Mechanisms of cellular clearance in the retinal pigment epithelium

视网膜色素上皮细胞清除机制

基本信息

批准号：
9769758
负责人：
Aparna Lakkaraju
金额：
$ 46.39万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9769758
关键词：
Acetylation Address Adult Affect Age related macular degeneration Autophagocytosis Autophagosome Biochemical Biogenesis Biology Blindness Cell physiology Cells Cellular Stress Complex Data Deacetylation Development Digestion Disease Drusen Elderly Electron Microscopy Engineering FRAP1 gene Functional disorder Garbage Genes Goals HDAC6 gene Health Histone Deacetylase Human Image Knowledge Lipofuscin Longevity Lysosomes Macular degeneration Membrane Metabolic Metabolism Microtubules Mitotic Modification Molecular Mus Nuclear Translocation Organelles Pathogenesis Pathway interactions Phagocytes Phagocytosis Phagosomes Pharmaceutical Preparations Phase Photoreceptors Physiological Post-Translational Protein Processing Process Proteins Publishing Regulation Research Retinal Degeneration Retinal Diseases Site Small Interfering RNA Speed Stargardt&apos s disease Structure of retinal pigment epithelium TFE3 gene Testing Time Tissues Tubulin Vision Work activating transcription factor base circadian experimental study flexibility human disease insight monolayer motor control mouse model new therapeutic target pathogen preservation prevent protein aggregate public health relevance recruit stressor therapeutic target trafficking transcription factor

项目摘要

DESCRIPTION (provided by applicant): The retinal pigment epithelium (RPE) is a highly metabolically active, post-mitotic tissue, which performs numerous functions that are indispensable for vision. A key function of the RPE is the daily phagocytosis and degradation of shed photoreceptor outer segments. These two features - high metabolism and circadian digestion of phagocytosed outer segments - place a heavy burden on cellular clearance mechanisms in the RPE. Inefficient disposal of debris by the RPE promotes the accumulation of insoluble aggregates called lipofuscin and drusen, which are implicated in the pathogenesis of macular degenerations. Autophagy is a clearance mechanism that removes damaged cellular components, pathogens and other debris. Although autophagy is an emerging central player in the health and dysfunction of the RPE, we have limited insight into how this essential catabolic function is accomplished in the RPE and how it can be therapeutically targeted in intractable diseases like age-related macular degeneration. The goal of this research is to dissect the molecular regulation and execution of autophagy in the RPE in mechanistic detail. We will use high-speed live imaging of polarized primary adult RPE monolayers, gene disruption and the Abca4-/- mouse model of Stargardt disease to test specific hypotheses regarding how autophagic, phagocytic and lysosomal pathways intersect in the RPE and how this is affected by innate stressors like lipofuscin bisretinoid accumulation. The execution of autophagy can be divided into three phases that our Aims are directed to: initiation of autophagy (Aim 1), biogenesis of autophagosomes (Aim 2) and completion of autophagy (Aim 3). Aim 1 will test the hypothesis that outer segment phagocytosis inhibits the mechanistic target of Rapamycin complex 1 (mTORC1) in the RPE to activate the transcription factors TFEB and TFE3, which induce the expression of a comprehensive network of lysosomal and autophagy genes. Aim 2 will investigate the step-wise biogenesis and maturation of autophagosomes in real-time and test the hypothesis that outer segment-containing phagosomes are recruited into nascent autophagosomes as autophagic cargo. Microtubule-based transport of autophagosomes and lysosomes is required for degradation of autophagic cargo. Aim 3 will test the hypothesis that post-translational modifications of tubulin regulate the efficiency of autophagosome/lysosome transport and thereby control autophagic flux. These experiments will answer fundamental questions about cellular clearance in the RPE by addressing how autophagic-lysosome functions are scaled to meet the burden of outer segment degradation, how autophagic and phago-lysosomal pathways interact to maintain "clean" RPE, and whether autophagy promotes or prevents RPE dysfunction. Lack of mechanistic insight has significantly hampered therapeutic targeting of autophagy in retinal degenerations. We anticipate that this research will yield crucia information regarding autophagic clearance in the RPE and aid the development of strategies to preserve RPE health and function over the human lifespan.

描述（由应用提供）：视网膜色素上皮（RPE）是一种高度代谢活跃的有线后组织，其执行许多功能对于视力是必不可少的。 RPE的关键功能是每日吞噬作用和脱落感光体外部段的降解。这两个特征 - 高代谢和吞噬外部段的昼夜节律消化 - 在RPE中放置了重燃烧的细胞清除机制。 RPE效率低下的碎屑促进了称为脂肪霉素和drusen的不溶性聚集体的积累，这些聚集体在黄斑退化的发病机理中实现。自噬是一种清晰的机制，可去除损坏的细胞成分，病原体和其他碎片。尽管自噬是RPE的健康和功能障碍的新兴中心参与者，但我们对在RPE中如何完成这种基本分解代谢功能以及如何在与年龄相关的黄斑变性（如年龄相关的黄斑变性等）中进行热靶向有限。这项研究的目的是在机械细节中剖析RPE中自噬的分子调节和执行。我们将使用两极分化的成人RPE单层，基因破坏和abca4 - / - 小鼠模型的高速实时成像来测试有关自噬，吞噬细胞和溶酶体途径在RPE中如何相交的特定假设，以及如何在诸如lipofofusccin compution comgutation computation-rpe中相互影响。自噬的执行可以分为我们的目的针对的三个阶段：自噬的启动（AIM 1），自噬体的生物发生（AIM 2）和自噬的完成（AIM 3）。 AIM 1将检验以下假设：外部段吞噬作用抑制RPE中雷帕霉素复合物1（MTORC1）的机械靶标，以激活转录因子TFEB和TFE3，从而诱导溶酶体和自噬基因的全面网络表达。 AIM 2将研究自噬体的逐步生物发生和实时的成熟，并检验以下假设：含外段的吞噬体被募集到新生的自噬体为自噬货物中。自噬体和溶酶体的基于微管的运输是自噬货物降解所必需的。 AIM 3将检验以下假设：小节蛋白调节的翻译后修饰自噬体/溶酶体传输的效率，从而控制自噬的磁通。这些实验将通过解决自噬 - 散肌体功能的缩放量表，以满足外部段降解的燃烧，自噬和phago-lysosomal途径如何保持“清洁” RPE以及自噬是否促进RPE Dysfunuction来维持“清洁” RPE，从而回答有关RPE中细胞清除率的基本问题。缺乏机械洞察力严重阻碍了自噬在视网膜变性中的治疗靶向。我们预计，这项研究将产生有关RPE中自噬清除率的Crucia信息，并有助于制定人类寿命中RPE健康和功能的策略。