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GSK3β regulates AKT-induced central nervous system axon regeneration via an eIF2Bε-dependent, mTORC1-independent pathway

  1. Xinzheng Guo
  2. William D Snider
  3. Bo Chen  Is a corresponding author
  1. Yale University School of Medicine, United States
  2. University of North Carolina at Chapel Hill, United States
Research Article
Cite this article as: eLife 2016;5:e11903 doi: 10.7554/eLife.11903
7 figures

Figures

Figure 1 with 2 supplements
AKT activation promotes axon regeneration and RGC survival.

(A) Detection of AKT Phosphorylation at Ser473 in Pten-deleted RGCs labeled by anti-Tuj1 immunohistochemistry, with or without (un) optic nerve injury. Scale bar, 20 µm. (B) Quantification of phospho-AKT immunofluorescence intensity following Pten deletion. Data are presented as mean ± s.d., n=4 per group. *p<0.001, Two-way ANOVA with Bonferroni post hoc test. (C) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after optic nerve injury from AAV-GFP or AAV-caAkt injected eyes. *, crush site. Scale bar, 200 µm. (D) Quantification of regenerating axons at different distances distal to the lesion site. Data are presented as mean ± s.d., n=5 per group. *p<0.01, +p<0.05, Two-way ANOVA with Bonferroni post hoc test. (E) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (F) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group. *p<0.01, Student’s t test.

https://doi.org/10.7554/eLife.11903.003
Figure 1—figure supplement 1
Developmental analysis of AKT phosphorylation in RGCs.

(A) Confocal images of retinal sections showing phospho-AKT immunoreactivity in Tuj1+ RGCs at postnatal day 7, 21, and 60. Scale bar, 20 µm. (B) Quantification of phospho-AKT immunofluorescence intensity relative to postnatal day 7. Data are presented as mean ± s.d., n=5 per group. *p<0.001, One-way ANOVA with Dunnett's test. (C) Confocal images of retinal sections showing PTEN immunoreactivity in Tuj1+ RGCs from AAV-GFP or AAV-Cre injected Ptenf/f eyes, Scale bar, 20 µm. (D) Quantification of PTEN immunofluorescence intensity. Data are presented as mean ± s.d., n=3 per group. *p<0.001, Student’s t test. (E) Confocal images of retinal sections showing double immunolabeling for Tuj1+ RGCs and AKT from AAV-GFP or AAV-caAkt injected eyes, Scale bar, 20 µm. (F) Quantification of AKT immunofluorescence intensity. Data are presented as mean ± s.d., n=3 per group. *p<0.01, Student’s t test.

https://doi.org/10.7554/eLife.11903.004
Figure 1—figure supplement 2
Inhibition of mTORC1 partially reduces AKT-induced axon regeneration.

(A) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after injury from AAV-GFP or AAV-caAkt injected eyes treated with vehicle or rapamycin. *, crush site. Scale bar, 200 µm. (B) Quantification of regenerating axons at different distances distal to the lesion site. Data are presented as mean ± s.d., n=6 per group. *p<0.01, +p<0.05, Two-way ANOVA with Bonferroni post hoc test. (C) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (D) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group. *p<0.001, One-way ANOVA with Dunnett's test. Data from AAV-GFP was used as comparison control.

https://doi.org/10.7554/eLife.11903.005
Figure 2 with 2 supplements
GSK3β is an essential downstream effector to mediate AKT-induced axon regeneration.

(A) Immunohistochemical detection of GSK3β phosphorylation at Ser9 in retinal sections from AAV-GFP or AAV-caAkt-injected eyes, either with or without optic nerve injury. Scale bar, 20 µm. (B) Quantification of phospho-GSK3β immunofluorescence intensity. Data are presented as mean ± s.d., n=4 per group. *p<0.001, Two-way ANOVA with Bonferroni post hoc test. (C) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after optic nerve injury from AAV-caAKT + AAV-GFP or AAV-caAkt + AAV-GSK3β S9A injected eyes. *, crush site. Scale bar, 200 µm. (D) Quantification of regenerating axons at different distances distal to the lesion site. Data are presented as mean ± s.d., n=5 per group. *p<0.01, +p<0.05, Two-way ANOVA with Bonferroni post hoc test. (E) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (F) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group.

https://doi.org/10.7554/eLife.11903.006
Figure 2—figure supplement 1
Developmental analysis of GSK3β phosphorylation in RGCs.

(A) Confocal images of retinal sections showing double immunolabeling for Tuj1+ RGCs and phospho-GSK3β at postnatal day 7, 21, and 60. Scale bar, 20 µm. (B) Quantification of phospho-GSK3β immunofluorescence intensity relative to postnatal day 7. Data are presented as mean ± s.d., n=5 per group. *p<0.001, One-way ANOVA with Dunnett's test.

https://doi.org/10.7554/eLife.11903.007
Figure 2—figure supplement 2
Pten deletion results in GSK3β phosphorylation in RGCs.

