REGULATION AND FUNCTION OF RETINAL PHOSPHOINOSITIDES

视网膜磷脂的调节和功能

• 批准号: 10653841
• 负责人: THEODORE G WENSEL
• 金额: $ 40万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10653841
• 关键词: Age related macular degeneration; Automobile Driving; Autophagocytosis; Autophagosome; Binding Proteins; Cell Line; Cell Survival; Cell membrane; Cells; Cellular Morphology; Charge; Development; Diabetic Retinopathy; Disease; Endocytosis; Enzymes; Event; Exocytosis; GTP-Binding Proteins; Goals; Health; Human; Knock-out; Light; Lipids; Lysosomes; Membrane; Modeling; Molecular; Mus; Nerve Degeneration; Neurons; PIK3CG gene; Pathway interactions; Phagocytosis; Phagosomes; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphotransferases; Phototransduction; Process; Protein Isoforms; Proteins; Publishing; Recycling; Regulation; Retina; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Rod; Role; Signal Transduction; Structure of retinal pigment epithelium; Testing; Therapeutic Intervention; Time; Transducin; Up-Regulation; Vision; cell type; cyclic-nucleotide gated ion channels; environmental stressor; experimental study; in vivo; novel therapeutic intervention; phosphatidylinositol 3-phosphate; phosphatidylinositol 4-phosphate; preservation; protein transport; recruit; response; retinal neuron; retinal rods; trafficking

The goal is to understand regulation of phosphoinositide synthesis, degradation and localization in the retina and retinal pigmented epithelium (RPE), and to understand the role of phosphoinositides in retinal signaling, development, health and disease. By illuminating molecular details of such processes as membrane trafficking, autophagy, phagocytosis, endocytosis and exocytosis, this understanding can help us better understand how these processes are disrupted in retinal disease and how therapeutic interventions could make use of them to preserve vision. The specific aims are: 1. Determine the relationship between each step in the phototransduction cascade and regulation of PI(3)P PI(4)P, and PI(4,5)P2 regulation in rods. Light, independently of time-of-day, drives massive increases in the levels of inner segment phosphoinositides. Preliminary results suggest the signal driving this increase is downstream of the phototransduction cascade, and our published results demonstrate a critical role for the Class III PI-3-kinase, Vps34. It remains unclear how light leads to upregulation of the activity of Vps34 or what drives PI(4,5)P2 increases. The following hypotheses will be tested: A. Transducin activation by photoexcited rhodopsin is essential for the light-driven increases. B. PDE6 activity is essential for light-driven increases. C. The cyclic nucleotide-gated channel is essential for the increases. 2. Determine the role of PI(4)P-5 kinase activity in PIP2 (phosphatidylinositol- (4,5)bisphosphate) regulation in the outer retina and the functional role there of PI(4,5)P2. We will generate mice with inducible rod-cell- or RPE-cell-specific knockouts of the principal enzyme responsible for synthesizing PI(4,5)P2 in neurons, PIP-5-kinaseγ, and determine the phenotype with respect to phosphoinositide levels, cell morphology and survival, protein trafficking, endocytosis, phagocytosis and autophagy. These experiments will test the following hypotheses: A. PI(4,5)P2 is primarily synthesized in rods and RPE by the action of the PIP-5-kinaseγ isoform using ATP and PI(4)P as substrates; B. PI(4,5)P2 synthesis is essential for a range of membrane trafficking functions and cell viability. If needed, we will also test the global knockouts of the α and β isoforms, which are viable, as well as double and triple. 3. Determine the distinct phosphoinositide-regulated mechanisms of LC3 recruitment and lysosome fusion in autophagy and phagocytosis. We have found deficiencies in the standard models of LC3 recruitment to autophagosomes and phagosomes, so we will combine in vivo experiments with experiments with RPE cell lines to determine which proteins are critical for LC3 recruitment in RPE cells and what sequence of events leads to this key event in both pathways. We will also identify the PI(3)P binding proteins that are essential for LC3 recruitment to phagosomes and those that are essential for lysosome fusion of both autophagosomes and phagosomes.

目的是了解肌醇磷脂在视网膜中的合成、降解和定位的调节。 和视网膜色素上皮(RPE)，并了解肌醇磷脂在视网膜信号中的作用， 发展、健康和疾病。通过阐明膜运输等过程的分子细节， 自噬、吞噬、内吞和胞吐，这种理解可以帮助我们更好地理解 这些过程在视网膜疾病中被破坏，以及治疗干预如何利用它们来 保护视力。具体目标是：1.确定各步骤之间的关系 光导级联反应及PI(3)P、PI(4)P和PI(4，5)P2的调控。灯, 独立于一天中的时间，推动内节段肌醇磷脂水平的大幅增加。 初步结果表明，驱动这种增加的信号位于光转导级联的下游， 我们发表的结果证明了III类PI-3-激酶Vps34的关键作用。目前仍不清楚 光如何导致Vps34活性上调或是什么驱动PI(4，5)P2增加。以下是 假设将被检验：a.光激发视紫红质激活的转导蛋白对于光驱动的 增加。B.PDE6的活性对于光驱动的增加是必不可少的。C.环核苷酸门控通道是 对于增长来说是必不可少的。2.确定PI(4)P-5激酶活性在PIP2(磷脂酰肌醇-2)中的作用 (4，5)二磷酸)在视网膜外的调节以及PI(4，5)P2在其中的功能作用。我们会 用可诱导的杆状细胞或RPE细胞特异性敲除负责 在神经元中合成PI(4，5)P2，PIP-5-激酶γ，并测定其表型 肌醇磷脂水平、细胞形态和存活率、蛋白质运输、内吞作用、吞噬作用和 自噬。这些实验将检验以下假设：A.Pi(4，5)P2主要是在杆状结构中合成的 和以三磷酸腺苷和PI(4)P为底物的PIP-5-激酶γ亚型的作用；B.PI(4，5)P2 合成对于一系列的膜转运功能和细胞活力是必不可少的。如果需要，我们还将测试 α和β亚型的全球敲除，这是可行的，以及双倍和三倍。3.确定 肌醇磷脂调节Lc3募集和溶酶体融合的不同机制 自噬和吞噬。我们发现立法会三级招聘的标准模式有不足之处。 自噬小体和吞噬小体，所以我们将把体内实验和RPE细胞实验结合起来 确定哪些蛋白质对RPE细胞中的LC3募集至关重要，以及什么序列的事件 在这两条道路上都会导致这一关键事件。我们还将确定PI(3)P结合蛋白，这些蛋白是 吞噬小体和自噬小体与溶酶体融合所必需的Lc3募集 吞噬小体。