IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2005;46:1765-1773.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1327
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miceli, M. V.
Right arrow Articles by Jazwinski, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miceli, M. V.
Right arrow Articles by Jazwinski, S. M.

Nuclear Gene Expression Changes Due to Mitochondrial Dysfunction in ARPE-19 Cells: Implications for Age-Related Macular Degeneration

Michael V. Miceli and S. Michal Jazwinski

From the Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To measure changes in nuclear gene expression resulting from mitochondrial dysfunction in retinal pigment epithelial cells.

METHODS. ARPE-19 retinal pigment epithelial cells were depleted of their mitochondrial (mt)DNA by passaging in a low concentration of ethidium bromide. Loss of mitochondrial DNA was determined by uridine auxotrophy and quantitative real-time polymerase chain reaction of isolated DNA. Loss of mitochondrial membrane potential was estimated by uptake of JC-1. Changes in nuclear gene expression were determined by quantitative real-time reverse transcription–polymerase chain reaction of isolated total RNA from ethidium-bromide–treated and untreated cells. Morphologic and phenotypic changes were determined by phase-contrast microscopy, sensitivity to the oxidant tert-butyl hydroperoxide (tBH), and invasion assay.

RESULTS. ARPE-19 cells became auxotrophic for growth on uridine after eight passages in 50 ng/mL ethidium bromide. Quantitative PCR revealed almost complete loss of mitochondrial DNA ({rho}0 cells). Uptake of JC-1 was reduced in the {rho}0 cells, indicating reduction of mitochondrial membrane potential. Quantitative RT-PCR measured increased expression of genes coding for drusen components, lipid transport, extracellular matrix components, and responses to inflammation in the {rho}0 cells. The {rho}0 cells also exhibited an increased sensitivity to killing by tBH and increased migration and invasion through solubulized basement membrane–coated tissue culture inserts.

CONCLUSIONS. ARPE-19 cells respond to loss of mitochondrial function by changes in nuclear gene expression that resemble changes observed in age-related macular degeneration. The results lead to the hypothesis that loss of mitochondrial function with age and resultant changes in nuclear gene expression may explain some of the changes in the macula that are associated with the known clinical manifestations of age-related macular degeneration.

The retinal pigment epithelium (RPE) is a single layer of cells in the vertebrate retina, and its continuous function has been shown to be necessary for vision.1 2 The RPE is a terminally differentiated tissue that ages in a unique fashion and in which changes with age have been implicated in the development of age-related macular degeneration (AMD),3 4 the leading cause of untreatable vision loss in persons older than 65 years in developed countries.5 6 Indeed, it has been estimated that at least 8 million people ≥65 years of age in the United States are at high risk for the development of advanced AMD.7

Some of the changes that have been shown to occur in the macula and that correlate with AMD include increased deposition in the RPE of the age pigment lipofuscin,8 9 the development of drusen between the RPE basal lamina and Bruch’s membrane,10 11 increased sterol deposition in the elastin layer of Bruch’s membrane,12 thickening of Bruch’s membrane and other basement membrane changes,13 14 choroidal neovascularization,15 16 and RPE cell apoptosis.17 Although the clinical manifestations of RPE aging are well described and several theories have been proposed to explain the cause of AMD, none of these theories has led to a satisfactory understanding of this unique aging phenotype.

We have been working to understand how changes in mitochondrial function contribute to aging phenotypes in eukaryotic cells. In one model of eukaryotic mitochondrial dysfunction, yeast cells that have been depleted of their mitochondrial (mt)DNA ({rho}0 cells) respond to loss of mitochondrial function with induction of a specific pathway that induces changes in nuclear gene expression.18 These changes, called retrograde regulation or retrograde response, respond to this loss in a way that not only compensates for the loss of respiratory function but also results in increased replicative lifespan in cells grown on fermentable substrates.19 20 Furthermore, our laboratory has demonstrated that mitochondrial dysfunction is a normal consequence of yeast aging and that the retrograde response increases with replicative age and titrates the progressive loss of mitochondrial function to enhance longevity.21

Mammalian cells also respond to loss of mitochondrial function with changes in nuclear gene expression. It has been shown that the mitochondrial-to-nuclear stress signal is probably due to a decrease in mitochondrial membrane potential, which results in increased cytosolic free [Ca]2+.22 The increased in [Ca]2+ in turn activates Ca2+-sensitive protein phosphatases, kinases, and calcium-calmodulin kinase IV (CaMKIV), which activate transcription factors, resulting in changes in nuclear gene expression.23 Although these changes in nuclear gene expression seem to be necessary for cell survival, we have shown that the specific genes induced are common to several cell types or are cell-type specific and appear to be dependent on the differentiated functions of the cell.24

