**3. Results**

To understand how mitochondria regulate their morphology and function, we first analyzed mitochondrial morphology quantitatively in ARPE-19 cells subjected to oxidative stress conditions using a systematic computational model (**Figure 1**). Representative mitochondrial images at selected time points (0.5, 1, 8, 24 h) are shown for clear comparison **Figure 1**). We examined mitochondrial area, circularity, perimeter, content as well as cellular area to identify changes between healthy and injured mitochondria. Previously, our *in vitro* data using RPE cells demonstrated the positive correlation between apoptotic signaling and mitochondria-nucleus prohibitin shuttling. Our previous studies suggest that cellular distribution and the total volume of mitochondria could be affected by microtubules, intermediate filaments and cardiolipin [24–27, 29, 30].

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Interacting Proteins (DIP), the Molecular Interaction Database (MINT), the Protein Interaction

Interaction mapping of prohibitin was determined using immunoprecipitation, followed by mass spectrometry analysis. Prohibitin binding proteins in the RPE were connected using

Prohibitin interactions were confirmed using eight sources that include neighborhood, gene fusion, co-occurence, high-throughput interaction experiments, databases, homology, conserved co-expression, and published knowledge, including ExPASy (http://www.expasy. org/proteomics /protein–protein\_interaction), MIPS (http://mips.helmholtz-muenchen.de/

AMD and oxidative biomarkers interactome were established using protein–protein interaction map software and databases, including STRING 10.0 (http://string-db.org/), MIPS and iHOP (http://www.ihop-net.org/UniPub/iHOP/) (**Figure 8**). 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), cooccurrence (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 interactome between AMD proteome was compared to the retina/

The genome regulatory network was connected to the proteome network using Uniprobe and JASPAR. Protein phosphorylations were examined by phosphoprotein/peptide enrichment, followed by mass spectrometry analysis. Phosphorylations were compared to Phospho. ELM, and PhosphoSite. The metabolome mapping was established using KEGG and BIGG

To understand how mitochondria regulate their morphology and function, we first analyzed mitochondrial morphology quantitatively in ARPE-19 cells subjected to oxidative stress conditions using a systematic computational model (**Figure 1**). Representative mitochondrial images at selected time points (0.5, 1, 8, 24 h) are shown for clear comparison **Figure 1**). We examined mitochondrial area, circularity, perimeter, content as well as cellular area to identify changes between healthy and injured mitochondria. Previously, our *in vitro* data using RPE cells demonstrated the positive correlation between apoptotic signaling and mitochondria-nucleus prohibitin shuttling. Our previous studies suggest that cellular distribution and the total volume of mitochondria could be affected by microtubules, intermediate filaments

proj/ppi/), and Pubmed database (http://www.ncbi.nlm.nih.gov/pubmed).

Database (IntAct), and STRING.

214 Mitochondrial Diseases

STRING 10.0 software (http://string-db.org/).

RPE proteome under stress conditions.

databases.

**3. Results**

and cardiolipin [24–27, 29, 30].

**Figure 1.** Quantitative analysis of mitochondrial morphology: representative images of MitoTracker Orange-labeled mitochondria from ARPE-19 cells exposed to *t*-BuOOH for 0.5–24 h or light for 1 h are shown here. A. ARPE-19 cells under oxidative stress were analyzed by immunocytochemistry using MitoTracker. B. Mitochondrial content was represented by 2D graph (radius/intensity) showing decreased size and fragmentation pattern under stress conditions. C. Mitochondria in ARPE-19 cells were presented in 3D structure using Image J software.

Our results showed that the connectivity, the number of mitochondrial branch points, and the interactive isosurfaces were altered at the contact sites between mitochondria and other organelles. Under extended oxidative stress (1, 8, 24 h) and intense light (7000 lx, 1 h), we observed a decrease in mitochondrial size, presence of fragmented filaments (red arrows), and holes on the organelle contact sites. Under intense light condition, mitochondria in ARPE-19 cells were decreased and fragmented as shown in oxidative stress (1–8 h).

