Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

Abstract

The microtubule-associated protein Tau is a key player in various neurodegenerative conditions, including Alzheimer's disease (AD) and Tauopathies, where its hyperphosphorylation disrupts neuronal microtubular lattice stability. Glaucoma, a neurodegenerative disorder affecting the retina, leads to irreversible vision loss by damaging retinal ganglion cells and the optic nerve, often associated with increased intraocular pressure. Prior studies have indicated Tau expression and phosphorylation alterations in the retina in both AD and glaucoma, yet the causative or downstream nature of Tau protein changes in these pathologies remains unclear. This study investigates the impact of Tau protein modulation on retinal neurons under normal and experimental glaucoma conditions. Employing AAV9-mediated gene therapy for Tau overexpression and knockdown, both manipulations were found to adversely affect retinal structural and functional measures as well as neuroprotective Akt/Erk survival signalling in healthy conditions. In the experimental glaucoma model, Tau overexpression intensified inner retinal degeneration, while Tau silencing provided significant protection against these degenerative changes. These findings underscore the critical role of endogenous Tau protein levels in preserving retinal integrity and emphasize the therapeutic potential of targeting Tau in glaucoma pathology.

Keywords: Glaucoma; Intraocular pressure; Microtubules; Neurodegeneration; Optic nerve; Retinal ganglion cells; Tau.

Fig. 1

Modulation of Tau expression in C57BL/6 mice retinas A Scheme of AAV plasmid vector containing GFP, mTau, scrambled shRNA, and mTau-shRNAmir sequences. mTau (overexpression) and mTau-shRNAmir (knockdown, KD) sequences were cloned in AAV9 viral vector plasmid fused to the ampicillin-resistance gene. For mTau protein expression, a T2A self-cleaving peptide sequence was used. B Schematic depicting the experimental design and AAV administration experimental timeline. C Immunofluorescence images of retinal sections from control, AAV-GFP, AAV-mTau, AAV-Scramble and AAV-mTau KD groups showing GFP (green, FITC), tau (Green, Alexa Fluor 488), and ptau^{(Ser199/Ser202)} (Red, Cy3). NeuN (red-Cy3) and nucleus (blue, DAPI) (representative images, Scale bar = 50 μm, arrows indicate the changes in the expressions; Antibody concentrations: GFP (1:1000), tau (1:800), ptau^{(Ser199/Ser202)} (1:800), NeuN (1:1000)). D Quantification of GFP relative fluorescence intensity (RFI) percentage (****P < 0.0001, n = 5). E Quantification of Tau RFI percentage (****P < 0.0001, n = 5) F Quantification of pTau^{(Ser199/Ser202)} RFI percentage (****P < 0.0001, n = 5). Statistical significance was assessed by employing One-way ANOVA analysis with Tukey’s multiple comparison test

Fig. 2

Electrophysiological and structural alterations in C57BL/6 mice retinas subjected to AAV-tau overexpression and AAV-tau knockdown under normal IOP conditions. A Positive scotopic threshold responses (pSTRs) of the control, AAV-GFP, and AAV-mTau injected mice. B Quantification of amplitudes indicates a significant decrease in pSTR amplitudes in AAV9-Tau overexpressing mice (n = 10, P < 0.0001 t-test). C pSTRs of the control, AAV-scrambled, and AAV-tau shRNA expressing mice and D their quantification indicates a significant decrease of pSTR amplitudes in AAV9-KD mice retinas (n = 10, ****P < 0.0001, t-test). No significant difference in pSTR amplitudes was observed between the control and AAV9-GFP or AAV9-scramble-shRNA sequence-expressing eyes. E H and E staining of retinal sections shows GCL cell density loss in Tau overexpression and Tau KD retinas compared to the controls, arrows indicate the changes in cell densities (representative images, Scale bar = 50 μm). F Quantification of cell density in the GCL from the retinal H and E-stained images (****P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test, n = 5 for each group)