In this study, we attempted to establish a link between the loss of mitochondrial function in RPE cells and changes that contribute to the development of AMD, by investigating changes in nuclear gene expression in ARPE-19 cells brought on by loss of mtDNA ({rho}0ARPE-19). These cells contain mitochondria but cannot respire, because of the absence of the subset of 13 respiratory transport chain proteins that are coded for by the mitochondria. We have previously reported that {rho}0ARPE-19 cells induce the transcription of glycolytic pathway genes to maintain energy production, genes to maintain cellular redox potential and mitochondrial membrane potential, and genes that may allow for increased stress resistance and protect the cell against cell death.24 Furthermore, there is evidence in the literature that a reduction in mitochondrial function may contribute to the development of AMD.25 26 Therefore, we hypothesized that changes in RPE nuclear gene expression due to loss of mitochondrial function may help explain some of the phenotypic changes in aged RPE cells that lead to the clinical manifestations in AMD. In this study, we more thoroughly characterized {rho}0ARPE-19 cells as a model system in which to study loss of mitochondrial function and demonstrate some specific changes in nuclear gene expression and in cell phenotype due to mitochondrial dysfunction.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
The retinal pigment epithelial cell line ARPE-19 was obtained from ATCC (Manassas, VA). ARPE-19 is an established cell line that has been shown to express many of the characteristics of RPE cells in culture.27 The cells were grown in DMEM/Ham’s F12 1:1 (Invitrogen-Gibco, Grand Island, NY) containing 24 mM sodium bicarbonate, 10% FBS, 10 µg/mL uridine, and 1.0 mM sodium pyruvate. All cells were maintained in a 5% CO2 humidified atmosphere at 37°C and grown without added antibiotics or fungicides.

The ARPE-19 cells were depleted of their mtDNA with ethidium bromide (EtBr).28 ARPE-19 cells were grown in T75 flasks and then passaged 1:3 into medium containing 50 ng/mL EtBr. Cells were grown until confluent and then serially passaged in medium containing EtBr. The cells were screened for loss of mtDNA by passaging into medium without added uridine containing 10% dialyzed FBS. Cells that have lost their mtDNA become auxotrophic for growth on uridine due to the loss of functional dihydroorotate dehydrogenase, which is located on the outer surface of the mitochondrial inner membrane. After five to seven passages, the ARPE-19 cells were unable to grow without added uridine. Loss of the mitochondrial genome was confirmed by quantitative real-time polymerase chain reaction (PCR), with a primer set that spanned the origin of replication of the mitochondrial heavy strand, as described later. Control cells were treated in an identical fashion, except that EtBr was omitted from the medium.

Nucleic Acid Isolation and Quantitative Real-Time RT-PCR
DNA and RNA were isolated from trypsinized cells with isolation kits (DNeasy or RNeasy; Qiagen, Valencia, CA). RNA was isolated with on-column DNase I digestion from recently confluent cells that had been fed 6 hours earlier. Final yields and concentrations were calculated by measuring the absorbance at 260 nm in 10 mM Tris, 1 mM EDTA (pH 7.5; TE buffer).

The genes of interest were chosen because their protein products have been identified as components of drusen, because they have been shown to be upregulated in RPE or retina of individuals with AMD, because of their association with extracellular components of Bruch’s membrane, or because of their association with outer segment phagocytosis or lipid transport into the RPE. Table 1 is a list of the genes analyzed and their descriptions.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Accepted Names of Genes Analyzed, Descriptions, and References

 
Real-time quantitative RT-PCR was performed on a commercial system (Prism 7000 Sequence Detection real-time PCR system; Applied Biosystems, Inc. [ABI], Foster City, CA) in a two-step reaction. Total RNA was reverse transcribed in a 50-µL volume with the RT reagents (ABI). Each reaction contained 1 µg of RNA, 5 µL of 10x RT buffer, 5.5 mM MgCL2, 2 mM dNTPs, 2.5 µM random hexamers, 20 U RNase inhibitor, and 62.5 U reverse transcriptase (MultiScribe; ABI). For real-time PCR, primers for the genes of interest were designed with commercial software (Primer Express; ABI) and purchased from Integrated DNA Technologies, Inc. (Coralville, IA). In general the primer pair with the lowest penalty score identified by the software was chosen. All primer pairs were check by a BLAST search against the human mRNA database, to assure homology only with the gene of interest. In cases in which there was more than one transcript variant, primer pairs were chosen that mapped to all known variants. Each 25-µL reaction contained 0.5 µL of the RT reaction as template, 0.2 µM each of the forward and reverse primers, and 12.5 µL 2x PCR mix (SYBR Green PCR Master Mix; ABI). All reactions were performed in triplicate and consisted of 1 cycle of 50°C for 2 minutes, 1 cycle of 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute (two-step PCR). The appropriate housekeeping gene to use as a control for DNA loading was determined by screening a set of commonly used housekeeping genes (ß-actin [ACTB] peptidylprolyl isomerase A [PPIA], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and ribosomal protein S9 [RPS9]) and choosing the gene that showed no significant difference in expression between control and experimental cells. With this method, we chose PPIA as the control gene. Standard curves were generated from dilutions of the cDNA template obtained from ARPE-19 cells, with primers for PPIA, and relative concentrations of the genes of interest were calculated from the standard curves after normalization with the housekeeping gene. In some cases, dilutions of the cDNA templates were analyzed with primer pairs of the genes of interest, to be certain that the genes of interest were amplified similarly to the housekeeping genes.

Estimation of Mitochondrial Membrane Potential
ARPE-19 cells were plated on two-well chamber slides (InterMed, Naperville, IL) and grown until confluent. The cells were incubated with 10 nM of the lipophilic dye JC-1 in complete medium for 10 minutes at 37°C,29 and either rinsed with PBS, coverslipped, and observed under a fluorescence microscope or trypsinized and analyzed in a flow cytometer. For microscopy the cells were observed by deconvolution microscope equipped with a mercury lamp and filters for epifluorescence applications (model DMRXA; Leica, Deerfield, IL). Digital images were then obtained (Sensicam QE 12-bit, cooled CCD system; PCO AG, Kelheim, Germany; and SlideBook image-processing software; Intelligent Imaging Innovations, Santa Monica, CA). Alternatively, the cells were analyzed with a flow cytometer (EPICS; Beckman Coulter, Inc., Fullerton, CA) and the data collected in both the FITC and rhodamine channels.

