mTORC1 is necessary but mTORC2 and GSK3β are inhibitory for AKT3-induced axon regeneration in the central nervous system
- Temple University Lewis Katz School of Medicine, United States
- University of Florida College of Medicine, United States
Figures
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, …
Figure 2 with 2 supplements
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 …
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 …
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 …
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 …
Figure 4 with 3 supplements
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 …
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 …
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 …
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 …
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 …
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 …
Figure 7 with 1 supplement
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 …
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 …
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 …