Fig. 3

Tau overexpressing mice retinas shows exacerbated degenerative changes in the inner retina in high-IOP glaucoma condition. A Chronic elevation of IOP in C57BL/6 mice eyes was induced by intracameral microbead injections in control, AAV-GFP and AAV-mTau administered mice for 2 months. B Positive scotopic threshold response (pSTR) traces from control, glaucoma, glaucoma + AAV-GFP, and glaucoma + AAV-mTau overexpression mice retinas, as indicated and C their pSTR amplitudes quantification indicated a significant decline of the pSTR in the glaucoma AAV9-tau overexpression animals. (n = 10, ***P < 0.001 ****P < 0.0001) D Western blot analysis of Tau, pTau^S199/202 and pTau^S404 levels in control and glaucomatous retina. β-actin was used as a loading control E Fold change of Tau, pTau^S199/202 and pTau.^S404 showed a significant increase in retinas in high IOP conditions (***P < 0.001, n = 3 each group, t-test). F H and E staining of retinal sections of control, glaucoma, glaucoma + AAV9-GFP, and glaucoma + AAV9-mTau mice (representative images, arrows indicate the changes in cell densities, Scale bar, 50 μm). G Quantification of the cell density in GCL indicated a significant decrease in the number of cells in glaucoma and AAV-tau overexpressed retinas compared to the controls (n = 5, ****P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test)

Fig. 4

Silencing of Tau expression using AAV protects the inner retinal function and laminar structure in glaucomatous eyes. A Chronic elevation of IOP in C57BL/6 mice eyes was induced by intracameral microbead injections for 2 months. B Positive scotopic threshold response (pSTR) traces from control, glaucoma, glaucoma + scrambled and glaucoma + Tau KD mice retinas, as indicated. C Quantification of the pSTR amplitudes from the glaucoma AAV9-Tau KD mice eyes compared to glaucoma scrambled sequence expressing controls indicated significant protection against pSTR amplitudes loss in the glaucoma-AAV-Tau KD animals. (n = 10, ***P < 0.001, One-way ANOVA analysis with Tukey’s multiple comparison test). D H and E staining of retinal sections of the control, glaucoma, glaucoma + AAV9-scramble, and glaucoma + AAV9-tau KD mice eyes. (representative images, arrows indicate the changes in cell densities, Scale bar, 50 μm). E Quantification of the cell density in GCL indicated significant protection against cell loss in glaucoma AAV9-tau KD-subjected retinas compared to the microbead-injected control eyes. (n = 5, ***P < 0.001, ****P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test)

Fig. 5

Western blotting (WB) analysis of tubulin and its post-translational modifications in AAV-mediated Tau modulated retinas. A WB of retinal lysates of the control and glaucomatous C57BL/6 mice eyes with Ac-tubulin (1:1000), Tyr-tubulin (1:1000), and total tubulin (1:1000) antibodies. B Quantification of relative intensities of the WB bands for Ac-tubulin and C Tyr-tubulin in retaliation to the total tubulin levels (n = 3, t-test, **P < 0.01, ***P < 0.001). D WB of retinal lysates of mice eyes transduced with AAV-GFP control and AAV-mTau construct (Tau overexpression) probed with Ac-tubulin, Tyr-tubulin, and total tubulin and their quantified E relative band intensities for Ac-tubulin and F Tyr-tubulin (**P < 0.01, ***P < 0.001, t-test, n = 3 per group, One-way ANOVA analysis with Tukey’s multiple comparison test,). G WB of retinal lysates from AAV-scrambled shRNA and AAV-mTau KD transduced mice probed with Ac-tubulin, Tyr-tubulin, and total tubulin antibodies. H Quantification of relative WB band intensities of Ac-tubulin and I Tyr-tubulin plotted to the total tubulin (n = 3) (***P < 0.001, ****P < 0.0001, t-test, n = 3 per group, One-way ANOVA analysis with Tukey’s multiple comparison test,)

