mTORC1 is necessary but mTORC2 and GSK3β are inhibitory for AKT3-induced axon regeneration in the central nervous system

Abstract

Injured mature CNS axons do not regenerate in mammals. Deletion of PTEN, the negative regulator of PI3K, induces CNS axon regeneration through the activation of PI3K-mTOR signaling. We have conducted an extensive molecular dissection of the cross-regulating mechanisms in axon regeneration that involve the downstream effectors of PI3K, AKT and the two mTOR complexes (mTORC1 and mTORC2). We found that the predominant AKT isoform in CNS, AKT3, induces much more robust axon regeneration than AKT1 and that activation of mTORC1 and inhibition of GSK3β are two critical parallel pathways for AKT-induced axon regeneration. Surprisingly, phosphorylation of T308 and S473 of AKT play opposite roles in GSK3β phosphorylation and inhibition, by which mTORC2 and pAKT-S473 negatively regulate axon regeneration. Thus, our study revealed a complex neuron-intrinsic balancing mechanism involving AKT as the nodal point of PI3K, mTORC1/2 and GSK3β that coordinates both positive and negative cues to regulate adult CNS axon regeneration.

Keywords: AKT; GSK3; axon regeneration; mTORC1; mTORC2; mouse; neuroscience; optic nerve.

Figure 1.. Overexpression of three constitutively active AKT isoforms in RGCs.

(A) Confocal images of flat-mounted retinas showing co-labeling of HA tag, Tuj1, pAKT-S473 and their merged images, and pS6 in a separate retina sample. Scale bar, 20 µm. (B) Quantification of HA, pAKT-S473 or pS6 positive RGCs. Data are presented as means ± s.e.m, n=6. (C) Western blot of retina lysates from three biological replicates showing expression levels of HA-AKT isoforms, and phosphorylation levels of AKT-T308, AKT-S473 and S6. (D) Quantification of Western blots. *p<0.05. Data are presented as means ± s.e.m, n=3. DOI: http://dx.doi.org/10.7554/eLife.14908.003

Figure 2.. Differential effects of three AKT isoforms on axon regeneration and RGC survival.

(A) Confocal images of ON longitudinal sections showing regenerating fibers labeled with CTB-Alexa 488 2 weeks after ON crush. Scale bar, 100 µm. *crush site. (B) Quantification of regenerating fibers at different distances distal to the lesion site. Data are presented as means ± s.e.m, n=10–20. (C) Confocal images of flat-mounted retinas showing co-labeling of HA-AKTs and Tuj1, 2 weeks after ON crush. Scale bar, 20 µm. (D) Quantification of surviving RGCs, represented as percentage of Tuj1 positive RGCs in the injured eye, compared to the intact contralateral eye. Data are presented as means ± s.e.m, n=8–10. *p<0.05, **p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.14908.004

Figure 2—figure supplement 1.. Illustration of the regenerating axon quantification procedure.

Perpendicular lines on the ON sections were drawn distal to the crush site in increments of 250 µm till 1000 µm, then every 500 µm till no fibers were visible. The regenerating fibers that crossed the perpendicular lines were counted. The width of the nerve (R) was measured at the point (d) at which the counts were taken and used together with the thickness of the section (t = 8 μm) to calculate the number of axons per µm² area of the nerve. The total number of axons per section was calculated based on the formula: ∑a_d= πr² * (axon number) / (R*t), which then was averaged over 3 sections per animal. All CTB signals that were in the range of intensity that was set from lowest intensity to the maximum intensity after background subtraction were counted as individual fibers by Nikon NIS Element R4 software. DOI: http://dx.doi.org/10.7554/eLife.14908.005

Figure 2—figure supplement 2.. Endogenous expression of three AKT isoforms in RGCs.

(A) In situ hybridization images of retina sections showing expression of three AKT isoforms in ganglion cell layer (GCL). Scale bar, 20 µm. (B) Western blots of retina lysates from three biological replicates showing expression of three AKT isoforms. (C) Confocal images of flat-mounted retinas showing co-labeling of HA tag and Tuj1. Scale bar, 20 µm. (D) RGC-specific ribosome-associated translating mRNA purified by Ribo-IP from three replicates of WT mice. (E) RNA-seq showing expression levels of three AKT isoforms in RGCs. DOI: http://dx.doi.org/10.7554/eLife.14908.006

Figure 3.. Over expression of three AKT3 mutants in RGCs.

(A) Confocal images of flat-mounted retinas showing co-labeling of HA tag, Tuj1, pAKT-S473 and their merged images, and pS6 in a separate retina sample. Scale bar, 20 µm. (B) Western blot of retina lysates from three biological replicates showing expression level of HA-AKT, and phosphorylation level of AKT-T308, AKT-S473 and S6. (C) Quantification of Western blots. *p<0.05, **p<0.01, ***p<0.001. Data are presented as means ± s.e.m, n=3. DOI: http://dx.doi.org/10.7554/eLife.14908.007

Figure 4.. Phosphorylation of AKT3-T305 is necessary but phosphorylation of AKT3-S472 by mTORC2 is inhibitory for axon regeneration.

(A) Confocal images of ON longitudinal sections showing lack of regenerating fibers 2 weeks after ON crush. Scale bar, 100 µm. *crush site. (B) Quantification of regenerating fibers at different distances distal to the lesion site. Data are presented as means ± s.e.m, n=6–8. (C) Confocal images of ON longitudinal sections showing regenerating fibers labeled with CTB 2 weeks after ON crush. Scale bar, 100 µm. *crush site. (D) Quantification of regenerating fibers at different distances distal to the lesion site. *p<0.05, ***p<0.001. Data are presented as means ± s.e.m, n=20–30. DOI: http://dx.doi.org/10.7554/eLife.14908.008

Figure 4—figure supplement 1.. AAV-Cre-mediated RGC-specific deletion of Rictor, Rptor, Mtor or Gsk3a did not induce axon regeneration.

