Cellular homeostasis maintains the intracellular environment against intra-, and/or extracellular stress. Autophagy, which is an important process to maintain the cellular homeostasis, degrades damaged organelles, unnecessary or dysfunctional proteins...
Cellular homeostasis maintains the intracellular environment against intra-, and/or extracellular stress. Autophagy, which is an important process to maintain the cellular homeostasis, degrades damaged organelles, unnecessary or dysfunctional proteins under cellular stress conditions such as starvation or oxidative damage. Senescence is caused by accumulation of cell damages due to loss of homeostasis under long-term exposure of stressful stimuli. Loss of homeostasis and accumulated cellular damage caused by autophagy disorder are known to lead to age-related disorders, such as Alzheimer's disease and Parkinson's disease.
Age-related macular degeneration (AMD), which causes the vision loss among people age 50 and older, is a disease that damages the macula in the central portion of the retina. Accumulation of detrimental metabolic products such as intracellular lipofuscin and extracellular drusen is possibly caused by imbalance between production of damaged cellular components and degradation. Early stage of AMD is established by atrophy delineated by retinal pigment epithelium (RPE) thinning or depigmentation that precede geographic atrophy. In advanced stages of AMD, geographic atrophy of RPE and/or development of new blood vessels (choroidal neovascularization, CNV) result in death of photoreceptors and central vision loss; the first is called dry type AMD and the second is called wet type AMD. The wet type AMD is characterized by an abnormal growth of new blood vessels that often causes leakage of blood and fluid. On the other hand, dry type AMD is characterized by accumulation of the extracellular waste products (i.e. drusen) in a layer of the retina. Because main therapeutic target for wet type AMD treatment is a vascular endothelial growth factor (VEGF), anti-VEGF agents have been used as a standard treatment for CNV. However, there is no currently proven effective treatment for dry type AMD, which accounts for nearly 90% of AMD patients. Although extensive research efforts have focused on understanding the basic of dry type AMD, pathogenesis of AMD remains to be elucidated.
In this study, I investigated the molecular pathogenesis of AMD through the maintenance of RPE under oxidative stress condition in terms of cellular homeostasis with an emphasis on roles of autophagy and intermediate cytoskeleton, Keratin 8 (KRT8). Keratin 8 has been selected as AMD pathogenesis marker protein from the previous proteomics study based on AMD patients’ aqueous humoral samples. In the first chapter, I investigated roles of autophagy on the cellular homeostasis in RPE under oxidative stress. Human RPE cells undergo mechanistic target of rapamycin (mTOR)-mediated autophagy pathway to protect RPE from apoptotic cell death under reactive oxygen species (ROS) stress conditions. Importantly, RPE cells under ROS stress condition enhances expression of KRT8 along with autophagy induction. Keratin 8 was observed to facilitate autophagosome-lysosome fusion in RPE cells under oxidative stress.
In the second chapter, role of KRT8 has been investigated on RPE homeostasis under ROS condition in terms of epithelial-mesenchymal transition (EMT). I found that phosphorylation of KRT8 induces RPE degeneration through pathologic EMT of RPE cells under oxidative stress. KRT8 is perinuclear reorganized through phosphorylation by extracellular signal-regulated kinases (ERK) under oxidative stress condition. Perinuclear reorganization of phosphorylated KRT8 leads to increase of cell migration and induction of EMT. I demonstrated that inhibition of KRT8 phosphorylation and its subsequent perinuclear reorganization by ERK inhibitor, PD98059, suppresses oxidative stress-induced cell degeneration both in vitro and in vivo.
In the last chapter, I studied a possible role of KRT8 on mitochondrial homeostasis in RPE under oxidative stress. KRT8 expression facilitates mitochondrial fission and mitophagy in RPE cells under oxidative stress, which leads to suppression of necrotic cell death. I found that mitophagy, a selective degradation process of mitochondria by autophagy, occurs sequentially after mitochondrial fission in RPE cells that express KRT8 under oxidative stress. In contrast, when mitochondrial fission does not occur properly, mitochondria were swollen to large shape (i.e. enlargement) due to accumulation of ROS stress. The cytoprotective effect of KRT8 is supposed to be achieved by mitochondrial fission and mitophagy via facilitation of the interaction between endoplasmic reticulum and mitochondria under oxidative stress condition.
Taken together, I studied homeostatic maintenance of RPE against oxidative stress with an emphasis on roles of autophagy and cytoskeletal protein KRT8. Through the doctorate study, I found three important results on molecular pathogenesis of AMD, as followings; 1) Autophagy protects retinal pigment epithelium from apoptotic cell death under oxidative stress, 2) Phosphorylated keratin 8 induces degeneration of retinal pigment epithelium through epithelial-mesenchymal transition, and 3) Keratin 8 suppresses necrotic cell death under oxidative stress through mitophagy. These findings will improve current understanding of the molecular pathology of AMD and suggest a therapeutic strategy for the treatment of AMD by preventing RPE degeneration under oxidative stress.