Fig. 6

Western blot (WB) analysis of retinal endoplasmic reticulum (ER) stress markers changes in glaucoma and Tau modulation conditions. A WBs of control and glaucoma C57BL/6 mice retinal lysates probed with GRP78(1:1000), CHOP(1:1000), P-PERK(1:1000), and β-actin (1:5000) antibodies and B-D their respective relative band intensities quantified as the fold change using β-actin as loading control (n = 3 ****P < 0.0001, t-test). E WB of retinal lysates of the control, glaucoma, AAV-GFP, AAV-mTau (overexpression), AAV-GFP + glaucoma, and AAV-mTau + glaucoma mice were probed with GRP-78, CHOP, P-PERK, and actin antibodies, and F-H their respective relative band intensities quantified as the fold change using β-actin as loading control (n = 3, **P < 0.01, ***P < 0.001, ****P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test,). I WB of retinal lysates from control, glaucoma, AAV9-scramble, AAV-mTau KD, AAV9-scramble + glaucoma, AAV-mTau KD + glaucoma mice were probed with GRP 78, CHOP, P-PERK, and actin antibodies, and (J-L) their respective band intensities quantified as fold change using β-actin as a loading control (***P < 0.001, ****P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test, n = 3 per group)

Fig. 7

Western blotting (WB) analysis of synaptic signaling proteins changes in glaucoma and AAV-Tau modulations in the retina. A WB of glaucoma and control retinal lysates probed with anti-synaptophysin(1:20,000), anti-PSD95(1:1000), and β-actin (1:5000), antibodies. The band intensities were quantified using β-actin as a loading control and relative fold change plot for B Synaptophysin and C PSD95 (***P < 0.001, ****P < 0.0001 n = 3 each group, t-test). D WB of retinal lysates of AAV-GFP control and AAV-mTtau overexpression-subjected mice eyes probed with anti-synaptophysin and anti-PSD-95 antibodies in healthy and glaucoma conditions. The band intensities were quantified using β-actin as a loading control and relative fold change plotted for E synaptophysin and F PSD95 (**P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3 each group, One-way ANOVA analysis with Tukey’s multiple comparison test). G WB of retinal lysates of AAV-Scramble control and AAV-Tau KD-subjected mice probed with anti-synaptophysin and anti-PSD-95 antibodies in normal and glaucoma conditions. The band intensities were quantified using β-actin as a loading control and relative fold change plotted for H Synaptophysin and I PSD95 (**P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3 each group, One-way ANOVA analysis with Tukey’s multiple comparison test)

Fig. 8

Western blot analysis of Akt, Erk and GSK3β signaling in the healthy and glaucoma retinas in response to AAV-Tau modulation. A Immunoblots of pAkt^(Ser473) (1:2000), Akt(1:1000), pErk^{(Thr202/Tyr204)} (1:2000), Erk(1:1000), pGSK3β^(Ser9)(1:1000), and GSK3β(1:1000) in Tau overexpressing retinas in normal and glaucoma conditions compared to the respective control retinas and β-actin (1:5000), used as a loading control. B Quantification of pAkt^(Ser473) band intensities measures as relative fold changes to the total Akt expression. C, D Quantitative analysis of pErk1^{(Thr202/Tyr204)} and pErk2^{(Thr202/Tyr 204)} levels compared to the total Erk1 and Erk2. E Quantitative analysis of pGSK3β^(Ser9) band intensities and their relative fold changes compared to the GSK3β expression (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3 each group, One-way ANOVA analysis with Tukey’s multiple comparison test). F Immunoblots of pAkt^(Ser473), Akt, pErk^{(Thr202/Tyr204)}, Erk, pGSK3β^(Ser9), and GSK3β in AAV-mTau knockdown retinas in normal and glaucoma conditions compared to the respective control retinas and β-actin used as a loading control. G Quantitative analysis of pAkt^(Ser473) band intensities and its relative fold change to total Akt expression. H, I Quantitative analysis of pErk1^{(Thr202/Tyr204)} and pErk2^{(Thr202/Tyr 204)} compared to the total Erk1 and Erk2 levels. J Quantitative analysis of pGSK3β^(Ser9) band intensities to the total GSK3β expression (***P < 0.001, ****P < 0.0001, n = 3 each group, One-way ANOVA analysis with Tukey’s multiple comparison test)