Molecular mechanisms of outer segment renewal

外节更新的分子机制

• 批准号: 9018962
• 负责人: SILVIA C FINNEMANN
• 金额: $ 39.65万
• 依托单位: FORDHAM UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9018962
• 关键词: Age; Age related macular degeneration; Animal Model; Animals; Apoptosis; Attenuated; Biochemical; Cell Death; Cell membrane; Cells; Circadian Rhythms; Collaborations; Data; Defect; Development; Dimerization; Distal; Eating; Ensure; Enzymes; Epithelial; Excision; Eye; Eye diseases; Frequencies; Functional disorder; Galectin 1; Genes; Health; Human; Image; Inherited; Integrins; Length; Life; Ligand Binding; Light; Lipids; Lipofuscin; Maintenance; Membrane; Membrane Lipids; Molecular; Mus; Neurons; Patients; Phagocytosis; Phenocopy; Phosphatidylserines; Phospholipids; Photoreceptors; Physiological Processes; Process; Proteins; Rattus; Recruitment Activity; Regulation; Research; Retina; Retinal; Retinal Degeneration; Retinal Pigments; Retinitis Pigmentosa; Rod Outer Segments; Rodent Model; Role; Side; Signal Transduction; Source; Specific qualifier value; Structure of retinal pigment epithelium; Techniques; Time; Vision; carbohydrate binding protein; cell type; design; extracellular; innovation; lipid transport; mutant; novel; oxidative damage; phospholipid scramblase; photoreceptor degeneration; prevent; public health relevance; quantitative imaging; receptor; response; retinal rods; uptake

 DESCRIPTION (provided by applicant): The proposed research aims to elucidate the cellular-molecular mechanisms in the mammalian retina that continuously rejuvenate the light-sensitive outer segment portions of photoreceptor neurons. This fundamental retinal process, known as outer segment renewal, is essential for life-long visual function: Experimental deletion of any gene known thus far to encode a protein of the conserved mammalian outer segment renewal machinery results in progressive loss of photoreceptor function and, eventually, photoreceptor degeneration in rodent models. Furthermore, inherited aberration in outer segment renewal causes forms of retinitis pigmentosa in human patients that are characterized by severe retinal degeneration. Finally, inefficiency of outer segment renewal with age causes lipofuscin accumulation in the RPE in the human eye, which impairs a number of RPE functions contributing to dysfunction associated with age-related macular degeneration. Mammalian outer segment renewal is a synchronized circadian process that involves collaboration of both photoreceptors and neighboring retinal pigment epithelial (RPE) cells. Diurnal exposure of the anionic membrane lipid phosphatidylserine (PS) at distal tips of rod outer segments triggers their shedding and simultaneous clearance phagocytosis by RPE cells. To date, PS exposure is the only confirmed molecular "eat-me" signal of spent cells (e.g. of any cell undergoing apoptosis) and of shed outer segment tips. Despite its universality, the molecular mechanisms triggering PS exposure are still largely obscure in any cell type. We propose two complementary but independent specific aims to identify molecules, activities and their functional interactions that control decorating distal rod outer segment tips with PS in a circadia rhythm in the mammalian retina. Employing innovative live and fixed imaging and quantitative biochemical approaches will allow tracking the entire outer segment renewal process in mutant animals that lack candidate proteins we hypothesize to govern rod PS exposure. In specific aim 1, focus will be on the role of the transmembrane phospholipid scramblase TMEM16F, which localizes to rod outer segment plasma membranes where we propose it to control membrane asymmetry to yield diurnal PS-marked tips. In support, we show: -TMEM16F is elevated in the retina at light onset; -inhibition of TMEM16F prevents ex vivo triggering of PS exposure at rod tips; -mice lacking TMEM16F phenocopy RCS rats and MerTK-/- mice defective in RPE phagocytosis, with normal outer segment development but abnormal RPE phagocytosis followed by progressive photoreceptor loss. In specific aim 2, focus will be on the role of the secreted carbohydrate binding protein galectin-1, which resides in the subretinal space where we propose it to contribute to PS exposure at rod outer segment tips from the extracellular side. In support, we show: -RPE cells are a source of galectin-1; -the PS-externalizing form of galectin-1 is elevated in the retina at light onset; -adding galectin-1 triggers PS externalizationat rod tips; -outer segment renewal in mice lacking galectin-1 is abnormal.

 描述（由适用提供）：拟议的研究旨在阐明哺乳动物视网膜中的细胞分子机制，这些机制不断使光感受器神经元的光敏感外部段部分恢复活力。这种称为外部段更新的基本视网膜过程对于终身视觉功能至关重要：迄今为止，任何已知的基因的实验缺失，用于编码保守的哺乳动物外部段更新机械的蛋白质会导致光感受器功能的逐步丧失，并最终在啮齿动物模型中的光感受器变性。此外，外部段更新中的遗传像差引起人类患者的视网膜炎形式，其特征在于严重的视网膜变性。最后，外部段更新随年龄的效率无效导致人眼中的RPE中脂肪霉素的积累，这会损害许多导致与年龄相关的黄斑变性相关的功能障碍的RPE功能。哺乳动物外节更新是一种同步的昼夜节律过程，涉及光感受器和邻近的视网膜色素上皮（RPE）细胞的合作。在杆外段的圆盘尖端上，阴离子膜脂质磷脂酰丝氨酸（PS）的昼夜暴露触发了RPE细胞的脱落和简单的清除吞噬作用。迄今为止，PS暴露是支出细胞的唯一确认的分子“饮食”信号（例如任何细胞凋亡的细胞）和棚外部段尖端。尽管具有普遍性，但在任何细胞类型中，触发PS暴露的分子机制仍然在很大程度上晦涩。我们提出了两种完整性但独立的特定目的，以识别分子，活动及其功能相互作用，以控制哺乳动物视网膜中的Circadia节奏中的PS远端外部段尖端。采用创新的实时成像和固定成像以及定量生化方法，将允许在缺乏候选蛋白的突变动物中跟踪整个外部段更新过程，我们假设控制ROD PS PS暴露。在特定的目标1中，将重点放在跨膜磷脂板酶TMEM16F的作用上，该磷脂酶TMEM16F定位于杆外段质膜，我们建议它以控制膜不对称，以产生昼夜ps ps的尖端。为了支持，我们表明：-TMEM16F在视网膜中以光发作升高； - 抑制TMEM16F可防止在杆尖端暴露的PS暴露； - 缺乏TMEM16F表观复制RCS大鼠和MERTK - / - 小鼠在RPE吞噬作用中有缺陷的小鼠，外部片段发育正常，但RPE吞噬作用异常，然后进行性光感受器丧失。在特定的目标2中，将重点放在分泌的碳水化合物结合蛋白lectectin-1的作用上，后者在视网膜下空间中居住，我们建议它为细胞外侧的杆外部段尖端的PS暴露。为了支持，我们表明：-RPE细胞是Galectin -1的来源； - lectectin-1的PS外面化形式在视网膜中以光发作升高； - 添加lectectin-1触发PS外部化杆尖； - 缺乏lectectin-1的小鼠中的段段更新是异常的。