Next, mitochondrial perimeter vs. circularity was examined to determine the correlation between mitochondrial morphology and oxidative stress (**Figure 2**). We hypothesized that some mitochondrial indexes that include circularity and perimeter ratio may represent mitochondrial dynamics. We calculated mitochondrial area, perimeter, minor axis, and circularity to conclude that specific mitochondrial ratio correlated positively with stress kinetics.

The average mitochondrial area/perimeter ratio normalized to the minor axis suggests that specific conditions may induce mitochondrial swelling (**Figure 3**). Time-dependent decrease of minor axis and mitochondrial area/perimeter normalized to the circularity was noticed under stress condition. Our previous proteomic study demonstrated that tubulin/vimentin depolymerization and phosphorylations increased in stressed mitochondria [25].

To understand mitochondrial dynamics in detail, mitochondrial trafficking complex was examined. Subcellular fractionation, immunoprecipitation using primary prohibitin antibody, native gel, and mass spectrometry analysis suggest that motor protein complex may determine mitochondrial dynamics and retrograde signaling under stress conditions. Molecular motor

complex contains plus-end-directed kinesin 19, myosin 9, protocadherin gA7, and prohibitin (**Figure 4**). The molecular motor/adaptor/receptor complex mediates mitochondrial dynamics. Motor proteins, including kinesin and myosin, facilitate mitochondrial trafficking along the cytoskeleton, mainly microtubules, actin polymers and intermediate filaments (**Figure 5**). Domain analysis of mitochondrial trafficking complex showed that plus-end-directed kinesin 19 (ATP and Mg2+ binding domains), myosin 9 (myosin head motor domain, SH3 domain, ATP binding domain), protocadherin gA7 (cadherin repeat and Ca2+ binding domain), and

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In order to correlate our *in vitro* findings with human pathology, we analyzed mitochondrial trafficking complex in human postmortem AMD eyes using a proteomic approach (**Figure 6**). RPE and retina tissues (central vs. peripheral) from AMD eyes and age-matching control eyes were analyzed by phosphoproteomics and mass spectrometry analysis. We observed different expression levels of prohibitin, inositol receptor, calponin, ankyrin, guanylate cyclase, and NADP ubiquinone oxidoreductase in the RPE and pyruvate kinase, PP2A, creatine kinase, PAK S/T kinase, vimentin, FES tyrosine kinase and dynamin like protein in the retina from AMD samples compared to control. Our results suggest that mitochondrial trafficking could be a significant determinant of RPE apoptosis by decreased prohibitin. Further, Ca2+, Fe2+, inositide, phosphorylation, and energy imbalance may lead to the accelerated pathogenesis

**Figure 4.** Mitochondrial Trafficking Complex *in vitro*: Retrograde vs. Anterograde Signal. Protein complex in ARPE-19 (RPE) and HRP (retina) cells were analyzed using immunoprecipitation and mass spectrometry. Under normal condition, trafficking complex including prohibitin translocalizes into mitochondria (anterograde) whereas trafficking complex moves into the nucleus (retrograde) under oxidative stress in RPE cells. The molecular motor/adaptor/receptor complex mediates mitochondrial anterograde vs. retrograde signaling. However, in the retina, retrograde signal (mitochondria to the nucleus) is dominant under normal condition, probably due to increased p53 signaling (prohibitin

prohibitin (PX domain, lipid binding pocket) exist in the trafficking complex.

toward AMD.

found in the nucleus).

