August 2012
Volume 53, Issue 9
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Retinal Cell Biology  |   August 2012
Moderately Reduced ATP Levels Promote Oxidative Stress and Debilitate Autophagic and Phagocytic Capacities in Human RPE Cells
Author Affiliations & Notes
  • Florian Schütt
    From the Department of Ophthalmology, INF 400; and the
  • Sebastian Aretz
    Department of Pathology, INF 220 69120, University of Heidelberg, Heidelberg, Germany.
  • Gerd U. Auffarth
    From the Department of Ophthalmology, INF 400; and the
  • Jürgen Kopitz
    Department of Pathology, INF 220 69120, University of Heidelberg, Heidelberg, Germany.
Investigative Ophthalmology & Visual Science August 2012, Vol.53, 5354-5361. doi:10.1167/iovs.12-9845
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      Florian Schütt, Sebastian Aretz, Gerd U. Auffarth, Jürgen Kopitz; Moderately Reduced ATP Levels Promote Oxidative Stress and Debilitate Autophagic and Phagocytic Capacities in Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(9):5354-5361. doi: 10.1167/iovs.12-9845.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Aging of the RPE is associated with a decrease of intracellular ATP levels and increased oxidative stress. We investigated the effects of moderate energy deficit on intracellular glutathione levels, oxidative damage of cellular proteins and DNA, and autophagy rates using an RPE cell culture model. Additionally, phagocytosis of photoreceptor outer segments was assayed as an example of an ATP-dependent normal function of the RPE.

Methods.: ATP synthesis of primary human RPE cells was moderately inhibited by atractyloside. Oxidative stress was induced by tert-butyl hydroperoxide (tBH). ATP, reduced glutathione (rG), malondialdehyde (MDA) adduct formation and 8-hydroxydeoxyguanosine (8OHdG) levels were measured. Autophagy and phagocytosis of photoreceptor outer segments were assayed by radiometric methods.

Results.: Atractyloside–treatment reduced cellular ATP levels by 30%, mimicking the energy status of aged RPE. tBH decreased rG in RPE cells with lowered ATP levels whereas cells with normal ATP content were not affected. tBH-induced oxidative stress resulted in substantial accumulation of MDA protein adducts in cells with lowered ATP while cells with regular ATP levels were only modestly affected. tBH induced more oxidative DNA damage (8OHdG formation) in cells with lowered ATP levels than in cells with regular ATP. In atractyloside-treated cells, autophagy rates decreased 3-fold as compared with controls. Phagocytic capacity for uptake and degradation of photoreceptor segments was reduced in RPE with low ATP.

Conclusions.: Moderately decreased ATP levels such as seen in aged individuals might contribute to the vulnerability of RPE to oxidative stress damage and to dysfunction.

