Pathophysiology and Treatment of Retinal Degenerations in Animal Models

动物模型视网膜变性的病理生理学和治疗

• 批准号: 7967004
• 负责人: Paul Sieving
• 金额: $ 64.52万
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967004
• 关键词: Address; Affect; Age related macular degeneration; Animal Model; Animals; Antibodies; Biochemistry; Blindness; Cause of Death; Cells; Cessation of life; Ciliary Neurotrophic Factor; Clinic; Clinical Trials; Coupled; Data; Degenerative Disorder; Development; Disease; Electron Microscopy; Electroretinography; Elements; Embryo; Environmental Risk Factor; Epithelial; Exhibits; Eye; Functional disorder; Gene Proteins; Generations; Genes; Genetic; Goals; Growth; Growth Factor; Heart; Heat Shock Protein 27; Homeostasis; Human; Human Genetics; Immunohistochemistry; Inherited; Ion Channel; Knock-out; Knockout Mice; Laboratories; Length; Light; Location; MERTK gene; Macular degeneration; Maintenance; Mediating; Membrane; Methods; Modeling; Molecular Biology; Molecular Genetics; Mus; Mutant Strains Mice; Mutate; Mutation; NADPH Oxidase; Neural Pathways; Neurodegenerative Disorders; Neuroglia; Neurons; Opsin; Oxidative Stress; Pathway interactions; Phenotype; Photochemistry; Photoreceptors; Physiological; Play; Potassium Channel; Process; Proteins; Rattus; Regulation; Retina; Retinal; Retinal Cone; Retinal Degeneration; Retinal Diseases; Retinal Pigments; Rhodopsin; Rodent; Role; Signal Transduction; Signaling Protein; Source; Stress; Structure; Structure of retinal pigment epithelium; Surgeon; Synapses; System; Targeted Research; Techniques; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Time; Tissues; Translational Research; Vertebrate Photoreceptors; Viral Vector; Vision; Visual Acuity; Work; behavior measurement; cell type; college; disease phenotype; gene therapy; human disease; in vivo; inherited retinal degeneration; knockout animal; lens; neurotrophic factor; novel; oxidative damage; photoreceptor degeneration; promoter; protein expression; receptor; relating to nervous system; response; retinal damage; retinal rods; small molecule; study characteristics; therapy development

This laboratory is appropriately titled Translational Research, as we use inherited retinal degenerations identified in the clinic as both a source of clues about retinal function and dysfunction and a target for research in therapeutic intervention. We have studied a number of mouse and rat models of human retinal degeneration diseases to elucidate the mechanisms of retinal neural signaling deficiencies and degeneration leading to blindness. We have investigated the role played by a number of retinal in proteins in the disease phenotypes and their possible use in treatment. We use normal rodents and rodents that are genetically altered to mimic human retinal disease to study the characteristics (phenotype), molecular genetics, physiological mechanisms and possible treatments of inherited retinal degenerations. Our laboratory applies the techniques of light and electron microscopy, immunohistochemistry, biochemistry, and molecular biology to human and animal retinal tissue, as well as the electroretinogram (ERG) and behavioral measurements to access retinal function in animals in ways similar to those used to evaluate human vision in the clinic. These studies address human conditions of retinal and macular degenerations and age-related macular degeneration. Using Antibodies To Investigate Retinal Pathophysiology: We found that inherited photoreceptor degeneration in the Royal College of Surgeons (RCS) rat is accompanied by an aberrant increase in the electroretinogram (ERG) response originating in neurons at least two synapses away from degenerating photoreceptors. ERG responses in these cells depend on potassium channel activity in associated glial cells. We developed a novel method for blocking specific membrane channels, including glial potassium channels, and receptors in the retina using antibodies. Using potassium channel antibodies injected in vivo, we explored the contribution of these channels to the aberrant ERG responses of the RCS rat and implicated potassium channel activity in their generation. Simultaneously, we could localize these antibodies to particular retinal cell types and subcellular locations. This approach provides a powerful opportunity to probe mechanisms of disease states using antibodies and non-invasive ERG response recordings to probe pathophysiologic mechanisms in vivo, even during treatment. Rod And Cone Pathway Interactions In Mutant Mice Lacking Rod Function: Retinal visual function depends on rod and cone photoreceptors and associated