**2. Materials and methods**

#### **2.1** *In vivo* **experimental 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

**169**

**2.3** *In vivo* **oxidative stress**

*Mechanistic Dissection of Macular Degeneration Using the Phosphorylation Interactome*

phosphorylations were confirmed by phospho-Western blotting analysis.

For *in vitro* experiments, retinal pigment epithelial cells (ARPE-19) were purchased from ATCC (Manassas, VA) and retinal progenitor cells (HRP) were kindly donated by Dr. Harold J. Sheeldo at the University of North Texas Health Science Center. ARPE-19 and HRP cells were cultured in a 5% CO2 incubator at 37°C in 100 mm dishes (Nalge Nunc International, Naperville, IL) using Dulbecco's modified Eagle's medium (DMEM) with fetal bovine serum (10%) and penicillin/streptomycin (1%). Confluent cells were trypsinized (5–7 min at 37°C) using a trypsin-EDTA buffer (0.1%, Sigma-Aldrich, St. Louis, MO), followed by centrifugation (300× *g,* 7 min). Cells (eight to nine passages) were grown to confluence for 2–4 days and then were treated with H2O2 (200 μM), intense light (7000–10,000 lux, 1–24 h) or constant light (700 lux, 48 h). Then, cells were rinsed (Modified Dulbecco's PBS) and lysed using IP lysis buffer containing Tris (25 mM), NaCl (150 mM), EDTA (1 mM), NP-40 (1%), glycerol (5%), and protease inhibitor cocktail at pH 7.4 by incubating on ice for 5 min with periodic sonication (3 × 5 min), followed by centrifugation (13,000× *g,* 10 min). Proteins (1 mg/ml, 200–400 μl) were loaded for immunoprecipitation and nonspecific bindings were avoided using control agarose resin cross-linked by 4% bead agarose. Amino-linked protein-A beads were used to immobilize antiprohibitin antibody with a coupling buffer (1 mM sodium phosphate, 150 mM NaCl, pH 7.2), followed by incubation (room temperature, 2 h) with sodium cyanoborohydride (3 μl, 5 M). Columns were washed using a washing buffer (1 M NaCl), and protein lysate was incubated in the protein A-antibody column with gentle rocking overnight at 4°C. The unbound proteins were spun down as flow-through, and the columns were washed three times using washing buffer to remove nonspecific binding proteins. The interacting proteins were eluted by incubating with elution buffer for 5 min at RT. The eluted proteins were equilibrated with Laemmli sample buffer (5X, 5% β-mercaptoethanol). Eluted proteins were separated using SDS-PAGE and stained using Coomassie blue (Pierce, IL) or silver staining kit (Bio-Rad, Hercules, CA). Immunoprecipitated proteins were reported previously [10] and were used to establish interactome in the current study.

Constant light experiment was conducted as previously reported [21]. 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

animals. Human *postmortem* donor eye tissues were used following the tenets of the Declaration of Helsinki. Diabetic retinopathy (DR) human retinal tissues (n = 9, biological triplicate × technical triplicate) were obtained from the Georgia Eye Bank (Atlanta, GA). AMD retina (8 mm macular and peripheral punches), RPE (8 mm central and peripheral punches), and age-matched control eyes (n = 9, biological triplicate × technical triplicate) were provided by the Lions Eye Bank (Moran Eye Center, University of Utah). Phosphoproteomes of macula (I), peripheral retina (II), central RPE (III), and peripheral RPE (IV) were compared to age-matched control donor eyes to determine region-specific, senescence-associated molecular mechanisms during AMD progression. Phosphoproteins were enriched by charge-based spin column chromatography and resolved by 2D gel electrophoresis as previously reported [11, 16]. Trypsin-digested phosphopeptides from whole lysates were enriched using Ga3+/TiO2 immobilized metal ion chromatography. Eluted phosphopeptides were analyzed using MALDI-TOF-TOF and ESI MS/MS. Serine, threonine, and tyrosine

*DOI: http://dx.doi.org/10.5772/intechopen.83016*

**2.2 ARPE-19 and HRP cells**

*Mechanistic Dissection of Macular Degeneration Using the Phosphorylation Interactome DOI: http://dx.doi.org/10.5772/intechopen.83016*

animals. Human *postmortem* donor eye tissues were used following the tenets of the Declaration of Helsinki. Diabetic retinopathy (DR) human retinal tissues (n = 9, biological triplicate × technical triplicate) were obtained from the Georgia Eye Bank (Atlanta, GA). AMD retina (8 mm macular and peripheral punches), RPE (8 mm central and peripheral punches), and age-matched control eyes (n = 9, biological triplicate × technical triplicate) were provided by the Lions Eye Bank (Moran Eye Center, University of Utah). Phosphoproteomes of macula (I), peripheral retina (II), central RPE (III), and peripheral RPE (IV) were compared to age-matched control donor eyes to determine region-specific, senescence-associated molecular mechanisms during AMD progression. Phosphoproteins were enriched by charge-based spin column chromatography and resolved by 2D gel electrophoresis as previously reported [11, 16]. Trypsin-digested phosphopeptides from whole lysates were enriched using Ga3+/TiO2 immobilized metal ion chromatography. Eluted phosphopeptides were analyzed using MALDI-TOF-TOF and ESI MS/MS. Serine, threonine, and tyrosine phosphorylations were confirmed by phospho-Western blotting analysis.

