RPE Cell Bystander Effects Contribute to AMD Pathology

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Age-related macular degeneration (AMD) is a slowly progressing multifactorial disease involving genetic ab- normalities and environmental insults. AMD is the leading cause of blindness for Americans over age sixty. Since smoking, a major cause of oxidative stress, significantly increases the risk of AMD and there is a 20% higher incidence of smoking in veterans than in the general U.S. civilian population, the VA system will have to provide care for potentially >7 million AMD cases. The current concepts of AMD recognize that chronic oxida- tive stress and inflammation (including complement activation) can trigger pathological changes in RPE, Bruch’s membrane (BrM) and choroid. Major efforts to develop new AMD therapeutics have been focused on complement inhibitors. However, to date, all targets within the complement cascade have failed. We pose that one complication not yet considered is the potential issue of intracellular complement signaling. The overarching goal is to determine how damage signals leading to mitochondrial dysfunction are spread throughout the RPE monolayer, and to elucidate the contribution of intracellular complement signaling. Using highly differentiated RPE cells we have made significant progress demonstrating that oxidative stress changes the cargo in exosomes that are released from cells and that those exosomes induce short- and long-term changes in recipient RPE. Importantly, we found that long-term changes triggered by exosomes included changes in mitochondrial homeostasis and have identified potential mediators. Likewise, we showed that oxidative stress triggers mitochondrial membrane potential and calcium changes in an individual stressed cell that is then propagated through gap junctions to receptive cells. A receptive cell was identified as a cell with high baseline calcium, less negative membrane potential and low melanin levels. Based on the new knowledge identified in the previous funding period, we wish to expand on the following hypotheses. 1) As it is now accepted that an overactive complement system is tied to AMD incidence, we propose to investi- gate the contribution of intracellular complement signaling in mitochondrial damage and ensuing pathology. And 2) as mitochondria are the organelles responsible for energy production and making life/death decisions in a cell, we wish to determine the contribution of exosomes to the transfer of information to recipient cells. Two aims were designed to answers those questions. Aim 1 tests the prediction that intracellular calcium and complement signaling contribute to mitochondrial stress in the RPE network; Aim 2 tests the prediction that exosomal content alters intracellular milieu resulting in changes in mitochondrial homeostasis in the RPE network. We will test questions in three sets of cells with different features: ARPE-19, which can easily be manipulated; second, we will use J and H mitochondrial cybrids, in which the J haplotype is associated with increased risk for AMD; and third, we will examine questions in iPSC-RPE cells with Chr1 versus Chr10 risk alleles and their isogenic controls. These studies will have important implication in AMD biology. First, knowledge about the role of intracellular complement signaling in mitochondrial metabolism, and second, elu- cidating how endogenous signaling transmitted via exosomes can alter mitochondrial homeostasis within the RPE, together will have important consequences for the future development of AMD therapeutics.