Cell Viability MTT Assay
Cell viability after oxidative stress was assessed with 3-(4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT). Cells plated in 24-well plates were grown to confluence and treated with 0 to 2000 µM tert-butyl hydroperoxide (tBH), for 6 hours in the absence of serum. After treatment, the cells were fed with growth medium and maintained overnight. The next day, the cells were incubated with DMEM without phenol red, containing 5% FBS and 1.0 mM MTT for 2 hours at 37°. Afterward, the cells and the formazan MTT reduction product were solubilized with 0.1 g/mL SDS in 0.01 M HCl and read at 570 nm.

Cell Invasion Assay
Cell invasion assays were performed in growth-factor–reduced, solubulized basement membrane (Matrigel)–coated, 24-well invasion chambers (BD Biosciences, Franklin Lakes, NJ). The precoated wells were rehydrated by incubation with 500 µL of culture medium in the lower chamber, and then 5 x 104 cells in 300 µL of medium were added to the upper chamber. The wells were incubated at 37°C for 48 hours under normal culture conditions. The medium was removed, and the cells adhering to the upper side of the membrane were removed by scrubbing three times with a cotton swab moistened with culture medium. The cells remaining on the underside of the membrane were then fixed with 0.5 mL 2% formaldehyde and 0.2% glutaraldehyde in PBS for 10 minutes. After they were rinsed, the cells were stained with 0.5 mL hematoxylin, counterstained with eosin, and rinsed with distilled water. The number of cells that migrated to the underside of the membrane was determined with an inverted microscope. In all, the assay was performed in duplicate two times, and three fields per insert were counted and averaged.

Statistical Analysis
Testing of statistical significance was performed on computer (StatMost; DataMost Corp., Sandy, UT). For the real-time RT-PCR data, Student’s paired t-test was performed on the raw values corrected for DNA loading as described earlier. P ≤ 0.05 was taken as the criterion of significance.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The EtBr treatment of ARPE-19 cells resulted in a morphologic change in the cells and an almost complete loss of the mtDNA. Figure 1A shows photomicrographs of ARPE-19 cells ({rho}+) and ARPE-19 cells that had been treated with EtBr for eight passages ({rho}0). In general {rho}0ARPE-19 cells grew more slowly, were unable to grow without added uridine, and appeared to spread out more. Partial characterization of the {rho}0ARPE-19 cells has been reported.24



View larger version (71K):
[in this window]
[in a new window]
  		FIGURE 1. Characterization of {rho}0ARPE-19 cells. (A) Phase-contrast photomicrographs showing control {rho}+ARPE-19 cells and {rho}0ARPE-19 cells at confluent density. {rho}0ARPE-19 cells were more spread out and grew to a lower confluent density than did control cells. (B) Quantification of mtDNA in {rho}0ARPE-19 cells. Amplification plots from a quantitative real-time PCR of DNA extracted from ARPE-19 cells and ARPE-19 cells that had been treated with 50 ng/mL EtBr ({rho}0ARPE-19). The plot is a graph of the baseline-corrected reaction fluorescence (Delta Rn) versus the cycle number. The primer pair was chosen to amplify a product spanning the origin of replication of the mitochondrial genome. Each sample was run in triplicate and contained 10 ng of total DNA. {rho}0ARPE-19 cells contained 2.5 x 10–4 the amount of mitochondrial DNA compared with the untreated control cells. Bars, 175 µm.

 
Figure 1B shows an amplification plot of a real-time quantitative PCR analysis of DNA isolated from {rho}+ARPE-19 and {rho}0ARPE-19 cells. The primers were chosen to amplify a region of the mtDNA that spans the origin of replication of the mtDNA heavy strand. Quantitative PCR showed mtDNA at <2.5 x 10–4 that of control cells.

We estimated mitochondrial membrane potential in the {rho}0ARPE-19 cells by using the lipophilic cationic fluorescent probe JC-1. Transport of JC-1 into mitochondria has been shown to be dependent on the mitochondrial membrane potential, and the fluorescence of JC-1 has been shown to change from green to red due to the formation of J aggregates at higher membrane potentials. Figure 2 shows fluorescence microscope images of {rho}+ARPE-19 and {rho}0ARPE-19 cells that were treated with JC-1. The micrographs demonstrate that the {rho}0ARPE-19 cells had reduced uptake of the dye, which indicates a lower mitochondrial membrane potential than in the untreated {rho}+ARPE-19 cells. This result was confirmed by flow cytometry.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 2. Fluorescence images of cells on chamber slides that were treated with the lipophilic dye JC-1. (A) {rho}+ARPE-19 cells observed with the FITC filter set. (B) The same field observed with the Texas red filter set, showing the formation of J aggregates in the mitochondria. (C) {rho}0ARPE-19 cells with the FITC filter set. (D) The same field observed with the Texas red filter set, showing very little J aggregate formation. The {rho}0ARPE-19 cells took up less dye than the untreated control cells, demonstrating reduced mitochondrial membrane potential. Bars, 20 µm.

 
When we measured changes in nuclear gene expression resulting from loss of mitochondrial function in a select set of genes, the results were dramatic. We arranged this select set of genes in four groups, and the results are shown in Figure 3 (see Table 1 for accepted gene names).



