Fig 2: Confirmation of DKO phenotype and genotype. (A) Representative immunofluorescence images of retinal cross-sections stained for rod a-transducin (top, Gnat1, gold) and cone a-transducin (bottom, Gnat2, gold) in a control mouse (left) and a DKO mouse (right). White dashed lines delineate the photoreceptor outer segments layer. For Gnat1 staining (top) red arrows point to the presence of robust Gnat1 staining in the outer segments of rod photoreceptors. This staining is undetectable in DKO mice (top, right, red arrow), suggesting a successful knockout of Gnat1 expression. In both images, a large amount of autofluorescence is present at the border between the photoreceptor layer and outer nuclear layer, which was attributed to non-specific staining by the secondary antibody (as shown by a no-primary antibody control, data not shown). For Gnat2 staining (bottom), cone a-transducin expression was clearly present in the oval-shaped outer segments of cone photoreceptors (indicated by red arrows) in the control animals (bottom, left). This staining pattern was totally absent from our DKO retinal slices (bottom, right), suggesting that Gnat2 expression was significantly reduced. (B) Example 1% agarose gels showing the genotyping of a control mouse (left) and a DKO mouse (right), as described in our methods. For gnat1, the control and knockout bands were located at ~300 and ~200 bp, respectively. For Gnat2, the control and knockout bands were located at ~480 and ~300 bp, respectively. Our DKO animals showed the proper homozygous band pattern we would expect.

Fig 3: Rod degeneration characterized with lower expression of rhodopsin and downstream phototransduction proteins as well as morphological alterations in APP23 mice at 12 and 18 months(A) Expression of rhodopsin in retinas from APP23 and WT mice was evaluated using Western blot analysis using anti-rhodopsin(1D4) antibody. Rhodopsin monomer levels were analyzed using densitometry and normalized by the GAPDH level.(B) Rhodopsin levels were significantly lower in APP23 mice than in WT mice (Up: 12 month old, Down: 18 month old).(C) Expression of rho in the retinas was validated by RT-PCR. Rho levels were significantly lower in APP23 mice than in WT mice at 12 months.(D) Expression of rod-specific phototransduction proteins GNAT1 and recoverin in retinas from APP23 and WT mice was evaluated using Western blot. Expression levels were analyzed using densitometry and normalized by the GAPDH level.(E) GNAT1 and recoverin levels were significantly lower in APP23 mice than in WT mice at 12 months.(F) GNAT1 and recoverin levels of APP23 and control WT mice at 18 months.(G) Representative images of rhodopsin labeling on retinal cross sections of APP23 and control WT mice at 12 months.(H) Left: Quantitative analysis of fluorescence intensities of rhodopsin showed a 50% decrease in APP23 mice compared with control WT mice at 12 months. Right: Thicknesses of rod outer segment (OS), inner segment (IS), and outer nuclear layer (ONL) of APP23 mice were not different from those of control WT mice at 12 months.(I) Representative images of rhodopsin labeling on retinal cross sections of APP23 and control WT mice at 18 months.(J) Left: Quantitative analysis of fluorescence intensities of rhodopsin showed a lower intensity in APP23 mice than in control WT mice at 18 months. Right: Thicknesses of rod OS, IS, and ONL of APP23 mice were not different from those of control WT mice at 18 months. Data are represented as mean ± SEM. ns: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, Student's t test. Scale bar = 20 µm

Fig 4: Rod degeneration in other types of AD transgenic mouse models(A and B) Expression of rhodopsin, GNAT1, and recoverin in retinas from 5xFAD and WT mice was evaluated using Western blot. Expression levels were analyzed using densitometry and normalized by the GAPDH level.(C) Rhodopsin levels were significantly lower in 5xFAD mice than in WT mice at 5 months.(D) GNAT1 and recoverin levels of 5xFAD and control WT mice at 5 months.(E and F) Expression of rhodopsin, GNAT1, and recoverin in retinas from APP NL-F (DKI) and WT mice was evaluated using Western blot. Expression levels were analyzed using densitometry and normalized by the GAPDH level.(G) Rhodopsin levels were significantly lower in DKI mice than in WT mice at 15 months.(H) GNAT1 and recoverin levels of DKI and control WT mice at 15 months.(I) Representative images of rhodopsin labeling on retinal cross sections from DKI and control WT mice at 15 months.(J) Left: Quantitative analysis of the fluorescence intensities of rhodopsin revealed that DKI mice had a lower intensity than control WT mice at 15 months. Right: There was no difference in the thicknesses of the outer segment (OS), inner segment (IS), and outer nuclear layer (ONL) of rods between DKI and control WT mice at 15 months. Data are represented as mean ± SEM. ns: not significant, *: p < 0.05, ****: p < 0.0001, Student's t test. Scale bar = 20 µm