Proteomics of a neurotransmitter recycling domain in glia of the visual system

描述(由申请人提供)：神经胶质细胞执行神经系统持续工作所需的许多特殊任务。其中最突出的两个例子是少突胶质细胞在髓磷脂形成中的作用和星形胶质细胞对神经递质稳态的贡献。为了执行这些功能，不同的胶质细胞精心设计了许多独特的细胞隔间，包括髓磷脂或“星形胶质细胞脚”，沿着血管附着在内皮细胞上。尽管这些和其他独特的神经胶质隔室很重要，但由于我们对其组成部分的不完全了解，对它们的形成和功能的了解仍然有限。这一目标的实现一直受到难以确定存在于如此复杂的胶质细胞隔室中的蛋白质的阻碍，这些蛋白质在体外培养的细胞中不能很好地概括。在这里，我们建议开发一种新的蛋白质组学工具，用于在体内对这种胶质细胞隔室进行系统分析。该方法是基于对存在于不同隔间中的蛋白质进行局部限制的生物素标记，并随后通过质谱学分离和鉴定。作为一个原理验证模型，我们将重点放在头状投射上，这是果蝇视觉系统中神经胶质细胞深入光感受器神经末梢的内陷。我们最近证明了神经递质循环中这个胶质细胞隔室的直接作用。利用这个模型系统，我们将在目标1中优化体内这个隔室中存在的蛋白质的生物素标记，并随后通过质谱学进行鉴定。因为对于任何新的蛋白质组学方法来说，关键地验证识别的候选蛋白质是重要的，我的目标2我们将专注于系统地测试识别的蛋白质在神经递质循环和头状投射形成中的作用。这种对候选基因的分析和对它们在头状突起功能中的作用的评估，将有助于我们进一步优化这种蛋白质组学方法，以了解不同胶质细胞隔室的组成和功能。 与公共健康相关：神经胶质细胞执行许多神经系统正常活动所必需的重要功能，包括电隔离轴突或清除分泌的神经递质并将其循环到神经末梢。为了执行这些功能，胶质细胞阐述了许多不同的细胞隔间，这些隔间的功能和组成仍然不完全清楚，部分原因是它们在培养的细胞中没有很好地概括。在这里，我们建议结合化学生物学、质谱学和蛋白质靶向神经胶质细胞的最新进展，开发一种新的方法来系统地测定体内确定的神经胶质细胞间隔中的所有蛋白质。

DESCRIPTION (provided by applicant): Glia cells perform numerous specialized tasks that are required for the continued working of the nervous system. Two of the most prominent examples are the role of oligodendrocytes in the formation of myelin sheets and the contribution of astrocytes to the homeostasis of neurotransmitters. For the execution of such functions different glia cells elaborate many unique cellular compartments including myelin sheets or the 'astrocyte feet' onto endothelial cells along blood vessels. Despite the importance of these and other distinctive glial compartments, the understanding of their formation and function is still limited due to our incomplete knowledge of their components. Achieving this goal has been hampered by the difficulty of determining the proteins present in such complex glia compartments, which are not well recapitulated in cultured cells in vitro. Here, we propose the development of a novel proteomics tool for a systematic analysis of such glia compartments in vivo. The method is based on the locally restricted biotin-labeling of proteins present in differen compartments and their subsequent isolation and identification by mass spectroscopy. As a proof-of-principle model we are focusing on capitate projections, which are deep invaginations of glial cells into the nerve endings of photoreceptors in the Drosophila visual system. We have recently demonstrated a direct role of this glia compartment in neurotransmitter recycling. Using this model system we will in Aim 1 optimize the biotin-labeling of proteins present in this compartment in vivo and their subsequent identification by mass spectroscopy. Because for any new proteomics approach it is important to critically validate the identified candidate proteins, i Aim 2 we will focus on systemically testing the role of identified proteins in neurotransmitter recycling and in the formation of capitate projections. This analysis of the candidates and the evaluation of their roles in the function of capitate projections will then aid us in the further optimization of this proteomics approach towards understanding the composition and function of different glia compartments. PUBLIC HEALTH RELEVANCE: Glia execute many important functions necessary for the normal activity of the nervous system, including the electrical isolation of axons or the sweeping up of secreted neurotransmitters and their recycling to nerve endings. To perform these functions glia elaborates many distinct cellular compartments that are still incompletely understood in their function and composition, in part because they are not well recapitulated in cultured cells. Here, we propose to combine recent advances in chemical biology, mass spectroscopy and protein targeting to glial compartments to develop a novel method to systematically determine all the proteins in defined glia cell compartments in vivo.