Fig 1: Silencing of S6k1 in pigs(A) Bar graphs from western blot quantifications showing a 72% reduction in S6K1 protein levels and a 34% reduction in pS6 protein levels when comparing the tetra-siRNAS6k1-injected eyes to the NTC-injected eyes at 3 months post-intravitreal injection of 300 μg in 100 μL volume of siRNA reagent. A total of five pigs were used, each having one eye injected with the tetra-siRNAS6k1 and the other with the tetra-siRNANTC. Eyes were dissected in four quadrants with the central portion used for histology (see sketch in A) and the quadrants used for quantification of silencing efficiency. Each dot in the bar graphs represents one quadrant from one animal (error bars = ± S.E.M.; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). The sketch details the silencing efficiency within each quadrant. S6K1 silencing was most efficient close to the injection site (marked in red in sketch; D, dorsal; V, ventral; T, temporal; N, nasal). (B) Representative antibody staining for S6K1 (first column, purple signal), GFAP (second column, green signal), and Iba1 (third column, red signal) on retinal cross-sections at 3 months post-intravitreal injection of 300 μg of each siRNA reagent. Sections originated from the central strip of the retina (see sketch in A). Blue, nuclear DAPI; green, GFAP; red Iba1; Scale bars: 100 μm; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; vertical bars in section mark height of different layers. (C) Pharmacokinetics of remaining siRNA in different eye tissues at 3 months post-injection. A PNA hybridization assay was performed with one quadrant per eye (N = 5, error bars = ± S.E.M.). Analyses of the retina, RPE, vitreous, lens, and cornea show that most of the siRNA is taken up by the retina and RPE and is cleared from the vitreous, with little migrating to the lens and cornea. Eye tissues from NTC-injected eyes did not show any signal and were therefore omitted from the figure. (D) Total phospholipid breakdown by class of phospholipid [PE: phosphatidylethanolamine; PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; PG: phosphatidylglycerol, BMP: bis(monoacylglycero)phosphate]. Shown is %-distribution of each class of phospholipid listed for wild-type mouse (rodTsc1+/+) and pig retinas with the sum of them representing 100%. (E) Changes in PC and PG phospholipids in retinas of rodTsc1−/− mice, rodTsc1+/+ mice, and pigs injected intravitreally with the tetra-siRNANTC or the tetra-siRNAS6k1. Error bars in (D and E) show S.E.M.; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; N = 5–6 mouse retinas and 8–10 pig retinal samples.
Fig 2: S6K1 activity is required for disease onset and progression in rodTsc1−/− mice(A) Top row: representative fundus, fluorescein angiography, and OCT images of an 18-month-old rodTsc1−/−S6k1+/+ mouse with focal RPE atrophy and neovascular pathology (yellow arrows). Bottom row: immunofluorescence and bright field images with red signal from immunofluorescence staining superimposed on bright field of a retinal cross-section of the eye above (section shown in the same orientation as the OCT image) showing loss of RPE65 (red signal) expression in the area of RPE atrophy (dashed line on choroid demarks region of RPE cells loss), indicating a disrupted RPE layer. RPE65 expression with RPE cells is visible on the left third of each panel (area between white arrowheads). Only RPE atrophy is shown on section, not the neovascular pathology. Scale bars: 100 μm; blue, nuclear DAPI; green, peanut agglutinin lectin (PNA) marking cone PR segments; red, RPE65 expression marking RPE cells. (B) Frequency in percentage of phenotypes scored in each genotype at 18 months of age, including microglia activation (white bar), retinal folds (gray bars), focal RPE atrophy (black bars), and neovascular pathologies (green bars). The number of mice examined in each group is indicated in parentheses. Error bar = margin of error (M.O.E.). (C) Representative image of APOE (green signal) accumulation at the RPE/BrM (white arrowheads) in mice with indicated genotype at 12 months of age (4–5 mice were examined in each group). Scale bars: 50 μm; blue, nuclear DAPI; red, peanut agglutinin lectin (PNA) marking cone PR segments; layers in (A and C): RPE, retinal-pigmented epithelium; PS, PR segment region covering inner and outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; vertical bars in sections mark height of different layers. (D) PR outer segment (POS) clearance in RPE cells of 2-month-old mice is shown as percentage of POS remaining at 11 am when compared to 8 am in genotypes indicated (N = 4–8 RPE flat mounts/genotype). (E) Percentage of di-DHA PE (left) and PC (right) phospholipids as a total of PE (left) and PC (right) phospholipids in genotypes indicated (N = 5–6 retinas/genotype). Results in (D and E) are shown as mean ± S.E.M. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001; n.s., not significant).