#### **2.2 ARPE-19 and HRP cells**

*Visual Impairment and Blindness - What We Know and What We Have to Know*

mechanisms are also dependent on mitochondria.

death that can contribute to the pathogenesis of AMD.

**2. Materials and methods**

**2.1** *In vivo* **experimental design**

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) [3–8]. Recently, mitochondrial alterations have drawn great deal of attention in understanding AMD [3]. 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 [9–11]. Insufficient bioenergetic processes may lead to drusen accumulation. A number of apoptotic regulators reside in mitochondria and various retrograde signaling

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 [9–20]. 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

All the animal procedures were performed in compliance with the Association for Research in Vision and Ophthalmology Statement for the humane use of laboratory

**168**

For *in vitro* experiments, retinal pigment epithelial cells (ARPE-19) were purchased from ATCC (Manassas, VA) and retinal progenitor cells (HRP) were kindly donated by Dr. Harold J. Sheeldo at the University of North Texas Health Science Center. ARPE-19 and HRP cells were cultured in a 5% CO2 incubator at 37°C in 100 mm dishes (Nalge Nunc International, Naperville, IL) using Dulbecco's modified Eagle's medium (DMEM) with fetal bovine serum (10%) and penicillin/streptomycin (1%). Confluent cells were trypsinized (5–7 min at 37°C) using a trypsin-EDTA buffer (0.1%, Sigma-Aldrich, St. Louis, MO), followed by centrifugation (300× *g,* 7 min). Cells (eight to nine passages) were grown to confluence for 2–4 days and then were treated with H2O2 (200 μM), intense light (7000–10,000 lux, 1–24 h) or constant light (700 lux, 48 h). Then, cells were rinsed (Modified Dulbecco's PBS) and lysed using IP lysis buffer containing Tris (25 mM), NaCl (150 mM), EDTA (1 mM), NP-40 (1%), glycerol (5%), and protease inhibitor cocktail at pH 7.4 by incubating on ice for 5 min with periodic sonication (3 × 5 min), followed by centrifugation (13,000× *g,* 10 min). Proteins (1 mg/ml, 200–400 μl) were loaded for immunoprecipitation and nonspecific bindings were avoided using control agarose resin cross-linked by 4% bead agarose. Amino-linked protein-A beads were used to immobilize antiprohibitin antibody with a coupling buffer (1 mM sodium phosphate, 150 mM NaCl, pH 7.2), followed by incubation (room temperature, 2 h) with sodium cyanoborohydride (3 μl, 5 M). Columns were washed using a washing buffer (1 M NaCl), and protein lysate was incubated in the protein A-antibody column with gentle rocking overnight at 4°C. The unbound proteins were spun down as flow-through, and the columns were washed three times using washing buffer to remove nonspecific binding proteins. The interacting proteins were eluted by incubating with elution buffer for 5 min at RT. The eluted proteins were equilibrated with Laemmli sample buffer (5X, 5% β-mercaptoethanol). Eluted proteins were separated using SDS-PAGE and stained using Coomassie blue (Pierce, IL) or silver staining kit (Bio-Rad, Hercules, CA). Immunoprecipitated proteins were reported previously [10] and were used to establish interactome in the current study.

#### **2.3** *In vivo* **oxidative stress**

Constant light experiment was conducted as previously reported [21]. 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× *g* for 30 min at 4°C. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed using Ettan IPGphor system with 11 cm of immobilized pH gradient (IPG) strips (pH 5–8, ReadyStrip, Biorad) and 8–16% gradient Precast Gel (Criterion Precast Gel, Biorad). About 200 μg of retinal proteins were diluted to 200-μl solution with rehydration buffer (4% CHAPS, 8 M urea, 1% pharmalytes 3–10, 10 mM DTT). The mixture (200 μl) was incubated with IPG strip at room temperature for 30 min. IPG strips were rehydrated for 14 h at 30 V, followed by isoelectric focusing (IEF) performed at 500 V for 1 h, 500–8000 V for 6 h, and 8000 V for 1 h. After IEF, IPG strips were equilibrated for 15 min in 10-ml equilibration buffer containing 50 mM Tris–HCl (1.5 M, pH 8.8), 6 M urea, 30% glycerol (v/v), 2% SDS (w/v), trace amount of Bromophenol Blue, and 0.05 g DTT, and then re-equilibrated for another 15 min in the same buffer containing 0.45 g of iodoacetamide. Equilibrated strips were then placed on top of precast gradient gels and embedded in 0.5% agarose. Proteins were electrophoresed at 100 volts for about 2 h until the dye had reached the bottom of the gel. After electrophoresis, separated proteins were visualized using Coomassie blue staining. The Commassie blue-stained gels were scanned with a transmission scanner and differential protein expression was analyzed. Differentially expressed protein spots were excised from gels and analyzed by MALDI-TOF and the selected proteins were further analyzed by MALDI-TOF-TOF mass spectrometry. All experiments were repeated in triplicate.
