Autophagic Lysosomal Pathway and Glaucoma

描述（由申请人提供）：在常规流出通道中发生的与眼压升高相关的根本异常，从而增加了发生青光眼的风险，青光眼是一种与年龄相关的疾病，全世界有超过7000万人受到影响，但目前仍不清楚。然而，包括我们在内的几个实验室的数据支持活性氧在青光眼发病机制中的关键作用，这些活性氧既存在于房水中，也在流出通道的正常衰老过程中产生。自噬是一种负责降解长寿命蛋白质和细胞器的溶酶体途径，已成为一种重要的细胞稳态机制，是抗氧化应激早期保护性细胞反应的一部分。自噬活性普遍下降，已观察到在一些组织与衰老和年龄相关的疾病。一个必然的问题是，在流出通道组织中，自噬功能是否会随着年龄的增长而下降，如果是这样，这是否会导致疾病的易感性。我们的初步数据表明，原代培养的小梁网（TM）细胞暴露于慢性氧化应激下，会导致溶酶体降解途径发生深刻的变化，包括：(1)溶酶体质量和溶酶体酶蛋白含量增加，(2)自噬空泡含量增加，(3)LC3-II水平上调，(4)溶酶体内氧化物质积累和线粒体损伤，(5)组织蛋白酶活性降低。此外，应激培养显示衰老相关的-2-半乳糖苷酶（SA-2-gal）升高，该标记物在青光眼供体的TM中也被发现上调。我们假设流出通路的老化伴随着自噬降解能力的下降，从而导致氧化成分的去除效率低下，导致细胞内非功能性异常细胞成分的积累，从而降低了TM细胞对自噬通路附加应激源（即突变心肌、色素）的反应能力，进一步损害了自噬细胞的功能。从而促进自噬溶酶体分泌到细胞外空间，这可能导致青光眼中观察到的异常沉积。为了验证这一假设，我们将研究(1)TM细胞的衰老是否与体外和体内自噬通量的减少有关；(2)实验诱导的TM细胞自噬能力下降是否导致受损蛋白和细胞器以及细胞外基质囊泡的积累；(3)在人眼和已建立的小鼠青光眼模型中，自噬功能障碍是否与青光眼表型的获得有关，包括细胞外基质囊泡的存在。

DESCRIPTION (provided by applicant): The fundamental abnormality occurring in the conventional outflow pathway associated with elevated intraocular pressure and therefore, increased risk of developing glaucoma, an age-related disease affecting more than 70 million people world wide still remains obscure. However, data from several laboratories, including ours, support a key role of reactive oxygen species, both present in the aqueous humor as well as generated during the normal aging process within the outflow pathway, in the pathogenesis of glaucoma. Autophagy, a lysosomal pathway responsible for the degradation of long-lived proteins and organelles, has emerged as an important cellular homeostatic mechanism that is part of the early protective cellular response against oxidative stress. A general decline in autophagic activity has been observed in several tissues with aging and in age-related disorders. A corollary question is whether autophagy function declines with age in the outflow pathway tissue, and if so, whether this could contribute to the susceptibility to disease. Our preliminary data show that exposure of primary cultures of trabecular meshwork (TM) cells to chronic oxidative stress causes profound changes in the lysosomal degradative pathway, including: (1) Increased lysosomal mass and lysosomal enzymes protein content, (2) increased autophagic vacuoles content, (3) upregulated levels of LC3-II, (4) accumulation of intralysosomal oxidized material and damaged mitochondria, and (5) decreased cathepsin activities. In addition, stressed cultures showed elevated senescence-associated- 2-galactosidase (SA-2-gal), a marker found to be also upregulated in the TM from glaucoma donors. We hypothesize that aging of the outflow pathway is accompanied by a decline in the autophagic degradative capacity, thus leading to the inefficient removal of oxidized components and to the intracellular accumulation of nonfunctional aberrant cellular components, which reduce the ability of TM cells to respond against additional stressors of the autophagic pathway (i.e. mutant myocilin, pigment) further compromises the autophagic cellular function, thus promoting the secretion of autophagolysosomes into the extracellular space, which can contribute to the abnormal deposition observed in glaucoma. To test this hypothesis, we will investigate (1) whether aging of TM cells is associated with a decrease in autophagic flux in vitro and in vivo; (2), whether the experimentally-induced decreased in autophagic capacity in TM cells results in the accumulation of damaged proteins and organelles, as well as extracellular matrix vesicles; and (3), whether autophagy dysfunction is associated with the acquisition of a glaucoma phenotype, including the presence of extracellular matrix vesicles, in human eyes and in established mice glaucoma models. PUBLIC HEALTH RELEVANCE: The objective of this grant proposal is to investigate whether aging of the outflow pathway tissue is associated with malfunction of the cellular machinery responsible for the degradation of waste material, thus resulting in accumulation of, so called, "biological garbage". We believe that, similar to what has been described in other age-related diseases, including Alzheimer's disease, Parkinson's disease, and atherosclerosis, the accumulation of that waste material may negatively affect other essential cellular functions and thus contribute to the pathology of Primary Open Angle Glaucoma (POAG). Understanding the mechanisms underlying a decline in the degradative capacity with aging in the outflow pathway might open new avenues for therapeutic target strategies for POAG