Introduction
Aging of post-mitotic tissues is associated with a continuous decrease of the mitochondrial capacity to produce ATP by oxidative phosphorylation resulting in a general decline of vital cellular functions. 1 Cellular dysfunction caused by reduced ATP/ADP ratios is accompanied by increased mitochondrial production of reactive oxygen species (ROS). Thus, decreased ATP production and enhanced oxidative stress are major triggers of senescent dysfunction of long-lived post-mitotic cells, such as neurons, cardiac myocytes, skeletal muscle fibers and RPE. 2 Various lines of evidence indicate that this functional decline severely impairs tissue function and substantially predisposes tissues to development and/or progression of age-related dysfunction, thereby contributing to the development of age-related disorders, including glaucoma, diabetic retinopathy (DR), and ARMD. 3 Glaucoma has been associated with mitochondrial dysfunction of retinal ganglion cells, 4 whereas in the etiology of diabetic retinopathy and ARMD, aging processes in the RPE seem to play a pivotal role. 5,6  
The RPE fulfills metabolic functions that are essential for proper action and survival of retinal photoreceptors. These vital functions include maintenance of the visual cycle by continuous uptake, processing, transport, and release of vitamin A; generation of ion gradients within the photoreceptor matrix; mediation of active transport of nutrients between the choroid and the photoreceptors; formation of the outer blood brain barrier; phagocytic uptake; and degradation of the constantly shed photoreceptor outer segments. 7 A failure of any one of these functions, which are highly dependent on the intracellular energy provision in the form of ATP, can lead to degeneration of the retina. He et al. described a 30% decrease of the intracellular ATP levels in aged RPE. 8 Reduced capacity of energy supply in aged cells is mainly due to decreased electron transfer in the mitochondrial respiratory chain. 9 According to the “free radical theory of aging,” accumulation of oxidative damage to mitochondrial macromolecules is considered the main cause of mitochondrial function to decline. As a consequence of electron leakage from the electron transport chain reactive oxygen species (ROS), including the superoxide anion radical and hydroxyl radical, form within mitochondria during normal respiration, thereby initiating oxidative damage to mitochondrial proteins, nucleic acids, and lipids. 10 Besides this “intrinsic” mechanism of ROS production, in RPE cells other factors may contribute to oxidative stress and mitochondrial dysfunction. Thus, RPE cells are at permanent risk for oxidative damage due to their location in the highly oxygenated environment of the outer retina and their exposure to light. Although lysosomal dysfunction causing buildup of undegradable material seems to be the initial incident, 1113 the accumulation of waste material finally results in the formation of lipofuscin granules that are highly phototoxic by generating ROS upon illumination. 14 ROS produced by such mechanism have been shown to impair mitochondrial function in RPE cells. 14 Furthermore, lysosomal accumulation of the ocular lipofuscin component A2E has been shown to reduce mitochondrial ATP synthesis in RPE cells. 15  
In aging cells, oxidative damage is fostered by decreasing activities of antioxidant enzymes, including dismutases and catalases and by a decline of intracellular low molecular weight scavengers of oxidizing species. Reduced glutathione represents the most abundant and most important intracellular antioxidant. Consequently, a high intracellular level of its reduced form is considered as a marker of cells that are well shielded from severe oxidative damage, whereas vast consumption of reduced glutathione is a hallmark of cells experiencing severe oxidative stress. Mitochondrial dysfunction, reducing ATP generation, is linked to glutathione deficiency by the Gibbs free energy equation. Thus, age-related reduction of intracellular ATP levels should result in an equivalent decrease of reduced glutathione levels thereby pushing the aging process. 
In the present study we applied atractyloside for inhibition of mitochondrial ATP synthesis to test the effects of moderate ATP depletion in cultured human RPE cells on cellular oxidative stress response, autophagy and related anti-aging mechanisms, and photoreceptor outer segment phagocytosis as an example for a vital RPE function. Atractyloside is a competitive inhibitor of the mitochondrial ADP/ATP carrier causing highly specific inhibition of mitochondrial ATP synthesis. 16 The application of atractyloside for reduction of cellular ATP production also induces the generation of ROS in the respiratory chain. 17 Thus, atractyloside-treated RPE cells appear as a suitable model to test whether a relatively small reduction of mitochondrial activities may impair normal RPE functions, in particular those that are considered to contribute to age-related changes in the retina, which finally may add to the multifactorial pathogenesis of retinal degenerations, such as diabetic retinopathy and ARMD. Although these most abundant retinopathies occur by different multifactorial mechanisms, reduced ATP levels and the resulting adverse effects addressed in our study may be common contributors to the etiologies of both disorders. 
