Regulation of PERK-eIF2α signalling by tuberous sclerosis complex-1 controls homoeostasis and survival of myelinating oligodendrocytes

Abstract

Tuberous sclerosis complex-1 or 2 (TSC1/2) mutations cause white matter abnormalities, including myelin deficits in the CNS; however, underlying mechanisms are not fully understood. TSC1/2 negatively regulate the function of mTOR, which is required for oligodendrocyte differentiation. Here we report that, unexpectedly, constitutive activation of mTOR signalling by Tsc1 deletion in the oligodendrocyte lineage results in severe myelination defects and oligodendrocyte cell death in mice, despite an initial increase of oligodendrocyte precursors during early development. Expression profiling analysis reveals that Tsc1 ablation induces prominent endoplasmic reticulum (ER) stress responses by activating a PERK-eIF2α signalling axis and Fas-JNK apoptotic pathways. Enhancement of the phospho-eIF2α adaptation pathway by inhibition of Gadd34-PP1 phosphatase with guanabenz protects oligodendrocytes and partially rescues myelination defects in Tsc1 mutants. Thus, TSC1-mTOR signalling acts as an important checkpoint for maintaining oligodendrocyte homoeostasis, pointing to a previously uncharacterized ER stress mechanism that contributes to hypomyelination in tuberous sclerosis.

Figure 1. Tsc1 ablation results in OL differentiation and maturation defects.

(a) Immunostaining for Tsc1 in OPCs (A2B5⁺), differentiating and maturating OLs (CNP⁺), terminally differentiated OL (MBP⁺) induced by treatment with T3 for 0, 1 and 3 days (left to right), respectively. Scale bar, 10 μm. (b) Expression of Tsc1 and Tsc2 assessed by western blot in OPCs treated with T3 for 0, 1 and 3 days. β-actin was used as loading control. (c) Schematic diagram of Cre-mediated excision of the floxed Tsc1 exons 17 and 18, a region of the gene that encodes an essential coiled-coil domain. (d) Expression of Tsc1 and Tsc2 in the corpus callosum of control and Tsc1cKO mice at P14 was examined by western blot. (e) Primary OPCs from control and Tsc1cKO mice were immunostained with antibodies to Tsc1 and Sox10. Scale bar, 10 μm. (f) The optic nerve of Tsc1cKO mice at P12 appears translucent compared with the control. (g–i) Expression of MBP and Plp1 mRNAs was examined by in situ hybridization in the (g,h) cortex and (i) spinal cord of Tsc1cKO and control mice at indicated ages. Scale bar, 100 μm. (j,k) MBP immunostaining in the (j) cerebellum and (k) cerebral cortex of control, and Tsc1cKO at P28 and P67. Scale bar, 100 μm. Arrows indicate white matter tracts. (l) Expression of MBP in the corpus callosum of control and Tsc1cKO mice at P14 was examined by western blot. β-actin was used as loading control.

Figure 2. Tsc1cKO mice develop myelinogenesis defects.

(a) Representative electron micrographs of optic nerves in control and Tsc1cKO mutants at P14, P28 and P60. Scale bar, 2 μm. (b) The percentages of myelinated axons in the optic nerves of control and Tsc1cKO mice at indicated stages. Data represent the mean±s.e.m. from three animals; ***P<0.001; Student's t-test. (c–e) g-ratio plotted versus axon diameters in the optic nerves of control and Tsc1cKO mice at P14 (c), P28 (d) and P60 (e). The data were displayed using scatter plots. n=3 animals for each genotype (≥150 myelinating axons were counted for each genotype). Student's t-test. (f) Representative electron micrographs of the spinal white matter in controls and Tsc1cKO mutants at indicated stages. Scale bar, 2 μm. (g) The percentages of myelinated axons in the spinal cord white matter of control and Tsc1cKO mice at indicated stages. Data represent the mean±s.e.m. from three animals; *P<0.05; ***P<0.001; Student's t-test. (h–j) g-ratio plotted versus axon diameters in the spinal white matter of control and Tsc1cKO mice at P14 (h), P28 (i) and P60 (j). The data were displayed using scatter plots. n=3 animals for each genotype (≥150 myelinating axons were counted for each genotype). Student's t-test.

Figure 3. Hyperactivation of mTOR signalling contributes to dysmyelination in Tsc1 mutants.

