stress in demyelinating disorders that are induced by CNS inflammation.

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Figures

Figure 1.

Figure 1.

IFN-γ–induced apoptosis in cultured rat oligodendrocytes is associated with ER stress. (A) Untreated oligodendrocytes that underwent differentiation for 7 d. (B) Oligodendrocytes that underwent differentiation for 5 d and treatment with 70 U/ml IFN-γ for 48 h, revealing cell shrinkage and aggregation of cell bodies (arrow). (C and D) TUNEL and CNP double labeling for untreated oligodendrocytes that underwent differentiation for 7 d (C) and for oligodendrocytes that underwent differentiation for 5 d and treatment with 70 U/ml IFN-γ for 48 h (D). (E) Quantitation of TUNEL and CNPase double positive cells; *, P < 0.05. (F) Caspase-3 activity assay in the oligodendrocyte lysates; *, P < 0.01. (G) Real-time PCR analyses of the expression of BIP, CHOP, and caspase-12 in oligodendrocytes treated with 70 U/ml IFN-γ; *, P < 0.05. (E–G) Error bars represent standard deviation. (H) Western blot analyses of total eIF-2α, p-eIF-2α, and caspase-12 in oligodendrocytes treated with 70 U/ml IFN-γ. All experiments were repeated at least three times. Bars: (A and B) 30 μM; (C and D) 20 μM.

Figure 2.

Figure 2.

Hypomyelination induced by ectopically expressed IFN-γ is associated with ER stress. (A) Real-time PCR analyses for detection of mRNA in the brains of 14-d-old mice ectopically expressing IFN-γ (n = 3); *, P < 0.05; **, P < 0.01. Error bars represent standard deviation. (B) Western blot analyses for caspase-12 in the CNS of 14-d-old double transgenic mice released from doxyclycline at E 14. (C and D) BIP and CC1 double immunostaining in the spinal cord of 14-d-old double transgenic mice that received doxycycline (C) or were released from doxycycline at E 14 (D). (E and F) p-eIF-2α and CC1 double immunostaining in the spinal cord of 14-d-old double transgenic mice that received doxycycline (E) or were released from doxycycline at E 14 (F). (G and H) Caspase-12 and CC1 double immunostaining in the spinal cord of 14-d-old double transgenic mice that received doxycycline (G) or were released from doxycycline at E 14 (H). (C–H) n = 3; bar, 30 μM.

Figure 3.

Figure 3.

Hypersensitivity of PERK+_/_− mice to the conditional misexpression of IFN-γ. (A) Mouse survival curve (n = 40 for each group). (B and C) p-eIF-2α and CC1 double labeling in the spinal cord of 14-d-old GFAP/tTA; TRE/IFN-γ; PERK+_/_− mice that received doxycycline (B) or were released from doxycycline at E 14 (C). (B and C) n = 3; bar, 30 μM. (D) Real-time PCR analyses of mRNA levels in the brain of 14-d-old mice (n = 3). Error bars represent standard deviation.

Figure 4.

Figure 4.

Double transgenic mice with a PERK+_/_− background develop severe hypomyelination. (A and C) MBP immunostaining in the spinal cord of 14-d-old double transgenic mice that received doxycycline (A) or were released from doxycycline at E 14 (C). (B and D) MBP immunostaining in the spinal cord of 14-d-old GFAP/tTA; TRE/IFN-γ; PERK+_/_− mice that received doxycycline (B) or were released from doxycycline at E 14 (D). (A–D) n = 3; bar, 150 μM.

Figure 5.

Figure 5.

Double transgenic mice with a PERK+_/_− background develop severe hypomyelination. (A and B) Ultrastructural examination showing normal myelination in the spinal cord of 14-d-old double transgenic mice (A) and GFAP/tTA; TRE/IFN-γ; PERK+_/− mice (B) that received doxycycline. (C and D) Ultrastructural examination showing minor hypomyelination in the spinal cord of 14-d-old double transgenic mice (C) and severe hypomyelination in the spinal cord of 14-d-old GFAP/tTA; TRE/IFN-γ; PERK+/_− mice (D) released from doxycycline at E 14. (A–D) n = 3; bars, 1 μM. (E) The percentage of unmyelinated axons in the white matter of the cervical spinal cord was calculated from three mice per time point; *, P < 0.01. Error bars represent standard deviation.

Figure 6.

Figure 6.

The levels of MBP, PLP, and CGT mRNA were significantly decreased in the CNS of double transgenic mice with a PERK+_/_− background. Real-time PCR analyses for myelin gene expression in the brain of 14-d-old mice (n = 3); *, P < 0.05. Error bars represent standard deviation.

Figure 7.

Figure 7.

Double transgenic mice with a PERK+_/_− background lose the majority of oligodendrocytes in the CNS. (A) Quantitation of CC1-positive cells in the CNS of 14-d-old mice (n = 3); *, P < 0.05. (B and C) TUNEL and CC1 double labeling in the spinal cord of 14-d-old double transgenic mice (B) and GFAP/tTA; TRE/IFN-γ; PERK+_/− mice (C) that received doxycycline. (D and E) TUNEL and CC1 double labeling in the spinal cord of 14-d-old double transgenic mice (D) and GFAP/tTA; TRE/IFN-γ; PERK+/_− mice (E) released from doxycycline at E 14. (B–E) n = 3; bar, 60 μM; red fluorescence shows CC1 immunoreactivity; green fluorescence shows TUNEL stain; and blue fluorescence shows DAPI countstain. (F) Quantitation of TUNEL and CC1 double positive cells in the spinal cord of 14-d-old mice (n = 3); *, P < 0.01. (A and F) Error bars represent standard deviation. (G) Ultrastructural examination showing that apoptotic oligodendrocytes contained highly condensed chromatin mass, intact membrane, shrunken cytoplasm, and apoptosis body; bar, 2 μM.

Figure 8.

Figure 8.

Oligodendrocytes in adult mice are less sensitive to IFN-γ than actively myelinating oligodendrocytes from younger mice. (A) Real-time PCR analyses of mRNA levels in the brains of 10-wk-old mice (n = 3); *, P < 0.05. Error bars represent standard deviation. (B and C) BIP and CC1 double immunostaining in the cerebellum of 10-wk-old double transgenic mice (B) and GFAP/tTA; TRE/IFN-γ; PERK+_/− mice (C) that received doxycycline. (D and E) BIP and CC1 double immunostaining in the cerebellum of 10-wk-old double transgenic mice (D) and GFAP/tTA; TRE/IFN-γ; PERK+/− mice (E) released from doxycycline at 4 wk of age. (B–E) n = 3; bar, 60 μM; red fluorescence shows CC1 immunoreactivity; absence of green fluorescence shows that no cells express detectable levels of BIP; and blue fluorescence shows DAPI countstain. (F and G) Ultrastructural examination showing normal myelination in the cerebellum of 10-wk-old double transgenic mice (F) and GFAP/tTA; TRE/IFN-γ; PERK+/− mice (G) that received doxycycline. (H and I) Ultrastructural examination showing normal myelination in the cerebellum of 10-wk-old double transgenic mice (H) and GFAP/tTA; TRE/IFN-γ; PERK+/_− mice (I) released from doxycycline at 4 wk of age. (F–I) n = 3; bars, 2 μM.

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