**Figure 2.** Quantitative analysis of mitochondrial morphology: perimeter vs. circularity. X axis represents time of ARPE-19 cells under oxidative stress and Y axis represents mitochondrial index on perimeter vs. circularity ratio in arbitrary units. Selected time points are 0, 8, 24 h were shown for clarity [24]. Our calculation demonstrated that mitochondria under oxidative stress change their morphology to circular shape for fusion, followed by fragmentation toward greater degree of roundness and circularity. Total area of mitochondria decreased to 40–50% and both perimeter/circular mitochondria were downregulated to 60–70%. Area/perimeter normalized to circularity ratio of mitochondria was decreased to 63% (1 h oxidative stress), showing a positive correlation between mitochondrial morphology changes and apoptotic RPE.

**Figure 3.** Mitochondrial index: mitochondrial area/perimeter/minor axis vs. minor axis vs. area/perimeter/circularity. X axis represents stressed time (hrs) of ARPE-19 under oxidants and Y axis represents mitochondrial index showing mitochondrial area/perimeter/minor axis (red), compared to mitochondrial minor axis (black), and mitochondrial area/ perimeter/mitochondrial circularity (blue) in arbitrary units.

complex contains plus-end-directed kinesin 19, myosin 9, protocadherin gA7, and prohibitin (**Figure 4**). The molecular motor/adaptor/receptor complex mediates mitochondrial dynamics. Motor proteins, including kinesin and myosin, facilitate mitochondrial trafficking along the cytoskeleton, mainly microtubules, actin polymers and intermediate filaments (**Figure 5**). Domain analysis of mitochondrial trafficking complex showed that plus-end-directed kinesin 19 (ATP and Mg2+ binding domains), myosin 9 (myosin head motor domain, SH3 domain, ATP binding domain), protocadherin gA7 (cadherin repeat and Ca2+ binding domain), and prohibitin (PX domain, lipid binding pocket) exist in the trafficking complex.

In order to correlate our *in vitro* findings with human pathology, we analyzed mitochondrial trafficking complex in human postmortem AMD eyes using a proteomic approach (**Figure 6**). RPE and retina tissues (central vs. peripheral) from AMD eyes and age-matching control eyes were analyzed by phosphoproteomics and mass spectrometry analysis. We observed different expression levels of prohibitin, inositol receptor, calponin, ankyrin, guanylate cyclase, and NADP ubiquinone oxidoreductase in the RPE and pyruvate kinase, PP2A, creatine kinase, PAK S/T kinase, vimentin, FES tyrosine kinase and dynamin like protein in the retina from AMD samples compared to control. Our results suggest that mitochondrial trafficking could be a significant determinant of RPE apoptosis by decreased prohibitin. Further, Ca2+, Fe2+, inositide, phosphorylation, and energy imbalance may lead to the accelerated pathogenesis toward AMD.

**Figure 2.** Quantitative analysis of mitochondrial morphology: perimeter vs. circularity. X axis represents time of ARPE-19 cells under oxidative stress and Y axis represents mitochondrial index on perimeter vs. circularity ratio in arbitrary units. Selected time points are 0, 8, 24 h were shown for clarity [24]. Our calculation demonstrated that mitochondria under oxidative stress change their morphology to circular shape for fusion, followed by fragmentation toward greater degree of roundness and circularity. Total area of mitochondria decreased to 40–50% and both perimeter/circular mitochondria were downregulated to 60–70%. Area/perimeter normalized to circularity ratio of mitochondria was decreased to 63% (1 h oxidative stress), showing a positive correlation between mitochondrial morphology changes and apoptotic RPE.

**Figure 3.** Mitochondrial index: mitochondrial area/perimeter/minor axis vs. minor axis vs. area/perimeter/circularity. X axis represents stressed time (hrs) of ARPE-19 under oxidants and Y axis represents mitochondrial index showing mitochondrial area/perimeter/minor axis (red), compared to mitochondrial minor axis (black), and mitochondrial area/

perimeter/mitochondrial circularity (blue) in arbitrary units.

