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Cited by 158Retinal Diseases and TreatmentsAutophagy in Disease and TherapyRetinal Development and Disorders

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The Age Lipid A2E and Mitochondrial Dysfunction Synergistically Impair Phagocytosis by Retinal Pigment Epithelial Cells

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Vives-Bauza, Cristofol, Anand, Monika, Shirazi, Arash K., Magrane, Jordi, Gao, Junping, Vollmer-Snarr, Heidi R., Manfredi, Giovanni, Finnemann, Silvia C.·2008·Journal of Biological Chemistry
DOI 10.1074/jbc.m800706200PMC PMC2529005
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Abstract

Accumulation of indigestible lipofuscin and decreased mitochondrial energy production are characteristic age-related changes of post-mitotic retinal pigment epithelial (RPE) cells in the human eye. To test whether these two forms of age-related impairment have interdependent effects, we quantified the ATP-dependent phagocytic function of RPE cells loaded or not with the lipofuscin component A2E and inhibiting or not mitochondrial ATP synthesis either pharmacologically or genetically. We found that physiological levels of lysosomal A2E reduced mitochondrial membrane potential and inhibited oxidative phosphorylation (OXPHOS) of RPE cells. Furthermore, in media with physiological concentrations of glucose or pyruvate, A2E significantly inhibited phagocytosis. Antioxidants reversed these effects of A2E, suggesting that A2E damage is mediated by oxidative processes. Because mitochondrial mutations accumulate with aging, we generated novel genetic cellular models of RPE carrying mitochondrial DNA point mutations causing either moderate or severe mitochondrial dysfunction. Exploring these mutant RPE cells we found that, by itself, only the severe but not the moderate OXPHOS defect reduces phagocytosis. However, sub-toxic levels of lysosomal A2E are sufficient to reduce phagocytic activity of RPE with moderate OXPHOS defect and cause cell death of RPE with severe OXPHOS defect. Taken together, RPE cells rely on OXPHOS for phagocytosis when the carbon energy source is limited. Our results demonstrate that A2E accumulation exacerbates the effects of moderate mitochondrial dysfunction. They suggest that synergy of sub-toxic lysosomal and mitochondrial changes in RPE cells with age may cause RPE dysfunction that is known to contribute to human retinal diseases like age-related macular degeneration.

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FIGURE1 LysosomalA2EinhibitsmitochondrialATPsynthesis. RPE-Jcells received 50 𝛍 MA2E(all black bars ) or solvent (all gray bars ) for 6 h followed by overnight incubation in growth medium prior to quantification of ATP content ( A ), ATP synthesis rate ( B ), or live lysosome and mitochondria organelle labeling ( C ). A , RPE-J cells received medium supplemented with 5% dialyzed fetal bovine serum and either 1 g/liter glucose or 1 mM pyruvate as indicated for 5 h. ATP content in trichloroacetic acid cell extracts was quantified. B , ATP synthesis rates of RPE-J cells with or without A2E were measured using substrates succinate (in the presence of 1 𝛍 Mrotenone;complexII)ormalateplus pyruvate (complex I) as indicated. Addition of 1 mM NAC antioxidant to growth medium during the overnight incubation following A2E load abolished the effect of A2E on mitochondrial ATP synthesis rate (dark gray bar , complex I). Bars show mean ± S.E. of three independent measurements. Asterisks indicate significant differences ( p < 0.05). C , live triple color confocal single x-y scans of A2E, LysoSensor, and MitoTracker as indicated of A2Eloaded RPE-J cells stained with LysoSensor only ( upper panels ) or with LysoSensor and MitoTracker ( lower panels ). A2E and LysoTracker signals colocalize like in our previous studies (17, 36). The lower field shows that mitochondria localize distinctly from lysosomes and from A2E. Acquisition parameters for upper and lower fields were identical and are provided under 'Experimental Procedures.' Scale bars , 10 𝛍 m.