Materials and Methods
Cell Culture
Primary human RPE cells were isolated from donor eyes according to published procedures 18 and maintained in a 1:1 mixture of Medium 199 and Ham's F-12 medium, supplemented with 1mM pyruvate, 200 U/mL penicillin, 100 μg/mL streptomycin,10% heat inactivated fetal bovine serum (all reagents by PAA Laboratories, Coelbe, Germany), and 2 μg/mL insulin (Sigma-Aldrich, Munich, Germany). All subjects were treated in accordance with the Declaration of Helsinki. 
Biochemical Assays
Intracellular ATP was determined by bioluminescent luciferase assay, including thermostable firefly luciferase and luciferin substrate. The ATP assay kit was obtained from Biaffin (Kassel, Germany). Reduced and total glutathione were determined with a commercial glutathione assay kit (Biovision, Mountain View, CA). Malondialdehyde-protein-adducts were quantified by an enzyme immunoassay (OxiSelect MDA Adduct ELISA Kit; Cell Biolabs, San Diego, CA). Total DNA from cell samples was extracted by a purification kit (DNeasy DNA; QIAGEN, Hilden, Germany). 8-Hydroxydeoxyguanosine in DNA was quantified by competitive enzyme immunoassay (OxiSelect Oxidative DNA Damage Quantification Kit 8-OHdG; Cell Biolabs). Lactate in conditioned medium was assayed according to Bergmeyer. 19 Protein concentrations were assayed by the Lowry procedure. 
Protein Degradation Assays
Turnover of endogenous proteins was measured in pulse-chase experiments as described previously. 20 Briefly, the cells, grown to confluency in 24-well tissue culture plates, were metabolically labeled by the inclusion of 500 kBq/mL 3H-leucine in the culture medium for 72 hours (pulse phase). After removal of the radioactive medium, the cell layers were washed and chased with nonradioactive medium (chase phase). Protein degradation was determined by measurement of the low-molecular (TCA-soluble) radioactivity released into the medium. 3-Methyladenine is a specific inhibitor for autophagic sequestration of cytoplasmic proteins. Therefore, the effect of this compound on intracellular protein turnover can be taken as a measure of autophagic sequestration. 21,22 The effect of 3-methyladenine was assayed by adding 10 mM 3-methyladenine at the beginning of the chase phase. Autophagic protein degradation was calculated by subtracting degradation rates in the presence from the rates observed in absence of 3-methyladenine. 13,23 Lysosomal protein degradation was blocked by adding 10 mM NH4Cl with the chase medium. Thus, lysosomal protein degradation (ammonia-sensitive degradation) was calculated by subtracting degradation rates in the presence of NH4Cl from the rates observed in absence of NH4Cl. 20  
Photoreceptor Outer Segment (POS) Isolation and Radiolabeling
POS was isolated from pig eyes (obtained from a local slaughter house) according to the method of Schraermeyer et al. 24 All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The anterior half of the eye was dissected, the vitreous and retina removed. Isolated retina were agitated in KCl-buffer (0.3 M KCl, 10 mM Hepes, 0.5 mM CaCl2, 1 mM MgCl2, 48% sucrose) at pH 7 and centrifuged at 7000 rpm in a table centrifuge (Sigma 3K10) for 5 minutes. The supernatant containing POS was filtered through gauze and diluted with KCl-buffer (1:1) and centrifuged at 5000 rpm for 7 minutes. For radiolabeling, the iodination beads (IODO-BEADS; Pierce) method was used. POS (500 μg total protein) were incubated at room temperature for 10 minutes in 50 mM sodium phosphate, pH 7.0, with 74 MBq carrier-free Na125I (Hartmann Analytic, Braunschweig, Germany) in the presence of three iodination beads (Pierce). Radiolabeling was stopped by removal of the iodination beads. Labeled POS were separated from free iodine by repeated washing in PBS and centrifugation at 14,000 g for 5 minutes. 23 Specific radioactivity was 53 MBq/mg protein. 
Phagocytosis and Degradation of Radiolabeled ROS