216 Mitochondrial Diseases

**Figure 4.** Mitochondrial Trafficking Complex *in vitro*: Retrograde vs. Anterograde Signal. Protein complex in ARPE-19 (RPE) and HRP (retina) cells were analyzed using immunoprecipitation and mass spectrometry. Under normal condition, trafficking complex including prohibitin translocalizes into mitochondria (anterograde) whereas trafficking complex moves into the nucleus (retrograde) under oxidative stress in RPE cells. The molecular motor/adaptor/receptor complex mediates mitochondrial anterograde vs. retrograde signaling. However, in the retina, retrograde signal (mitochondria to the nucleus) is dominant under normal condition, probably due to increased p53 signaling (prohibitin found in the nucleus).

**Figure 5.** Domain analysis of mitochondrial trafficking complex: ATP, Ca2+, and lipid-dependent signaling. Prohibitin binding proteins were analyzed using bioinformatics software and databases, including iHOP, InterPro (https://www. ebi.ac.uk/interpro/), Expasy/Prosite (https://prosite.expasy.org/), conserved domain search (https://www.ncbi.nlm.nih. gov/Structure/cdd/wrpsb.cgi), Motif (https://molbiol-tools.ca/Motifs.htm) and Pfam (http://pfam.xfam.org/). Kinesin 19 contains ATP binding domain and kinesin motor domain, whereas Myosin 9 has SH3, ATP binding, TATA binding, and myosin motor domains. Protocadherin γA7 includes repeat domain and calcium binding sequences. Prohibitin contains PX domain and the second lipid binding domain.

In order to understand AMD protein network, the new AMD interactome with oxidative biomarkers was established using phosphoproteomics data and a computational model. The current AMD interactome demonstrated that several earlier unrelated to AMD proteins, including ubiquitin, peroxiredoxin, MAP kinase, BUB 1/3, vimentin and crystalline could be involved in AMD progression, suggesting that cytoskeletal protein phosphorylation, crystalline aggregation, and mitochondrial signaling may contribute to RPE apoptosis (**Figure 8**). To confirm oxidative stress biomarkers, specific cytoskeletal protein changes were determined *in vivo* using animal model previously (C3H female mice, 7 weeks old) [25, 30, 38]. Neurofilament, vimentin, and tubulin were upregulated under 24 h constant light compared

**Figure 6.** Mitochondrial signaling in the retina and RPE *in vivo* using human AMD eyes: mitochondrial trafficking is a significant determinant of RPE apoptosis. RPE and retina tissues (central vs. peripheral) from AMD eyes and agematching control eyes were analyzed using phosphoproteomics and mass spectrometry analysis. We observed different expressions of inositol receptor, calponin, ankyrin, guanylate cyclase, and NADP ubiquinone oxidoreductase in the RPE and pyruvate kinase, PP2A, creatine kinase, PAK S/T kinase, vimentin, FES tyrosine kinase and dynamin like protein in the retina from AMD samples compared to control. Altered concentrations of mitochondrial complex, phosphoproteins,

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The current study determined the mitochondrial morphology quantitatively using a mathematic model and mitochondrial trafficking complex under stress conditions. Our data suggest that the kinesin-myosin-cadherin-prohibitin complex could be involved in anterograde mitochondrial trafficking, whereas PKUb S/T kinase-myosin-PI3K-lamin B2 bindings may regulate an energy demanding retrograde transport of mitochondria [25, 39–43]. Prohibitin binding with a trafficking protein complex may regulate the bidirectional transport of mitochondria along actin microfilaments, intermediate filaments, and microtubules. The mitochondrial trafficking complex implies that a specific mechanism of communication may exist in the ATP

to 12 h dark/12 h light condition [38].

and ATP/ADP may lead to premature senescence in RPE cells.

