Autophagy and exosomes in the aged retinal pigment epithelium: possible relevance to drusen formation and age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch's membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch's membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly.

Figure 1. Immunolocalization of autophagy marker, Atg5, in the human RPE/choroid.

In these confocal immunofluorescence images: an-anti-Atg5 antibody labels particles in drusen (arrows). (A) AMD eye from 94-year-old male; (B) AMD eye from 97-year-old male; (C) AMD eye from 74-year-old male; (D) non-AMD eye from 75-year-old male ; (E) non-AMD eye from 60-year-old male; (F) non-AMD eye from 87-year-old male. Dr, drusen. Scale bar = 20 µm. Blue: DAPI; red: Atg-5; Green: autofluorescence.

Figure 2. Increased autophagy markers in old mice.

(A–B): Localization of Atg12 in young and old RPE/choroid in the mouse eye. (A) In the eyes from young mice, there was no Atg12 labeling (red) in the RPE/choroid tissue. (B) In the RPE/choroids from old eyes, there was spotted labeling (red, arrows) in the RPE. Scale bar = 20 µm. (C–E): Comparison of protein levels of (C) Atg12-Atg5 conjugates and (D) LC3B in RPE/choroid of young and old mice by immunoblot. Atg12-Atg5 conjugates and LC3B were all increased in RPE/choroid from old animals. (E) β-actin was used as a loading control. (F): The differences in expression levels were determined by multiple scans of blots to ensure a maximium and minimum response range for the measured areas and the integrated areas of the bands were calculated by using Image-J software. Data were expressed as normalized ratios (Young  = 1). There were significant increases in aged retinas of Atg12-Atg5 (p<0.05, n = 3) and LC3B (p<0.05, n = 3), compared to young retinas. Values are the mean±SEM. Appropriate background subtraction and normalization of the data to actin was done for each blot.

Figure 3. Induction of autophagy in ARPE-19 cells.

(A): Cell viability assay and ATP assay for ARPE-19 cells treated with rotenone. Concentrations in the media of 0.08 to 2.5 µM did not cause a significant decrease of cell viability or ATP levels. (B): Rotenone at concentrations in the media of 0.6 to 2.5 µM did not cause a significant decrease in CoQ levels. (C): Rotenone at concentrations in the media of 0.6 to 2.5 µM damaged mtDNA, but not nDNA. (D): Comparison of protein levels of Atg12-Atg5 conjugates and LC3B in ARPE-19 cells by immunoblots. Atg12-Atg5 conjugates and LC3B were increased in ARPE-19 cells after 2.5 µM rotenone treatment. β-actin was used as a loading control.

Figure 4. Phagocytotic activity of ARPE-19 cells.

Rotenone at concentrations in the media of 0.3–2.5 µM did not change phagocytic activity when cells were exposed to photoreceptor outer segments for 3 hr (p<0.05, n = 4).

Figure 5. Lysosomal activity.

(A) Comparison of protein levels of mature cathepsin D (33kD) and pre-pro-cathepsin D (48–52 kD) in ARPE-19 cells by immunoblot. β-actin was used as a loading control. (B) Cathepsin D enzymatic activity from cell extracts were significantly decreased at 1.25–2.5 µM concentrations of rotenone (p<0.05). (C) immunofluorescence of in vivo cathepsin D substrate showing uptake into the lysosomes of ARPE-19 cells (red: total substrate; green: cleaved substrate; blue: DAPI). (D) In vivo lysosomal activity assay showing lysosomal activity against a peptide substrate for cathepsin D was significantly decreased at 2.5 µM rotenone (p<0.05). Scale bar = 20 µm.

Figure 6. Exosome markers in ARPE-19 cells.

