Inner retinal injury in experimental glaucoma is prevented upon AAV mediated Shp2 silencing in a caveolin dependent manner

Abstract

SH2 domain containing tyrosine phosphatase 2 (Shp2; PTPN11) regulates several intracellular pathways downstream of multiple growth factor receptors. Our studies implicate that Shp2 interacts with Caveolin-1 (Cav-1) protein in retinal ganglion cells (RGCs) and negatively regulates BDNF/TrkB signaling. This study aimed to investigate the mechanisms underlying the protective effects of shp2 silencing in the RGCs in glaucomatous conditions. Methods: Shp2 was silenced in the Cav-1 deficient mice and the age matched wildtype littermates using adeno-associated viral (AAV) constructs. Shp2 expression modulation was performed in an acute and a chronic mouse model of experimental glaucoma. AAV2 expressing Shp2 eGFP-shRNA under a strong synthetic CAG promoter was administered intravitreally in the animals' eyes. The contralateral eye received AAV-eGFP-scramble-shRNA as control. Animals with Shp2 downregulation were subjected to either microbead injections or acute ocular hypertension experimental paradigm. Changes in inner retinal function were evaluated by measuring positive scotopic threshold response (pSTR) while structural and biochemical alterations were evaluated through H&E staining, western blotting and immunohistochemical analysis of the retinal tissues. Results: A greater loss of pSTR amplitudes was observed in the WT mice compared to Cav-1^-/- retinas in both the models. Silencing of Shp2 phosphatase imparted protection against inner retinal function loss in chronic glaucoma model in WT mice. The functional rescue also translated to structural preservation of ganglion cell layer in the chronic glaucoma condition in WT mice which was not evident in Cav-1^-/- mice retinas. Conclusions: This study indicates that protective effects of Shp2 ablation under chronic experimental glaucoma conditions are dependent on Cav-1 in the retina, suggesting in vivo interactions between the two proteins.

Keywords: Caveolin; Glaucoma; Retinal Ganglion cells, Shp2 phosphatase; TrkB..

Figure 1

Cav 1^-/- mice retinas were relatively protected against the functional loss induced upon Shp2 upregulation. A, B) Average trace of pSTR signal from AAV-Shp2 treated WT and Cav-1^-/- mice and corresponding controls 2 months following treatment. (C) Quantification indicates that AAV-Shp2 injected WT mice had significantly lower pSTR amplitudes in comparison to AAV-GFP counterparts. (****p < 0.0001, WT; Cav-1^-/- mice: p = 0.17; n = 16) (C) Average traces of pSTR amplitudes in WT and Cav-1 KO retinas. (D-F) No significant changes were observed in pSTR amplitudes in either WT or Cav-1 KO groups retinas following Shp2 downregulation under normal healthy conditions.

Figure 2

Shp2 upregulation resulted in retinal TrkB receptor dephosphorylation (A) Western blot indicating pTrkB Y⁵¹⁵/TrkB, pShp2 and Shp2 expression under control and AAV-Shp2 modulated conditions. (B). Multiple comparisons using Bonferroni post-hoc test showed that there was a significant decrease in pTrkB (**p < 0.01) which was concurrent with higher pShp2 levels (***p < 0.001) (C) in WT retinas following Shp2 upregulation. Cav-1^-/- GCL did not show significant decline in pTrkB and total TrkB levels upon Shp2 downregulation. (D) Significant reduction in Shp2 expression was observed upon using AAV-mShp2 shRNA in both WT and Cav-1 ^-/- mice (*p < 0.05, one-way ANOVA; n = 24) while increased expression levels were observed following AAV-Shp2 administration (****p < 0.0001, WT; ***p < 0.001, Cav-1 ^-/-; one-way ANOVA; n = 24).

Figure 3

Histochemical evaluation of WT and Cav-1^-/- retinas two months following Shp2 modulation (A) H&E staining of paraffin-embedded retinal sections indicating different retinal layers including GCL in WT and Cav-1^-/- groups (Scale bar = 50 µm). (B) Quantification revealed significantly reduced GCL density in AAV-Shp2 treated WT mice compared to controls (***P < 0.001, one-way ANOVA; n = 4 in each group) while no significant changes in GCL density were identified in case of AAV-Shp2 injected Cav-1 KO mice. (A; panel 2). Further, no significant changes were observed upon Shp2 KD in the WT or Cav-1^-/- mice.

Figure 4

Silencing of Shp2 protected the inner retinal function in WT but not Cav-1 KO retinas. (A, B) pSTR responses in WT and Cav-1^-/- retinas in control and experimental groups treated with AAV-Shp2 scramble (sc) and AAV-Shp2 KD under chronic glaucoma conditions. (C) A significant decline in pSTR observed in AAV-Sc treated WT mice exposed to chronic elevation of IOP (****p < 0.0001; one-way ANOVA; Bonferroni post-hoc test; n = 16) as against the AAV-Shp2 KD retinas that were rescued under similar conditions (***p < 0.001; one-way ANOVA; Bonferroni post-hoc test; n = 16). AAV-Sc treated Cav-1^-/- retinas demonstrated reduced loss of pSTR amplitudes compare to WT (*p < 0.05, Cav-1^-/- and Cav-1^-/- microbead) and Shp2 knockdown did not induce a protection of the retinal function.

