Ultrastructural Analysis of a Form of Macular Degeneration - Macular Telangiectasia

项目摘要/摘要： 人们对人类黄斑/中心凹内的细胞相互作用知之甚少。这一点对于 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)，这是一种年龄相关性黄斑 退行性变(AMD)，据推测，Müler神经胶质细胞的缺陷可能在其中起作用。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，构成 血-视网膜屏障，以及其他多种基本的神经保护功能。越来越多的关注已经 自视网膜神经元(AS)以来，视网膜神经元对Müler细胞L丝氨酸生物合成的依赖 中枢神经系统中的DO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是必不可少的。不仅代谢组学研究 L-丝氨酸代谢途径在斑点猴全基因组发育过程中的变化 协会/Gwas的研究已经将PHGDH基因的变化与早发性Mactel联系起来。不 令人惊讶的是，神经元和神经胶质细胞之间正常关系的改变在 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有关。 近年来，电子显微镜(EM)技术已经发展到现在 有可能将视网膜组织的碎片重建到膜水平。被称为连接学，它是 可以在磁带上剪切和收集数以千计的连续部分，并对部分的特定区域进行图像处理 使用具有多个波束的EM，允许同时获得61个图像。软件方法 用于将图像对准成单个3D体积的技术也已开发出来。到目前为止，这种连接 这种方法还没有被用来了解其背后的病理变化 视网膜退行性疾病。我们目前提案的第一个目标是确定结构 与Mactel有关的神经胶质-神经元关系和线粒体健康的特征/变化。这些研究 将重点放在线粒体脊结构、线粒体退化及其关系上 Müler细胞与光感受器轴突和突触终末之间。我们已经取得了重大进展 为了使通常冗长的连接工作流更有效率，并为分析 神经退行性疾病。使用我们的目标高吞吐量连接方法，我们的第二个目标是 对我们基因相似的79岁供体眼和其他视网膜进行平行分析 从我们与48岁的捐献者眼睛的工作经验中提炼出来的。总而言之，我们相信我们的 定向高通量连接组学方法将使我们能够高效地提取相关的超微结构数据 从多种疾病的视网膜样本和对照视网膜样本中提取。在未来，有能力进行高效的大规模 超微结构研究将为了解视网膜退行性疾病的细胞学基础提供一条途径。

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.