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

描述（由申请人提供）：神经胶质细胞执行许多特殊的任务，这些任务是神经系统持续工作所必需的。两个最突出的例子是少突胶质细胞在髓磷脂层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，不同的神经胶质细胞精心制作了许多独特的细胞室，包括髓磷脂片或“星形胶质脚”，位于血管内皮细胞上。尽管这些和其他独特的胶质隔室很重要，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理解仍然有限。实现这一目标的困难之处在于，很难确定这种复杂胶质细胞区室中存在的蛋白质，而这些蛋白质在体外培养的细胞中不能很好地重现。在这里，我们建议开发一种新的蛋白质组学工具来系统地分析这种胶质细胞室。该方法是基于局部限制的生物素标记的蛋白质存在于不同的室和他们随后的分离和鉴定质谱。作为一个原理验证模型，我们关注的是头状突起，这是果蝇视觉系统中神经胶质细胞进入光感受器神经末梢的深度内翻。我们最近证明了这种胶质隔室在神经递质循环中的直接作用。使用该模型系统，我们将在Aim 1中优化生物素标记存在于该隔室中的蛋白质，并随后通过质谱进行鉴定。因为对于任何新的蛋白质组学方法，重要的是严格验证已鉴定的候选蛋白质，在Aim 2中，我们将专注于系统地测试已鉴定的蛋白质在神经递质循环和头状突起形成中的作用。对候选蛋白的分析和它们在头状突起功能中的作用的评估将帮助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室的组成和功能。

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.