**3.1. Discussion**

It is proposed that specific organelles, including mitochondria, melanosome, and phagosome, may use different kinesin and myosin motors for their distribution and trafficking in the RPE [25, 31–33]. Mitochondrial trafficking could be determined by ATP, Ca2+, and lipid interactions based on their domain analysis [29, 34–38] and mitochondrial trafficking is a significant determinant of RPE apoptosis [24, 37]. Altered concentrations of mitochondrial complex, phosphoproteins, and ATP/ADP may lead to premature senescence in RPE cells [27]. Our enriched phosphoproteins and phosphopeptides analysis demonstrated that altered inositol triphosphate receptor, ankyrin, NADP reactions exist in AMD (**Figure 6**). Regulation of mitochondrial complex/lipid ratio and the energy producing machinery may enable enhanced longevity of RPE cells.

Next, a 3D surface model was used to analyze mitochondrial nodes, edges, branches, and tubular filaments (**Figure 7**). Cellular distribution and the total volume were affected by microtubules, intermediate filaments and cardiolipin. A 3D model showed that mitochondrial contact sites with endoplasmic reticulum (ER) and/or the nucleus were opened irreversibly under extended stress (24 h).

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**Figure 6.** Mitochondrial signaling in the retina and RPE *in vivo* using human AMD eyes: mitochondrial trafficking is a significant determinant of RPE apoptosis. RPE and retina tissues (central vs. peripheral) from AMD eyes and agematching control eyes were analyzed using phosphoproteomics and mass spectrometry analysis. We observed different expressions of inositol receptor, calponin, ankyrin, guanylate cyclase, and NADP ubiquinone oxidoreductase in the RPE and pyruvate kinase, PP2A, creatine kinase, PAK S/T kinase, vimentin, FES tyrosine kinase and dynamin like protein in the retina from AMD samples compared to control. Altered concentrations of mitochondrial complex, phosphoproteins, and ATP/ADP may lead to premature senescence in RPE cells.

In order to understand AMD protein network, the new AMD interactome with oxidative biomarkers was established using phosphoproteomics data and a computational model. The current AMD interactome demonstrated that several earlier unrelated to AMD proteins, including ubiquitin, peroxiredoxin, MAP kinase, BUB 1/3, vimentin and crystalline could be involved in AMD progression, suggesting that cytoskeletal protein phosphorylation, crystalline aggregation, and mitochondrial signaling may contribute to RPE apoptosis (**Figure 8**). To confirm oxidative stress biomarkers, specific cytoskeletal protein changes were determined *in vivo* using animal model previously (C3H female mice, 7 weeks old) [25, 30, 38]. Neurofilament, vimentin, and tubulin were upregulated under 24 h constant light compared to 12 h dark/12 h light condition [38].

#### **3.1. Discussion**

**Figure 5.** Domain analysis of mitochondrial trafficking complex: ATP, Ca2+, and lipid-dependent signaling. Prohibitin binding proteins were analyzed using bioinformatics software and databases, including iHOP, InterPro (https://www. ebi.ac.uk/interpro/), Expasy/Prosite (https://prosite.expasy.org/), conserved domain search (https://www.ncbi.nlm.nih. gov/Structure/cdd/wrpsb.cgi), Motif (https://molbiol-tools.ca/Motifs.htm) and Pfam (http://pfam.xfam.org/). Kinesin 19 contains ATP binding domain and kinesin motor domain, whereas Myosin 9 has SH3, ATP binding, TATA binding, and myosin motor domains. Protocadherin γA7 includes repeat domain and calcium binding sequences. Prohibitin contains

It is proposed that specific organelles, including mitochondria, melanosome, and phagosome, may use different kinesin and myosin motors for their distribution and trafficking in the RPE [25, 31–33]. Mitochondrial trafficking could be determined by ATP, Ca2+, and lipid interactions based on their domain analysis [29, 34–38] and mitochondrial trafficking is a significant determinant of RPE apoptosis [24, 37]. Altered concentrations of mitochondrial complex, phosphoproteins, and ATP/ADP may lead to premature senescence in RPE cells [27]. Our enriched phosphoproteins and phosphopeptides analysis demonstrated that altered inositol triphosphate receptor, ankyrin, NADP reactions exist in AMD (**Figure 6**). Regulation of mitochondrial complex/lipid

ratio and the energy producing machinery may enable enhanced longevity of RPE cells.