FIGURE1 LysosomalA2EinhibitsmitochondrialATPsynthesis. RPE-Jcells received 50 𝛍 MA2E(all black bars ) or solvent (all gray bars ) for 6 h followed by overnight incubation in growth medium prior to quantification of ATP content ( A ), ATP synthesis rate ( B ), or live lysosome and mitochondria organelle labeling ( C ). A , RPE-J cells received medium supplemented with 5% dialyzed fetal bovine serum and either 1 g/liter glucose or 1 mM pyruvate as indicated for 5 h. ATP content in trichloroacetic acid cell extracts was quantified. B , ATP synthesis rates of RPE-J cells with or without A2E were measured using substrates succinate (in the presence of 1 𝛍 Mrotenone;complexII)ormalateplus pyruvate (complex I) as indicated. Addition of 1 mM NAC antioxidant to growth medium during the overnight incubation following A2E load abolished the effect of A2E on mitochondrial ATP synthesis rate (dark gray bar , complex I). Bars show mean ± S.E. of three independent measurements. Asterisks indicate significant differences ( p < 0.05). C , live triple color confocal single x-y scans of A2E, LysoSensor, and MitoTracker as indicated of A2Eloaded RPE-J cells stained with LysoSensor only ( upper panels ) or with LysoSensor and MitoTracker ( lower panels ). A2E and LysoTracker signals colocalize like in our previous studies (17, 36). The lower field shows that mitochondria localize distinctly from lysosomes and from A2E. Acquisition parameters for upper and lower fields were identical and are provided under 'Experimental Procedures.' Scale bars , 10 𝛍 m.CC-BY · From the PDF
FIGURE 2 A2Ereduces binding and internalization of OS by RPE-J cells in thepresenceofphysiologicallevelsofglucoseorpyruvate. A and B , RPE-J cells with ( black bars ) or without ( gray bars ) A2E received FITC-labeled OS for 5 h in carbon energy source-free medium containing 4.5 g/liter glucose, 1 g/liter glucose, or 1 mM pyruvate with or without 20 𝛍 M Trolox as indicated. Bars show relative binding ( A ) and internalization ( B ) compared with cells in 4.5 g/liter glucose, which was set at 100%. C and D , experiments as in A and B were repeated with inclusion of 0.1 𝛍 M oligomycin in the 5-h uptake assay. Bars show percent inhibition of binding ( C ) and internalization ( D ) compared with the same condition without drug. A to D , bars represent mean ± S.D., of three independent assays with triplicate samples each. Asterisks indicate significant differences of cells with A2E compared with cells without A2E in the same condition ( p < 0.05).
FIGURE 2 A2Ereduces binding and internalization of OS by RPE-J cells in thepresenceofphysiologicallevelsofglucoseorpyruvate. A and B , RPE-J cells with ( black bars ) or without ( gray bars ) A2E received FITC-labeled OS for 5 h in carbon energy source-free medium containing 4.5 g/liter glucose, 1 g/liter glucose, or 1 mM pyruvate with or without 20 𝛍 M Trolox as indicated. Bars show relative binding ( A ) and internalization ( B ) compared with cells in 4.5 g/liter glucose, which was set at 100%. C and D , experiments as in A and B were repeated with inclusion of 0.1 𝛍 M oligomycin in the 5-h uptake assay. Bars show percent inhibition of binding ( C ) and internalization ( D ) compared with the same condition without drug. A to D , bars represent mean ± S.D., of three independent assays with triplicate samples each. Asterisks indicate significant differences of cells with A2E compared with cells without A2E in the same condition ( p < 0.05).CC-BY · From the PDF
Image 3
CC-BY · From the PDF
FIGURE4 AntioxidantsrestorebothloweredRPEmitochondrialmembrane potential ( 𝚫𝚿 m) and decreased OS uptake by primary rat RPE caused by A2E load. A , RPE-J cells loaded with A2E or not and incubated with 1 mM NAC overnight or not as indicated in the panels were loaded with the membrane potentiometric dye TMRM and imaged live. Representative maximal projections are shown. Scale bars , 20 𝛍 m. B , each bar represents mean ± S.E. of total TMRM signal intensities quantified from individual cells in arbitrary units ( A.U. ) by Metamorph H23006 , n = 35, cells imaged during three independent experiments. Gray bars , control cells; black bars , cells loaded with A2E. C and D , TMRMlivelabeling, imaging,andevaluationwasperformedasdescribedfor A and B usingunpassagedrat RPE cells in primary culture with or without A2E load as indicated. C , representative maximal projections are shown. Scale bars , 20 𝛍 m. D , each bar represents mean ± S.E. of total cellular TMRM signal intensities, n = 30, cells imaged during three independent experiments. Gray bars , control cells; black bars , cells loaded withA2E. Asterisks in B and D indicatesignificantdifferencesbetweenA2E-loaded cells with and without antioxidant ( p < 0.001). NAC did not significantly alter TMRMintensity of control cells ( p > 0.05). Imaging and quantification details are provided in the experimental procedures section. E and F , primary, unpassaged rat RPE loaded ( black bars ) or not ( graybars ) with A2E were challenged with FITClabeledOSinmediumcontaining1mMpyruvatewithorwithout20 𝛍 MTroloxas indicated for 1.5 h before quantification of bound ( E ) and internalized OS ( F ). All bars represent mean OS per cell ± S.D., of three independent experiments with duplicate samples each. Asterisks indicate significantly different OS uptake by A2E-loaded cells with and without antioxidant ( p < 0.01).
