Peroxiredoxin 6 and Cataractogenesis

描述(申请人提供)：最近的证据表明，氧化应激在老年性白内障的病因中发挥了作用，并表明老化的晶状体细胞由于抗氧化剂表达减少而容易受到这种损害。由于眼球晶状体不断暴露在环境压力下，它会不断产生活性氧(ROS)，如果不清除，细胞微环境中ROS的形成和局部积累的增加会引发广泛的有害信号，从而导致晶状体细胞损伤，如果这种情况持续下去，可能会导致白内障的发生。过氧化还蛋白6(PRDX6)是一种兼有GSH过氧化物酶和aiPLA2(酸性钙非依赖性磷脂酶A2)活性的兼职蛋白，在晶状体中高度表达。我们的研究表明，在衰老过程中，晶状体细胞中PRDX6的表达减少，细胞内ROS水平升高。利用Prdx6基因在小鼠体内的靶向失活，我们发现Prdx6缺失的晶状体和/或晶状体上皮细胞(LECs)含有高水平的ROS和生物活性的TGF21；表现出表型变化，TGF21诱导的基因如1-SM-肌动蛋白和2ig-h3过度调节，这些基因参与了白内障的病理生理学。我们设想了一个恶性的前馈过程(ROS？？)TGF？1？？ROS=基因过度调节？？)发生在老化的LECs或面临氧化应激的LECs的局部微环境中，因此，我们假设通过阻断ROS介导的有害信号应该通过阻断细胞微环境中局部高水平的ROS和激活的TGF21启动的恶性循环来减少白内障的进展。我们认为这些事件是因果相关的，即环境应激和晶状体组织中与年龄相关的PRDX6的减少导致了ROS诱导的膜或胞浆因子的损伤，结果是这种损伤导致了细胞内平衡系统的破坏。因此，这项建议的总体目标是揭示氧化应激在白内障形成的病理生理学中的作用，并通过三个具体目的显示PRDX6在治疗/延缓白内障发生中的能力：1)了解PRDX6的功能意义及其在氧化应激和衰老过程中的调节作用。2)通过使用TAT-HA-PRDX6携带PRDX6蛋白，在Prdx6/-耗竭小鼠、百草枯诱导的氧化应激小鼠以及Shumiya白内障大鼠(SCR)中评估PRDX6在体内外保护面临氧化应激的细胞的抗氧化潜力，并评估PRDX6是否减缓白内障的进展。3)研究PrDX6在正常和老化的晶状体上皮细胞及氧化应激下的调控机制，明确其下游氧化还原信号在调控其基因转录中的作用(S)。这些研究将为氧化应激在白内障形成中的作用提供新的见解，并将为合理使用以抗氧化剂为基础的治疗药物治疗或预防/延缓白内障的发生提供基础。老年性白内障(ARC)是一种常见的眼病，是导致失明的主要原因之一。尽管有证据表明，活性氧物种驱动的氧化应激在包括ARC在内的年龄相关退行性疾病的进展和病因中发挥了作用，但氧化应激诱导的有害信号的机制尚不清楚，这是导致疾病状态的细胞损伤的原因。因此，抗氧化剂疗法或联合疗法的临床应用充其量也是模棱两可的。利用眼晶状体作为衰老相关疾病的模型，我们将揭示氧化应激或衰老过程中组织/器官病理生理学的潜在机制。拟议的研究将为开发以抗氧化剂为基础的疗法或综合疗法提供可靠的科学基础，以预防白内障形成和与年龄相关的退行性疾病。

DESCRIPTION (provided by applicant): Recent evidence implicates that the oxidative stress plays a role in the etiology of Age-Related Cataract, and suggests that aged lens cells are prone to this damage due to reduced expression of antioxidants. Because ocular lens is constantly exposed to environmental stress, it continuously generates reactive oxygen species (ROS), if not removed, increased formation and local accumulation of ROS in the cellular microenvironment causes lens cell damage-by initiating wide-spectrum of deleterious signaling and if this situation prolongs may lead to cataractogenesis. Peroxiredoxin 6 (PRDX6), a 'moonlighting protein' with both GSH peroxidase and aiPLA2 (acidic Ca2+independent phospholipase A2) activities, is highly expressed in lens. Our studies have shown diminution of PRDX6 expression and higher intracellular ROS levels in lens cells during aging. Using targeted inactivation of Prdx6 gene in mice, we found that Prdx6-depleted lenses and/or lens epithelial cells (LECs) contain elevated levels of ROS and bio-activeTGF21; exhibit phenotypic changes with overmodulation of TGF21 inducible genes such as 1-SM-actin and 2ig-h3, and these genes are implicated in pathophysiology of cataractogenesis. We envisage a vicious feed-forward-process (ROS?? TGF?1??ROS = overmodulation of genes??) taking place within the local microenvironment of aging LECs or LECs facing oxidative stress, and therefore, we hypothesized that by blocking ROS mediated deleterious signaling should reduce progression of cataractogenesis, by interrupting the vicious cycle initiated by locally high levels of ROS and activated TGF21 within cellular microenvironment. We believe that these events are causally related, i.e., that the environmental stress and age-related reduction in PRDX6 in lens tissues leads to ROS-induced damage of membrane or cytosolic factors, as a consequence of this damage, cell homeostatic system fails. The over all goal of this proposal, therefore, is to unveil the roles of oxidative stress in pathophisiology of cataract formation and to show PRDX6 ability in treating/delaying cataractogenesis through three specific aims: 1) Understand the functional significance of PRDX6 and its regulatory role during oxidative stress and aging. 2) Assess the antioxidant potential of PRDX6 in protecting cells facing oxidative stress in vitro and in vivo using TAT-HA- PRDX6 to cargoing PRDX6 protein in Prdx6-/- depleted mice and mice with Paraquat-induced oxidative stress as well as Shumiya cataract rat (SCR), and assess whether cataract progression is slowed by PRDX6. 3) Investigate the regulatory mechanisms of PRDX6 in normal and aging LECs and cells under oxidative stress and define role(s) of downstream redox signaling in controlling its gene transcription. These studies should provide novel insights into the role of oxidative stress in cataract formation and will provide a foundation for rational use of antioxidant based therapeutics for treating or preventing/delaying cataractogenesis. A common disorder of the eye, Age-Related Cataract (ARC) is among the leading causes of blindness. Although evidence suggests a role for reactive oxygen species-driven oxidative stress in the progression and etiology of age-related degenerative diseases including ARC, the mechanism of oxidative stress-induced deleterious signaling, a cause of cellular damage that leads to the disease state, is not clear. Thus clinical application of antioxidant therapy or combination of therapies has been at best equivocal. Using eye lens as a model for age-associated disorders, we will unveil the underlying mechanism involved in the pathophysiology of tissues/organs during oxidative stress or aging. The proposed studies will provide a sound scientific basis for developing an antioxidant-based therapy or combination of therapies for preventing cataractogenesis and age-associated degenerative diseases in general.