Fig 3: S6k1 silencing in mouse reverses early disease pathologies in rodTsc1−/− mice(A and B) Long-term siRNA retention and silencing efficacy in rodTsc1−/− mice examined at 3, 6, and 9 months post-injection. Mice received one intravitreal injection of 15 μg of siRNA reagents at 3 months of age. (A) Distribution of tetra-siRNAS6k1 and S6K1 protein expression in rodTsc1−/− mouse retinas. Left: tiled retinal sections showing either tetra-siRNANTC (top) or tetra-siRNAS6k1 (bottom, visualized with RNAScope, red signal) at 3 months post-injection (scale bars: 500 μm). Right: higher magnification of tetra-siRNAS6k1 distribution on retinal sections and S6K1 protein expression at time points indicated. Tetra-siRNAS6k1 is visualized with RNAScope (red signal), and S6K1 protein expression is visualized by immunohistochemistry (purple signal). Staining for tetra-siRNAS6k1 and S6K1 was performed on separate slides. Scale bars: 50 μm; PS, PR segment region covering inner and outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; vertical bars in sections mark height of different layers. (B) Silencing efficiency of tetra-siRNAS6k1 (blue bars) at time points indicated post-intravitreal injection when compared to the NTC (green bars). Silencing was measured by western blotting with retinal protein extracts. Mice were all injected at 3 months of age (N = 5–8 retinas/group). (C) Percentage silencing in PR vs. non-PR cells that were enriched by FACS at 2 months post-intravitreal delivery of siRNA. The percentage of protein expression level is normalized to the tetra-siRNANTC treated group. (D) PR outer segment (POS) clearance in RPE cells of 4-month-old mice shown as percentage of POS remaining at 11 am when compared to the peak of shedding at 8 am in the genotypes indicated. rodTsc1−/− mice were injected at 2 months of age with siRNA reagents indicated (N = 4–7 eyes/group). (E and F) Reversal of APOE accumulation at the BrM in tetra-siRNAS6k1-treated mice. (E) APOE protein expression level measured by western blotting with RPE/choroid protein extracts of 15-month-old rodTsc1−/− mice that are untreated or treated with either tetra-siRNANTC or tetra-siRNAS6k1 for 3 months (treatment started at 12 months of age). Expression levels are compared to 15-month-old littermate control rodTsc1+/+ mice (N = 5–10 eyes/group). (B–E) Results are shown as mean ± S.E.M. Each dot represents one retina or RPE/choroid from one mouse. Only one eye per mouse was used for each analysis (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; green bars represent tetra-siRNANTC and blue bars tetra-siRNAS6k1-injected eyes). (F) Retinal cross-section of rodTsc1−/− eyes showing reduction in the accumulation APOE (green signal) at the BrM (white arrowheads) of tetra-siRNAS6k1-injected eyes (right panel). Mice were injected at 12 months of age and analyzed 3 months post-injection. Scale bars: 50 μm; blue, nuclear DAPI; red, peanut agglutinin lectin (PNA) marking cone PR segments; RPE, retinal-pigmented epithelium; PS, PR segment region covering inner and outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; vertical bars in sections mark height of different layers.
from Cell Signaling Technology for PathScan ® Total p70 S6 Kinase Sandwich ELISA Kit