RPE Cell Bystander Effects Contribute to AMD Pathology

RPE 细胞旁观者效应有助于 AMD 病理学

• 批准号: 10293593
• 负责人: Baerbel Rohrer
• 金额: --
• 依托单位: RALPH H JOHNSON VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10293593
• 关键词: Affect; Age; Age related macular degeneration; American; Biology; Blindness; Bruch' s basal membrane structure; Bystander Effect; Calcium; Caring; Cells; Cellular Stress; Cessation of life; Choroid; Choroidal Neovascularization; Chronic; Communication; Complement; Complement 3a; Complement Activation; Complement Inactivators; Complex; Complication; DNA; Development; Disease; Drusen; Elastases; Epigenetic Process; Family suidae; Funding; Future; Gap Junctions; Genetic; Goals; HDAC6 gene; Haplotypes; Homeostasis; Hybrids; Incidence; Individual; Inflammation; Knowledge; Life; Location; Mediating; Mediator of activation protein; Melanins; Membrane Potentials; Metabolic; Metabolism; Mitochondria; Mitochondrial DNA; Molecular Abnormality; Normalcy; Organelles; Oxidative Stress; Pathologic; Pathology; Population; Prevalence; Production; Receptor Signaling; Research; Retinal Photoreceptors; Risk; Role; Severities; Signal Transduction; Smoking; Stress; Structure of retinal pigment epithelium; System; Testing; Therapeutic; Veterans; base; cell injury; complement C3a receptor; complement system; design; exosome; experimental study; geographic atrophy; induced pluripotent stem cell; mitochondrial dysfunction; mitochondrial membrane; mitochondrial metabolism; monolayer; receptor; response; risk variant; uptake

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.

与年龄相关的黄斑变性（AMD）是一种缓慢进展的多因素疾病，涉及遗传 正常和环境侮辱。 AMD是60岁以上美国人失明的主要原因。 自吸烟以来，这是氧化应激的主要原因，大大增加了AMD的风险，并且有20％ 在退伍军人中吸烟的事件要比美国普通民众高的吸烟事件，VA制度将必须 为可能> 700万例AMD案件提供护理。当前的AMD概念认识到慢性氧气 压力和注射（包括补体激活）可以触发RPE的病理变化， Bruch的膜（BRM）和脉络膜。开发新AMD疗法的重大努力一直集中在 补充抑制剂。但是，迄今为止，完成级联的所有目标都失败了。我们姿势 尚未考虑的一种并发症是细胞内完成信号传导的潜在问题。这 总体目标是确定导致线粒体功能障碍的损伤信号如何传播 通过RPE单层，并阐明细胞内完成信号的贡献。使用 高度分化的RPE细胞我们取得了重大进展，表明氧化应激 改变从细胞释放的外泌体中的货物，这些外泌体影响短和 接收者RPE的长期变化。重要的是，我们发现外泌体触发的长期变化 包括线粒体稳态的变化，并确定了潜在的介体。同样，我们 表明氧化应激会触发线粒体膜电位和钙的变化 然后通过间隙连接到接受细胞的间隙连接传播的压力细胞。鉴定了一个接受单元 作为具有高基线钙的细胞，负膜电位较小和黑色素水平低。基于 在上一个资金期间确定的新知识，我们希望扩展以下假设。 1） 由于现在接受了一个过度活跃的完成系统与AMD事件有关，我们建议投资 栅极在线粒体损伤和随之而来的病理学中的细胞内完成信号传导的贡献。 2）由于线粒体是负责能源生产并做出生命/死亡决定的细胞器 在单元格中，我们希望确定外泌体对信息传递向受体细胞的贡献。 设计了两个目标来回答这些问题。 AIM 1测试细胞内钙的预测 补体信号传导导致RPE网络中的线粒体应力； AIM 2测试预测 外泌体含量改变了细胞内环境，导致线粒体体内平衡的变化 RPE网络。我们将在具有不同特征的三组单元中测试问题：ARPE-19，可以轻松 被操纵；其次，我们将使用J和H线粒体圆柱，其中J单倍型相关 随着AMD的风险增加；第三，我们将检查具有CHR1与CHR10的IPSC-RPE细胞中的问题 风险等位基因及其同源控制。这些研究将对AMD生物学具有重要意义。第一的， 关于细胞内完成信号传导在线粒体代谢中的作用的知识，其次 依从性内源信号如何通过外泌体传播如何改变线粒体稳态。 RPE，将对AMD治疗的未来发展产生重要的影响。