neural pathways. We are studying the cone pathway responses in mutant mice that lack functional rods due to knockout of either of two genes: NRL, which is necessary for the development of rods, or rhodopsin, which is necessary for the development of their photoreceptive membrane and capacity to respond to light. Both have been shown to still have anatomically intact rod pathways. We have found time course and amplitude changes in the cone pathway responses during light exposure in these mutant mice that, in normal mice, have been attributed to light activation of the rod pathway. Our data indicates that rods without photoreceptive membranes constitutively activate the rod pathway, and that when rods are replaced by cones (in the NRL knockout mouse) the cones activate the normal rod pathway. These results show that (1) the rod pathway that develops in the absence of functional rods is physiologically intact, which could be important for a therapy that replaces the non functioning rods and (2) cones that develop in place of rods in the NRL knockout model signal through the rod pathway, which is a physiological demonstration of retinal plasticity, the ability to modify neuronal connections in response to developmental and disease changes. Gene Therapy with Retinal Survival Factors: In addition to therapy for retinal degenerations by replacement a specific mutated gene , this year we completed a study oof viral vector mediated over expression of endogenous retinal growth and survival factors lens epithelial derived growth factor (LEDGF) and heat shock protein 27 (HSP27) in slowing retinal degeneration in rat models. These factors could be useful as therapy for a broad range of retinal degenerations. We cloned each of these genes into viral vectors for retinal delivery into the intact rodent eye. Both agents slowed the natural course of degeneration in the RCS rat model of inherited retinal degeneration due to a mutation in the MERTK gene expressed in retinal pigment epithelial cells. We also analyzed the concomitant changes in gene and protein expression in relation to the disease state and treatment with these agents. Photoreceptor Plasticity and Homeostasis In Normal and Diseased Retina: A critical facet of retinal neurodegenerative disease involves the structural changes, particularly to the photoreceptor outer segments (OS) that precede photoreceptor death, causing loss of vision. As photoreceptor cells undergo primary degeneration through progressive outer segment (OS) shortening in many of these conditions, a critical question is whether the outer segment may exhibit sufficient structural plasticity to support elongation of OS that have been shortened by disease states and whether this would promote survival of the photoreceptor cell. The goal of the work is to investigate the molecules that are important in the regulation of OS length under light stress and genetic degenerative conditions. We are focusing on neurotrophic factors, such as CNTF, and on small molecules that regulate cytoskeletal growth, including RAC1. The Role of RAC1 In Oxidative Stress-Induced Retinal Damage: In addition to its role as a cytoskeletal element, RAC1 is a component of NADPH oxidase, which is known to cause oxidative damage in some tissues such as the heart. Some retinal degenerations caused by inherited and environmental factors, such as light, are thought to involve stress-induced oxidative damage. This year we completed a study of light-induced photoreceptor damage in Rac1 conditional knockout mice. As RAC1 knockout is embryonic lethal, we developed a conditional knockout of photoreceptor RAC1 using the Cre-loxP system coupled to the opsin promoter and then crossed these mice with a light damage sensitive strain. Mice with an estimated 50% knockdown in photoreceptor Rac1 had reduced photoreceptor death due to bright light exposure. The knockdown did not affect any other retinal function, including visual acuity, electroretinographic response and visual pigment photochemistry, or development and maintenance of retinal structure. In addition, we analyzed the levels of RAC1 and other components of NADPH oxidase and of light damage pathways during light exposure and found no difference between control and knockout animals except in membrane associated RAC1. This evidence points to Rac1 as a key component of the pathway leading to light-induced photoreceptor death through activation of NADPH oxidase. Since normal retinal function was not affect by the knockdown of RAC1, inhibition of RAC1 may be a viable therapeutic strategy for some types of retinal degeneration.

