In the current study, we suggest that phosphorylation reactions of specific proteins in mitochondria and the nucleus are a key step in the progression of age-related macular degeneration (AMD). To determine the molecular mechanism of AMD, we examined proteomic changes under oxidative stress to establish the protein interaction map using in vitro and in vivo models that mimic the complex and progressive characteristics of AMD. We postulated that apoptosis can be initiated by phosphorylation reactions under chronic oxidative stress in a region-specific and tissue-specific manner. The analysis of AMD interactome and oxidative biomarker network demonstrated that the presence of tissue- and region-dependent post-translational mechanisms may contribute toward AMD progression through the mitochondrial-nuclear communication. The AMD interactome suggests that new therapeutic targets, including prohibitin, erythropoietin, vitronectin, crystalline, nitric oxide synthase, ubiquitin, and complement inhibition may exist as a proteome network. Further, immunocytochemistry demonstrated that mitochondria could enter the nucleus in the retinal pigment epithelium (RPE) under oxidative stress. The current interactome map implies that a positive correlation may exist between oxidative stress-mediated phosphorylation and AMD progression. The unbiased proteome network provides a basis for understanding oxidative stress-induced mitochondrial dysfunction in AMD and exploring effective therapeutic approaches to treat age-related neurodegeneration.
- protein interactome
- retrograde signaling
- AMD target
Age-related macular degeneration (AMD) is the leading cause of legal blindness in developed countries [1, 2]. Although the vision loss directly results from dysfunction and cell death of photoreceptors in the central retina, it has been demonstrated that the early stage of AMD involves the pathological changes in retinal pigment epithelium (RPE), the cell layer that plays pivotal roles in supporting photoreceptors. Due to the inevitable roles of RPE in supporting retinal function, it is critical to understand the physiological and pathological events in the RPE to prevent the development of AMD.
AMD symptoms include RPE atrophy, drusen accumulation, pigmentary changes, and choroidal neovascularization [1, 2]. Progressive cell death of post-mitotic RPE can lead to rod and cone apoptosis, resulting in AMD eventually. As AMD is a complex and multifactorial disease, AMD mechanisms could be discussed under environmental and genetic factors, including oxidative stress (smoking, light exposure, and hypoxia), RPE dysfunction (retinoid recycling, phagocytosis, aging, and apoptosis), accumulation of visual cycle waste, chronic inflammation (involving CFH, CFB/C2, C3, CF1, C5, and C9), drusen formation (lipid metabolism involving APOE, LIPC, and CETP), geographic atrophy, and choroidal neovascularization (VEGF signaling) [3, 4, 5, 6, 7, 8].
Recently, mitochondrial alterations have drawn great deal of attention in understanding AMD . Mitochondrion is the main cell compartment for cell respiration and cell signaling. Many studies have shown that RPE mitochondria undergo severe structural and functional changes during aging and AMD [3, 4]. The mitochondrial dysfunction causes the excessive generation and leaking of reactive oxidative species (ROS) from the respiration chain and RPE is one of the most susceptible cells to ROS. RPE is also responsible for phagocytosis of rod outer segments, where polyunsaturated fatty acids abound. Phagocytosis and oxidation of unsaturated fatty acids generate additional ROS. Further, RPE cells are exposed to chronic oxidative stress, including constant exposure to intense light and oxidants from mitochondria due to high levels of oxygen demand and consumption. The increased oxidative stress in RPE may in turn deteriorates mitochondria and causes RPE cell death. Our data suggested that mitochondrial morphology and functional integrity are closely related to apoptosis and cellular aging [9, 10, 11]. Insufficient bioenergetic processes may lead to drusen accumulation. A number of apoptotic regulators reside in mitochondria and various retrograde signaling mechanisms are also dependent on mitochondria.
Oxidative stress facilitates the formation of toxic lipids and protein peroxidation, resulting in drusen deposition. There are excessive generation and leaking of oxidants from the respiratory chain under oxidative stress. This explains why AMD is associated with the accumulation of advanced lipid peroxidation end products, leading to apoptosis of photoreceptors and RPE cells. In addition, phosphorylation of crystalline and vimentin may participate in the pathogenesis of AMD by forming soft drusen with longer chain of phosphatidylcholine and cholesteryl esters. With aging, lipids and cholesterol accumulate underneath the RPE and contribute toward drusen formation. The excessive drusen deposition may damage the RPE and lead to degeneration of collagen or elastin in Brook’s membrane, the outer retina, and the choroid vasculature.
