Autophagic Lysosomal Pathway and Glaucoma

描述(申请人提供)：传统流出途径中发生的基本异常与眼压升高相关，因此发展为青光眼的风险增加。青光眼是一种影响全球超过7000万人的与年龄相关的疾病，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，在青光眼的发病机制中，存在于房水中的以及在流出通道内正常衰老过程中产生的活性氧物种在青光眼发病中起着关键作用。自噬是一种溶酶体途径，负责降解长寿命的蛋白质和细胞器，已成为一种重要的细胞内稳态机制，是早期保护细胞免受氧化应激反应的一部分。随着年龄的增长和与年龄相关的疾病，在几个组织中观察到自噬活性的普遍下降。一个必然的问题是，流出途径组织中的自噬功能是否会随着年龄的增长而下降，如果是的话，这是否会导致疾病的易感性。我们的初步数据显示，小梁网(TM)细胞的原代培养暴露在慢性氧化应激下，导致溶酶体降解途径的深刻变化，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受损，以及(5)组织蛋白酶活性降低。此外，压力培养显示衰老相关2-半乳糖苷酶(SA-2-GAL)升高，这一标记在青光眼捐赠者的TM中也被发现上调。我们假设流出途径的老化伴随着自噬降解能力的下降，从而导致氧化成分的低效去除和细胞内无功能的异常细胞成分的积累，这降低了TM细胞对自噬途径的额外应激源(即突变的肌球蛋白、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，这可能是青光眼观察到的异常沉积的原因。为了验证这一假说，我们将调查(1)TM细胞的衰老是否与体内外自噬通量的减少有关；(2)在实验中，TM细胞的自噬能力降低是否导致受损的蛋白质和细胞器以及细胞外基质小泡的积累；以及(3)在人眼和已建立的青光眼模型中，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。 与公共健康相关：这项赠款提案的目的是调查流出途径组织的老化是否与负责废物降解的细胞机械故障有关，从而导致所谓的“生物垃圾”的堆积。我们认为，与阿尔茨海默病、帕金森氏病和动脉粥样硬化等其他年龄相关疾病的描述类似，这些废物的积累可能会对其他基本细胞功能产生负面影响，从而导致原发性开角型青光眼(POAG)的病理。了解外流通路中降解能力随年龄增长而下降的机制可能会为POAG的治疗靶点策略开辟新的途径

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