Next, a 3D surface model was used to analyze mitochondrial nodes, edges, branches, and tubular filaments (**Figure 7**). Cellular distribution and the total volume were affected by microtubules, intermediate filaments and cardiolipin. A 3D model showed that mitochondrial contact sites with endoplasmic reticulum (ER) and/or the nucleus were opened irreversibly

PX domain and the second lipid binding domain.

218 Mitochondrial Diseases

under extended stress (24 h).

The current study determined the mitochondrial morphology quantitatively using a mathematic model and mitochondrial trafficking complex under stress conditions. Our data suggest that the kinesin-myosin-cadherin-prohibitin complex could be involved in anterograde mitochondrial trafficking, whereas PKUb S/T kinase-myosin-PI3K-lamin B2 bindings may regulate an energy demanding retrograde transport of mitochondria [25, 39–43]. Prohibitin binding with a trafficking protein complex may regulate the bidirectional transport of mitochondria along actin microfilaments, intermediate filaments, and microtubules. The mitochondrial trafficking complex implies that a specific mechanism of communication may exist in the ATP

**Figure 7.** A 3D surface model and a graph representation by nodes, edges, branches, and tubular filaments: Mitochondrial filaments in three dimensions in RPE cells were calculated quantitatively. The connectivity, the number of mitochondrial branch points, and the interactive 3D visualization of isosurfaces were examined to identify the contact point between mitochondria and other organelles, including ER and the nucleus (white arrow). Mitochondria under oxidative stress (24 hrs) decreased their average perimeter (49%), average area (28%), area/perimeter (56%), and minor axis (32%).

and Ca2+ demanding regions. Mitochondrial dysfunction, altered dynamics, impaired transport, and turnover perturbation are associated with AMD.

Oxidative stress-induced apoptosis is the final cell death pathway in many irreversible ocular diseases that include AMD. While the end point of apoptosis is well established, the knowledge of early biochemical reactions and specific molecular players has been elusive. We have examined early biosignatures and mechanisms of retinal and RPE cell death under oxidative stress [44]. Our previous studies demonstrated that not only intense light but also constant moderate light and mild oxidative stress may trigger induction of anti-apoptotic Bcl-xL and erythropoietin (EPO) as well as pro-apoptotic caspases [24, 25, 27–29, 37, 38, 45–47]. We determined that protein modifications, including nitration and phosphorylation, were altered under oxidative stress possibly due to excess of NO production [26, 48–50].

The analysis of AMD interactome using proteome-genome-metabolome network suggests that there is a positive correlation between mitochondrial retrograde signaling and AMD progression. The AMD interactome suggests: (1) network-based interactions among AMD-related hub proteins that include UBC, MMP2, BCL, PRDX, ATP5O, C3, TF, and CRYAB, (2) increased local interactions between oxidative stress, complement activation, transcription, metabolism, (3) AMD module as a cluster in the same network neighborhood, (4) poten-

**Figure 8.** Genome-proteome-metabolome mapping in AMD: retrograde mitochondrial signaling. The protein interactome was established using STRING software and our proteomics data. The genome regulatory network was connected to the proteome network using Uniprobe and JASPAR. Protein phosphorylations were examined by phosphoprotein/peptide enrichment, followed by mass spectrometry analysis. Phosphorylations were compared to Phospho.ELM, and PhosphoSite. The metabolome mapping was established using KEGG and BIGG databases. Based on our proteomics and the interactome data that identified altered signaling of apoptosis in the retina and RPE both *in vitro and in vivo*, the pathological pathway determined by the AMD interactome could yield suitable targets for antiapoptotic and anti-angiogenic therapy: (1) mitochondrial dysfunction in the peripheral RPE (prohibitin, ATP synthase); (2) oxidative stress including intense and constant light (peroxiredoxin, thioredoxin, glutathione S-transferase); (3) cytoskeletal remodeling by microtubule, actin filament, and intermediate filament (vimentin, actin, tubulin); (4) high concentration of nitric oxide (nitric oxide synthase), (5) hypoxia (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)

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2−, lipids, (5) altered cytoskeleton,

tial causal molecules including Ca2+, Fe2+, ATP/ADP, OPO4

microtubule, abnormal mitochondrial signaling.

inflammation (CFH, C3, collagen, vitronectin).

