The Rheb-mTOR pathway is upregulated in reactive astrocytes of the injured spinal cord

Abstract

Astrocytes in the CNS respond to tissue damage by becoming reactive. They migrate, undergo hypertrophy, and form a glial scar that inhibits axon regeneration. Therefore, limiting astrocytic responses represents a potential therapeutic strategy to improve functional recovery. It was recently shown that the epidermal growth factor (EGF) receptor is upregulated in astrocytes after injury and promotes their transformation into reactive astrocytes. Furthermore, EGF receptor inhibitors were shown to enhance axon regeneration in the injured optic nerve and promote recovery after spinal cord injury. However, the signaling pathways involved were not elucidated. Here we show that in cultures of adult spinal cord astrocytes EGF activates the mTOR pathway, a key regulator of astrocyte physiology. This occurs through Akt-mediated phosphorylation of the GTPase-activating protein Tuberin, which inhibits Tuberin's ability to inactivate the small GTPase Rheb. Indeed, we found that Rheb is required for EGF-dependent mTOR activation in spinal cord astrocytes, whereas the Ras-MAP kinase pathway does not appear to be involved. Moreover, astrocyte growth and EGF-dependent chemoattraction were inhibited by the mTOR-selective drug rapamycin. We also detected elevated levels of activated EGF receptor and mTOR signaling in reactive astrocytes in vivo in an ischemic model of spinal cord injury. Furthermore, increased Rheb expression likely contributes to mTOR activation in the injured spinal cord. Interestingly, injured rats treated with rapamycin showed reduced signs of reactive gliosis, suggesting that rapamycin could be used to harness astrocytic responses in the damaged nervous system to promote an environment more permissive to axon regeneration.

Figure 1.

EGF stimulation of astrocytes from adult spinal cord activates the mTOR pathway. A, EGF promotes phosphorylation of the EGF receptor and S6 kinase. Astrocytes were stimulated for 15 min with EGF (+) or vehicle (−), and lysates were probed by immunoblotting with antibodies to phosphorylated EGF receptor (p-EGFR) and phosphorylated S6 kinase (p-S6K) and reprobed with antibodies to the EGF receptor and S6 kinase. B, The Erk MAP kinase pathway is not involved in EGF-dependent S6 kinase phosphorylation. Astrocytes treated with the PD98059 inhibitor (+) or vehicle (−) were stimulated with EGF, and lysates were probed by immunoblotting with antibodies to phosphorylated S6 kinase (p-S6K) or phosphorylated Erk1 and Erk2 MAP kinases (p-Erk) and reprobed with antibodies detecting the nonphosphorylated forms of the proteins. The levels of phosphorylated S6 kinase determined from the optical density of the bands were normalized to total S6 kinase protein levels. The histogram shows average levels of phosphorylated S6 kinase relative to the level after EGF treatment. The error bars represent the SE from three independent experiments. PD98059 did not cause significant differences in S6 phosphorylation as determined by one-way ANOVA and Bonferroni's post hoc test. C, The PI3 kinase–Akt pathway mediates EGF-dependent Tuberin phosphorylation. Astrocytes incubated with the LY294002 PI3 kinase inhibitor (+) or vehicle (−) were stimulated with EGF, and lysates were probed by immunoblotting with the indicated antibodies. The levels of phosphorylated proteins were quantified as described in B for S6 kinase. The histogram shows average levels of phosphorylated Tuberin and phosphorylated Akt relative to the levels after EGF treatment. The error bars represent the SE from three independent experiments. ***p < 0.001 for the comparison between untreated and LY294002-treated by one-way ANOVA and Bonferroni's post hoc test. D, The mTORC1-selective inhibitor rapamycin blocks S6 kinase phosphorylation but not Akt phosphorylation. Astrocytes incubated with rapamycin (+) or vehicle (−) were stimulated with EGF, and the lysates were probed by immunoblotting with the indicated antibodies.

Figure 2.