Figure 5

AOH induced inner retinal impairment was not rescued by Shp2 silencing. (A, B) pSTR traces in WT and Cav-1^-/- eyes exposed to acute IOP increase and Shp2 downregulation using AAV-Shp2 KD. (C) Quantification of pSTR amplitudes revealed statistically significant decrease in inner retinal function in WT and Cav-1^-/- mice (****p < 0.0001, WT; *p < 0.05, Cav-1^-/-; one-way ANOVA; Bonferroni post-hoc test; n = 16) which was not affected upon Shp2 KD.

Figure 6

Shp2 silencing protected inner retinal laminar structure in WT mice in chronic but not acute glaucoma model (A, B) H and E staining of WT and Cav-1 null retinas two months after chronic high IOP and (C) 2 weeks following exposure to acute IOP (Scale bar = 50 µm). (B) Quantification of GCL density revealed that under chronically elevated IOP, a significant reduction occurred in WT (****P < 0.0001, one-way ANOVA, n = 8,) and Cav-1 ablated mice (*p < 0.05, one-way ANOVA, n = 8). Upon subjecting the RGCs to AAV-Shp2 KD, significant protection of GCL density was observed in WT (*p < 0.05, one-way ANOVA, n = 8) compared to Cav-1^-/- group. (D) H and E staining also indicated a significant loss in GCL density in acute high IOP (**P < 0.01, WT; *p < 0.05, Cav-1^-/-. one-way ANOVA, n = 8) while downregulation of Shp2 resulted in no significant change in either WT or Cav-1^-/- mice.

Figure 7

Shp2 expression knockdown partially rescued IOP-induced loss of Brn3a positive cells in WT mice. (A, C) Retinal sections immunostained with RGC marker Brn3a, two months following chronic and 2 weeks after acute glaucoma exposure (Scale bar = 50 µm). (B, D) WT retinas indicated a higher Brn3a⁺ loss following chronic high IOP exposure compared to the Cav-1 KO mice (****p < 0.0001, WT; *p < 0.05, Cav-1^-/-. one-way ANOVA, n = 8). This loss was significantly diminished (**p < 0.01, one-way ANOVA, n = 8), in the WT group administered AAV-Shp2 KD construct compared to Cav-1^-/- animal retinas where no significant changes were observed under similar conditions.

Figure 8

Reduced levels of pTrkB in chronic glaucoma model were mitigated upon Shp2 silencing. (A-D) Retinal sections immunostained with pTrkB (A, B and pShp2 (C, D) in WT and Cav-1^-/- retinas 2 months following chronic IOP elevation. DAPI was used for staining the nuclei. (Scale bar = 50 µm). (E) WB analysis of pTrkB and pShp2 levels in retinal ONH lysates. (F) Densitometric analysis indicated markedly reduced pTrkB immunoreactivity following chronic IOP elevation in WT and Cav-1^-/- GCL (**p < 0.01, WT, *p < 0.05, Cav-1^-/- one-way ANOVA, n = 6) which was concurrent with increased pShp2 (G) in WT group (*p < 0.05, one-way ANOVA, n = 6). Shp2 silencing protected against pTrkB decline (G) (*p < 0.05, one-way ANOVA, n = 6) in WT mice while no significant effects on pTrkB levels were observed in Cav-1 deficient retinas following similar levels of Shp2 KD.

Figure 9

Reduced pTrkB levels in acutely elevated IOP condition was independent of Shp2 silencing effects. (A-D) IF images of retinal sections subjected to immunostaining with pTrkB (A, B) and pShp2 (C, D) in WT and Cav-1^-/- retinas two weeks following acute IOP exposure (Scale bar = 50 µm). DAPI used for staining the nuclei. (Scale bar = 50 µm). (E) Western blot indicating pTrkB and pShp2 levels in high IOP exposed retinal lysates. (F) TrkB receptor significantly lost its phosphorylation following acute IOP elevation in WT or Cav-1 null retinas (*p < 0.05, one-way ANOVA, n = 6). (G) pShp2 levels were significantly reduced in AOH model in Shp2 KD conditions.

