Autophagic Lysosomal Pathway and Glaucoma

摘要 与抬高相关的常规流出通道中发生的基本异常 因此，发生青光眼的风险增加，这是一种与年龄相关的疾病，影响 全世界有7000多万人仍然默默无闻。然而，来自几个实验室的数据， 包括我们的，支持活性氧物种的关键作用，既存在于房水中也存在于 在青光眼的发病机制中，在流出通道内的正常衰老过程中产生。 自噬是一种溶酶体途径，负责降解长寿的蛋白质和细胞器。 作为一种重要的细胞内稳态机制出现，它是早期保护性细胞反应的一部分 对抗氧化应激。在几个组织中观察到自噬活性普遍下降 衰老和与年龄相关的疾病。一个必然的问题是，自噬功能是否会随着年龄的增长而下降 外流途径组织，如果是这样，这是否可能有助于疾病的易感性。 我们的初步数据显示，小梁网络(TM)细胞的原代培养暴露于慢性 氧化应激引起溶酶体降解途径的深刻变化，包括：(1)增加 溶酶体质量和溶酶体酶蛋白含量，(2)自噬空泡含量增加，(3) (4)溶酶体内氧化物质积聚和线粒体损伤； (5)组织蛋白酶活性降低。此外，压力培养显示出与衰老相关的升高- -半乳糖苷酶(SA-GAL)，一种在青光眼捐献者的TM中也上调的标志物。 我们假设流出通道的老化伴随着自噬功能的下降。 降解能力，从而导致氧化成分和细胞内的去除效率低下 无功能异常细胞成分的积累，这降低了TM细胞的反应能力 对抗自噬途径的额外应激源(即突变的肌红素、色素)进一步损害 自噬细胞功能，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。为了检验这一假设，我们将 研究(1)在体外和体内，TM细胞的老化是否与自噬通量的减少有关； (2)实验诱导的TM细胞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堆积；以及(3)是否 自噬功能障碍与青光眼表型的获得有关，包括 细胞外基质囊泡，在人眼和已建立的小鼠青光眼模型中。

ABSTRACT 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- ¿-galactosidase (SA-¿-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.