RPE Cell Bystander Effects Contribute to AMD Pathology

年龄相关性黄斑变性(AMD)是一种进展缓慢的多因素疾病，涉及基因突变。 常态和对环境的侮辱。老年性黄斑变性是60岁以上美国人失明的主要原因。 由于吸烟是氧化应激的一个主要原因，它显著增加了患AMD的风险，而且有20% 退伍军人的吸烟发生率高于普通美国平民，退伍军人管理局将不得不 为潜在的&gt；700万AMD病例提供护理。目前AMD的概念认识到慢性氧化- 积极的应激和炎症(包括补体激活)可以触发RPE的病理变化， Bruch膜(BRM)和脉络膜。开发新的AMD疗法的主要努力集中在 补体抑制剂。然而，到目前为止，补充级联中的所有目标都失败了。我们摆姿势 一个尚未考虑的并发症是细胞内补体信号的潜在问题。这个 首要目标是确定导致线粒体功能障碍的损伤信号是如何传播的 整个RPE单层，并阐明细胞内补体信号的贡献。vbl.使用 高度分化的RPE细胞我们取得了重大进展，证明了氧化应激 改变从细胞中释放的外切体中的货物，这些外切体诱导短和 收件人RPE的长期变化。重要的是，我们发现由外显子引发的长期变化 包括线粒体动态平衡的变化，并已确定了潜在的介体。同样，我们 显示氧化应激触发个体线粒体膜电位和钙的变化 受压力的细胞，然后通过缝隙连接传播到接受细胞。鉴定出一种感受性细胞。 作为一种具有高基线钙、较少负膜电位和低黑色素水平的细胞。基于 鉴于在上一个供资期间发现的新知识，我们希望在以下假设基础上加以扩展。1) 既然现在公认过度活跃的补体系统与AMD的发病率有关，我们建议研究- GATE细胞内补体信号在线粒体损伤和随后的病理中的作用。 2)因为线粒体是负责产生能量和决定生死的细胞器 在细胞中，我们希望确定外切体对向受体细胞传递信息的贡献。 有两个目标旨在回答这些问题。目的1验证细胞内钙离子的预测 和补体信号对RPE网络中的线粒体应激起作用；Aim 2验证了这一预测 胞外体含量改变细胞内环境，导致线粒体内稳态改变 RPE网络。我们将在三组具有不同功能的单元格中测试问题：ARPE-19，它可以很容易地 第二，我们将使用J和H线粒体杂交体，其中J单倍型是相关的 增加了患AMD的风险；第三，我们将用ChR1和Chr10来检查IPSC-RPE细胞中的问题 风险等位基因及其等位基因对照。这些研究将对AMD生物学有重要的意义。第一, 第二，了解细胞内补体信号在线粒体代谢中的作用。 研究内源性信号如何通过外切体传递改变线粒体内的动态平衡 RPE将对AMD治疗学的未来发展产生重要影响。

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.