RPE cultures were grown to confluence on 96-well tissue culture plates and maintained for 4 weeks. Then 80 kBq 125I-labeled POS were added to each well and incubated for 6, 12, 18, or 24 hours and phagocytosis and degradation rates were assayed according to a published method.23 The medium was removed, mixed with the same volume of 10% TCA, centrifuged (14,000g, 5 minutes, 4°C) and the supernatant (S1), containing TCA-soluble low-molecular weight protein degradation products, collected. The remaining pellet was solubilized in 400 μL NaOH (P1). The remaining cells in the wells were washed twice with 250 μL PBS, the washing fractions treated with 10% TCA and centrifuged as described above, yielding supernatants S2 and S3 and protein pellets P2 and P3. P2 and P3 were dissolved with 200μL 0.5M NaOH. The cells in the wells were solubilized with 200 μL 0.5M NaOH and treated again with TCA as mentioned before (S4 and P4). The fractions were combined in the following manner: TCA-soluble (low-molecular weight) radioactivity released to the medium: TM = S1 + S2 + S3; Protein-bound radioactivity in the medium: PM = P1 + P2 + P3; intracellular TCA-soluble (low-molecular weight) radioactivity: TZ = S4; Intracellular protein-bound radioactivity: PZ = P4. TM, PM, TZ, and PZ were mixed with 10 mL of liquid scintillation cocktail (Ultima Gold; PerkinElmer, Waltham, MA) and their radioactivity determined in a liquid scintillation counter. The following calculations were made:     
Results
Applying atractyloside for inhibition of mitochondrial ATP synthesis at a concentration of 1 μM reduced cellular ATP levels by approximately 30%, mimicking the energy status of aged RPE (Fig. 1). The amount of lactate detected in the conditioned medium after three days of culture was 2.77 mg/mL (±0.11, n = 6) for untreated cells and 3.51mg/mL (±0.12, n = 6) for cells cultured in the presence of atractyloside. Thus, the cells are able to partially compensate for the atractyloside-induced reduction of mitochondrial ATP supply by nonmitochondrial ATP generation. Consequently, ATP synthesis is affected more than 30%. In addition to moderately reduced cellular ATP levels, we intended to induce conditions of increased oxidative stress. To this end, the cells were treated with tert-butyl hydroperoxide (tBH). Neither treatment with atractyloside or tBH alone nor the combination of both induced significant changes in the morphology of the cells (data not shown). No increase of LDH leakage to the culture medium of the treated cell was observed, excluding acute cell death during the experiments. Specific activities of the lysosomal marker β–hexosaminidase and the mitochondrial marker succinate dehydrogenase were not altered during treatment (data not shown). As shown in Figure 2, tBH at moderate concentrations of 100 μM only marginally decreases intracellular levels of reduced glutathione in cells with normal ATP levels. However, when ATP levels were reduced according to the conditions described above, a striking decrease of reduced glutathione was observed upon tBH treatment. In order to test whether these conditions of elevated oxidative stress may cause harm to cellular proteins, the amount of malondialdehyde (MDA) modifications on cellular protein was tested by ELISA. MDA is a widely accepted marker for protein damage caused by oxidative stress. During oxidative stress, MDA is generated as a lipid peroxidation byproduct that covalently attaches to cellular proteins. The treatment with atractyloside alone did not increase the formation of MDA adducts to levels above the detection limit of 2 pmol/mg protein. However, when tBH, an inducer of oxidative stress, was included in the culture medium to induce conditions of elevated oxidative stress, an increase of protein damage was observed in cells with reduced ATP levels, whereas the amount of MDA modification was only moderately elevated in cultures with regular ATP levels (Fig. 3). Oxidative DNA damage was tested by 8-hydroxydeoxyguanosine (8OHdG) ELISA. 8OHdG is a modified base that occurs in DNA due to attacks by hydroxyl radicals that are formed as byproducts and intermediates of aerobic metabolism and during oxidative stress. Again, 8OHdG was not elevated above the detection limit of 2.5 ng/mL in atractyloside-treated cells. Induction of additional oxidative stress by tBH induced oxidative DNA damage in RPE cells with reduced ATP levels, whereas cells with normal ATP levels were only slightly affected (Fig. 4). A major mechanism in post-mitotic cells to prevent aging and oxidative damage of cellular biomolecules and structures is autophagy. Since autophagy is a highly energy-dependent process, we assessed the effects of atractyloside-induced reduction of intracellular ATP levels on intracellular autophagic degradation rates by measuring 3-methyladenine-sensitive protein degradation after labeling intracellular proteins with 3H-leucine. In the atractyloside-treated cells autophagic activity was approximately 3-fold lower as compared with the untreated controls (Fig. 5). For comparison, also ammonia-sensitive degradation (total lysosomal degradation) is shown. The differences between ammonia-sensitive and 3MA-sensitive degradation indicate the contribution of nonautophagic lysosomal degradation, which appears relatively small and not affected by the decreased ATP level. However, phagocytic capacity for uptake and degradation of photoreceptor outer segments was reduced in cells with lowered ATP-levels (Fig. 6). 
Figure 1. 
 