Rheb is required for EGF-dependent S6 kinase phosphorylation in spinal cord astrocytes. A, The FTI-277 farnesyltransferase inhibitor decreases phosphorylation of S6 kinase. Astrocytes incubated with FTI-277 were stimulated with EGF and lysates were probed by immunoblotting with antibodies to phosphorylated S6 kinase (p-S6K) or Rheb, and reprobed with antibodies to S6 kinase. Note that nonfarnesylated Rheb has a different molecular weight, so Rheb immunoreactivity appears weaker because the protein is no longer concentrated in a single band. The levels of phosphorylated S6 kinase determined from the optical density of the bands were normalized to total S6 kinase protein levels. The histogram shows average levels of phosphorylated S6 kinase relative to the level after EGF treatment. The error bars represent the SE from three independent experiments, each with duplicate measurements. **p < 0.01 for the comparison between untreated and FTI-277-treated astrocytes by one-way ANOVA and Bonferroni's post hoc test. B, C, Lentiviral delivery of Rheb shRNA decreases Rheb mRNA and protein levels in spinal cord astrocytes. Astrocytes were infected with a lentivirus encoding Rheb shRNA or a control (GFP) shRNA. In B, Rheb mRNA was quantified by quantitative real-time PCR, and the histogram shows the averages ± SE from three independent experiments, each with duplicate measurements. ***p < 0.001 for the comparison with cells treated with control shRNA by one-way ANOVA and Bonferroni's post hoc test. In C, astrocytes were incubated with rapamycin (+) or vehicle (−) and stimulated with EGF. The levels of Rheb protein were determined from the optical density of the bands and normalized to the levels of β-tubulin in the lysates. The histogram shows average levels of Rheb relative to the level in unstimulated control cells. The error bars represent the SE from four independent experiments, each with duplicate measurements. ***p < 0.001 for the comparison of control shRNA- versus Rheb shRNA-treated samples by two-way ANOVA. D, Rheb knockdown decreases S6 kinase phosphorylation. Astrocytes infected with lentivirus encoding Rheb shRNA or a control shRNA were incubated with rapamycin (+) or vehicle (−) and stimulated with EGF. The histogram shows the average levels of phosphorylated S6 kinase normalized to total S6 kinase and relative to the level in EGF-stimulated control astrocytes. The error bars represent the SE from four independent experiments, each with duplicate samples. ***p < 0.001 for the comparison of control shRNA versus Rheb shRNA by one-way ANOVA and Bonferroni's post hoc test.

Figure 3.

mTORC1 is required for the growth of cultured spinal cord astrocytes. A, Fetal bovine serum promotes the growth of astrocytes. Astrocytes were grown for the indicated number of days and their growth was quantified using the MTT assay. The graph shows averages ± SE from three independent experiments, each with triplicate measurements. ***p < 0.001 for the comparison with control-treated astrocytes by two-way ANOVA and Bonferroni's post hoc test. B, C, Rapamycin inhibits astrocyte growth. The graph in B shows averages ± SE from three independent experiments, each with triplicate measurements using an MTT assay. The graph in C shows averages ± SE from triplicate measurements of cell numbers counted with a hemocytometer. **p < 0.01 and ***p < 0.001 for the comparison between serum- and rapamycin-treated astrocytes by two-way ANOVA and Bonferroni's post hoc test. D, Confluency and rapamycin inhibit S6 kinase phosphorylation and enhance Akt phosphorylation. Lysates from untreated and rapamycin-treated cells were probed by immunoblotting with the indicated antibodies. All the lanes are from the same gel, and a lane between untreated and rapamycin-treated samples was digitally removed. E, Rapamycin treatment increases the levels of IRS-2 and does not increase the levels of cleaved caspase 3. Lysates from untreated and rapamycin-treated cells were probed by immunoblotting with the indicated antibodies.

