Figure 4.

Autophagy-Lysosomal pathways are impaired in the RPE and retina of APOE-mice and treatment with trehalose and metformin ameliorate aspects of this deficit. Autophagy–lysosomal pathways in RPE and photoreceptors were investigated using transverse sections labeled for autophagosomes with an LC3 antibody (red) and lysosomes with a LAMP1 antibody (green). (A-D) Representative images of RPE from (A) WT-control mice, with magnified view of (Ai) LC3-puncta, (Aii) LAMP1-puncta and (Aiii) colocalization (white arrowheads); (B) APOE-control mice; and APOE mice treated with (C) trehalose and (D) metformin are shown. (E) The number of LC3-puncta in the RPE was reduced in APOE-control mice relative to WT-control mice. This loss of LC3-puncta was not apparent in APOE-mice treated with trehalose or metformin relative to their treated WT counterparts. (F) LAMP1-puncta were also reduced in the RPE of APOE-control relative to WT-control mice and this was not altered by treatment with trehalose or metformin. (G) Both trehalose and metformin were found to enhance the number of colocalized LC3- and LAMP1-puncta, suggesting active autophagy was increased in the RPE of both WT and APOE mice relative to their genetic controls. (H-K) Representative images of the photoreceptor nuclei layer in the retina from (H) WT-control mice, (I) APOE-control mice, and APOE mice treated with (J) trehalose and (K) metformin are shown. (L) The number of LC3-puncta in the photoreceptors were reduced in APOE-control mice relative to WT-control mice but not in APOE-mice treated with trehalose or metformin. (M) LAMP1-puncta number and (N) colocalized LC3- and LAMP1-puncta were not altered by either genotype or treatment. For all groups n = >6; Two way ANOVA with post-hoc significance of p < 0.05 shown for genotype (*) and treatment (#). A-D, scale: 5 µm; Ai-Aiii, scale: 5 µm; H-K, scale: 10 µm; RPE, retinal pigment epithelium; ONL, outer nuclear layer.