We have studied the mechanism of RPE cell death under various stress conditions [9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20]. Our data demonstrated that mitochondrial morphological changes and mitochondrial-nuclear shuttling of prohibitin are significant responses in the RPE under oxidative stress [9, 10, 18]. Herein, we discuss AMD mechanisms based on four distinctive subnetworks of protein interactome, including complement activation, transcriptional regulation, mitochondrial signaling, and apoptosis. We propose that altered retrograde mitochondrial-nuclear crosstalk may initiate the pathological reactions observed in aging and oxidative stress-mediated RPE cell death that can contribute to the pathogenesis of AMD.
2. Materials and methods
In vivoexperimental design
All the animal procedures were performed in compliance with the Association for Research in Vision and Ophthalmology Statement for the humane use of laboratory animals. Human
2.2 ARPE-19 and HRP cells
In vivooxidative stress
Constant light experiment was conducted as previously reported . Female C3HeB/FeJ mice (12 weeks of age) were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed under a 12-h light/12-h dark cyclic lighting condition (250–300 lux of full spectra fluorescent room light) for 2 weeks. The first group of mice (light/dark group) was housed in the 12-h light/12-h dark condition and the second group (constant light group) was housed in constant room light (250–300 lux) for 7 days. After euthanasia, eyes were rapidly removed from animals and retinas were isolated by microscopic dissection. Retinas were then washed with a solution of 250 mM sucrose, 10 mM Tris–HCl, at pH 7.0 to remove contaminants before lysis in 30 mM Tris–HCl, 2 M thiourea, 7 M urea, 4% CHAPS, and protease inhibitors. Samples were then sonicated intermittently until cells were lysed. The crude lysate was centrifuged at 20817×
2.4 Protein identification by MALDI-TOF and TOF-TOF mass spectrometry
Protein spots, manually excised from the gel, were de-stained with 100 mM NH4HCO3/50% acetonitrile (MeCN) at 37°C for 45 min twice. Gel slices were then incubated with 100% MeCN at room temperature for 5 min. After dehydration and drying, gel slices were incubated with 250 ng of trypsin (20 μg/20 μl, Promega) in 40 mM NH4HCO3/10% MeCN at 37°C overnight. Trypsin/digestion buffer (50 μl) was added so that the gel slices were completely covered. After trypsin treatment, peptides were collected and gel slices were washed for 1 h with extraction buffer (50% MeCN, 0.1% Trifluoroacetic acid (TFA)) with gentle agitation. Peptides were combined and concentrated in a speed vacuum. Ziptip has been used to purify peptides. About 0.6 μl of purified peptides were mixed with 0.6 μl of alpha-Cyano-4-hydroxy-cinnamic acid matrix solution saturated in 50% MeCN/0.1% TFA solution (1:1 vol/vol) onto a MALDI 100-well target plate. It was analyzed through matrix-assisted laser/desorption ionization time of flight mass spectrometry (Bruker Ultraflex MALDI-TOF-TOF Mass spectrometer) in a reflector mode. Mass spectra and tandem mass spectra were acquired manually with laser intensity at 2400 and 200 shots per spectrum in MS mode and laser intensity at 3800 and 400 shots in MS/MS mode. The spectra were analyzed using Flex analysis 2.0 and Biotools 2.2 software. Peptide mass was calibrated internally using two trypsin auto digest ions (m/z 842.509, m/z 2211.104). Protein identification was performed using Mascot software (www.matrixscience.com) to search the National Center for Biotechnology Information (NCBI) database with mouse taxonomy. A missed trypsin cleavage was not allowed and 100 ppm of mass tolerance was applied for the search for the matching peptide. For MALDI-TOF and peptide fingerprinting, the probability-based Mowse score is used −10*Log (P), where P is the probability that the observed peptide match is a random event. Protein scores greater than 55 are considered significant (p < 0.05).
2.5 Immunocytochemical analysis
Cells were grown on sterile glass cover slips using DMEM/F12 medium with 10% FBS and 1% penicillin/streptomycin (Hyclone) in 5% CO2 incubator at 37°C. Cells were treated under oxidative stress or intense light as previously reported [9, 10, 11, 18, 19, 20]. Cells were washed with PBS and incubated with MitoTracker Orange CMTMRos (100 nM, Molecular Probes, Carlsbad, CA) in serum-free culture medium (30 min, 37°C), followed by washing (PBS) and fixing (10% formaldehyde, 30 min, room temperature). Next, cells were treated using Triton X-100 (0.2%, Sigma-Aldrich, St. Louis, MO) in PBS (30 min) for permeabilization and blocked using complete medium (10% FBS, 0.05% Tween-20, 1 h). To stain cells, anti-actin, anti-tubulin, anti-vimentin antibody (1:100; Santa Cruz), and anti-prohibitin antibody (1:500, Genemed Synthesis Inc., San Antonio, TX, overnight, 4°C) were used; then, the cells were washed with PBS, followed by incubation with Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (1:700; Molecular Probes, Carlsbad, CA, 1 h, room temperature). VECTASHIELD medium with DAPI (4,6-diamidino-2-phenylindole, the nucleus) was applied to mount the samples which were visualized using a Zeiss AxioVert fluorescent microscope (200 M Apo Tome, 63× magnification). Images were analyzed using ImageJ software (NIH).