FIGURE4 AntioxidantsrestorebothloweredRPEmitochondrialmembrane potential ( 𝚫𝚿 m) and decreased OS uptake by primary rat RPE caused by A2E load. A , RPE-J cells loaded with A2E or not and incubated with 1 mM NAC overnight or not as indicated in the panels were loaded with the membrane potentiometric dye TMRM and imaged live. Representative maximal projections are shown. Scale bars , 20 𝛍 m. B , each bar represents mean ± S.E. of total TMRM signal intensities quantified from individual cells in arbitrary units ( A.U. ) by Metamorph H23006 , n = 35, cells imaged during three independent experiments. Gray bars , control cells; black bars , cells loaded with A2E. C and D , TMRMlivelabeling, imaging,andevaluationwasperformedasdescribedfor A and B usingunpassagedrat RPE cells in primary culture with or without A2E load as indicated. C , representative maximal projections are shown. Scale bars , 20 𝛍 m. D , each bar represents mean ± S.E. of total cellular TMRM signal intensities, n = 30, cells imaged during three independent experiments. Gray bars , control cells; black bars , cells loaded withA2E. Asterisks in B and D indicatesignificantdifferencesbetweenA2E-loaded cells with and without antioxidant ( p < 0.001). NAC did not significantly alter TMRMintensity of control cells ( p > 0.05). Imaging and quantification details are provided in the experimental procedures section. E and F , primary, unpassaged rat RPE loaded ( black bars ) or not ( graybars ) with A2E were challenged with FITClabeledOSinmediumcontaining1mMpyruvatewithorwithout20 𝛍 MTroloxas indicated for 1.5 h before quantification of bound ( E ) and internalized OS ( F ). All bars represent mean OS per cell ± S.D., of three independent experiments with duplicate samples each. Asterisks indicate significantly different OS uptake by A2E-loaded cells with and without antioxidant ( p < 0.01).CC-BY · From the PDF
FIGURE 5 Generation and genetic validation of d407 RPE-derived cybrids by PCR/fragment length polymorphism analysis. A , rhodamine-6G-treated parental d407 RPE cells devoid of mitochondria were fused with actinomycin D-enucleated 143B-derived osteosarcoma cybrids that carried wild-type, NARP, or MERRF mutant mitochondria to generate d407 RPE cybrid clones that carry exclusively donor-derived mitochondria. B , the restriction enzyme AvaI cleaves a529-bpNARPmutantmtDNAPCRfragmentintothreefragmentsof301,130,and92bp,whereaswild-typemtDNAdigestyieldstwofragmentof392and130 bp. u = uncut PCR product; d , d407 parental cell line; w , wild-type 143B-derived cybrid; n , 143B-derived NARP cybrid; N1 and N2 , d407-derived NARP mutant clones. C , the restriction enzyme NaeI cleaves a 118-bp PCR fragment of MERRF mtDNA but not parental d407 mtDNA into fragments of 100 bp and 18 bp. u = uncut PCR product; d , d407 parental cell line; m , 143B-derived MERRF cybrid; M , d407-derived MERRF mutant clone. D , PCR/fragment length polymorphism analysis of the neutral polymorphism T16519C. The restriction enzyme HaeIII cleaves a PCR product of 174 bp into three fragments of 64, 56, and 54 bp if the template is d407 parental mtDNA but into only two fragments of 120 and 54 bp if the template is 143B-derived mtDNA. Lane designations are the same as in B and C .
