**Abstract**

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.

**Keywords:** protein interactome, mitochondria, phosphoproteomics, prohibitin, retrograde signaling, AMD target

#### **1. Introduction**

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) [3–8].

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 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 [9–20]. 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.