2.6 AMD interactome map
Oxidative biomarker and AMD interactome were established using protein-protein interaction map software and databases, including STRING 10.0 (http://string-db.org/), MIPS (http://mips.helmholtz-muenchen.de/proj/ppi/), and iHOP (http://www.ihop-net.org/UniPub/iHOP/). Proteins found in AMD or oxidative stress conditions were added to establish the AMD interactome. Protein interactions were presented using eight categories, including neighborhood (green), gene fusion (red), co-occurrence (dark blue), co-expression (black), binding experiments (purple), databases (blue), text mining (lime), and homology (cyan). Protein interactions were determined and confirmed by genomic context, high-throughput experiments, co-expression, and previous publications in Pubmed. Protein database analysis showed the region-specific phosphorylation of specific proteins in AMD eyes. The AMD interactome was compared to the retina/RPE proteome under stress conditions.
First, we determined the phosphorylation reactions in AMD samples to understand mitochondrial signaling, immune response, energy metabolism, and apoptosis under oxidative stress. The molecular network of altered phosphorylation is essential for determining molecular targets to treat AMD in the early stage. We built a comprehensive interaction map by combining several independent sets of
The AMD interactome was connected together using phosphoproteomics data from AMD tissues,
The AMD interactome with oxidative biomarkers demonstrated that several proteins that were previously characterized as unrelated to AMD, including ubiquitin, peroxiredoxin, MAP kinase, BUB 1/3, vimentin, and crystalline, were involved in AMD progression. Our interactome map suggests that mitochondrial protein trafficking, crystalline aggregation, and protein degradation may contribute to AMD pathway. To confirm oxidative stress biomarkers, specific cytoskeletal protein changes were determined
Based on our proteomics data showing altered signaling of apoptosis in the retina and RPE, new targets for anti-apoptotic and anti-angiogenic therapy can be determined. The current interactome suggests the new biomarkers and targets in AMD including: (1) mitochondrial dysfunction in the peripheral RPE (depleted prohibitin, increased ATP synthase); (2) oxidative stress including intense and constant light (peroxiredoxin, thioredoxin, glutathione S-transferase); (3) cytoskeletal/mitochondrial remodeling by microtubule, actin filament, and intermediate filament (tubulin, actin, vimentin); (4) high concentration of nitric oxide (nitric oxide synthase); (5) hypoxia responses (HIF1, erythropoietin, VEGF); (6) disrupted circadian clock (melatonin); (7) apoptotic downstream (pJAK2, pSTAT3, Bclxl, caspases); (8) altered lipid concentrations (cardiolipin, cholesterol); (9) altered visual cycle (CRABP, CRALBP, RPE65); (10) altered energy metabolism (S/T vs. Y kinases, carnitine, pyruvate, ATP synthase); (11) aggregation of heat shock proteins and crystallins; and (12) inflammation switch (CFH, C3, collagen, vitronectin).
Next, we examined light-induced protein regulation to determine oxidative stress biomarkers
To determine the interdependence of mitochondrial trafficking versus oxidative stress, we examined β-tubulin and vimentin dynamics under stress conditions (Figure 3A). Constant or intense light accelerated β-tubulin aggregation as well as nuclear localization. Mitochondrial trafficking was colocalized with tubulin polymerization, whereas vimentin was more likely to determine mitochondrial morphology in the dark. Intense light also led to actin filament aggregation in cytosol in the RPE. Cytosolic β-actin in the dark entered the nucleus under stress conditions. Immunocytochemistry of tubulin and actin demonstrated oxidative stress-mediated mitochondrial aggregation and size changes along with mitochondrial decay. Vimentin was shown as an extended filamentous structure in control; however, it was aggregated around the nucleus under stress conditions, that is oxidative stress and intense light (7000 lux). Mitochondrial proteins and cytoskeletal proteins, including prohibitin, actin, tubulin, and vimentin moved toward the nucleus under oxidative stresses (Figure 3B).
