Oxidative Damage in Retinal Pigment Epithelium---The Role Played by Mitochondria and the Protecting Effect of Mitochondria-Targeted Peptide Antioxidants

Age-related macular degeneration (AMD) is the leading cause of irreversible visual impairment in the elderly people in developed countries. Although the exact etiology remains obscure, oxidative stress from reactive oxygen species (ROS) on nretinal pigment epithelium (RPE) is proposed to play an important role in the pathogenesis of AMD. Mitochondria consume nearly 85% to 90% of a cell’s oxygen to support oxidative phosphorylation by connecting oxidized fuel to the synthesis of ATP which serves as energy source of the cells. Oxygen normally serves as the ultimate electron acceptor and is reduced to water. However, electron leak to oxygen through complexes I and III can generate superoxide anion (one kind of ROS). Therefore, mitochondria are highly probable to be exposed to ROS. Besides, mitochondrial DNA are more vulnerable than nuclear DNA to oxidative damage because they lack protective histones and have much more limited base excision repair mechanisms than they do nuclear repair mechanisms In our preliminary study, we have established an oxidative stress toxicity model in RPE with hydrogen peroxide treatment. The specific aim #1 in this proposal is to investigate the effects of oxidative stress on mitochondria in RPE cells (including mitochondrial DNAmitochondrial membrane potential, mitochondrial morphology and ATP production) and compare with the effects on nuclear DNA. It has been shown that mitochondria play an important role in cell death process. Cytochrome c released from mitochondria activates caspases which are executors of apoptosis process. On the other hand, ATP produced in mitochondria plays an critical role in determining which cell death pathway the cell would take (apoptosis when ATP is adequate; necrosis when ATP is insufficient). If oxidative stress would damage mitochondria, then it would be interesting to know the cell death mechanism(s) involved in oxidative stress-induced RPE damage (whether cytochrome c released from mitochondria on oxidative stress lead the cell to apoptosis, or inhibition of ATP production make the cell necrotic?). This would be the specific aim #2 of this proposal. Since oxidative damage was implied in the pathogenesis of AMD, many studies have focused on the efficacy of various antioxidants in preventing or retarding the progression of AMD. Contradictory results were obtained from different clinical trials. In reality, many antioxidants are poorly cell-permeable, requiring high concentrations to prevent oxidative cell death and some antioxidants may not reach the relevant sites of free radical generation. Recently, a novel class of small cell–permeable peptide antioxidants that target mitochondria in a potential-independent manner was reported recently. The structural motif of these peptides centers on alternating aromatic residues and basic amino acids (aromatic-cationic peptides). In our previous study, we found quercetin (one kind of flavonoids) is effective in protecting RPE from acridine orange-induced photosensitizing toxicity (intracellular ROS were increased in this model). In our model of acridine orange-associated photosensitizing toxicity, the EC50 for quercetin in protecting RPE from the toxicity was in the range of 25 to 30 M. With the advent of these peptide antioxidants, as mentioned above, we will investigate the effectiveness of these mitochondria-targeted peptides (e.g. D-Arg-Tyr-Lys-Phe-NH2, Tyr-D-Arg-Phe-Lys-NH2) in protecting RPE from oxidative stress.We will also compare the potency of the protecting effect of these peptides with other antioxidants, such as quercetin. This constitutes the specific aim #3 of this proposal. At the same time, we will also determine the toxic threshold concentration of these peptides on RPE cells (specific aim #4). We hope to find peptides which are protective for RPE against oxidative stress and at the same time, are also non-toxic to RPE cells. Only those peptides conforming to these requirements can be considered for clinical use in the future. While the cellular uptake and intracellular distribution of these peptide antioxidants have been studied in Caco-2 cells (human intestinal epithelium cells) and N2A cells (murine neuroblastoma cells), the cellular uptake and intracellular localization of these peptides in RPE cells are still unknown.We will investigate the cellular uptake and intracellular localization of these so called mitochondria-targeted peptides in RPE cells. Mitochondrial