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

Wang AL, Lukas TJ, Yuan M, Du N, Tso MO, Neufeld AH - PLoS ONE (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Forsythe Laboratory for the Investigation of the Aging Retina, Northwestern University School of Medicine, Chicago, Illinois, United States of America.

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.

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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.
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pone-0004160-g002: 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.

Mentions: In mammalian cells, an Atg12-Atg5 conjugate associates with the membranes of precursor autophagosomes [23]. We examined the autophagy conjugation system by detecting Atg12-Atg5 using a specific monoclonal antibody raised against a full-length recombinant Atg12. There was immunolabeling for ATG12 in the RPE of old mice but no immunolabeling in the RPE of young mice (Fig. 2A and B). By Western blots (Fig 2C–F), we found that autophagy markers, Atg12-Atg5 and LC3B, were increased in RPE/choroids in old mice (24–28 mos) compared to young mice (4 mos).


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

Wang AL, Lukas TJ, Yuan M, Du N, Tso MO, Neufeld AH - PLoS ONE (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2612751&req=5

pone-0004160-g002: 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.
Mentions: In mammalian cells, an Atg12-Atg5 conjugate associates with the membranes of precursor autophagosomes [23]. We examined the autophagy conjugation system by detecting Atg12-Atg5 using a specific monoclonal antibody raised against a full-length recombinant Atg12. There was immunolabeling for ATG12 in the RPE of old mice but no immunolabeling in the RPE of young mice (Fig. 2A and B). By Western blots (Fig 2C–F), we found that autophagy markers, Atg12-Atg5 and LC3B, were increased in RPE/choroids in old mice (24–28 mos) compared to young mice (4 mos).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Forsythe Laboratory for the Investigation of the Aging Retina, Northwestern University School of Medicine, Chicago, Illinois, United States of America.

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.

Show MeSH
Related in: MedlinePlus