Translocalization of prohibitin might be related to post-transcriptional regulation and mitochondrial membrane depolarization. Down- or up-regulation of prohibitin in specific concentrations of H2O2 may imply one of several anti-apoptotic or pro-apoptotic responses depending on the intensity and temporal pattern of the stress. In our previous experiment, NF-κB was translocated into the nucleus in oxidative stress as a survival factor. It is important to note that prohibitin and NF-κB moved in parallel or opposite directions between the nucleus and mitochondria under various conditions. This coordinated translocalization may determine cell viability and apoptotic population in the retina and RPE.
We also examined the nuclear function of prohibitin by immunoprecipitation. Prohibitin binds with many transcription factors and nucleotide-binding proteins, including TFIIIB, DNA mismatch repair protein, ski2-type helicase, Cyclin-D-binding Myb-like transcription factor 1, DNA ligase, elongation factor, and BRCA1-A complex subunit RAP80. Additional interacting proteins have been reported such as E2F, retinoblastoma-associated protein, cellular tumor antigen p53, Heatshock 70: Stress-70 protein, and histone deacetylase (HDAC1). We confirmed transcriptional regulation of prohibitin by immunoprecipitation and protein-nucleotide binding assay.
Next, we established the signaling network of AMD using the interactome results. Based on our proteomics and interactome data, the potential AMD mechanisms were integrated as shown in Figure 4, suggesting altered energy metabolism, mitochondrial dysfunction, retinoid metabolism, circadian clock, inflammation, angiogenesis, lipid metabolism, and apoptosis.
Previous data demonstrated that Hsp70 (c-Jun N-terminal kinase), crystallins (Akt), and the increased expression of VDAC might be involved in AMD progression [22, 23, 24, 25, 26]. Altered phosphorylations of mitochondrial heat shock protein mtHsp70, αA/aB crystalline, vimentin, and ATP synthase were observed in RPE cell death under oxidative stress [9, 22, 27, 28, 29]. Retinoid-binding proteins, including CRABP, RPE65, and RLBP1, could be involved in the advanced stages of AMD [30, 31, 32]. It was reported that accumulation of all-
We observed altered lipid compositions that include increased carbon number of fatty acids, double bond saturation, higher cholesterol, and phosphatidylcholine, whereas cardiolipin levels decreased in RPE apoptosis [9, 10, 18]. Changes in lipid concentrations seem to diminish the membrane fluidity and accelerate protein aggregation in the RPE [38, 39, 40, 41, 42, 43, 44].
A positive correlation between the levels of PP2A and vimentin under light-induced stress suggests that cytoskeletal dynamics are regulated by vimentin phosphorylation. We postulate that light may induce post-translational modifications of vimentin. Stabilized vimentin may act as an anti-apoptotic agent when cells are under stress.
The current interactome suggests that altered phosphoproteome interactions, including pyruvate kinase, tyrosine kinase, and vimentin, exist in the retina and RPE in AMD. Phospho-Western blotting analysis revealed that phosphorylations of intermediate filament vimentin (Ser38, Ser55) and mitochondrial heat shock protein mtHsp70 were modulated in the RPE
We used bioinformatics approaches to integrate molecular events associated with the progression of AMD. Proteomics data obtained using the oxidative stress animal model as well as the
The current interactome mapping also suggests that changes in phosphoprotein levels in response to oxidative stress may induce complement activation, transcriptional regulation, mitochondrial dysfunction, and apoptosis. The phosphorylation signaling may explain why AMD is induced by oxidative stress and how the downstream of phosphorylations are associated with the changes of mitochondrial protein expressions, cytoskeleton reorganization, membrane remodeling, and lipid oxidation. For example, previous observations of vimentin derived from human choroidal neovascular membranes in AMD, as well as in drusen and melanolipofuscin, support the positive correlation between the biomarkers we characterized in RPE cells under stress and the AMD proteomics.
The AMD interactome map also elucidates the regulatory mechanism of apoptotic cell death governed by phosphorylation. Changes in the global phosphoproteome could be one of indications of early signaling events, including an increase of longer chain fatty acids, especially phosphatidylcholine and cholesteryl esters. The phosphoprotein interactome also provides a connection between oxidative stress-induced mitochondrial trafficking changes and AMD.