uptake of these peptides, if any, will also be examined. These constitute the specific aim #5 of this proposal. Some photosensitizers in RPE, such as lipofuscin, are localized in the lysosomes of aging RPE. Lipofuscin may act as a photosensitizing agent causing damage to RPE from accumulating exposure to light. It has been demonstrated that the lifetime of ROS within cells is very short; making the mean diffusion distance for ROS on the order of 14 nm [a63]. Therefore, ROS generated by photosensitization of lipofuscin would be in the vicinity of the lysosomes. To simulate the lipofuscin-associated photosensitizing toxicity in RPE, we selected acridine orange which, just like lipofuscins, is known to be localized in lysosomes as the photosensitizer in the model of photosensitizing toxicity.We have established the model of photosensitizing toxicity of RPE by acridine orange treatment followed by blue light illumination. If the peptide antioxidants are really highly concentrated in mitochondria of RPE, then, it would be interesting to know if these peptide antioxidants are effective in protecting RPE cells in the acridine orange-associated photosensitizing toxicity, in which ROS are generated mainly at the lysosomes. This will be the specific aim #6 of this proposal.We will also compare the potency of the protecting effect of these peptides with other antioxidants, such as quercetin. In our previous study on the ICG-induced RPE toxicity, we found ICG caused loss of cristae in the mitochondria and mitochondria swelling. It also resulted in depolarization of mitochondrial membrane potential. ATP production on RPE cells was reduced when exposed to ICG treatment. In addition, it has been shown that ICG inhibited mitochondrial respiration when incubated with isolated mitochondria.We speculate that the mechanism of ICG-induced RPE toxicity may involve ROS and ROS-mediated mitochondial damage. In facts, we found in our preliminary study that intracellular ROS increased when ARPE-19 cells were treated with ICG. In the next step, we are interested in knowing whether the mitochondria-targeted peptides can reduce the ICG-associated RPE toxicity. This will be the specific aim #7 of this proposal. The results of this part of study will help to judge if our hypothesis is correct or not and to elucidate more deeply the pathways involved in the ICG-induced RPE toxicity. Besides, if the results of the experiment showed that these mitochondria-targeted peptides could reduce ICG-associated RPE toxicity in non-toxic concentration, it will be a clinically promising approach to prevent the clinically observed ICG-induced RPE toxicity. As the treatment for AMD is not yet satisfactory clinically at present time, more endeavors are being made to develop approaches for preventing the initial insults that lead to disease occurrence and progression and for rescuing the RPE and photoreceptor cells that have been damaged. The study results of this proposal will help us to achieve the clinical goal of improving the prevention and management of AMD. In summary, this proposal is planed to accomplish the following specific aims in 3 years: First year. 1. Specific Aim #1. Investigation of the effect of oxidative stress on mitochondria of RPE cells (including mitochondrial DNAmitochondrial membrane potential, mitochondrial morphology and ATP production) and compare with the effects on nuclear DNA. 2. Specific aim #2. Determine the cell death mechanism(s) involved in the oxidative stress-induced RPE damage and the dose effect of oxidative stress on determination of the mechanisms. Second year. 3. Specific Aim #3. Investigation of the effectiveness of peptide antioxidants (e.g. D-Arg-Tyr-Lys-Phe-NH2, Tyr-D-Arg-Phe-Lys-NH2) in protecting RPE from oxidative stress.We will also compare the potency of the protecting effect of these peptides with other antioxidants, such as quercetin. 4. Specific Aim #4. Determine the toxic threshold concentration of theses mitochondria-targeted peptides on RPE cells. Third year. 5. Specific Aim #5. Investigating the cellular uptake and intracellular localization of the peptide antioxidants in RPE cells. Mitochondrial uptake of these peptides, if any, will also be examined. 6. Specific Aim #6. Exploring the protective effects of peptide antioxidants in RPE cells against the acridine orange-associated photosensitizing toxicity.We will also compare the potency of the protecting effect of these peptides with other antioxidants, such as quercetin. 7. Specific Aim #7. Determining whether the peptide antioxidants can reduce the ICG-associated RPE toxicity.