Ultrastructural Analysis of a Form of Macular Degeneration - Macular Telangiectasia

项目概要/摘要： 关于人类黄斑/中央凹内的细胞相互作用知之甚少。更是如此 视网膜神经胶质细胞和视网膜神经元之间的相互作用-相互作用可能对理解 视网膜退行性疾病，包括黄斑毛细血管扩张症（MacTel），一种年龄相关性黄斑变性的形式， 在AMD中，已经提出Müller神经胶质细胞中的缺陷可能起作用。米勒 神经胶质细胞依赖于调节离子平衡和神经传递，维持代谢停滞， 血视网膜屏障，以及其他多种重要的神经保护功能。越来越多的关注 自从视网膜神经元（如神经细胞）依赖于基于Müller细胞的L-丝氨酸生物合成以来， 整个CNS的神经元）缺乏限速生物合成酶PHGDH。L-丝氨酸的合成是 对脂质代谢和维持线粒体功能至关重要。不仅代谢组学研究 在MacTel的发展中涉及L-丝氨酸代谢途径的改变， 关联/GWAS研究已经将PHGDH基因的改变与早发性MacTel联系起来。不 令人惊讶的是，神经元和神经胶质细胞之间的正常关系的改变在神经元和神经胶质细胞之间的关系中具有显著的特征。 维持正常的视网膜功能，并与多种形式的视网膜变性的病因学有关。 近年来，已经开发了电子显微镜（EM）技术， 可以重建视网膜组织的碎片，直到膜水平。这就是所谓的连接组学 可以在磁带上切割和收集数千个连续切片，并对切片的特定区域进行成像 使用具有多个光束的EM，允许同时获得61个图像。软件方法 用于将图像对准到单个3D体积中的方法也已经被开发出来。迄今为止， 这种方法还没有被应用于了解病理变化的基础， 视网膜变性疾病我们目前建议的第一个目标是确定结构 MacTel中涉及的神经胶质-神经元关系和线粒体健康的特征/变化。这些研究 将特别关注线粒体的嵴结构，线粒体降解，以及 Müller细胞与感光轴突和突触末梢之间的连接。我们取得了重大进展 在使通常冗长的连接组学工作流程更有效和更适合于分析 神经退行性疾病使用我们有针对性的高通量连接组学方法，我们的第二个目标是 对我们79岁的基因相似的供体眼睛和其他视网膜进行平行分析， 从我们与48岁捐赠者的眼睛合作的经验中提炼出来。总之，我们相信， 有针对性的高通量连接组学方法将使我们能够有效地提取相关的超微结构数据 来自多个患病和对照视网膜样本。在未来，有能力进行有效的大规模 超微结构的研究将提供一个途径，以了解视网膜变性疾病的细胞基础。

Project Summary/Abstract: Little is known about the cellular interactions within the human macula/fovea. This is especially true for interactions between retinal glial cells and retinal neurons – interactions likely important to understanding retinal degenerative diseases including Macular Telangiectasia (MacTel), a form of age-related macular degeneration (AMD) in which it has been proposed that a defect in the Müller glial cells may be at play. Müller glia cells are relied upon to regulate ionic balance and neurotransmission, maintain metabolic stasis, constitute the blood-retinal barrier, among multiple other essential neuroprotective functions. An increasing focus has been on the dependence of retinal neurons on Müller cell based L-serine biosynthesis since retinal neurons (as do neurons throughout the CNS) lack the rate limiting biosynthetic enzyme PHGDH. L-serine synthesis is essential for lipid metabolism and maintenance of mitochondrial function. Not only have metabolomics studies implicated alterations in the L-serine metabolic pathway in the development of MacTel but genome-wide association/GWAS studies have linked alterations in the PHGDH gene with early onset MacTel. Not surprisingly, alterations in the normal relationships between neurons and glial cells feature prominently in maintaining normal retinal function and are implicated in the etiology of multiple forms of retinal degeneration. In recent years, electron microscopic (EM) techniques have been developed such that it is now possible to reconstruct pieces of retinal tissue down to the membrane level. Termed connectomics, it is possible to cut and collect on tape, thousands of serial sections, and image specific regions of the sections with an EM that has multiple beams, allowing for 61 images to be obtained simultaneously. Software methods for aligning the images into a single 3D volume have also been developed. To date, this connectomics approach has not been applied to gaining an understanding of pathological changes that underlie retinal degenerative diseases. The first aim in our current proposal is to determine the structural features/changes in glial-neuronal relationships and mitochondrial health involved in MacTel. These studies will focus especially on the cristae structure of mitochondria, mitochondrial degradation, and the relationship between Müller cells and the photoreceptor axons and synaptic terminals. We have made significant progress in making the typically lengthy connectomics workflow more efficient and tailored to analyzing the basis of a neurodegenerative disease. Using our targeted high-throughput connectomics approach our second aim is to perform parallel analysis of our genetically similar 79-year-old donor eye and other retinas using methods refined from our experience working with our 48-year-old donor eye. In summary, we are confident that our targeted high-throughput connectomics approach will enable us to efficiently extract relevant ultrastructural data from multiple diseased and control retinal samples. In future, having the ability to perform efficient large-scale ultrastructural studies will provide a pathway to understand the cellular basis of retinal degenerative diseases.