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: 8939474
• 负责人: Paul Sieving
• 金额: $ 11.56万
• 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/8939474
• 关键词: Address; Adolescent; Age; Age related macular degeneration; Ally; Animal Model; Animals; Biochemical; Biochemistry; Biological; Biology; Blindness; Brain; Cause of Death; Cell Death; Cell Nucleus; Cells; Ciliary Neurotrophic Factor; Clinic; Clinical; Defect; Degenerative Disorder; Development; Disease; Electron Microscopy; Electroretinography; Exhibits; Functional disorder; Gene Targeting; Gene-Modified; Genes; Genetic; Goals; Growth; Homeostasis; Human; Human Genetics; Immunohistochemistry; Inherited; Invertebrate Photoreceptors; Knockout Mice; Laboratories; Learning; Left; Length; Life; Light; Lighting; Location; Macular degeneration; Mammals; Measures; Membrane; Methods; Modeling; Molecular; Molecular Biology; Molecular Genetics; Morphogenesis; Morphology; Movement; Mus; Mutation; Neurodegenerative Disorders; Neurons; Partner in relationship; Pathway interactions; Patients; Phenotype; Photophobia; Photoreceptors; Physiological; Play; Process; Protein translocation; Proteins; Publishing; Rattus; Regulation; Reporting; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Rhodopsin; Rodent; Role; Side; Signal Transduction; Signaling Protein; Source; Stress; Structure; Structure of retinal pigment epithelium; Synapses; Targeted Research; Techniques; Testing; Therapeutic Intervention; Time; Tissues; Transgenic Organisms; Translational Research; Vision; Work; X Chromosome; X-Linked Retinoschisis; XLRS1 protein; base; behavior measurement; cell type; human disease; human male; inherited retinal degeneration; light microscopy; mouse model; neurotrophic factor; oxidative damage; postnatal; rac1 GTP-Binding Protein; receptor; relating to nervous system; repaired; response; retinal rods; small molecule; study characteristics; therapy design; therapy development; tomography; transcription factor; vector; visual information

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. The broad direction for our laboratory involves the biology of photoreceptor rescue and repair and opportunities to initiate human clinical rescue trials for RP and allied diseases based on animal studies. 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 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 these 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), ocular coherence tomography (OCT) and behavioral measurements in living animals to access retinal structure and function 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. Mechanisms of Retinal Degeneration: 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. This year we continue a molecular approach to studying retinal disease mechanisms by investigating the role Rac1 in photoreceptor plasticity and homeostasis in normal and diseased retinas using Rac1 transgenic and conditional knockout mice. Rac1 is a protein that can function as an intracellular molecular switch, which is activated by various types of membrane receptors and produce a variety of downstream biological effects in many different cell types. We use a method call conditional gene targeting to modify the gene for Rac1 to learn about its role in photoreceptors. By this method only the gene in these cells is altered, leaving the Rac1 gene in other cell types unaffected. One of the photoreceptor specific functions of Rac1 in invertebrate photoreceptors is to regulate photoreceptor morphogenesis, and in particular the photoreceptive membrane analogous to outer segments in mammals. This was discovered using conditional gene targeting to produce depletion of Rac1 or constitutive activation of Rac1 in photoreceptors. We showed that conditional knockdown of Rac1 in mouse photoreceptors protected them from cell death resulting from overexposure to light, which indicates Rac1 is involved in one form of oxidative damage in photoreceptors. This may be useful in understanding the mechanisms of some types of inherited or environmental retinal degenerations and in designing treatments. To further explore the role of Rac1 in mammalian photoreceptors, we used conditional gene targeting to make a mouse which expresses a constitutively active form of Rac1 in rod photoreceptors. This transgenic Rac1 was constructed so that its expression in photoreceptors coincided with the major outer segment protein rhodopsin, which begins about postnatal day 4. This allowed us to test its effect on postnatal development. Three lines of mice expressing different levels of this transgenic Rac1 are being studied. By 14 days of age, the amount of modified Rac1 protein in these lines is between 2 times and the level of normal protein. Results so far indicate that the modified Rac1 disturbs the development of the normal laminar structure of the photoreceptor layer and some cell nuclei were mislocalized to the layer on either side of the photoreceptor layer. In addition, the number of photoreceptors was reduced in the medium and high expressing lines by postnatal day 21, but all lines had folds and whorls in the photoreceptor layer with some cells oriented toward the inner retina rather than toward the outer margin formed by the retinal pigmented epithelial cells. The outer segment portion of the displaced cells was either absent or severely shortened. We are now investigating genetic and biochemical identity of the mislocalized cells to determine the pathways by which transgenic Rac1 altered their morphology. This will give us information about the role Rac1 in postnatal retinal layer formation and photoreceptor morphogenesis. Retinoschisnin Function in Photoreceptors: Mutations in the gene for retinoschisin protein (RS1) found on the X chromosome cause X-linked retinoschisis (XLRS). XLRS is an inherited retinal disease and is a leading cause of juvenile macular degeneration in human males. The RS1 is found primarily on the outer membrane of photoreceptor inner segments. However, the role of RS1 in photoreceptor function is not known. We showed that young mice lacking retinoschisin have a specific defect in how their photoreceptors respond to light. While their electrical response to a light flash measured with the ERG is normal, the process of light activated protein translocation in photoreceptors (the movement of proteins from one compartment of the cell to another) in response to continuous illumination is ten times less sensitive in these mice at a young age than in litter mates who have the RS1. When the mice are a few weeks older, however, the light sensitivity of translocation is near normal. Furthermore, during this period, the photoreceptor outer segments in the mice lacking RS1 grow from much shorter than normal to near normal. This suggests that the photoreceptors in these mice have a delay in their maturation. Our published report describes how these changes may be related to changes in transcription factors which determine the level of the proteins involved in photoreceptor transduction during maturation. In addition, we are finding out that RS1 may play an important role in the localization of proteins at the synaptic connection between photoreceptors and the next neuron in the chain of neurons passing visual information on to the brain. Dysfunction at this connection would help explain some of the vision loss and abnormal electrophysiological response in XLRS patients. Treating the Rs1-KO mouse model of XLRS with a vector delivering the missing gene partial restores the synaptic proteins to their normal location.