FIGURE 5 Generation and genetic validation of d407 RPE-derived cybrids by PCR/fragment length polymorphism analysis. A , rhodamine-6G-treated parental d407 RPE cells devoid of mitochondria were fused with actinomycin D-enucleated 143B-derived osteosarcoma cybrids that carried wild-type, NARP, or MERRF mutant mitochondria to generate d407 RPE cybrid clones that carry exclusively donor-derived mitochondria. B , the restriction enzyme AvaI cleaves a529-bpNARPmutantmtDNAPCRfragmentintothreefragmentsof301,130,and92bp,whereaswild-typemtDNAdigestyieldstwofragmentof392and130 bp. u = uncut PCR product; d , d407 parental cell line; w , wild-type 143B-derived cybrid; n , 143B-derived NARP cybrid; N1 and N2 , d407-derived NARP mutant clones. C , the restriction enzyme NaeI cleaves a 118-bp PCR fragment of MERRF mtDNA but not parental d407 mtDNA into fragments of 100 bp and 18 bp. u = uncut PCR product; d , d407 parental cell line; m , 143B-derived MERRF cybrid; M , d407-derived MERRF mutant clone. D , PCR/fragment length polymorphism analysis of the neutral polymorphism T16519C. The restriction enzyme HaeIII cleaves a PCR product of 174 bp into three fragments of 64, 56, and 54 bp if the template is d407 parental mtDNA but into only two fragments of 120 and 54 bp if the template is 143B-derived mtDNA. Lane designations are the same as in B and C .CC-BY · From the PDF
FIGURE 6 NARP and MERRF mutant d407 RPE cybrids retain phagocytic protein expression and form mitochondria of normal appearance. MERRF but not NARP mutant cells lose COX-1 expression. A , comparative immunoblots of lysates prepared from wild-type cybrids W1 and W2, NARP mutant cybrids N1 and N2, and MERRF mutant cybrid clone M show similar protein expression levels of actin, tubulin, 𝛂 v and 𝛃 5 integrins, MerTK, focal adhesion kinase, and porin. As expected, COX-1 (encoded by mtDNA) was severely decreased in MERRF cells but remained normal in NARP mutant cells. B , immunofluorescence labeling of COX-1 confirmed reduction of COX-1 exclusively in MERRF mutant cybrids. C , immunofluorescence labeling of porin demonstrated similar abundance of mitochondrial organelles in all cybrids. Representative images acquired and processed identically are shown. Imaging details are provided under 'Experimental Procedures.' All scale bars : 10 𝛍 m.
FIGURE 6 NARP and MERRF mutant d407 RPE cybrids retain phagocytic protein expression and form mitochondria of normal appearance. MERRF but not NARP mutant cells lose COX-1 expression. A , comparative immunoblots of lysates prepared from wild-type cybrids W1 and W2, NARP mutant cybrids N1 and N2, and MERRF mutant cybrid clone M show similar protein expression levels of actin, tubulin, 𝛂 v and 𝛃 5 integrins, MerTK, focal adhesion kinase, and porin. As expected, COX-1 (encoded by mtDNA) was severely decreased in MERRF cells but remained normal in NARP mutant cells. B , immunofluorescence labeling of COX-1 confirmed reduction of COX-1 exclusively in MERRF mutant cybrids. C , immunofluorescence labeling of porin demonstrated similar abundance of mitochondrial organelles in all cybrids. Representative images acquired and processed identically are shown. Imaging details are provided under 'Experimental Procedures.' All scale bars : 10 𝛍 m.CC-BY · From the PDF
FIGURE7 Theseverityofmitochondrialdeficiencydeterminessensitivity to A2E. We compared mitochondrial respiration ( A ), ATP synthesis rates ( B ), ATP content ( C ), and phagocytic activity with and without A2E ( D ) among parental d407 cells (parent, dark gray bars ), wild-type d407 RPE cybrids (W1 andW2, white bars ), NARP mutant d407 RPE-derived cybrids (N1 and N2, light gray bars ), and MERRF mutant d407 RPE clone M ( black bars ). A , bars show oxygenconsumptionbyintactcellsusingpyruvateassubstrate(mean ± S.D., n = 5). Asterisks indicate significant differences compared with parental cells ( p < 0.05). NARP mutant clones respire significantly less than parental or wild-type cells but significantly more than MERRF cells. B , bars show ATP synthesis rates with malate plus pyruvate as substrates (mean ± S.D., n = 5). Asterisks indicate significant differences compared with wild-type clone W1 ( p < 0.05). C , bars show ATP steady-state levels for cybrids following feeding for 3.5 h with physiological concentrations of glucose ( left ) or pyruvate ( right ) (mean ± S.E., n = 5). Asterisks indicate significant differences compared with wild-type clone W1 ( p < 0.05). D , bars show relative OS internalization for each clone in the presence of pyruvate as compared with in the presence of glucose. For cells without A2E ( left panel ) asterisks indicate significant differencescomparedwithwild-typecloneW1( p < 0.05). For cells loaded with A2E at about one-fifth of the level of aging human RPE ( right panel ), asterisks indicate significant differences compared with relative internalization without A2Eforthesameclone(compare left with right bars ). Even low concentrations of A2E were highly toxic to MERRF mutant cells precluding phagocytosis measurements for clone M.
