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.

该实验室被恰当地命名为“转化研究”，因为我们使用临床中发现的遗传性视网膜变性作为有关视网膜功能和功能障碍的线索来源，以及治疗干预研究的目标。我们实验室的大方向涉及光感受器拯救和修复的生物学，以及基于动物研究启动针对 RP 和相关疾病的人体临床拯救试验的机会。我们研究了多种人类视网膜变性疾病的小鼠和大鼠模型，以阐明视网膜神经信号缺陷和变性导致失明的机制。我们使用正常啮齿动物和经过基因改造模仿人类视网膜疾病的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。我们的实验室将光学和电子显微镜、免疫组织化学、生物化学和分子生物学技术应用于人和动物视网膜组织，以及活体动物的视网膜电图（ERG）、眼相干断层扫描（OCT）和行为测量，以了解视网膜结构其功能与临床上评估人类视力的方式类似。这些研究针对人类视网膜和黄斑变性以及与年龄相关的黄斑变性的状况。 视网膜变性的机制：视网膜神经退行性疾病的一个关键方面涉及结构变化，特别是光感受器外节（OS）的变化，这些变化先于光感受器死亡，导致视力丧失。由于在许多这些情况下，感光细胞通过进行性外节（OS）缩短而经历原发性变性，一个关键问题是外节是否可能表现出足够的结构可塑性来支持因疾病状态而缩短的 OS 的延长，以及这是否会促进感光细胞的存活。这项工作的目标是研究在光应激和遗传退化条件下调节 OS 长度的重要分子。我们专注于神经营养因子（例如 CNTF）和调节细胞骨架生长的小分子（包括 Rac1）。 今年，我们继续采用分子方法研究视网膜疾病机制，使用 Rac1 转基因和条件敲除小鼠研究 Rac1 在正常和患病视网膜的光感受器可塑性和稳态中的作用。 Rac1是一种可以充当细胞内分子开关的蛋白质，它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效应。我们使用一种称为条件基因打靶的方法来修改 Rac1 基因，以了解其在光感受器中的作用。通过这种方法，仅改变这些细胞中的基因，而其他细胞类型中的 Rac1 基因不受影响。 Rac1 在无脊椎动物光感受器中的光感受器特异性功能之一是调节光感受器形态发生，特别是类似于哺乳动物外节的光感受器膜。这是通过使用条件基因靶向来消除光感受器中 Rac1 或 Rac1 的组成型激活而发现的。我们发现，小鼠光感受器中 Rac1 的条件性敲低可以保护它们免受过度暴露于光导致的细胞死亡，这表明 Rac1 参与了光感受器中的一种形式的氧化损伤。这可能有助于了解某些类型的遗传性或环境性视网膜变性的机制以及设计治疗方法。 为了进一步探索 Rac1 在哺乳动物光感受器中的作用，我们使用条件基因打靶来制备在视杆光感受器中表达 Rac1 组成型活性形式的小鼠。这种转基因 Rac1 的构建使其在光感受器中的表达与主要外段蛋白视紫红质一致，该表达大约在出生后第 4 天开始。这使我们能够测试其对出生后发育的影响。正在研究表达不同水平的转基因 Rac1 的三​​个品系的小鼠。到 14 日龄时，这些品系中修饰的 Rac1 蛋白含量达到正常蛋白水平的 2 倍。迄今为止的结果表明，修饰的 Rac1 干扰了光感受器层正常层状结构的发育，并且一些细胞核错误定位到光感受器层两侧的层。此外，到出生后第21天，中表达系和高表达系中的光感受器数量减少，但所有系的光感受器层中都有褶皱和螺旋，其中一些细胞朝向视网膜内层而不是视网膜形成的外缘。色素上皮细胞。移位细胞的外节部分要么缺失，要么严重缩短。我们现在正在研究错误定位细胞的遗传和生化特性，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 在出生后视网膜层形成和光感受器形态发生中的作用的信息。 视网膜劈裂蛋白在光感受器中的功能：X 染色体上发现的视网膜劈裂蛋白 (RS1) 基因突变会导致 X 连锁视网膜劈裂 (XLRS)。 XLRS 是一种遗传性视网膜疾病，是人类男性青少年黄斑变性的主要原因。 RS1 主要存在于光感受器内节的外膜上。然而，RS1 在光感受器功能中的作用尚不清楚。我们发现，缺乏视网膜分裂素的年轻小鼠的光感受器对光的反应存在特定缺陷。虽然它们对用 ERG 测量的闪光的电反应是正常的，但光感受器中光激活蛋白质易位的过程（蛋白质从细胞的一个隔室移动到另一个隔室）响应连续照明的敏感度在这些细胞中要低十倍。年轻时的小鼠比携带 RS1 的同窝小鼠更年轻。然而，当小鼠长大几周后，易位的光敏感性接近正常。 此外，在此期间，缺乏RS1的小鼠的光感受器外节从比正常短得多生长到接近正常。这表明这些小鼠的光感受器成熟延迟。 我们发表的报告描述了这些变化如何与转录因子的变化相关，转录因子决定成熟过程中参与光感受器转导的蛋白质的水平。此外，我们发现 RS1 可能在光感受器和神经元链中将视觉信息传递到大脑的下一个神经元之间的突触连接处的蛋白质定位中发挥重要作用。这种连接的功能障碍有助于解释 XLRS 患者的一些视力丧失和异常电生理反应。用传递部分缺失基因的载体处理 XLRS 的 Rs1-KO 小鼠模型，可将突触蛋白恢复到正常位置。