Autophagic Lysosomal Pathway and Glaucoma

自噬溶酶体途径与青光眼

• 批准号: 8461206
• 负责人: Paloma Liton
• 金额: $ 32.01万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8461206
• 关键词: Affect; Age; Aging; Aging-Related Process; Alzheimer' s Disease; Anterior; Applications Grants; Aqueous Humor; Atherosclerosis; Autophagocytosis; Autophagolysosome; Autophagosome; Biological; Cadaver; Cathepsins; Cell Culture Techniques; Cell physiology; Cells; Chronic; Data; Degradation Pathway; Deposition; Disease; Endocytosis; Enzymes; Excision; Extracellular Matrix; Extracellular Space; Eye; Functional disorder; Galactosidase; Garbage; Genes; Genetic; Glaucoma; Human; In Vitro; Laboratories; Life; Lysosomes; Mediating; Mitochondria; Modeling; Molecular; Monitor; Mus; Organelles; Oxidative Stress; Parkinson Disease; Pathogenesis; Pathology; Pathway interactions; Phenotype; Physiologic Intraocular Pressure; Pigments; Predisposition; Primary Open Angle Glaucoma; Proteins; Proteolysis; Rattus; Reactive Oxygen Species; Risk; Stress; Structure of sinus venosus of sclera; Testing; Tissues; Trabecular meshwork structure; Transgenic Organisms; Vacuole; Vesicle; age related; base; in vivo; mouse model; mutant; myocilin; normal aging; response; senescence; stressor; therapeutic target; tool; wasting

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.

抽象的 与升高相关的常规流出途径中发生的基本异常 眼压，因此，患青光眼的风险增加，青光眼是一种与年龄相关的疾病，影响 全世界仍有超过 7000 万人仍不清楚。 包括我们在内，支持活性氧的关键作用，它们都存在于房水和 在正常衰老过程中产生的流出途径，在青光眼的发病机制中。 自噬是一种负责长寿蛋白质和细胞器降解的溶酶体途径， 作为一种重要的细胞稳态机制出现，是早期保护性细胞反应的一部分 在一些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的一个必然问题是自噬功能是否随着年龄的增长而下降。 流出组织途径，如果是，是否会导致疾病易感性。 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 氧化应激导致溶酶体降解途径发生深刻变化，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) LC3-II 水平上调，(4) 溶酶体内氧化物质的积累和线粒体受损， (5)组织蛋白酶活性降低，此外，应激培养物显示衰老相关蛋白升高。 ¿ -半乳糖苷酶 (SA-¿-gal)，一种在青光眼供体的 TM 中也上调的标记物。 我们发现流出途径的老化伴随着自噬的下降 降解能力，从而导致氧化成分的去除效率低下以及细胞内的 非功能性异常细胞成分的积累，降低 TM 细胞的反应能力 针对自噬途径的额外应激源（即突变肌纤蛋白、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，我们将进行测试。 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有关，包括存在 细胞外基质囊泡，在人眼和已建立的小鼠青光眼模型中。