RPE cells were grown to confluence in 12-well plates. Then 1 μM atractyloside was added to the culture medium and cells were harvested at the indicated time points after addition of the inhibitor. Cells were homogenized in ATP-releasing reagent (Sigma-Aldrich). ATP and protein concentration was assayed as described in “Materials and Methods.” ATP levels were normalized to protein content. ATP concentration at time point 0h was set to 100%. Results are the means of three independent experiments (±SD).
Figure 1. 
 
RPE cells were grown to confluence in 12-well plates. Then 1 μM atractyloside was added to the culture medium and cells were harvested at the indicated time points after addition of the inhibitor. Cells were homogenized in ATP-releasing reagent (Sigma-Aldrich). ATP and protein concentration was assayed as described in “Materials and Methods.” ATP levels were normalized to protein content. ATP concentration at time point 0h was set to 100%. Results are the means of three independent experiments (±SD).
Figure 2. 
 
Cells were treated with atractyloside as described in Figure 1 and total and reduced glutathione was assayed after homogenization of the cells at the indicated time points by ELISA. The percentage of reduced glutathione as compared to total glutathione is shown. Results are the means of three independent experiments (±SD).
Figure 2. 
 
Cells were treated with atractyloside as described in Figure 1 and total and reduced glutathione was assayed after homogenization of the cells at the indicated time points by ELISA. The percentage of reduced glutathione as compared to total glutathione is shown. Results are the means of three independent experiments (±SD).
Figure 3. 
 
Cells were treated with atractyloside as described in Figure 2. Oxidative stress was induced by adding tBH to the culture medium. Cells were harvested and homogenized at the indicated time points and MDA-adduct formation on cellular proteins was assayed by ELISA. Results are the means of three independent experiments (±SD).
Figure 3. 
 
Cells were treated with atractyloside as described in Figure 2. Oxidative stress was induced by adding tBH to the culture medium. Cells were harvested and homogenized at the indicated time points and MDA-adduct formation on cellular proteins was assayed by ELISA. Results are the means of three independent experiments (±SD).
Figure 4. 
 
Cells were treated with tBH as described in Figure 3. Total DNA was extracted from the cell homogenates by DNeasy DNA purification kit. 8-Hydroxydeoxyguanosine in the extracted DNA was quantified by competitive enzyme immunoassay. Results are the means of three independent experiments (±SD).
Figure 4. 
 
Cells were treated with tBH as described in Figure 3. Total DNA was extracted from the cell homogenates by DNeasy DNA purification kit. 8-Hydroxydeoxyguanosine in the extracted DNA was quantified by competitive enzyme immunoassay. Results are the means of three independent experiments (±SD).
Figure 5. 
 
Cells were metabolically labeled by the inclusion of 500 kBq/mL 3H-leucine in the culture medium for 72 hours (pulse phase). Then the cultures were chased in nonradioactive medium in the absence (•) or presence (▵) of 1μM atractyloside. 3-Methyladenine-sensitive (autophagic protein degradation, solid line) and NH4Cl-sensitive (total lysosomal degradation, dotted line) protein degradation rates were assayed at the indicated time points of the chase phase as described in “Materials and Methods.” Results are the means of three independent experiments (±SD).
Figure 5. 
 
Cells were metabolically labeled by the inclusion of 500 kBq/mL 3H-leucine in the culture medium for 72 hours (pulse phase). Then the cultures were chased in nonradioactive medium in the absence (•) or presence (▵) of 1μM atractyloside. 3-Methyladenine-sensitive (autophagic protein degradation, solid line) and NH4Cl-sensitive (total lysosomal degradation, dotted line) protein degradation rates were assayed at the indicated time points of the chase phase as described in “Materials and Methods.” Results are the means of three independent experiments (±SD).
Figure 6. 
 
Cells were grown in the absence or presence of 1μM atractyloside for 48 hours. Then labeled POSs were added without change of medium. (A) Uptake (clearance from the medium), (B) Intracellular accumulation, (C) Degradation of labeled POS was assayed as described in “Materials and Methods.” Results are the means of three independent experiments.
Figure 6. 
 
