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（由申请人提供）：拟议的研究旨在阐明哺乳动物视网膜中不断使感光神经元的光敏感外节部分恢复活力的细胞分子机制。这种基本的视网膜过程被称为外节更新，对于终生视觉功能至关重要：实验性删除迄今为止已知的编码保守哺乳动物外节更新机制蛋白质的任何基因，都会导致光感受器功能逐渐丧失，并最终导致啮齿动物模型中的光感受器退化。此外，外节更新的遗传性畸变会导致人类患者出现色素性视网膜炎，其特征是严重的视网膜变性。最后，随着年龄的增长，外节更新效率低下，导致脂褐质在人眼 RPE 中积累，从而损害许多 RPE 功能，导致与年龄相关的黄斑变性相关的功能障碍。哺乳动物外节更新是一个同步的昼夜节律过程，涉及光感受器和邻近视网膜色素上皮（RPE）细胞的协作。杆外节远端的阴离子膜脂质磷脂酰丝氨酸 (PS) 的昼夜暴露会触发其脱落并同时被 RPE 细胞清除吞噬。迄今为止，PS暴露是唯一证实的废细胞（例如任何正在凋亡的细胞）和脱落的外节尖端的分子“吃我”信号。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很大程度上不清楚。 我们提出了两个互补但独立的具体目标，以识别分子、活动及其功能相互作用，这些分子、活动及其功能相互作用控制着哺乳动物视网膜中昼夜节律中用 PS 装饰远端杆外段尖端。采用创新的实时和固定成像以及定量生化方法将允许跟踪突变动物的整个外节更新过程，这些突变动物缺乏我们假设控制视杆 PS 暴露的候选蛋白。在具体目标 1 中，重点将放在跨膜磷脂扰乱酶 TMEM16F 的作用上，该酶定位于杆外节质膜，我们建议它控制膜不对称性以产生昼夜 PS 标记尖端。作为支持，我们显示： -TMEM16F 在光照开始时在视网膜中升高； -抑制 TMEM16F 可防止在杆尖端离体触发 PS 暴露； -缺乏TMEM16F表型的小鼠 RCS大鼠和MerTK-/-小鼠RPE吞噬作用有缺陷，外节发育正常，但RPE吞噬作用异常，随后进行性光感受器丧失。在具体目标 2 中，重点将放在分泌型碳水化合物结合蛋白半乳糖凝集素-1 的作用上，该蛋白位于视网膜下腔，我们建议它有助于细胞外侧视杆外节尖端的 PS 暴露。作为支持，我们证明： -RPE 细胞是半乳糖凝集素-1 的来源； -在光照开始时，视网膜中半乳糖凝集素-1的 PS-外化形式升高； -添加半乳糖凝集素-1触发PS在杆尖的外化； -缺乏半乳糖凝集素-1的小鼠的外节更新是异常的。