该实验室的标题为转化研究，因为我们使用诊所中确定的遗传性视网膜退化是有关视网膜功能和功能障碍的线索来源，也是治疗干预研究的目标。我们研究了许多小鼠和人类视网膜变性疾病的大鼠模型，以阐明视网膜神经信号传导缺陷的机制和变性，从而导致失明。我们已经调查了许多视网膜在蛋白质表型中的作用及其在治疗中的可能使用。我们使用对模拟人类视网膜疾病的基因改变的正常啮齿动物和啮齿动物来研究特征（表型），分子遗传学，生理机制以及遗传性视网膜变性的可能治疗方法。我们的实验室将光和电子显微镜，免疫组织化学，生物化学和分子生物学应用于人类和动物视网膜组织，以及电子图（ERG）以及行为测量以及与在临时型人类视野中相似的动物中访问视网膜功能的行为测量。这些研究涉及视网膜和黄斑变性以及与年龄相关的黄斑变性的人类条件。 使用抗体研究视网膜病理生理学：我们发现，皇家外科医生学院（RCS）大鼠的遗传感受器变性伴随着源自反应感光受感染者至少两个突触的神经元的电视图（ERG）反应的异常增加。这些细胞中的ERG反应取决于相关神经胶质细胞中的钾通道活性。我们开发了一种新的方法，用于阻止特定的膜通道，包括使用抗体的视网膜中的神经胶质钾通道和受体。使用注入体内的钾通道抗体，我们探讨了这些通道对RCS大鼠异常ERG反应的贡献，并与其一代相关的钾通道活性。同时，我们可以将这些抗体定位于特定的视网膜细胞类型和亚细胞位置。这种方法为使用抗体和非侵入性ERG反应记录探测疾病状态的机制提供了有力的机会，即使在治疗期间，也可以在体内探测病理生理机制。 缺乏杆功能的突变小鼠中的杆和锥途径相互作用：视网膜视觉功能取决于棒和锥形感光体以及相关的神经途径。我们正在研究由于两种基因中的任何一个敲除杆的敲除而缺乏功能性杆的突变小鼠中的锥途径响应，而NRL是杆的发育或Rhodopsin所必需的，这对于开发其光感受性膜和能力的能力是必要的。两者都已证明仍然具有解剖学完整的杆途径。在这些突变小鼠中，在光暴露期间，我们发现了时间过程和振幅变化，而在正常小鼠中，这些反应归因于杆途径的光激活。我们的数据表明，没有感光膜的杆组成性地激活了杆途径，并且当杆被圆锥（在NRL敲除小鼠中）取代时，锥体会激活正常的杆途径。 These results show that (1) the rod pathway that develops in the absence of functional rods is physiologically intact, which could be important for a therapy that replaces the non functioning rods and (2) cones that develop in place of rods in the NRL knockout model signal through the rod pathway, which is a physiological demonstration of retinal plasticity, the ability to modify neuronal connections in response to developmental and disease changes. 带有视网膜生存因子的基因疗法：除了通过替换特定突变基因进行视网膜变性的治疗外，今年，我们完成了一项研究的病毒载体，该研究介导了内源性视网膜生长和生存因子的镜头上皮衍生生长因子（LEDGF）和热休克蛋白27（HSP27）（HSP27）（HSP27）的镜头上皮衍生因子（HSP27）。这些因素可以作为对多种视网膜变性的治疗有用的。我们将这些基因的每个基因都克隆到病毒载体中，以使视网膜递送到完整的啮齿动物眼中。由于在视网膜色素上皮细胞中表达的MERTK基因突变，两种药物在遗传性视网膜变性的RCS大鼠模型中的自然变性降低了。我们还分析了与疾病状态相关的基因和蛋白质表达的伴随变化以及与这些药物的治疗。 正常和患病视网膜中的感光受体可塑性和体内平衡： 视网膜神经退行性疾病的关键方面涉及结构变化，特别是对感光受体死亡之前的光感受器外部段（OS），导致视力丧失。随着感光细胞通过进行性外部节段（OS）在许多情况下缩短的一级变性，一个关键的问题是，外部段是否可以表现出足够的结构可塑性，以支持疾病状态缩短的OS的延长，以及这是否会促进光感受器细胞的存活。这项工作的目的是研究在光应力和遗传退化条件下调节OS长度重要的分子。我们关注的是神经营养因子，例如CNTF，以及调节包括Rac1在内的细胞骨架生长的小分子。 Rac1在氧化应激引起的视网膜损伤中的作用：除了其作为细胞骨架元件的作用外，Rac1还是NADPH氧化酶的组成部分，已知在某些组织（例如心脏）中会造成氧化损伤。遗传和环境因素（例如光）引起的一些视网膜变性涉及压力引起的氧化损伤。今年，我们完成了一项针对Rac1有条件敲除小鼠的光诱导的光感受器损伤的研究。由于Rac1敲除具有胚胎致命，我们使用与Opsin启动子耦合的CRE-LoxP系统开发了有条件的光感受器Rac1敲除，然后用轻损伤敏感性菌株越过了这些小鼠。估计光感受器RAC1估计敲低的小鼠由于明亮的光照暴露而减少了光感受器死亡。敲低不影响任何其他视网膜功能，包括视敏度，电子图像响应和视觉色素光化学或视网膜结构的开发和维护。此外，我们分析了在光照暴露期间NADPH氧化酶的Rac1和其他成分的水平以及光损伤途径的水平，除了在膜相关的Rac1中，对照动物和基因敲除动物之间没有差异。该证据表明Rac1是通过激活NADPH氧化酶导致光诱导的光感受器死亡的途径的关键组成部分。 由于RAC1的敲低不影响正常的视网膜功能，因此Rac1的抑制可能是某些类型的视网膜变性的可行治疗策略。