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**Figure 8.** Genome-proteome-metabolome mapping in AMD: retrograde mitochondrial signaling. The protein interactome was established using STRING software and our proteomics data. The genome regulatory network was connected to the proteome network using Uniprobe and JASPAR. Protein phosphorylations were examined by phosphoprotein/peptide enrichment, followed by mass spectrometry analysis. Phosphorylations were compared to Phospho.ELM, and PhosphoSite. The metabolome mapping was established using KEGG and BIGG databases. Based on our proteomics and the interactome data that identified altered signaling of apoptosis in the retina and RPE both *in vitro and in vivo*, the pathological pathway determined by the AMD interactome could yield suitable targets for antiapoptotic and anti-angiogenic therapy: (1) mitochondrial dysfunction in the peripheral RPE (prohibitin, ATP synthase); (2) oxidative stress including intense and constant light (peroxiredoxin, thioredoxin, glutathione S-transferase); (3) cytoskeletal remodeling by microtubule, actin filament, and intermediate filament (vimentin, actin, tubulin); (4) high concentration of nitric oxide (nitric oxide synthase), (5) hypoxia (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 (CFH, C3, collagen, vitronectin).

and Ca2+ demanding regions. Mitochondrial dysfunction, altered dynamics, impaired trans-

**Figure 7.** A 3D surface model and a graph representation by nodes, edges, branches, and tubular filaments: Mitochondrial filaments in three dimensions in RPE cells were calculated quantitatively. The connectivity, the number of mitochondrial branch points, and the interactive 3D visualization of isosurfaces were examined to identify the contact point between mitochondria and other organelles, including ER and the nucleus (white arrow). Mitochondria under oxidative stress (24 hrs) decreased their average perimeter (49%), average area (28%), area/perimeter (56%), and minor axis (32%).

Oxidative stress-induced apoptosis is the final cell death pathway in many irreversible ocular diseases that include AMD. While the end point of apoptosis is well established, the knowledge of early biochemical reactions and specific molecular players has been elusive. We have examined early biosignatures and mechanisms of retinal and RPE cell death under oxidative stress [44]. Our previous studies demonstrated that not only intense light but also constant moderate light and mild oxidative stress may trigger induction of anti-apoptotic Bcl-xL and erythropoietin (EPO) as well as pro-apoptotic caspases [24, 25, 27–29, 37, 38, 45–47]. We determined that protein modifications, including nitration and phosphorylation, were altered

The analysis of AMD interactome using proteome-genome-metabolome network suggests that there is a positive correlation between mitochondrial retrograde signaling and AMD progression. The AMD interactome suggests: (1) network-based interactions among AMD-related

under oxidative stress possibly due to excess of NO production [26, 48–50].

port, and turnover perturbation are associated with AMD.

220 Mitochondrial Diseases

hub proteins that include UBC, MMP2, BCL, PRDX, ATP5O, C3, TF, and CRYAB, (2) increased local interactions between oxidative stress, complement activation, transcription, metabolism, (3) AMD module as a cluster in the same network neighborhood, (4) potential causal molecules including Ca2+, Fe2+, ATP/ADP, OPO4 2−, lipids, (5) altered cytoskeleton, microtubule, abnormal mitochondrial signaling.

The mitochondrial interactome provides a base for better understanding of oxidative stressinduced apoptosis and the mechanism of age-related diseases, including AMD. As a consequence, an effective treatment of neurodegenerative diseases based on the modulation of mitochondrial network is expected to result.

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