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 3. Changes in gene expression determined by quantitative real-time RT-PCR. Shown is the percentage change in gene expression of the experimental ({rho}0) cell population compared with the normal control ({rho}+). Data are the average of three to four determinations from at least three RNA isolations. Gene groups: (A) those coding for proteins involved in ROS uptake of components of drusen; (B) those coding for components of the extracellular matrix; (C) those coding for proteins associated with lipid transport; and (D) those associated with responses to inflammatory or oxidative stress. Gene descriptions are given in Table 1 . Error bars, ±SEM. Statistical significance was calculated from raw data normalized to cyclophilin A as the housekeeping gene. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

 
Group 3A contained genes that code for proteins that have been shown to be involved in outer segment phagocytosis or to be components of drusen. There was an increase in expression of ITGAV, ITGB5, CLU, and FBLN and a decrease in expression of VTN in {rho}0ARPE-19 cells compared with ARPE-19 cells.

Group 3B contained genes that code for components of the extracellular matrix. We measured a large increase in expression of COL1A1, COL5A1, MMP2, and PLAU; modest increased expression of PLAUR and LOX; and decreased expression of TIMP3.

Group 3C contained genes associated with lipid transport in the RPE. We noted a large increase in APOE expression, and increased expression of VLDLR, SCARB1, SCARB2, and CD68.

Group 3D contained genes associated with a response to inflammation or oxidative stress. We noted a large increase in expression of VEGF and increases in TGFB1, FGF5, CD97, MGST1, EPHX1 and SOD2.

Because oxidative stress is considered one of the contributing factors in AMD, we also were interested in whether {rho}0ARPE-19 cells were more or less sensitive to oxidative stress than their {rho}+ counterparts. Figure 4 shows the results of a cell viability assay on cells treated with the oxidant tBH. These results show that the {rho}0ARPE-19 cells are much more sensitive to the oxidant than are the control cells, indicating that the {rho}0ARPE-19 cells are more sensitive to oxidative stress than their {rho}+ counterparts.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 4. Cell viability assay for cells treated with tBH for 6 hours and assayed after 24 hours with MTT. Results are the average of six determinations ± SEM. The {rho}0ARPE-19 cells were much more susceptible to the oxidant than were the {rho}+ARPE-19 cells.

 
Finally, because pigmentary changes due to clumping and migration of the RPE are characteristic of AMD and because of the observed changes in extracellular matrix and matrix metalloproteinase gene expression observed in this study, we were interested in whether we could demonstrate a change in invasive phenotype in our {rho}0 cells. Figure 5 shows the result of a migration assay using solubulized basement membrane–coated tissue culture inserts. The results show an approximate sixfold increase in the invasive properties of the {rho}0 cells compared with the control cells.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 5. RPE cell invasion assay. (A, B) Images of fixed and stained cells that had migrated to the underside of solubulized basement membrane–coated tissue culture inserts. (C) Summary graph of the photomicrograph data for the two cell types. Data are the average number of cells per field from two separate experiments performed in duplicate. Three fields per insert were counted. Data are the mean ± SEM. The {rho}0ARPE-19 cells showed a more invasive phenotype than did the {rho}+ARPE-19 cells. Bars, 55 µm.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The results show {rho}0ARPE-19 cells had a lower mitochondrial membrane potential than the {rho}+ARPE-19 cells. It has been suggested that it is the reduced mitochondrial membrane potential in {rho}0 cells and the resultant increase in intracellular free [Ca]2+ that activates Ca2+-sensitive kinases, resulting in changes in nuclear gene expression.22

Furthermore, {rho}0ARPE-19 cells upregulated several nuclear genes known to be associated with an aging RPE phenotype and/or with AMD. Some of the changes in nuclear gene expression in response to the loss of mitochondrial function are most likely an attempt to compensate for the energy deficit. This may explain the upregulation of genes for proteins involved in lipid transport such as APOE and VLDLR, and increased expression of the genes for the phagocytosis receptors ITGAV and ITGB5. This may be because the RPE, in contrast to the neural retina, apparently derives a large portion of its energy needs from mitochondrial fatty acid ß-oxidation.54 55 Because mitochondrial fatty acid ß-oxidation is normally linked to electron transport, nonmetabolized lipids would have to be excreted or they would accumulate intracellularly. This could account for the increase of esterified lipid shown to accumulate in basal linear deposits in AMD. The cell may also increase expression of VEGF and other angiogenic factors to attempt to compensate for low energy levels, in a fashion similar to tumor cells, which upregulate angiogenic cytokines as a response to hypoxia. This upregulation of VEGF in RPE cells would contribute to the choroidal neovascularization in advanced AMD. Other upregulated genes may be due to a state of chronic inflammatory response brought on by increased cellular oxidative stress. Increased oxidative stress was evidenced by the increased sensitivity of the {rho}0ARPE-19 cells to an oxidant challenge (Fig. 4) and by the increased expression of SOD2, EPHX1, and MGST1 (Fig. 3D) . Polymorphisms in SOD2 and EPHX1 have also been shown to be associated with AMD.52

Although the effect of increased gene expression on protein translation was not measured, it is probably not coincidental that genes coding for proteins that have been identified as components of drusen were shown to be upregulated. This includes APOE, CLU, and FBLN. Vitronectin gene expression, the product of which is found in high concentrations in drusen, was shown to be markedly decreased in {rho}0ARPE-19 cells. However, it has been shown that vitronectin is abundantly expressed in the neural retina as well as the RPE,56 and the vitronectin receptor genes ITGAV and ITGB5 were upregulated in {rho}0ARPE-19 cells. Therefore, vitronectin may be deposited in drusen as nonmetabolized receptor-vitronectin complexes. A more complete picture of the effect of mitochondrial dysfunction on cellular metabolism awaits further studies that should include measurements of changes in protein expression and enzyme activity.