(A) Detection of GSK3β phosphorylation in RGCs by double immunolabeling for phospho-GSK3β (Ser9) and Tuj1 in retinal sections from Ptenf/f mice injected with AAV-GFP or AAV-Cre, with or without optic nerve crush. Scale bar, 20 µm. (B) Quantification of phospho-GSK3β immunofluorescence intensity. Data are presented as mean ± s.d., n=4 per group. *p<0.001, Two-way ANOVA with Bonferroni post hoc test.

https://doi.org/10.7554/eLife.11903.008
Figure 3 with 3 supplements
Deletion or inactivation of GSK3β promotes axon regeneration.

(A) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after optic nerve crush from Gsk3a−/− mice, Gsk3bf/f mice injected with AAV-GFP or AAV-Cre, or AAV-GSK3β K85A injected wild-type mice. *, crush site. Scale bar, 200 µm. (B) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (C) Quantification of regenerating axons at different distances distal to the lesion site. Data are presented as mean ± s.d., n=5 per group. *p<0.01, Two-way ANOVA with Bonferroni post hoc test. (D) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group.

https://doi.org/10.7554/eLife.11903.009
Figure 3—figure supplement 1
Cre-mediated Gsk3b deletion in RGCs.

(A) Confocal images of retinal sections showing double immunolabeling for Tuj1+ RGCs and GSK3β from AAV-GFP or AAV-Cre-injected Gsk3bf/f eyes, Scale bar, 20 µm. (B) Quantification of GSK3β immunofluorescence intensity. Data are presented as mean ± s.d., n=3 per group. *p<0.01, Student’s t test.

https://doi.org/10.7554/eLife.11903.010
Figure 3—figure supplement 2
A time course study of axon regeneration in Gsk3b-deleted RGCs.

(A-C) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 1, 3, or 7 days from Gsk3bf/f mice injected with AAV-GFP or AAV-Cre. *, crush site. Scale bar, 100 µm. (D) Quantification of regenerating axons at 7 days after injury from Gsk3bf/f mice injected with AAV-GFP or AAV-Cre at different distances distal to the lesion site. Data are presented as mean ± s.d., n=4 per group. *p<0.01, Two-way ANOVA with Bonferroni post hoc test. (E) Confocal images of optic nerve sections showing double immunolabeling for GAP43+ regenerating axons and chondroitin sulfate proteoglycan (CSPG). Scale bar, 20 µm. (F) Confocal images of optic nerve sections showing double immunolabeling for GAP43+ regenerating axons and glial fibrillary acidic protein (GFAP). Scale bar, 20 µm.

https://doi.org/10.7554/eLife.11903.011
Figure 3—figure supplement 3
AKT activation increases GSK3α phosphorylation.

(A) Immunohistochemical detection of GSK3α phosphorylation at Ser21 in retinal sections from AAV-GFP or AAV-caAkt injected eyes, with or without optic nerve injury. Scale bar, 20 µm. (B) Quantification of RGC phospho-GSK3α immunofluorescence intensity. Data are presented as mean ± s.d., n=4 per group. *p<0.001, Two-way ANOVA with Bonferroni post hoc test.

https://doi.org/10.7554/eLife.11903.012
Figure 4 with 1 supplement
Gsk3b deletion does not alter the mTORC1 pathway.

(A) Confocal images of retinal sections showing double immunolabeling for Tuj1+ RGCs and phospho-S6 from AAV-GFP, AAV-caAkt, or AAV-Cre (Gsk3bf/f)-injected eyes, with or without optic nerve injury. Scale bar, 20 µm. (B) Quantification of phospho-S6 immunofluorescence intensity. Data are presented as mean ± s.d., n=4 per group. *p<0.001, Two-way ANOVA with Bonferroni post hoc test.

https://doi.org/10.7554/eLife.11903.013
Figure 4—figure supplement 1
Gsk3b deletion-induced axon regeneration is sensitive to protein synthesis inhibition but not to mTORC1 inhibition.

(A) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after injury in Gsk3bf/fmice injected with AAV-Cre with vehicle, rapamycin, or anisomycin treatment. *, crush site. Scale bar, 200 µm. (B) Quantification of regenerating axons at different distances distal to the lesion site. Data are presented as mean ± s.d., n=6 per group. *p<0.01, +p<0.05, Two-way ANOVA with Bonferroni post hoc test. (C) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (D) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group.

https://doi.org/10.7554/eLife.11903.014
Figure 5 with 2 supplements
eIF2Bε is required for Gsk3b deletion-induced axon regeneration.