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。我们实验室的广泛方向涉及光感受器救援和修复的生物学以及基于动物研究的RP和相关疾病的人类临床救援试验的机会。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这些遗传性视网膜变性的特征（表型），分子遗传学，生理机制和可能的治疗方法。我们的实验室将光与电子显微镜，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），眼相干性断层扫描（OCT）（OCT）以及与人类使用类似于人类的视野相似的方式，以访问人类的视觉结构和功能。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。 视网膜变性的机制：视网膜神经退行性疾病的关键方面涉及结构变化，特别是对光感受器外部段（OS）的结构变化，该疾病（OS）在光感受器死亡之前导致视力丧失。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。 今年，我们通过使用RAC1转基因和有条件的敲除小鼠研究RAC1在正常和患病视网膜中的光感受器可塑性和稳态中的作用，继续研究视网膜疾病机制。 Rac1是一种可以用作细胞内分子开关的蛋白质，该蛋白质被各种类型的膜受体激活，并在许多不同的细胞类型中产生多种下游生物学作用。我们使用调用条件基因靶向的方法来修改Rac1的基因，以了解其在感光体中的作用。通过这种方法，只有这些细胞中的基因被改变，而Rac1基因在其他细胞类型中不受影响。 RAC1在无脊椎动物感光体中的感光体特异性功能之一是调节光感受器的形态发生，尤其是与哺乳动物外部片段相似的光感受器膜。这是使用条件基因靶向发现的，以产生Rac1的耗竭或Rac1在光感受器中的构成激活。我们表明，Rac1在小鼠光感受器中的有条件敲低保护它们免受过度暴露导致细胞死亡的影响，这表明Rac1在感光体中涉及一种形式的氧化损伤。这可能有助于理解某些类型的遗传或环境视网膜变性以及设计治疗方法的机制。 为了进一步探索Rac1在哺乳动物感光体中的作用，我们使用条件基因靶向制造小鼠，该小鼠在杆感光体中表达Rac1的组成型活性形式。该转基因Rac1的构建是为了使其在光感受器中的表达与主要的外部段蛋白视紫红质相吻合，该蛋白呈蛋白紫红素，该蛋白开始于产后第4天。这使我们能够测试其对产后发育的影响。正在研究表达不同水平的转基因Rac1的三条小鼠。到14天大时，这些系中修饰的RAC1蛋白的量在2倍和正常蛋白水平之间。到目前为止的结果表明，修饰的Rac1扰乱了光感受器层的正常层流结构的发展，并且某些细胞核被错误地定位在光感受器层的两侧的层上。此外，到产后21日，在培养基和高表达线上减少了感光体的数量，但是所有线条在光感受器层中均具有折叠和螺旋，其中一些细胞朝向内部视网膜，而不是朝向视网膜上皮细胞形成的外缘。不存在或严重缩短了位移细胞的外部段部分。我们现在正在研究错误定位的细胞的遗传和生化身份，以确定转基因Rac1改变其形态的途径。这将为我们提供有关Rac1在产后视网膜后层形成和光感受器形态发生中的作用的信息。 视网膜感受器中的视网膜感应功能：在X染色体上发现的视黄蛋白蛋白（RS1）的突变引起X连接的视网膜静脉（XLRS）。 XLRS是一种遗传性的视网膜疾病，是人类男性少年黄斑变性的主要原因。 RS1主要在光感受器内部段的外膜上发现。但是，RS1在光感受器功能中的作用尚不清楚。我们表明，缺乏视网膜感染的年轻小鼠在光感受器对光的反应方式方面具有特定的缺陷。尽管它们对用ERG测量的光闪光的电响应是正常的，但光感受器中光活化蛋白易位的过程（蛋白质从一个细胞的一个隔室转移到另一个小区的运动）响应连续照明的过程中，在这些小鼠中，在这些小鼠中的敏感性比有RS1的垃圾伴侣低十倍。但是，当小鼠年龄大几周时，易位的光灵敏度接近正常。 此外，在此期间，缺乏RS1的小鼠中的光感受器外段从比正常短得多到接近正常的小鼠生长。这表明这些小鼠中的感光体的成熟延迟。 我们已发表的报告描述了这些变化如何与转录因子的变化有关，这些变化决定了成熟过程中涉及光感受器转导的蛋白质水平。此外，我们发现RS1可能在蛋白质在光感受器和神经元链之间的突触连接处的定位中起重要作用，将视觉信息传递到大脑。在这种联系下功能障碍将有助于解释XLRS患者的某些视力丧失和异常电生理反应。用XLR的RS1-KO小鼠模型使用向量输送丢失基因的载体将突触蛋白恢复到其正常位置。