In our study, {rho}0ARPE-19 cells had a more invasive phenotype that was the result of increased expression of inflammatory cytokines such as TGFß and of changes in ECM and MMP gene expression. These changes could explain observations of pigmentary changes in the maculas of patients with AMD and the observation that RPE cells have been identified as components of choroidal neovascular (CNV) membranes.57 RPE cells in CNV membranes have also been reported to be immunoreactive for VEGF which correlates with the findings in the current study. Because other genes that are associated with an inflammatory response are also upregulated in {rho}0ARPE-19 cells, this VEGF immunoreactivity may indicate that these cells contribute to a chronic low level of inflammation in the retina and Bruch’s membrane. There is ample evidence that drusen deposition in the RPE-choroid is associated with an inflammatory response.58

The question is, what is the evidence of mitochondrial dysfunction in the aging human RPE cell? mtDNA deletions are clearly associated with human disease. This diverse group of diseases collectively termed mitochondrial myopathies include Kearns-Sayre syndrome (KSS); chronic progressive external ophthalmoplegia (CPEO); mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episode (MELAS); myoclonic epilepsy and ragged-red fibers (MERRF); and Leber hereditary optic neuropathy (LHON). These diseases are generally fatal, and it is the brain and muscles that are the tissues principally affected. However, it has also been reported that the retina, particularly the macular RPE, is also likely to be involved in these diseases.59 60

We have demonstrated that mitochondrial dysfunction is a normal consequence of aging in yeast.21 The loss of mitochondrial function with age has been postulated to contribute to aging in higher eukaryotes as well, although definitive demonstration of widespread mitochondrial dysfunction with age has not been shown, perhaps because of the large number of mtDNA mutations known and the difficulty of quantifying all potential mtDNA mutations within a single sample. It has been demonstrated that mutations in mtDNA occur with aging in postmitotic tissues and can reach high levels in old individuals,61 especially in the control region responsible for mtDNA replication and transcription.62

The RPE mtDNA is particularly susceptible to oxidative damage. It is terminally differentiated, and damaged cells cannot be replaced by mitosis, whereas damage to the mtDNA can be propagated as the mitochondria in the cells divide. The RPE has a high metabolic demand and may produce high levels of oxidants in the mitochondria due to ß oxidation of fatty acids. We showed an increase in expression of SOD2 in our {rho}0ARPE-19 cells, suggesting increased superoxide production in the mitochondria. It has been reported that SOD2 activity increases with donor age in freshly isolated RPE cells,63 and there is one report of an association of SOD2 polymorphism with exudative AMD.52 Although there has been speculation as to how oxidative damage to mitochondria may lead to an RPE energy deficit or to RPE apoptotic or necrotic cell death, there has been no previous consideration of the response of nuclear gene expression to the mitochondrial dysfunction caused by oxidative damage to protein or DNA and how changes in nuclear gene expression may relate to clinical manifestations of AMD.

We postulate that accumulation of oxidative damage in mtDNA is the primary event leading to mitochondrial dysfunction and an inflammatory phenotype in the macula, but it is also possible that other cellular events could lead to drusen deposition that could precipitate an inflammatory response in the RPE or Bruch’s membrane. The inflammatory response in turn could lead to the accumulation of toxic proteins such as ß-amyloid which may be targeted to the mitochondria and also precipitate changes in nuclear gene expression. It has been recently reported that amyloid-P may be targeted to the mitochondria, resulting in mitochondrial dysfunction and loss of calcium homeostasis.64 Because there is ample evidence of calcium deregulation in Alzheimer’s disease, a similar scenario could be envisioned for the RPE-choroid, although the contribution of inflammatory protein deposition to mitochondrial dysfunction in the RPE must be demonstrated.

In Figure 6 we present a schematic representation of our hypothesis that the manifestations of AMD can be explained by damage to the mtDNA of the RPE, resulting in mitochondria containing dysfunctional electron transport chains. This dysfunction results in mitochondria with a lower mitochondrial membrane potential and increased superoxide production and in release of Ca2+ into the cytoplasm. This in turn upregulates calcium-dependent kinases and nuclear transcription factors, which upregulate genes that aid in the survival of the RPE. However, although these changes in gene expression may contribute to RPE survival, they can also contribute to a pathologic state in the retina.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 6. Schematic representation of a hypothesis of how mitochondrial dysfunction could lead to a RPE phenotype, resulting in AMD. In this view, accumulated damage to mtDNA results in a defective electron transport chain due to an imbalance of transport chain components. This defect causes lower mitochondrial membrane potential ({Delta}{psi}m), increased superoxide production, and increased cytosolic [Ca2+]. The resultant increase in Ca2+ concentration activates calcium-dependent transcription factors that signal a nuclear response in an attempt to compensate for loss of energy production and to increase RPE cell viability. These gene expression changes contribute to some of the manifestations in AMD, such as RPE cell migration angiogenesis and lipid deposition. The excess superoxide production also contributes to an increased level of inflammation in the retina and Bruch’s membrane.

 


   Footnotes
 
Supported by grants from the National Institute on Aging of the National Institutes of Health.