(A) Confocal images of ON longitudinal sections showing regenerating fibers labeled with CTB, 2 weeks after ON crush. Scale bar, 100 µm. *crush site. (B) Quantification of regenerating fibers at different distances distal to the lesion site. Data are presented as means ± s.e.m, n=6. **p<0.01, ***p<0.001 DOI: http://dx.doi.org/10.7554/eLife.14908.009

Figure 4—figure supplement 2.. Translocation of AKT in ON.

(A) Confocal images of ON longitudinal sections showing axons co-labeled with HA tag and Tuj1. Scale bar, 100 µm. (B) Confocal images of ON longitudinal sections showing regenerating fibers co-labeled with HA tag and CTB, 2 weeks after ON crush. Scale bar, 100 µm. *crush site. DOI: http://dx.doi.org/10.7554/eLife.14908.010

Figure 4—figure supplement 3.. The effects of AKT3 mutants on RGC survival.

(A) Confocal images of flat-mounted retinas showing Tuj1 positive RGCs, 2 weeks after ON crush. Scale bar, 20 µm. (B) Quantification of surviving RGCs, represented as percentage of Tuj1 positive RGCs in the injured eye, compared to the intact contralateral eye. *p<0.05, ***p<0.001. Data are presented as means ± s.e.m, n=8–10. DOI: http://dx.doi.org/10.7554/eLife.14908.011

Figure 5.. Increased phosphorylation of GSK3β-S9 after blocking AKT-S473 phosphorylation in RGCs.

(A) Confocal images of flat-mounted retinas showing co-labeling of HA tag, Tuj1, pAKT-S473 and their merged images, and pS6 in a separate retina sample. Scale bar, 20 µm. (B) Western blots of retina lysates from three biological replicates showing expression level of HA, and phosphorylation levels of AKT-T308, AKT-S473, S6 and GSK3β-S9. (C) Western blots of retina lysates from three biological replicates showing phosphorylation levels of GSK3β-S9. (D-H) Quantification of Western blots. *p<0.05, **p<0.01, ***p<0.001. Data are presented as means ± s.e.m, n=3. DOI: http://dx.doi.org/10.7554/eLife.14908.012

Figure 6.. mTORC1 and its downstream effectors are essential for AKT3-induced axon regeneration.

(A) Confocal images of ON longitudinal sections showing regenerating fibers labeled with CTB 2 weeks after ON crush. Scale bar, 100 µm. *crush site. (B) Quantification of regenerating fibers at different distances distal to the lesion site. *p<0.05, **p<0.01, ***p<0.001. Data are presented as means ± s.e.m, n=8–10. (C, D) Western blots of retina lysates from three biological replicates showing expression levels of HA-AKT3, and phosphorylation levels of S6. (E) Confocal images of flat-mounted retinas showing pS6 levels and Tuj1 positive RGCs, 2 weeks after ON crush. Scale bar, 20 µm. (F) Quantification of surviving RGCs, represented as percentage of Tuj1 positive RGCs in the injured eye, compared to the intact contralateral eye. Data are presented as means ± s.e.m, n=8–14. DOI: http://dx.doi.org/10.7554/eLife.14908.013

Figure 7.. GSK3β phosphorylation and inhibition by AKT is necessary and sufficient for axon regeneration.

(A,C,E) Confocal images of ON longitudinal sections showing regenerating fibers labeled with CTB 2 weeks after ON crush. Scale bar, 100 µm. *crush site. (B,D,F) Quantification of regenerating fibers at different distances distal to the lesion site. *p<0.05, **p<0.01, ***p<0.001. Data are presented as means ± s.e.m, n=8–15. DOI: http://dx.doi.org/10.7554/eLife.14908.014

Figure 7—figure supplement 1.. AKT3 expression and RGC survival after GSK3β manipulation.

(A) Western blots of retina lysates from three biological replicates showing expression level of HA-AKT3. (B) Quantification of Western blots. *p<0.05, **p<0.01, ***p<0.001. Data are presented as means ± s.e.m, n=3. (C) Confocal images of flat-mounted retinas showing individual labeling of pGSK3β, pS6 and Tuj1, 2 weeks after ON crush. Scale bar, 20 µm. (D) Quantification of surviving RGCs, represented as percentage of Tuj1 positive RGCs in the injured eye, compared to the intact contralateral eye. Data are presented as means ± s.e.m, n=8–10. DOI: http://dx.doi.org/10.7554/eLife.14908.015

Figure 8.. A schematic illustration depicting the interplay between AKT, mTORC1/2 and GSK3β in CNS axon regeneration.

The predominant isoform of AKT in brain and retina, AKT3, generates the most robust axon regeneration. Its function in axon regeneration is positively regulated by the PI3K/PDK1 pathway through phosphorylation of T308 (T305 in AKT3), and negatively regulated by the PI3K/mTORC2 pathway through phosphorylation of S473 (S472 in AKT3), through at least partially regulation of GSK3β phosphorylation and inhibition. Both AKT downstream effectors, activation of mTORC1 and phosphorylation/inhibition of GSK3β, synergistically promote axon regeneration; inhibition of GSK3β alone is also sufficient for axon regeneration. The question mark represents other upstream regulators of GSK3β that may also promote axon regeneration. Green color-coated molecules are pro-axon regeneration and dark color-coated molecules are anti-axon regeneration. DOI: http://dx.doi.org/10.7554/eLife.14908.016