REGULATION AND FUNCTION OF RETINAL PHOSPHOINOSITIDES

视网膜磷脂的调节和功能

• 批准号: 10441540
• 负责人: THEODORE G WENSEL
• 金额: $ 38.8万
• 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/10441540
• 关键词: 1-Phosphatidylinositol 3-Kinase; 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; Pathway interactions; Phagocytosis; Phagosomes; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphotransferases; Phototransduction; Process; Protein Isoforms; Proteins; Publishing; Recycling; Regulation; Retina; Retinal Degeneration; Retinal Diseases; Retinal Pigments; 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.

目的是了解调节磷酸肌醇的合成，降解和定位在视网膜 和视网膜色素上皮（RPE），并了解磷酸肌醇在视网膜信号传导中的作用， 发展、健康和疾病。通过阐明膜运输等过程的分子细节， 自噬，吞噬作用，内吞作用和胞吐作用，这种理解可以帮助我们更好地了解如何 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 保持视力。具体目标是：1.中每一步之间的关系 光转导级联和PI（3）P、PI（4）P和PI（4，5）P2调节。光， 独立于一天中的时间，驱动内节磷酸肌醇水平的大量增加。 初步结果表明，驱动这种增加的信号是光转导级联的下游， 我们发表的结果证明了III类PI-3-激酶Vps 34的关键作用。目前还不清楚 光如何导致Vps 34活性的上调或什么驱动PI（4，5）P2增加。以下 假设将被测试：A.光激发视紫红质激活转导蛋白对于光驱动的 增大B。PDE 6活性对于光驱动的增加至关重要。C.环核苷酸门控通道是 对于增长至关重要。2.确定PI（4）P-5激酶活性在PIP 2（磷脂酰肌醇-磷脂酰肌醇 （4，5）二磷酸）调节和PI（4，5）P2在其中的功能作用。我们将 产生具有可诱导的杆状细胞或RPE细胞特异性敲除的主要酶的小鼠， 在神经元中合成PI（4，5）P2，PIP-5-激酶γ，并确定关于 磷酸肌醇水平、细胞形态和存活、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主要在杆中合成 和RPE通过PIP-5-激酶γ同种型的作用，使用ATP和PI（4）P作为底物; B. PI（4，5）P2 合成对于一系列膜运输功能和细胞活力是必需的。如果需要，我们还将测试 α和β同种型的全局敲除，其是可行的，以及双重和三重敲除。3.确定 LC 3募集和溶酶体融合的不同磷酸肌醇调节机制 自噬和吞噬作用。我们发现LC 3征聘的标准模式存在缺陷， 自噬体和吞噬体，因此我们将联合收割机体内实验与RPE细胞实验相结合 细胞系，以确定哪些蛋白质对RPE细胞中的LC 3募集至关重要，以及事件的顺序 导致了这两个途径中的关键事件。我们还将鉴定PI（3）P结合蛋白，其对于 LC 3向吞噬体的募集以及自噬体和巨噬细胞的溶酶体融合所必需的那些。 吞噬体