We also emphasize that mitochondria play a significant role in stress response in RPE cells, and this in turn influences the progression of AMD. The number of RPE mitochondria decreases with aging . Through the observation by scanning electron microscope, mitochondrial shape becomes more oval in normal aged samples, while it is more bacillus-like in young eyes. The number of cristae decreases and cristae structure is less organized in aged tissue. In addition, mitochondrial matrix shows less electron density along with aging. In AMD samples, loss of cristae structure and matrix density is more obvious. Bleb formation on mitochondrial membrane and loss of mitochondrial membrane integrity were also observed in AMD [32, 49, 50].
Previously, quantitative analysis of mitochondrial morphology reveals that mitochondrial structure and functionality are closely associated with aging and AMD [9, 22, 32, 51, 52, 53, 54, 55, 56, 57]. In both aging and AMD samples, mitochondrion number per cell, cristae per mitochondrion, and mean area of mitochondrion per cell are declined. A similar trend was also observed in oxidative stressed ARPE-19, where mitochondrial distribution also changed. It is important to note that a mitochondrion is a highly dynamic organelle in response to different environmental factors, and those responses may determine the cell fate. RPE mitochondria undergo loss of structural integrity under oxidative stress and aging, indicating that mitochondrial signaling could be interrupted in AMD.
The retrograde communication from mitochondria to the nucleus has been demonstrated by our previous data by tracking the chaperon protein prohibitin using fluorescent microscopy techniques [10, 18, 58]. Mitochondrial components may also determine cell signaling in the nucleus, including change of mitochondrial membrane potential, mitochondrial DNA (mtDNA), and mitochondrial protein expressions. When cumulative damages hit the threshold where mitochondria cannot maintain their structural integrity, there is a decrease of mitochondrial membrane potential, along with a number of mitochondrial components released into the cytosol, including cardiolipin, cytochrome c, and Ca2+. Release of cytochrome c initiates the caspase-dependent apoptosis and triggers more Ca2+ release from endoplasmic reticulum, whereas elevation of cytosolic Ca2+ level can cause more cytochrome c release and activation of caspase-9.
Mitochondrial DNA (mtDNA) is susceptible to oxidative stress damage. Compared to nuclear DNA (nDNA), mtDNA is located in mitochondrial matrix in close proximity to the ROS source in the cell. MtDNA is lack of histones and contains no introns that also increases its susceptibility to oxidative damage. Meanwhile, mtDNA mainly encodes electron transport chain proteins, including ATP synthase, cytochrome b, cytochrome c oxidase, and NADH dehydrogenase. Damaged mtDNA will lead to impaired electron transport chain proteins, further deteriorating cell energy production, generating more ROS, and inducing extra damages to mtDNA. Previous studies have revealed that abnormal mtDNA leads to reduced energy production and initiation of apoptosis [59, 60, 61, 62]. Loss of mtDNA in ARPE-19 cells led to the change of nuclear gene expression, especially the up-regulation of genes related to glycolysis [63, 64, 65].
The mitochondrial-nuclear crosstalk could be a mechanism that the RPE cell uses to compensate the insufficient energy productions due to the mitochondrial dysfunction. Other changes in nuclear gene expressions caused by loss of mtDNA include up-regulation of proteins related to uptake of ROS and drusen, extracellular matrix and matrix enzymes, lipid transport-related proteins, and inflammation-related regulators. Therefore, damaged mtDNA has been considered as an important biomarker of oxidative stressed RPE and progression of AMD [50, 66, 67]. Fragments of mtDNA have been found to migrate to the nucleus and be inserted into the nuclear genome [68, 69, 70, 71]. The entrance of mitochondria into the nucleus has been reported to promote both the attack of mitochondria by nuclear protein and the attack of nuclear DNA and protein by protein of the mitochondrial intermembrane space [65, 68, 69, 70, 71, 72, 73, 74]. Mitochondria move to the nucleus under stress to fulfill energy demand of the nucleus. Therefore, our observation that mitochondria entering the nucleus could be one of the mechanisms to explain mitochondrial diseases and the aging process.
Our AMD interactome map implies that a positive correlation exists between AMD mechanism and early oxidative stress biomarkers, as well as inflammation switch, apoptosis, transcriptional regulation, and mitochondrial dysfunction [26, 32, 52, 75, 76, 77]. The mechanistic dissection of our AMD interactome map is the initial delineation of the underlying physiology of oxidative stress-mediated phosphorylation signaling in RPE apoptosis which can lead to AMD progression. In addition, the phosphoprotein interactome provides a stimulus for understanding oxidative stress-induced mitochondrial changes and the mechanism of aggregate formation induced by protein phosphorylations. As a consequence, an effective therapeutic approach to treat AMD based on the modulation of phosphorylation reactions is expected to result.