Submitted for publication November 12, 2004; revised January 8, 2005; accepted January 24, 2005.

Disclosure: M.V. Miceli, None; S.M. Jazwinski, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Michael V. Miceli, Louisiana State University Health Sciences Center, Department of Biochemistry and Molecular Biology, 1901 Perdido Street, Box P7–2, New Orleans, LA 70112; mmicel{at}lsuhsc.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Young RW, Bok D. Participation of the retinal pigment epithelium in the rod outer segment renewal process. J Cell Biol. 1969;42:392–403.[Abstract/Free Full Text]
  2. Bok D, Hall MO. The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J Cell Biol. 1971;49:664–682.[Abstract/Free Full Text]
  3. Hogan MJ. Role of the retinal pigment epithelium in macular disease. Trans Am Acad Ophthalmol Otolaryngol. 1972;76:64–80.[Medline][Order article via Infotrieve]
  4. Young RW. Pathophysiology of age-related macular degeneration. Surv Ophthalmol. 1987;31:291–306.[CrossRef][ISI][Medline][Order article via Infotrieve]
  5. Klein R, Klein BE, Linton KL. Prevalence of age related maculopathy. The Beaver Dam Eye Study. Ophthalmology. 1992;99:933–943.[ISI][Medline][Order article via Infotrieve]
  6. van Leeuwen R, Klaver CC, Vingerling JR, Hofman A, de Jong PT. Epidemiology of age-related maculopathy: a review. Eur J Epidemiol. 2003;18:845–854.[CrossRef][ISI][Medline][Order article via Infotrieve]
  7. Age-Related Eye Disease Study Research Group. Potential public health impact of age-related eye disease study results. Arch Ophthalmol. 2003;121:1621–1624.[Abstract/Free Full Text]
  8. Eldred GE. Retinoid reaction products in age related retinal degeneration. Hollyfield JG Anderson RE LaVail MM eds. Retinal Degeneration: Clinical and Laboratory Manifestations. 1993;15–24. Plenum Press New York.
  9. Van der Schaft TL, Mooy CM, de Bruijn WC, de Jong PTVM. Early stages of age-related macular degeneration: an immunofluorescence and electron microscopy study. Br J Ophthalmol. 1993;77:657–661.[Abstract/Free Full Text]
  10. Sarks SH. Drusen patterns predisposing to geographic atrophy of the retinal pigment epithelium. Aust J Ophthalmol. 1982;10:91–97.[ISI][Medline][Order article via Infotrieve]
  11. Bressler MN, Silva JC, Bressler SB, Fine SL, Green WR. Clinicopathologic correlation of drusen and retinal pigment epithelial abnormalities in age-related macular degeneration. Retina. 1994;14:130–142.[CrossRef][ISI][Medline][Order article via Infotrieve]
  12. Curcio CA, Millican CL, Bailey T, Kruth HS. Accumulation of cholesterol with age in human Bruch’s membrane. Invest Ophthalmol Vis Sci. 2001;42:265–274.[Abstract/Free Full Text]
  13. Green WR. Histopathology of age-related macular degeneration. Mol Vis. 1999;5:27.[Medline][Order article via Infotrieve]
  14. Spraul CW, Lang GE, Grossniklaus HE, Lang GK. Histologic and morphometric analysis of the choroid, Bruch’s membrane, and retinal pigment epithelium in postmortem eyes with age-related macular degeneration and histologic examination of surgically excised choroidal neovascular membranes. Surv Ophthalmol. 1999;44:S10–S32.
  15. Sarks SH. Ageing and degeneration in the macular region: a clinico-pathological study. Br J Ophthalmol. 1976;60:324–341.[Abstract/Free Full Text]
  16. Zarbin MA. Age-related macular degeneration: review of pathogenesis. Eur J Ophthalmol. 1998;8:199–206.[ISI][Medline][Order article via Infotrieve]
  17. Dunaief JL, Dentchav T, Ying GS, Milam AH. The role of apoptosis in age-related macular degeneration. Arch Ophthalmol. 2002;120:1435–1442.[Abstract/Free Full Text]
  18. Traven A, Wong JM, Xu D, Sopta M, Ingles CJ. Interorganellar communication: altered nuclear gene expression profiles in a yeast mitochondrial DNA mutant. J Biol Chem. 2001;276:4020–4027.[Abstract/Free Full Text]
  19. Butow RA. Cellular responses to mitochondrial dysfunction: it’s not always downhill. Cell Death Differ. 2002;9:1043–1045.[CrossRef][ISI][Medline][Order article via Infotrieve]
  20. Kirchman PA, Kim S, Lai CY, Jazwinski SM. Interorganelle signaling is a determinant of longevity in Saccharomyces cerevisiae. Genetics. 1999;152:179–190.[Abstract/Free Full Text]
  21. Borghouts C, Benguria A, Wawryn J, Jazwinski SM. Rtg2 protein links metabolism and genome stability in yeast longevity. Genetics. 2004;16:765–777.
  22. Biswas G, Adebanjo OA, Freedman BD, et al. Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter-organelle crosstalk. EMBO J. 1999;18:522–533.[CrossRef][ISI][Medline][Order article via Infotrieve]
  23. Butow RA, Avadhani NG. Mitochondrial signaling: the retrograde response. Mol Cell. 2004;14:1–15.[CrossRef][ISI][Medline][Order article via Infotrieve]