Cells were grown in the absence or presence of 1μM atractyloside for 48 hours. Then labeled POSs were added without change of medium. (A) Uptake (clearance from the medium), (B) Intracellular accumulation, (C) Degradation of labeled POS was assayed as described in “Materials and Methods.” Results are the means of three independent experiments.
Discussion
Recent studies indicate that the ATP content in human RPE decreases with age. Complex changes in mitochondrial structure and function, including disorganization of mitochondrial structure, decline in the activity of enzymes involved in mitochondrial ATP synthesis, accumulation of mtDNA mutations, increased damage of mitochondrial proteins and lipids by reactive oxygen species are considered to cause a decrease in cellular ATP production in aged RPE cells. 8,25,26 Thus, in RPE cells from aged donors (>60 years), an approximately 30% decrease of the intracellular ATP level was observed. Likewise an increased susceptibility to oxidative stress was found in RPE cells from aged donors. 8 These findings suggest a link between the energy status of the cell, oxidative stress sensitivity, and impaired RPE function. However, in these comparative studies of RPE cells from individuals of different age, it is difficult to sort out single mechanisms of the multifaceted aging process. In our present study, we investigated a link between moderately decreased intracellular ATP levels, stress response, autophagy as a major repair mechanism for age-related injury, and normal phagocytic RPE function. To this end, RPE cells from a 38-year-old individual were treated with a concentration of atractyloside, an inhibitor of mitochondrial ATP supply, that induces a decrease of intracellular ATP levels comparable to what is seen in aged RPE. Even under aerobic conditions, cultured human RPE cells exhibit high levels of lactate production. 27 This has also been reported for intact retina. 28 Thus, the cultured cells may be able to at least partially compensate for the atractyloside-induced reduction of mitochondrial ATP supply by increased nonmitochondrial ATP generation as indicated by increased lactate production in the presence of the inhibitor. However, considering that ATP synthesis via lactate production is much less effective than mitochondrial ATP production, the compensation is scarce. It is not known to which extent age-related decline of mitochondrial energy production may be balanced by increased lactate production in vivo. Therefore, our experimental model may not perfectly mimic mitochondrial dysfunction in vivo, rather it focuses on the consequences of reduced ATP supply in the RPE. Atractyloside was chosen to reduce intracellular ATP levels since it is a highly specific competitive inhibitor of the mitochondrial ADP/ATP exchange carrier. 16 Consequently, it allows easy adjustment of the desired degree of inhibition of ATP production and it does not directly interfere with the mitochondrial electron transport chain nor with metabolic pathways. Nevertheless, atractyloside may cause side effects, (e.g., alterations in mitochondrial membrane potential), but the use of atractyloside directs the obtained results mainly to ATP-related effects. 
The use of a moderate concentration of atractyloside enabled reproducible reduction of ATP levels in RPE cells by approximately 30% resembling conditions as observed in aged RPE. Thus, such atractyloside treatment devises a suitable model to study the effects of moderate energy deficit on cellular functions and structures. Applying this model, we show that intracellular ATP level reduction diminishes the RPE cells' capabilities to cope with oxidative stress. Decreased level of reduced glutathione is a sensitive indicator of oxidative damage risk. While induced oxidative stress by tBH did not affect reduced intracellular glutathione in RPE cells with regular ATP levels, a 30% reduction of ATP caused a tBH-induced drop of reduced glutathione. Besides the lowered activities of antioxidative enzymes in aged RPE, also a direct link between declining ATP levels and the reduction of nonenzymatic defense mechanisms against free radicals might promote oxidative damage. Accordingly in our glutathione measurements, the major cellular free radical scavenger, was decreased in atractyloside-treated cells. 
Of the many cellular targets of oxidative stress, lipids are the most involved class of biomolecules. In particular free radical-induced oxidation of polyunsaturated fatty acids, termed lipid peroxidation, gives rise to a number of toxic secondary products, mainly reactive electrophilic aldehydes, that may cause severe cellular damage. MDA is a principle and most studied example of these toxic lipid peroxidation derived aldehydes. It is a sensitive marker of oxidative stress and lipid peroxidation. 29 MDA is a highly toxic molecule that is considered to be involved in the pathogenesis of RPE degenerations in retinal disorders. 12,13,30 The toxic effects of MDA and related lipid peroxidation products are related to their ability to form protein adducts and crosslinks as well as to their mutagenic properties. 11,29,31,32 Thus, the detection of MDA protein adducts is a sensitive measure of peroxidation-triggered damage. In our experimental model, where oxidative stress was induced by tBH, already moderate reduction of cellular ATP was associated with increased MDA adduct formation on cellular proteins. Lipid peroxidation is known to generate specific epitopes on cellular proteins that are targets of cellular and humoral immune reactions. 33 ARMD is a complex disease caused by the combination of environmental factors and genetic predisposition. Among the diverse genes implicated in ARMD, various immune and inflammation relevant genes were found. 3436 Following this, concerted action of genetic predisposition, mitochondrial dysfunction and oxidative protein damage may contribute to the pathogenesis of macular degeneration. 