Figure 10

Shp2 silencing alleviated apoptotic activation in GCL in a chronic model of glaucoma (A, B) Retinal sections immunostained with both cleaved c-casp3 and TUNEL apoptotic markers (Scale bar = 50 µm). WT retinas depicted a higher TUNEL⁺ and c-casp3 immunoreactivity (A) following chronic IOP exposure when compared to their (B) Cav-1 KO counterparts. Significant decline in apoptotic staining and immunoreactivity of c-casp3 was observed subsequent to Shp2 silencing in WT retinas. (C) Quantification of TUNEL⁺ cells in GCL showed higher apoptotic activity in WT retinas after chronic elevation of IOP (****p < 0.0001, one-way ANOVA; Bonferroni post-hoc test; n = 8) which was considerably attenuated in the AAV-Shp2 KD subjected group (**p < 0.01, one-way ANOVA; Bonferroni post-hoc test; n = 8). Cav-1 ablated retinas illustrated apoptosis that remained relatively unaltered after shp2 loss (*p < 0.05, one-way ANOVA; Bonferroni post-hoc test; n = 8). (D) c-casp3 immunostaining established increased apoptosis in WT retinas subsequent to chronic IOP elevation (****p < 0.0001, one-way ANOVA; Bonferroni post-hoc test; n = 8). This increase in c-casp3 was mitigated in the WT group with Shp2 silencing effects (*p < 0.05, one-way ANOVA; Bonferroni post-hoc test; n = 8). Cav-1^-/- retinas exhibited lower c-casp3 immunoreactivity that remained unaltered in response to Shp2 silencing (*p < 0.05, one-way ANOVA; Bonferroni post-hoc test; n = 8). (E, F) TUNEL and c-casp 3 staining in WT and Cav-1 ablated retinas following acute model of experimental glaucoma (Scale bar = 50 µm). (G, H) Quantification analysis of TUNEL and c-casp3 staining established that both of these apoptotic cell markers were significantly enhanced in both WT (***p < 0.001, one-way ANOVA; Bonferroni post-hoc test; n = 8) and Cav-1^-/- retinas when subjected to acutely elevated IOP (*p < 0.05, one-way ANOVA; Bonferroni post-hoc test; n = 8). AAV mediated shp2 downregulation did not result in significant amelioration of apoptotic staining in either of these groups.

Figure 11

Effects of Shp2 modulation on integrin β1/ FAK expression in WT and Cav-1^-/- retinas. Immunofluorscence analysis of the retinal sections demonstrating integrin β1and its downstream effector FAK distribution within the retina in WT (A) and Cav-1 deficient (B) mice (Scale bar = 50 µm). (C) ONH lysates probed for integrin and phospho-FAK (Y³⁹⁷) using their specific antibodies with β-actin as loading control. (D, E) ANOVA Multiple analysis using Bonferroni post-hoc test revealed that a there was decline in integrin β1 (**p < 0.01, one-way ANOVA; Bonferroni post-hoc test; n = 6) and pFAK levels (**p < 0.01, one-way ANOVA; Bonferroni post-hoc test; n = 6) in WT retinas after exposure to chronic IOP stress. (E) Loss of FAK phosphorylation in WT animals was significantly protected upon subjecting retinas to AAV-Shp2 KD (*p < 0.05, one-way ANOVA; Bonferroni post-hoc test; n = 6). No significant differences were detected in the level of integrin β1 expression (D) or FAK activity (E) in Cav-1 deficient retinas under similar conditions. (F, G) Immunohistochemical images of the retinal sections stained integrin β1/ FAK expression following acute model of High IOP (Scale bar = 50 µm). (H-J) Immunoblotting and densitometric quantification indicated that similar to chronic type of high IOP, in acute IOP elevation there was significant decline in integrin β1 (**p < 0.01, one-way ANOVA; Bonferroni post-hoc test; n = 6) (I) and pFAK (*p < 0.05, one-way ANOVA; Bonferroni post-hoc test; n = 6) (J) in WT retinas compared to controls and Cav-1^-/- mice.

Figure 11

Effects of Shp2 modulation on integrin β1/ FAK expression in WT and Cav-1^-/- retinas. Immunofluorscence analysis of the retinal sections demonstrating integrin β1and its downstream effector FAK distribution within the retina in WT (A) and Cav-1 deficient (B) mice (Scale bar = 50 µm). (C) ONH lysates probed for integrin and phospho-FAK (Y³⁹⁷) using their specific antibodies with β-actin as loading control. (D, E) ANOVA Multiple analysis using Bonferroni post-hoc test revealed that a there was decline in integrin β1 (**p < 0.01, one-way ANOVA; Bonferroni post-hoc test; n = 6) and pFAK levels (**p < 0.01, one-way ANOVA; Bonferroni post-hoc test; n = 6) in WT retinas after exposure to chronic IOP stress. (E) Loss of FAK phosphorylation in WT animals was significantly protected upon subjecting retinas to AAV-Shp2 KD (*p < 0.05, one-way ANOVA; Bonferroni post-hoc test; n = 6). No significant differences were detected in the level of integrin β1 expression (D) or FAK activity (E) in Cav-1 deficient retinas under similar conditions. (F, G) Immunohistochemical images of the retinal sections stained integrin β1/ FAK expression following acute model of High IOP (Scale bar = 50 µm). (H-J) Immunoblotting and densitometric quantification indicated that similar to chronic type of high IOP, in acute IOP elevation there was significant decline in integrin β1 (**p < 0.01, one-way ANOVA; Bonferroni post-hoc test; n = 6) (I) and pFAK (*p < 0.05, one-way ANOVA; Bonferroni post-hoc test; n = 6) (J) in WT retinas compared to controls and Cav-1^-/- mice.