Molecular mechanisms of outer segment renewal

 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持续恢复感光神经元光敏外节部分的细胞分子机制。这个基本的视网膜过程，称为外节更新，是终身视觉功能所必需的：迄今为止已知的任何编码保守的哺乳动物外节更新机制的蛋白质的基因的实验性缺失导致感光细胞功能的进行性丧失，最终导致啮齿动物模型中的感光细胞变性。此外，外段更新中的遗传性畸变在人类患者中引起以严重视网膜变性为特征的视网膜色素变性形式。最后，随着年龄的增长，外段更新的效率低下导致人眼中的RPE中的脂褐质积累，这损害了许多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，涉及光感受器和相邻的视网膜色素上皮（RPE）细胞的合作。昼夜暴露的阴离子膜脂质磷脂酰丝氨酸（PS）在杆外节的远端尖端触发他们的脱落和同时清除RPE细胞的吞噬作用。到目前为止，PS暴露是消耗细胞（例如经历细胞凋亡的任何细胞）和脱落的外节尖端的唯一确认的分子“吃我”信号。尽管其普遍性，触发PS暴露的分子机制在任何细胞类型中仍然很大程度上是模糊的。 我们提出了两个互补的，但独立的具体目标，以确定分子，活动和它们的功能相互作用，控制装饰远杆外段提示与PS在哺乳动物视网膜的昼夜节律。采用创新的活的和固定的成像和定量生化方法将允许跟踪整个外节更新过程中的突变动物，缺乏候选蛋白质，我们假设管理杆PS曝光。在具体的目标1，重点将是跨膜磷脂scramblase TMEM 16 F的作用，它定位于杆外段质膜，我们建议它控制膜的不对称性，以产生昼夜PS标记的提示。作为支持，我们展示：- TMEM 16 F在光开始时在视网膜中升高; -TMEM 16 F的抑制防止离体触发在杆尖端处的PS暴露; -缺乏TMEM 16 F表型的RCS大鼠和MerTK-/-小鼠的RPE吞噬缺陷，具有正常的外节发育，但异常的RPE吞噬，随后是进行性感光细胞损失。在具体目标2中，重点将是分泌的碳水化合物结合蛋白半乳糖凝集素-1的作用，其位于视网膜下腔，我们建议其有助于从细胞外侧在杆外段尖端处的PS暴露。作为支持，我们展示：-RPE细胞是半乳糖凝集素-1的来源; -半乳糖凝集素-1的PS外化形式在光开始时在视网膜中升高; -添加半乳糖凝集素-1触发杆尖端的PS外化; -缺乏半乳糖凝集素-1的小鼠的外节更新异常。

 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.