Autophagic Lysosomal Pathway and Glaucoma

摘要 在常规流出道中发生的基本异常与升高的 眼内压，因此增加了患青光眼的风险，青光眼是一种与年龄有关的疾病， 全世界仍有7 000多万人下落不明。然而，几个实验室的数据， 包括我们的，支持活性氧的关键作用，既存在于房水中， 在青光眼的发病机制中，在外流途径内的正常老化过程中产生。 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， 作为一种重要的细胞内稳态机制出现，是早期保护性细胞反应的一部分 对抗氧化应激自噬活性的普遍下降已经在几种组织中观察到， 衰老和与年龄有关的疾病。一个必然的问题是自噬功能是否随着年龄的增长而下降， 流出道组织，如果是这样，这是否会导致对疾病的易感性。 我们的初步数据表明，小梁网（TM）细胞的原代培养物暴露于慢性 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）自噬泡含量增加，（3） LC 3-II水平上调，（4）溶酶体内氧化物质的积累和受损的线粒体， （5）组织蛋白酶活性降低。此外，应激培养物显示衰老相关的 半乳糖苷酶（SA-<$-gal），这是一种在青光眼供体的TM中也被上调的标志物。 我们假设外流通路的老化伴随着自噬细胞的减少， 降解能力，从而导致氧化组分的低效去除和细胞内的 非功能性异常细胞成分的积累，这降低了TM细胞的反应能力 针对自噬途径的额外应激物（即突变的肌球蛋白、色素）的免疫抑制进一步损害了细胞的免疫功能。 自噬细胞功能，从而促进自噬溶酶体分泌到细胞外空间， 这可能导致在青光眼中观察到的异常沉积。为了验证这个假设，我们将 研究（1）TM细胞的老化是否与体外和体内自噬通量的减少有关; (2)实验诱导的TM细胞自噬能力的降低是否会导致 受损蛋白质和细胞器以及细胞外基质囊泡的积累;以及（3），是否 自噬功能障碍与青光眼表型的获得相关，包括 细胞外基质囊泡，在人眼和建立的小鼠青光眼模型。

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.