FIGURE7 Theseverityofmitochondrialdeficiencydeterminessensitivity to A2E. We compared mitochondrial respiration ( A ), ATP synthesis rates ( B ), ATP content ( C ), and phagocytic activity with and without A2E ( D ) among parental d407 cells (parent, dark gray bars ), wild-type d407 RPE cybrids (W1 andW2, white bars ), NARP mutant d407 RPE-derived cybrids (N1 and N2, light gray bars ), and MERRF mutant d407 RPE clone M ( black bars ). A , bars show oxygenconsumptionbyintactcellsusingpyruvateassubstrate(mean ± S.D., n = 5). Asterisks indicate significant differences compared with parental cells ( p < 0.05). NARP mutant clones respire significantly less than parental or wild-type cells but significantly more than MERRF cells. B , bars show ATP synthesis rates with malate plus pyruvate as substrates (mean ± S.D., n = 5). Asterisks indicate significant differences compared with wild-type clone W1 ( p < 0.05). C , bars show ATP steady-state levels for cybrids following feeding for 3.5 h with physiological concentrations of glucose ( left ) or pyruvate ( right ) (mean ± S.E., n = 5). Asterisks indicate significant differences compared with wild-type clone W1 ( p < 0.05). D , bars show relative OS internalization for each clone in the presence of pyruvate as compared with in the presence of glucose. For cells without A2E ( left panel ) asterisks indicate significant differencescomparedwithwild-typecloneW1( p < 0.05). For cells loaded with A2E at about one-fifth of the level of aging human RPE ( right panel ), asterisks indicate significant differences compared with relative internalization without A2Eforthesameclone(compare left with right bars ). Even low concentrations of A2E were highly toxic to MERRF mutant cells precluding phagocytosis measurements for clone M.CC-BY · From the PDF

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  1. Feeney, 1978, Invest. Ophthalmol. Vis. Sci., 17, 583
  2. Sparrow, 1999, Invest. Ophthalmol. Vis. Sci., 40, 2988
  3. Del Priore, 2002, Invest. Ophthalmol. Vis. Sci., 43, 3312
  4. Barron, 2001, Invest. Ophthalmol. Vis. Sci., 42, 3016
  5. Holt, 1990, Am. J. Hum. Genet., 46, 428
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Metadata

Authors
Vives-Bauza, C., Anand, M., Shirazi, A. K., Magrane, J., Gao, J., Vollmer-Snarr, H. R., Manfredi, G., & Finnemann, S. C.
Affiliations
  • Cornell University (US)
  • Brigham Young University (US)
Corresponding author
Finnemann, Silvia C.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2008-09
Volume
283
Issue
36
Pages
24770-24780
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m800706200
PMC
PMC2529005
OpenAlex
W2021234912
Cited by
155
References
54
Citations by year
2026: 4 · 2025: 6 · 2024: 6 · 2023: 7 · 2022: 7 · 2021: 8 · 2020: 13 · 2019: 6 · 2018: 12 · 2017: 8 · 2016: 4 · 2015: 9 · 2014: 17 · 2013: 9 · 2012: 13
FWCI
8.78
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Fulbright Program
  • American Health Assistance Foundation
  • Research to Prevent Blindness
  • National Institutes of Health
  • National Eye Institute (R01 EY13295, R01EY013295)
  • National Institute of Neurological Disorders and Stroke (K02 NS 47306, K02NS047306)
Topics
  • Health Sciences › Medicine › Ophthalmology › Retinal Diseases and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Health Sciences › Medicine › Ophthalmology › Retinal Development and Disorders
Keywords
retinal pigment epithelium, phagocytosis, mitochondrial dysfunction, oxidative phosphorylation, mitochondrial membrane potential, aging, mitochondrial DNA mutations, lysosomal accumulation, oxidative stress, age-related macular degeneration, RPE cell death
MeSH terms
Adenosine Triphosphate, Aging, Animals, Antioxidants, Cell Line, DNA, Mitochondrial, Epithelial Cells, Glucose, Humans, Lipofuscin, Lysosomes, Macular Degeneration, Mitochondria, Mitosis, Oxidative Phosphorylation, Phagocytosis, Pigment Epithelium of Eye, Pyridinium Compounds, Retinoids, Cell Death, Point Mutation, Pyruvic Acid, Rats, Long-Evans, Rats, Membrane Potential, Mitochondrial

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