(A) Localization of CD63 in ARPE-19 cells without treatment. (B) 2.5 µM rotenone for 24 hrs or exposure to POS 12 hrs alone did not change CD63 expression at 36 hrs. However, when the cells were treated with rotenone for 24 hrs to damage their mtDNA and then fed POS for 12 hrs, CD63 was significantly increased (p<0.05). (C) Localization of LAMP2 in ARPE-19 cells without treatment. (D) Exposure to 2.5 µM rotenone for 24 hrs alone or POS for 12 hrs alone did not change LAMP2 expression. However, when the cells were treated with rotenone for 24 hrs to damage their mtDNA and then fed POS for 12 hrs, LAMP2 was significantly increased at 36 hrs (p<0.05).

Figure 7. Exosome markers in RPE/choroid of mice.

(A–B): Localization of CD63 in young and old RPE/choroid. (A) In the young RPE/choroid, there was no CD63 labeling in the RPE layer or Bruch's membrane. (B) In the old RPE/choroids, there was labeling in the RPE layer in old animals (small arrow heads). CD63 deposition at Bruch's membrane showed large and discontinuous clumps (arrows). (C–E): Comparison of protein levels of CD63 and LAMP2 in RPE/choroid of young and old mice by immunoblot. (C) CD63 and (D) LAMP2 were increased in the RPE/choroid from old animals. (E) β-actin was used as a loading control. (F–G): Co-localization of CD63 (green) and C3 (red) in young and old RPE/choroids. (F) In the young RPE/choroid, there was no co-localization of CD63 and C3 in the RPE layer or Bruch's membrane. (G) In the old RPE/choroid, C3 co-localized with CD63 at Bruch's membrane (arrows). Figure F and G were overlay images with the brightfield image. Scale bar = 20 µm.

Figure 8. Immunolocalization of exosome markers in the RPE/choroid complex.

(A): CD63 (red); AMD eye from a 74-year-old male. Anti-CD63 antibody labels large amorphous areas (CD63: arrows) in drusen. (B):CD81 (red); AMD eye from a 96-year-old male (CD81, arrows). (C): LAMP2 (red). AMD eye from a 93-year-old (LAMP2, arrow). (D): CD63 (red); Non-AMD eye from a 75-year-old male as an age-matched control. No CD63 labeling was seen in the drusen from any age-matched control (n = 10). (E–H): CD63 (green) co-localization with proteins that are known to be in drusen. (E) amyloid β (red) showed no co-localization with CD63 (arrow). (F) α B crystalline (red) showed co-localization with CD63 (arrowhead). (G) C5b-9 (red) showed no co-localization with CD63 (arrow). (H) CFH (red) showed co-localization with CD63 (arrowhead). Dr, drusen. Blue: DAPI.

Figure 9. Analyses of exosomes by FACS.

(A) Exoxomes are CD63, LAMP2, CD81 and C3 positive, but CFH negative. Isotype control: Black; CFH: light green; CD63: blue; LAMP2: red; CD81: dark green; C3: magenta. (B) Added CFH bound to exosomes in a dose-dependent manner. (C) Added CFH did not alter the amount of C3 on exosomes. For B and C: CFH 0.5 mg/mL: black; CFH 0.05 mg/mL: blue; CFH 0.005 mg/mL: purple; CFH 0.0005 mg/mL: green; CFH 0 mg/mL: red.

Figure 10. Cytokines secreted from RPE cells and macrophage infiltration into old mouse RPE.

(A) Human Cytokine Array of media from ARPE-19 cells. MCP-1, MIF, CXCL1, IL-8 and PAI-1 were detectable in both the control group and after rotenone treatmen t and appear to increase under conditions of increased mtDNA damage. (B) MCP-1 ELISA. MCP-1 protein from cell media was significantly increased at 1.25–2.5 µM concentrations of rotenone (p<0.05). (C) MIF ELISA. MIF protein from cell media was significantly increased at 0.6–2.5 µM concentrations of rotenone (p<0.05). (D) Absence of F4/80 labeled macrophages in young RPE/choroid tissue (overlaid with a brightfield image; (E) F4/80 labeled macrophages infiltrated into old RPE/choroid tissue (red). Arrows indicate that most of the macrophages are on the choroid side of Bruch's membrane.