Fig 1.
A map of AAV2 construct used to knockdown PTEN.
The shRNA.PTEN was inserted into the gap between BamHI and EcoRI, expressed under the control of an U6 promoter. A robust and non-cell-specific chicken β-actin (CBA) promoter controlled the expression of ZsGreen acting as control reporter.
Fig 2.
FluoroGold retrograde labeling of RGCs identifying the complete axotomy.
No RGCs was labeled with FluoroGold in retinal longitudinal sections (A) and whole-mounts (C) of axotomized rats while numerous RGCs were labeled with FluoroGold in retinal longitudinal sections (B)and whole-mounts (D) of intact rats. Scale bar, 100μm.
Fig 3.
FluoroGold retrograde labeling evaluating the transduction efficiency of AAV2 vectors 4 weeks after injection.
Retinal whole-mounts displayed FluoroGold-labeled (white) RGCs and gene-transduced (green) RGCs from the same retinal regions of rats intravitreally injected Wt AAV2-shRNA.PTEN-GFP or Y444F AAV2-shRNA.PTEN-GFP vector (A-H). Quantifying the percentage of GFP-positive cells in FluoroGold-labeled cells revealed the transduction capacity of Y444F AAV2-shRNA.PTEN-GFP was significantly stronger than that of Wt AAV2-shRNA.PTEN-GFP (I). ** P < 0.01 as tested by Student’s t-test. Scale bar, 100μm.
Fig 4.
Immunofluorescence displaying RGCs and Müller cells transgene expressing GFP 4 weeks after intravitreal injection.
Merged image showed colocalization of GFP fluorescence and TUJ1 staining in retinal flat-mounts from eyes treated with Y444F AAV2-shRNA.PTEN-GFP (A-C) or Wt AAV2-shRNA.PTEN-GFP (D-F), showed colocalization of GFP fluorescence and GS staining in retinal sections from eyes treated with Y444F AAV2-shRNA.PTEN-GFP (G-I) or Wt AAV2-shRNA.PTEN-GFP (J-L). Scale bar, 50μm.
Fig 5.
Western-blot assessing the expression of PTEN, pS6, and GLAST in retina 4 weeks after intravitreal injection of AAV2 vectors.
Expression of PTEN in retinas transduced with Y444F AAV2-shRNA.PTEN or Wt AAV2-shRNA.PTEN significantly decreased compared with Wt AAV2- GFP or intact control, and that the knockdown extent with Y444F AAV2-shRNA.PTEN was higher than that with Wt AAV2-shRNA.PTEN (A, B). As a result, transduction with Y444F AAV2-shRNA.PTEN significantly promoted the expression of pS6 (A, C). Levels of GLAST were not altered in the retinas transduced with any kinds of vectors compared with intact control (A, D). *P < 0.05, **P < 0.01 in ANOVA followed by Bonferroni’s post-test.
Fig 6.
Immunofluorescence testing PTEN in retinas 4 weeks after intravitreal injection of AAV2 vectors.
Immunofluorescence of retinal sections showed PTEN expression in retinas following Wt AAV2-GFP (A-C), Y444FA AV2-shRNA.PTEN (D-F), or Wt AAV2-shRNA.PTEN (G-I) injection. Quantification of PTEN expression, measured by ImageJ densitometry method, revealed a significant difference in GCL and INL treated with Wt AAV2-GFP, Wt AAV2-shRNA.PTEN, and Y444F AAV2-shRNA.PTEN respectively (J). **P < 0.01 in ANOVA followed by Bonferroni’s post-test. Scale bar, 100μm.
Fig 7.
pS6 immunoreactivity in retinas 4 weeks after AAV2 vectors intravitreal injection.
Immunofluorescence of sections showed pS6 positive cells in retina following Wt AAV2-GFP (A-C), Wt AAV2-shRNA.PTEN (D-F), or Y444F AAV2-shRNA.PTEN (G-I) injection. Quantifying the percentage of pS6 positive cells in GCL indicated a significant difference in Wt AAV2-GFP, Wt AAV2-shRNA.PTEN, and Y444F AAV2-shRNA.PTEN-treated retina (J). The fluorescence of pS6 also presented in inner nuclear layer (INL) of Y444F AAV2-shRNA.PTEN-treated retinas, which was almost absent in Wt AAV2-shRNA.PTEN or Wt AAV2-GFP-treated retinas. **P < 0.01 in ANOVA followed by Bonferroni’s post-test. Scale bar, 50μm.
Fig 8.
Western-blotting for the expression of GLAST in retina 6 weeks after axotomy.
Compared to intact control, ONA resulted in dramatic down-regulation of GLAST, yet Y444F AAV2-shRNA.PTEN, compared with Wt AAV2-shRNA.PTEN or Wt AAV2-GFP, inhibited the reduction significantly. *P < 0.05, **P < 0.01 in ANOVA followed by Bonferroni’s post-test.
Fig 9.
TUJ1 immuno-labeling evaluating RGCs survival 6 weeks after axotomy.
The number of survived RGCs decreased significantly 6 weeks after axotomy. Compared with Wt AAV2-GFP, both Y444F AAV2-shRNA.PTEN and Wt AAV2-shRNA.PTEN significantly prompted RGCs survival, and the pro-survival effect of Y444F AAV2-shRNA.PTEN was stronger than that of Wt AAV2-shRNA.PTEN. **P < 0.01 in Student’s t-test or ANOVA followed by Bonferroni’s post-test. Scale bar, 50μm.
Fig 10.
PTEN knockdown promoting axons regeneration in optic nerve 6 weeks after axotomy.
Fluorescent images of optic nerve longitudinal sections showed CTB-FITC labeled regenerating axons of rats treated with Y444F AAV2-shRNA.PTEN (A), Wt AAV2-shRNA.PTEN (B), and Wt AAV2-GFP (C) respectively. Quantification of the fluorescence intensity at different distances proximal to and distal to the ONA site showed the significant difference in Y444F AAV2-shRNA.PTEN, Wt AAV2-shRNA.PTEN and Wt AAV2-GFP groups (D). *P < 0.05, **P < 0.01 in ANOVA followed by Bonferroni’s post-test. Scale bar, 100μm. Arrow, ONA site.
Fig 11.
Regenerating axons in optic chiasm labeled with CTB-FITC 6 weeks after axotomy.
Fluorescent images of optic chiasm coronal sections displayed normal CTB-FITC-labeled axons of intact rats (A), considerable regenerating axons of Y444F AAV2-shRNA.PTEN-treated rats (B), no regenerating axons of Wt AAV2-shRNA.PTEN-treated rats (C) or Wt AAV2-GFP-treated rats (D). Scale bar, 100μm.