DNA is among the most biologically significant targets of oxidative attack, and it is widely thought that continuous oxidative damage to DNA is a significant contributor to the age-related disorders. 3,37 The formation of 8-hydroxydeoxyguanosine (8-OHdG) is a sensitive marker of oxidative stress induced DNA damage. Therefore, our results suggest a link between reduced ATP levels and DNA mutations. In particular, mitochondrial genomic instability due to susceptibility of mtDNA to oxidative stress and failure of mtDNA protection and repair may be important factors in retinal degeneration. 6 Remarkably, although atractyloside treatment has been described to increase mitochondrial free radical production, in our experiments no increase in MDA-protein adduct formation or 8OHdG generation was observed. This indicates that the increase in oxidative stress damage after combined atractyloside/tBH treatment cannot be explained by a simple additive effect of two inducers of radical formation. Rather, the atractyloside induced energy deficit seems to enhance tBH damage by weakening the cells stress protection (e.g., by lowering levels of reduced glutathione). Glutathione synthesis is a highly ATP-dependent process and the reduction of ATP as a consequence of hypoxia has been shown to impair biosynthesis of glutathione in hepatocytes. 38 However, this would cause a decrease in total glutathione but most likely without affecting the GSH/GSSG ratio. In another study, reduction of cellular ATP correlated with an efflux of reduced GSH from the cells. 39 Indeed, we also observed a slight decrease in total glutathione of approximately 10%. But this effect was too small to completely explain the striking reduction of reduced glutathione in our study. The data suggest a direct link between the cellular ATP levels and the redox state of glutathione in RPE cells. NADPH supply for glutathione reduction via the pentose phosphate pathway or the glutathione reductase reaction may be affected. Although the exact molecular causes remain to be established, our results indicate a causative link between reduced intracellular energy supply and the vulnerability of cellular proteins and DNA to oxidative stress induced damage in human RPE. 
Inhibition of autophagy has been shown to induce degenerative changes in mammalian tissues that resemble those associated with aging, and normal and pathological aging are often associated with a reduced autophagic potential. 40 Autophagy is the major mechanism through which all cytoplasmic parts of post-mitotic cells can be renewed. Therefore, it is considered a vital cytoprotective process that prevents the accumulation of damaged cellular biomolecules and structures. 41 This also applies to oxidative stress. If antioxidant enzyme systems and cellular antioxidants fail to prevent oxidative damage, autophagy takes effect as a second line protection response by degrading oxidatively damaged cellular structures. 42 Accordingly oxidative stress and lipid peroxidation products have been shown to upregulate autophagy for prevention damage accumulation. 43 However, autophagy is a highly energy consuming process which may be affected upon ATP deficit. 44 The results obtained in our study suggest that already moderately reduced ATP levels may affect autophagy in RPE cells, thereby weakening this stress response. This effect seems to be restricted to classical macroautophagy. The difference between total lysosomal protein degradation rates (ammonia-sensitive) and macroautophagy (3-methyladenine-sensitive) represents the contribution of alternative autophagic pathways, including chaperone-mediated autophagy and microautophagy. This difference was not influenced by the atractyloside-induced energy deficit. 
Phagocytosis and degradation of shed photoreceptor outer segment degradation is a vital maintenance function performed by the RPE to preserve phototransduction activity and viability of photoreceptors. Since it is an enormously energy consuming process, inhibition of mitochondrial ATP generation has already been linked to reduced phagocytic degradative capacities of RPE cells. Therefore such RPE dysfunction has been suggested to contribute to human retinal diseases like ARMD. 15 Our experiments on the phagocytic degradation of POS in atractyloside-treated RPE cells are in line with these previous results. 
Altogether our in vitro results suggest that moderately decreased levels of ATP might contribute to the vulnerability of RPE cells to oxidative stress damage and to dysfunction. This was observed at ATP levels that also occur in vivo in the RPE of aged individuals. Therefore, future in vivo studies should address a possible link between reduced ATP production in aged RPE and age-related degenerative eye disorders. 
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Footnotes
 Supported by a Fritz-Tobler-Foundation grant (SA, JK).
Footnotes
 Disclosure: F. Schütt, None; S. Aretz, None; G.U. Auffarth, None; J. Kopitz, None
Figure 1. 
 
