Autophagic Lysosomal Pathway and Glaucoma

自噬溶酶体途径与青光眼

• 批准号: 7862236
• 负责人: Paloma Liton
• 金额: $ 19.5万
• 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/7862236
• 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; Role; 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; public health relevance; response; senescence; stressor; therapeutic target; tool; wasting

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

描述（由申请人提供）：传统流出途径中发生的与眼内压升高相关的基本异常，因此导致患青光眼的风险增加，青光眼是一种影响全世界超过 7000 万人的年龄相关疾病，但仍然不清楚。然而，包括我们在内的多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，活性氧既存在于房水中，也存在于正常衰老过程中的流出途径中产生。 自噬是一种负责长寿蛋白质和细胞器降解的溶酶体途径，已成为一种重要的细胞稳态机制，是针对氧化应激的早期保护性细胞反应的一部分。在一些衰老组织和与年龄相关的疾病中，观察到自噬活性普遍下降。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。 我们的初步数据表明，小梁网（TM）细胞原代培养物暴露于慢性氧化应激会导致溶酶体降解途径发生深刻变化，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）溶酶体内积累 氧化物质和线粒体受损，(5) 组织蛋白酶活性降低。此外，应激培养物显示衰老相关 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去除效率低下以及非功能性异常细胞成分在细胞内的积累，从而降低了TM细胞对自噬途径的额外应激源（即突变肌纤蛋白、色素）的反应能力，进一步损害了自噬 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）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质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。 公共健康相关性：本拨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器故障有关，从而导致所谓的“生物垃圾”的积累。我们认为，与其他与年龄相关的疾病（包括阿尔茨海默病、帕金森病和动脉粥样硬化）中所描述的类似，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）的病理学。了解流出途径中降解能力随老化而下降的机制可能为 POAG 的治疗靶点策略开辟新途径