  24. Miceli MV, Jazwinski SM. Common and cell type-specific responses of human cells to mitochondrial dysfunction. Exp Cell Res. 2005;302:270–280.[CrossRef][ISI][Medline][Order article via Infotrieve]
  25. Liang FQ, Godley BF. Oxidative stress-induced mitochondrial DNA damage in human retinal pigment epithelial cells: a possible mechanism for RPE aging and age-related disease. Exp Eye Res. 2003;76:397–403.[CrossRef][ISI][Medline][Order article via Infotrieve]
  26. Feher J, Papale A, Mannino G, Gualdi L, Gabrieli CB. Mitotropic compounds for the treatment of age-related macular degeneration. Ophthalmologica. 2003;217:351–357.[CrossRef][ISI][Medline][Order article via Infotrieve]
  27. Dunn KC, Aotaki-Keen AE, Putkey F, Hjelmeland LM. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res. 1996;62:155–169.[CrossRef][ISI][Medline][Order article via Infotrieve]
  28. King MP, Attardi G. Isolation of human cell lines lacking mitochondrial DNA. Methods Enzymol. 1996;264:304–313.[Medline][Order article via Infotrieve]
  29. Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun. 1993;197:40–45.[CrossRef][ISI][Medline][Order article via Infotrieve]
  30. Miceli MV, Newsome DA, Tate DJ, Jr. Vitronectin is responsible for serum-stimulated uptake of rod outer segments by cultured retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1997;38:1588–1597.[Abstract/Free Full Text]
  31. Finnemann SC. Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors. EMBO J. 2003;22:4143–4154.[CrossRef][ISI][Medline][Order article via Infotrieve]
  32. Crabb JW, Miyagi M, Gu X, et al. Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc Natl Acad Sci USA. 2002;99:14682–14687.[Abstract/Free Full Text]
  33. Blackburn J, Tarttelin EE, Gregory-Evans CY, Moosajee M, Gregory-Evans K. Transcriptional regulation and expression of the dominant drusen gene FBLN3 (EFEMP1) in mammalian retina. Invest Ophthalmol Vis Sci. 2003;44:4613–4621.[Abstract/Free Full Text]
  34. Hageman GS, Mullins RF, Russell SR, Johnson LV, Anderson DH. Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells. FASEB J. 1999;13:477–484.[Abstract/Free Full Text]
  35. Kimoto K, Nakattsuka K, Matsuo N, Yoshioka H. p38 MAPK mediates the expression of type I collagen induced by TGF-ß2 in human retinal pigment epithelial cells ARPE-19. Invest Ophthalmol Vis Sci. 2004;45:2431–2437.[Abstract/Free Full Text]
  36. Scheiffarth OF, Kampik A, Gunther H, von der Mark K. Proteins of the extracellular matrix in vitreoretinal membranes. Graefes Arch Clin Exp Ophthalmol. 1988;226:357–361.[CrossRef][ISI][Medline][Order article via Infotrieve]
  37. Kvanta A, Shen WY, Sarman S, Seregard S, Steen B, Rakcozy E. Matrix metalloproteinase (MMP) expression in experimental choroidal neovascularization. Curr Eye Res. 2000;21:684–690.[CrossRef][ISI][Medline][Order article via Infotrieve]
  38. Elner SG. Human retinal pigment epithelial lysis of extracellular matrix: functional urokinase plasminogen activator receptor, collagenase and elastase. Trans Am Ophthalmol Soc. 2002;100:273–299.[Medline][Order article via Infotrieve]
  39. Rakic JM, Lambert V, Munaut C, et al. Mice without uPA, tPA or plasminogen genes are resistant to experimental choroidal neovascularization. Invest Ophthalmol Vis Sci. 2003;44:1732–1739.[Abstract/Free Full Text]
  40. Omori K, Fujiseki Y, Omori K, Suzkawa J, Inagaki C. Regulation of the expression of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium cells. Matrix Biol. 2002;21:337–348.[CrossRef][ISI][Medline][Order article via Infotrieve]
  41. Kamei M, Hollyfield JG. TIMP-3 in Bruch’s membrane: changes during aging and in age-related macular degeneration. Invest Ophthalmol Vis Sci. 1999;40:2367–2375.[Abstract/Free Full Text]
  42. Anderson DH, Ozaki S, Nealon M, et al. Local cellular sources of apolipoprotein E in the human retina and retinal pigmented epithelium: implications for the process of drusen formation. Am J Ophthalmol. 2001;131:767–781.[CrossRef][ISI][Medline][Order article via Infotrieve]
  43. Malek G, Li CM, Guidry C, Medeiros NE, Curcio CA. Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with age-related maculopathy. Am J Pathol. 2003;162:413–425.[Abstract/Free Full Text]
  44. Heckenlively JR, Hawes NL, Friedlander M, et al. Mouse model of subretinal neovascularization with choroidal anastomosis. Retina. 2003;23:518–522.[CrossRef][ISI][Medline][Order article via Infotrieve]
  45. Elner SG, Elner VM, Nielsen JC, Torczynski E, Yu R, Franklin WA. CD68 antigen expression by human retinal pigment epithelial cells. Exp Eye Res. 1992;55:21–28.[ISI][Medline][Order article via Infotrieve]
  46. Duncan KG, Bailey KR, Kane JP, Schwartz DM. Human retinal pigment epithelial cells express scavenger receptors BI and BII. Biochem Biophys Res Commun. 2002;292:1017–1022.[CrossRef][ISI][Medline][Order article via Infotrieve]