RPE cells were grown to confluence in 12-well plates. Then 1 μM atractyloside was added to the culture medium and cells were harvested at the indicated time points after addition of the inhibitor. Cells were homogenized in ATP-releasing reagent (Sigma-Aldrich). ATP and protein concentration was assayed as described in “Materials and Methods.” ATP levels were normalized to protein content. ATP concentration at time point 0h was set to 100%. Results are the means of three independent experiments (±SD).
Figure 1. 
 
RPE cells were grown to confluence in 12-well plates. Then 1 μM atractyloside was added to the culture medium and cells were harvested at the indicated time points after addition of the inhibitor. Cells were homogenized in ATP-releasing reagent (Sigma-Aldrich). ATP and protein concentration was assayed as described in “Materials and Methods.” ATP levels were normalized to protein content. ATP concentration at time point 0h was set to 100%. Results are the means of three independent experiments (±SD).
Figure 2. 
 
Cells were treated with atractyloside as described in Figure 1 and total and reduced glutathione was assayed after homogenization of the cells at the indicated time points by ELISA. The percentage of reduced glutathione as compared to total glutathione is shown. Results are the means of three independent experiments (±SD).
Figure 2. 
 
Cells were treated with atractyloside as described in Figure 1 and total and reduced glutathione was assayed after homogenization of the cells at the indicated time points by ELISA. The percentage of reduced glutathione as compared to total glutathione is shown. Results are the means of three independent experiments (±SD).
Figure 3. 
 
Cells were treated with atractyloside as described in Figure 2. Oxidative stress was induced by adding tBH to the culture medium. Cells were harvested and homogenized at the indicated time points and MDA-adduct formation on cellular proteins was assayed by ELISA. Results are the means of three independent experiments (±SD).
Figure 3. 
 
Cells were treated with atractyloside as described in Figure 2. Oxidative stress was induced by adding tBH to the culture medium. Cells were harvested and homogenized at the indicated time points and MDA-adduct formation on cellular proteins was assayed by ELISA. Results are the means of three independent experiments (±SD).
Figure 4. 
 
Cells were treated with tBH as described in Figure 3. Total DNA was extracted from the cell homogenates by DNeasy DNA purification kit. 8-Hydroxydeoxyguanosine in the extracted DNA was quantified by competitive enzyme immunoassay. Results are the means of three independent experiments (±SD).
Figure 4. 
 
Cells were treated with tBH as described in Figure 3. Total DNA was extracted from the cell homogenates by DNeasy DNA purification kit. 8-Hydroxydeoxyguanosine in the extracted DNA was quantified by competitive enzyme immunoassay. Results are the means of three independent experiments (±SD).
Figure 5. 
 
Cells were metabolically labeled by the inclusion of 500 kBq/mL 3H-leucine in the culture medium for 72 hours (pulse phase). Then the cultures were chased in nonradioactive medium in the absence (•) or presence (▵) of 1μM atractyloside. 3-Methyladenine-sensitive (autophagic protein degradation, solid line) and NH4Cl-sensitive (total lysosomal degradation, dotted line) protein degradation rates were assayed at the indicated time points of the chase phase as described in “Materials and Methods.” Results are the means of three independent experiments (±SD).
Figure 5. 
 
Cells were metabolically labeled by the inclusion of 500 kBq/mL 3H-leucine in the culture medium for 72 hours (pulse phase). Then the cultures were chased in nonradioactive medium in the absence (•) or presence (▵) of 1μM atractyloside. 3-Methyladenine-sensitive (autophagic protein degradation, solid line) and NH4Cl-sensitive (total lysosomal degradation, dotted line) protein degradation rates were assayed at the indicated time points of the chase phase as described in “Materials and Methods.” Results are the means of three independent experiments (±SD).
Figure 6. 
 
Cells were grown in the absence or presence of 1μM atractyloside for 48 hours. Then labeled POSs were added without change of medium. (A) Uptake (clearance from the medium), (B) Intracellular accumulation, (C) Degradation of labeled POS was assayed as described in “Materials and Methods.” Results are the means of three independent experiments.
Figure 6. 
 
Cells were grown in the absence or presence of 1μM atractyloside for 48 hours. Then labeled POSs were added without change of medium. (A) Uptake (clearance from the medium), (B) Intracellular accumulation, (C) Degradation of labeled POS was assayed as described in “Materials and Methods.” Results are the means of three independent experiments.
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