Figure 4.

mTORC1 promotes EGF-dependent cytoskeletal reorganization and migration of spinal cord astrocytes. A, EGF induces morphological changes in astrocytes. Astrocytes treated with rapamycin were stimulated for 15 min with EGF, fixed, and stained for paxillin (green), F-actin (red), and DNA (blue). Scale bar, 20 μm. B, Rapamycin inhibits astrocyte chemotactic migration toward EGF. Astrocytes were seeded on Transwell filters coated with laminin and allowed to migrate through the filters toward EGF for 4 h. The histogram shows the average number of cells that migrated to the lower side of the filters in the different conditions relative to control. Error bars indicate the SE from three independent experiments, each with triplicate samples. C, Lysates from astrocytes treated for 4 h with rapamycin and untreated controls were probed by immunoblotting with antibodies to phosphorylated Akt (p-Akt) and phosphorylated S6 kinase (p-S6 kinase) and reprobed with antibodies to Akt and S6 kinase.

Figure 5.

The EGF receptor is activated in vivo in the injured spinal cord. A, The increased vimentin and glial fibrillary acidic protein (GFAP) immunoreactivity reveals the presence of reactive astrocytes in the white matter of the lumbar spinal cord (asterisks) following an ischemic injury. Scale bar, 500 μm. B, Immunolabeling for phosphorylated EGF receptor (green) is increased in the white matter of the spinal cord after ischemia and shows substantial colocalization with the astrocytic glutamate transporter GLT-1 (red) as well as partial colocalization with the cytoskeletal protein vimentin (blue) in triple-labeled sections. Scale bars, 20 μm.

Figure 6.

Rheb is upregulated and mTORC1 is activated in vivo in the injured spinal cord. A, Immunolabeling for phosphorylated S6 ribosomal protein (green) is dramatically upregulated in the vimentin-positive astrocytes (red) within the white matter of injured spinal cord. Scale bar, 20 μm. B, Immunolabeling for Rheb (green) is also dramatically upregulated in the vimentin-positive astrocytes (red) in the white matter of the injured spinal cord after ischemia (asterisks). Scale bars, 500 μm (top) and 20 μm (bottom).

Figure 7.

Rapamycin administration decreases reactive gliosis in the injured spinal cord. A, Lysates from the lumbar spinal cord of uninjured naive rats (N1 through N4), and rats treated with vehicle control (−) or rapamycin (+) after spinal cord ischemia, were probed by immunoblotting with antibodies to vimentin and GFAP. The levels of vimentin and GFAP determined from the optical density of the bands were normalized for protein loading based on two Coomassie Blue-stained protein bands (the ∼75 kDa protein band shown in the figure and the ∼18 kDa band shown in supplemental Fig. 4, available at www.jneurosci.org as supplemental material). The histograms show average vimentin and GFAP levels relative to the levels in the injured, vehicle control-treated rats. *p < 0.05 and **p < 0.01 by one-way ANOVA followed by Dunnett's post hoc test for the comparison with vehicle-treated injured spinal cord. B, Vimentin and GFAP immunoreactivity in the spinal cords of naive and vehicle control-treated (Ischemic-Vehicle) or rapamycin-treated (Ischemic-Rapamycin) spinal cords. Reduction in overall vimentin and GFAP immunoreactivity in the rapamycin-treated compared with vehicle-treated spinal cords suggests decreased reactive gliosis. The arrows in the bottom left panels indicate the epicenter of injury in the rapamycin-treated spinal cord. The right panels, showing higher magnification views of the intermediate zone, reveal that there are few GFAP-positive cells in the injury epicenter of the rapamycin-treated spinal cord; examples of vimentin-positive but GFAP-negative cells in the injury epicenter are indicated by arrows. The scale bar represents 500 μm in the left panels and 70 μm in the right panels. C, The scheme on the left illustrates the spinal cord regions used for quantification: VC, ventral column; IZ, intermediate zone; LC, lateral column. The histograms show average vimentin and GFAP levels in the ventral column, intermediate zone, and lateral column, normalized to the levels in the injured, vehicle-treated spinal cords. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc test for the comparison with vehicle-treated injured spinal cord.