(A) Confocal images of retinal sections showing double immunolabeling for Tuj1+ RGCs and phospho-eIF2Bε from Gsk3bf/fmice injected with AAV-GFP (Control) or AAV-Cre (Gsk3b-/-), with or without optic nerve injury. Scale bar, 20 µm. (B) Quantification of phospho-eIF2Bε immunofluorescence intensity. Data are presented as mean ± s.d., n=5 per group. *p<0.001, Two-way ANOVA with Bonferroni post hoc test. (C) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after optic nerve crush from Gsk3bf/fmice injected with AAV-Cre + AAV-GFP or AAV-Cre + AAV-eIF2Bε E572A. *, crush site. Scale bar, 200 µm. (D) Quantification of regenerating axons from Gsk3bf/f mice injected with AAV-Cre + AAV-GFP or AAV-Cre + AAV-eIF2Bε E572A at different distances distal to the lesion site. Data are presented as mean ± s.d., n=9 per group. *p<0.01, Two-way ANOVA with Bonferroni post hoc test. (E) OPP Alexa Fluor 594 protein synthesis assay in retinal whole-mounts from Gsk3bf/f mice injected with AAV-GFP (Control), AAV-Cre (Gsk3b-/-) + AAV-GFP, or AAV-Cre + AAV-eIF2Bε E572A or treated with anisomycin. Scale bar, 25 µm. (F) Quantification of OPP fluorescence intensity. Data are presented as mean ± s.d., n=5 per group. *p<0.01, One-way ANOVA with Tukey’s test. (G) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (H) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group.

https://doi.org/10.7554/eLife.11903.015
Figure 5—figure supplement 1
Developmental analysis of eIF2Bε phosphorylation in RGCs.

(A) Confocal images of retinal sections showing double immunolabeling for Tuj1+ RGCs and phospho-eIF2Bε at postnatal day 7, 21, and 60. Scale bar, 20 µm. (B) Quantification of phospho-eIF2Bε immunofluorescence intensity relative to postnatal day 7. Data are presented as mean ± s.d., n=5 per group. *p<0.001, One-way ANOVA with Dunnett's test.

https://doi.org/10.7554/eLife.11903.016
Figure 5—figure supplement 2
Analysis of eIF2Bε phosphorylation in Gsk3a knockout mice.

(A) Confocal images of retinal sections showing double immunolabeling for Tuj1+ RGCs and phospho-eIF2Bε in Gsk3a-/- mice or their wild type litter mates as a control, with or without optic nerve injury. Scale bar, 20 µm. (B) Quantification of phospho-eIF2Bε immunofluorescence intensity. Data are presented as mean ± s.d., n= 5 per group.

https://doi.org/10.7554/eLife.11903.017
eIF2Bε is required for AKT-induced axon regeneration.

(A) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after injury from AAV-caAKT + AAV-GFP or AAV-caAkt + AAV-eIF2Bε E572A injected eyes. *, crush site. Scale bar, 200 µm. (B) Quantification of regenerating axons from AAV-caAKT + AAV-GFP or AAV-caAkt + AAV-eIF2Bε E572A injected eyes at different distances distal to the lesion site. Data are presented as mean ± s.d., n=5 per group. *p<0.05, +p<0.05, Two-way ANOVA with Bonferroni post hoc test. (C) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (D) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group.

https://doi.org/10.7554/eLife.11903.018
Figure 7 with 2 supplements
eIF2Bε activation promotes axon regeneration.

(A) OPP Alexa Fluor 594 protein synthesis assay in retinal whole-mounts from AAV-GFP or AAV-eIF2Bε S535A injected eyes. Scale bar, 25 µm. (B) Quantification of OPP fluorescence intensity. Data are presented as mean ± s.d., n=5 per group. *p<0.01, Student’s t test. (C) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after injury from wild-type mice injected with AAV-GFP or AAV-eIF2Bε S535A. *, crush site. Scale bar, 200 µm. (D) Quantification of regenerating axons from retinas injected with AAV-GFP or AAV-eIF2Bε S535A at different distances distal to the lesion site. Data are presented as mean ± s.d., n=5 per group. *p<0.01, +p<0.05, Two-way ANOVA with Bonferroni post hoc test. (E) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (F) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group.

https://doi.org/10.7554/eLife.11903.019
Figure 7—figure supplement 1
eIF2Bε-induced axon regeneration is sensitive to protein synthesis inhibition but not to mTORC1 inhibition.

(A) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after injury from AAV-eIF2Bε S535A injected eyes with vehicle, rapamycin, or anisomycin treatment. *, crush site. Scale bar, 200 µm. (B) Quantification of regenerating axons at different distances distal to the lesion site. Data are presented as mean ± s.d., n=5 per group. *p<0.01, Two-way ANOVA with Bonferroni post hoc test. (C) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (D) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group.

https://doi.org/10.7554/eLife.11903.020
Figure 7—figure supplement 2
A schematic illustration of GSK3β/eIF2Bε and mTORC1 signaling in AKT-induced CNS axon regeneration.
https://doi.org/10.7554/eLife.11903.021

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