(a) Representative images of the corpus callosum of control and Tsc1cKO mutants treated with vehicle and rapamycin from P5 to P14 immunostained with p-S6 and CC1. Scale bar, 50 μm. (b) Lysates of control and Tsc1cKO corpus callosum at P14 assayed by western blot using antibodies to MBP, p-mTOR, p-S6 and S6. β-actin was used as loading control. (c) The number of CC1⁺ cells per mm² from the corpus callosum of control and Tsc1cKO mice-treated vehicle and rapamycin. Data represent the mean±s.e.m from four animals per genotype. *P<0.05; **P<0.01; *** P<0.001, One-way analysis of variance (ANOVA) with Tukey's multiple-comparison test. (d) The corpus callosum of control and Tsc1cKO mutants at P14-treated vehicle and rapamycin from P5 to P14 was immunostained with MBP. Scale bar, 100 μm. (e) Percentage of MBP area in the cortex of control and Tsc1cKO mice treated with vehicle or rapamycin. Data represent the mean±s.e.m from four animals per genotype. *P<0.05; **P<0.01; ***P<0.001. One-way ANOVA with Tukey's multiple-comparison test. (f) OPCs isolated from control and Tsc1cKO mice were cultured in the differentiation medium for 24 h with vehicle and rapamycin, and immunostained with GalC and p-S6. Scale bar, 50 μm. (g) The percentage of GalC⁺ cells in cultures of control and Tsc1cKO OPCs. Data represent the mean±s.e.m. from three independent experiments. *P<0.05; **P<0.01; ***P<0.001. One-way ANOVA with Tukey's multiple-comparison test.

Figure 4. Tsc1 ablation leads to OPC loss and inhibits cell cycle exit.

(a) Expression of PDGFRα examined by in situ hybridization in the cortex of control and Tsc1cKO mutants at E17.5 and P0. Scale bar, 100 μm. (b) Numbers of PDGFRα⁺ cells in the cortex per unit area (0.4 mm²) in control and Tsc1cKO mutants at indicated stages. Data represent the mean±s.e.m. from three animals. * P<0.05; Student's t-test. (c) Control and Tsc1cKO mice at P2 were pulse-labelled with BrdU for 24 h. Cortical sections collected at P3 were immunostained with antibodies to BrdU, Ki67 and Olig2. (d) The percentage of BrdU⁺ and Olig2⁺ OPCs that are Ki67⁺ in control and Tsc1cKO cortices. Data represent the mean±s.e.m. from three animals. *P<0.05; Student's t-test. (e) Cortical sections at P3 were immunostained with BrdU, Ki67 and Olig2. Scale bar, 50 μm. (f) Expression of PDGFRα by in situ hybridization in cortices of control and Tsc1cKO mutants at indicated stages. Scale bar, 100 μm. (g) Quantification of PDGFRα⁺ OPCs in the cortex per unit area (0.4 mm²) in control and Tsc1cKO mutants at indicated stages. Data represent the mean±s.e.m. from three animals. **P<0.01; Student's t-test. (h,i) Representative images (h) and percentage (i) of cells expressing cleaved Caspase 3 (cl-Casp3) among Olig2⁺ cells in the cortex of control and Tsc1cKO mutants at P3. Data represent the mean±s.e.m from three animals. **P<0.01; Student's t-test. Scale bar, 25 μm. (j,k) Representative electron microscopy images (j) and quantification (k) of dying OLs in the spinal cord of Tsc1cKO mutants at P14. Data represent the mean±s.e.m from three animals. Scale bar, 2 μm.

Figure 5. Transcriptome profiling reveals enrichment of the cell survival and apoptotic pathway genes in Tsc1cKO mutants.

(a) Heatmap depicts gene expression of representative myelination-associated and apoptosis-related genes in control and Tsc1cKO optic nerves at P12. (b,c) Gene ontology analyses of pathway enrichment among (b) downregulated and (c) upregulated genes in Tsc1cKO as compared with control. (d–f) qRT–PCR analysis of representative (d) myelination-related genes and differentiation inhibitors, (e) lipid synthesis genes, and (f) apoptosis and stress response genes from optic nerves of control and Tsc1cKO mice at P12. Data represent the mean±s.e.m. from three animals. * P<0.05; ** P<0.01; *** P<0.001; Student's t-test.

Figure 6. Tsc1 ablation activates cell apoptotic program.

(a,b) Representative images showing the corpus callosum of P14 control and Tsc1cKO mice immunostained with antibodies to (a) Fas and CC1, and (b) CHOP and Olig2. Scale bars, 25 μm. (c) Percentage of CHOP⁺/Olig2⁺ cells in control and Tsc1cKO cortices at P14. Data represent the mean±s.e.m. from three animals. *P<0.05; **P<0.01; ***P<0.001; Student's t-test. (d) Primary OPCs from control and Tsc1cKO pups cultured in T3-containing differentiation medium for 24 h were immunostained with CHOP and Fas. Scale bars, 25 μm. (e) Western blot analysis of extracts from primary OPCs isolated from control and Tsc1cKO animals with antibodies to Fas, CHOP, p-JNK, p-S6 and p-4EBP1; β-actin: loading control. (f) Primary OPCs from wild-type mice were treated with solvent and 100 ng ml⁻¹ FasL for 24 h, and immunostained with antibodies to cl-Casp3 and Sox10. Scale bar, 25 μm. (g) Percentage of cl-Casp3⁺/Sox10⁺ cells from above wild-type OPCs treated with FasL. Data represent the mean±s.e.m. from three independent experiments. ***P<0.001; Student's t-test.

Figure 7. Elevation of adaptive stress responses enhances OL survival.

(a) OPCs from control and Tsc1cKO mutants at P5 were cultured in PDGFAA proliferation (Pro) or T3 differentiation media (Diff) for 24 h and stained with EthD-1 and Calcein AM. Corresponding upper and lower panels were taken in the same field. Scale bar, 50 μm. (b) The percentage of dying cells among OPCs isolated from control and Tsc1cKO mutants in proliferation or differentiation medium for 24 h. Data represent the mean±s.e.m. from three independent experiments; ***P<0.001, Student's t-test. (c–f) Primary OPCs from control and Tsc1cKO pups were cultured under (c,d) proliferation or (e,f) differentiation conditions for 24 h. The cell lysates were subject to western blot analysis using antibodies to p-PERK, p-eIF2α and eIF2α; α-tubulin: loading control. p-eIF2α and p-PERK levels were normalized to α-tubulin. The graphs in d and f depict the fold change of expression levels in Tsc1cKO over control cells (data represent the mean±s.e.m. from three independent experiments). *P<0.05; Student's t-test. (g) qRT–PCR of Gadd34 from control and Tsc1cKO OPCs under proliferation or differentiation conditions for 24 h. Data represent the mean±s.e.m from three independent experiments. *P<0.05; **P<0.01; Student's t-test. (h) Expression of Gadd34 and p-PERK in the spinal cord of control and Tsc1cKO mice at P14 was examined by western blot. α-Tubulin was used as loading control. (i) Primary OPCs from control and Tsc1cKO mice were treated with Gadd34 siRNA or scramble control siRNA 24 h and immunostained with antibodies to cl-Casp3 and Sox10. Scale bar, 25 μm. (j) qRT–PCR analysis of Gadd34 mRNA expression from OPCs transfected with Gadd34 siRNA or scramble control siRNA for 48 h. (k) Percentage of cl-Casp3⁺ Sox10⁺ cells from above-treated OPCs in i. Data represent the mean±s.e.m from three independent experiments. ***P<0.001; one-way analysis of variance test with Tukey's multiple-comparison test.

Figure 8. Activation of p-eIF2α-mediated signalling by guanabenz protects OLs and enhances myelination.

(a) OPCs from control and Tsc1cKO mice treated with 5 μM guanabenz for 24 h in T3-containing differentiation medium immunostained with cl-Casp3 and Sox10. Scale bar, 25 μm. (b) The percentage of cl-Casp3⁺/Sox10⁺ cells from guanabenz-treated OPCs from control and Tsc1cKO mice. Data represent the mean±s.e.m. from three independent experiments. ***P<0.001, Student's t-test. (c) Western blot analysis of p-eIF2α and eIF2α in OPCs from guanabenz or vehicle-treated Tsc1cKO animals; α-tubulin, loading control. (d) The corpus callosum of control and Tsc1cKO mice treated with vehicle and guanabenz from P7 to P14 was removed at P15, and immunostained with PDGFRα and CC1. Scale bar, 50 μm. (e,f) Numbers of CC1⁺ cells in the corpus callosum (e) and PDGFRα⁺ cells in the cortex (f) from vehicle or guanabenz-treated control and Tsc1cKO mice. Whiskers in boxplots show the minimum and maximum, boxes extend from the first to the third quartiles with cross lines at the medians. n=6 animals per group. *P<0.05. One-way analysis of variance with Tukey's multiple-comparison test. (g) Percentage of Ki67⁺ and Olig2⁺ cells from the cortex of control and Tsc1cKO mice treated with vehicle or guanabenz. Data represent the mean±s.e.m. from six animals. (h) Representative images showing MBP in the corpus callosum of control and Tsc1cKO mice treated with vehicle or guanabenz. Scale bar, 50 μm. (i) Western blot analysis of MBP in the cortices from control and Tsc1cKO animals treated with vehicle or guanabenz; β-actin, loading control. (j) A schematic diagram of Tsc1/2 mutation-induced ER stress in OL homoeostasis. Tsc1 loss leads to mTOR activation, resulting in excessive protein translation and ER stress by activating the PERK–eIF2α–ATF4 adaptation pathway and Fas/p-JNK apoptotic programs. Sustained ER stress coupled with apoptotic pathway activation contributes to the cell death during OL differentiation in Tsc1 mutants. Inhibition of Gadd34-PP1 by guanabenz (GBZ) enhances p-eIF2α-mediated adaptive responses and partially rescues OL death and myelination defects in Tsc1 mutants.