  47. Hayes KC, Lindsey S, Stephan ZF, Brecker D. Retinal pigment epithelium possesses both LDL and scavenger receptor activity. Invest Ophthalmol Vis Sci. 1989;30:225–232.[Abstract/Free Full Text]
  48. Mitsuhiro MR, Eguchi S, Yamashita H. Regulation mechanisms of retinal pigment epithelial cell migration by the TGF-beta superfamily. Acta Ophthalmol Scand. 2003;81:630–638.[CrossRef][ISI][Medline][Order article via Infotrieve]
  49. Kitaoka T, Morse LS, Schneeberger S, Ishigooka H, Hjelmeland LM. Expression of FGF5 in choroidal neovascular membranes associated with ARMD. Cur Eye Res. 1997;16:396–399.
  50. Gray JX, Haino M, Roth MJ, et al. CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation. J Immunol. 1996;157:5438–5447.[Abstract]
  51. Kwak N, Okamoto N, Wood JM, Campochiaro PA. VEGF is major stimulator in model of choroidal neovascularization. Invest Ophthalmol Vis Sci. 2000;41:3158–3148.[Abstract/Free Full Text]
  52. Kimura K, Isashiki Y, Sonoda S, Kakiuchi-Matsumoto T, Ohba N. Genetic association of manganese superoxide dismutase with exudative age-related macular degeneration. Am J Ophthalmol. 2000;130:769–773.[CrossRef][ISI][Medline][Order article via Infotrieve]
  53. Strunnkikova N, Zhang C, Teichberg , et al. Survival of retinal pigment epithelium after exposure to prolonged oxidative injury: a detailed gene expression and cellular analysis. Invest Ophthalmol Vis Sci. 2004;45:3767–3777.[Abstract/Free Full Text]
  54. Miceli MV, Newsome DA. Cultured retinal pigment epithelium cells from donors with type I diabetes show an altered insulin response. Invest Ophthalmol Vis Sci. 1991;32:2847–2853.[Abstract/Free Full Text]
  55. Tyni T, Johnson M, Eaton S, Pourfarzam M, Andrews R, Turnbull DM. Mitochondrial fatty acid beta-oxidation in the retinal pigment epithelium. Pediatr Res. 2002;52:595–600.[CrossRef][ISI][Medline][Order article via Infotrieve]
  56. Ozaki S, Johnson LV, Mullins RF, Hageman GS, Anderson DH. The human retina and retinal pigment epithelium are abundant sources of vitronectin mRNA. Biochem Biophys Res Commun. 1999;258:524–529.[CrossRef][ISI][Medline][Order article via Infotrieve]
  57. Lopez PF, Sippy BD, Lambert HM, Thach AB, Hinton DR. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. Invest Ophthalmol Vis Sci. 1996;37:855–868.[Abstract/Free Full Text]
  58. Anderson DH, Mullins RF, Hageman GS, Johnson LV. A role for local inflammation in the formation of drusen in the aging eye. Am J Ophthalmol. 2002;134:411–431.[CrossRef][ISI][Medline][Order article via Infotrieve]
  59. Mullie MA, Harding AE, Petty RK, Ikeda H, Morgan-Hughes JA, Sanders MD. The retinal manifestations of mitochondrial myopathy: a study of 22 cases. Arch Ophthalmol. 1985;103:1825–1830.[Abstract]
  60. Isashiki Y, Nakagawa M, Ohba N, et al. Retinal manifestations in mitochondrial diseases associated with mitochondrial DNA mutation. Acta Ophthalmol Scand. 1998;76:6–13.[CrossRef][ISI][Medline][Order article via Infotrieve]
  61. Barga G. Free radicals and aging. Trends Neurosci. 2004;27:595–600.[CrossRef][ISI][Medline][Order article via Infotrieve]
  62. Chomyn A, Attardi G. MtDNA mutations in aging and apoptosis. Biochem Biophys Res Commun. 2003;304:519–529.[CrossRef][ISI][Medline][Order article via Infotrieve]
  63. Newsome DA, Miceli MV, Tate DJ, Jr, Alcock NW, Oliver PD. Zinc content of human retinal pigment epithelium decreases with age and macular degeneration, but superoxide dismutase increases. J Trace Elem Exp Med. 1995;8:193–199.
  64. Anandatheerthavarada HK, Biswas G, Robin MA, Avadhani NG. Mitochondrial targeting and a novel transmembrane arrest of Alzheimer’s amyloid precursor protein impairs mitochondrial function in neuronal cells. J Cell Biol. 2003;161:41–54.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Genome ResHome page
J. Chen, X. Shi, R. Padmanabhan, Q. Wang, Z. Wu, S. C. Stevenson, M. Hild, D. Garza, and H. Li
Identification of novel modulators of mitochondrial function by a genome-wide RNAi screen in Drosophila melanogaster
Genome Res., January 1, 2008; 18(1): 123 - 136.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
L. Jia, Z. Liu, L. Sun, S. S. Miller, B. N. Ames, C. W. Cotman, and J. Liu
Acrolein, a Toxicant in Cigarette Smoke, Causes Oxidative Damage and Mitochondrial Dysfunction in RPE Cells: Protection by (R)-{alpha}-Lipoic Acid
Invest. Ophthalmol. Vis. Sci., January 1, 2007; 48(1): 339 - 348.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miceli, M. V.
Right arrow Articles by Jazwinski, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miceli, M. V.
Right arrow Articles by Jazwinski, S. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS