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

描述（由申请人提供）：胶质细胞执行神经系统持续工作所需的许多专门任务。两个最突出的例子是少突胶质细胞在髓鞘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区室，包括沿着血管在内皮细胞上的髓鞘片或“星形胶质细胞足”。尽管这些和其他独特的神经胶质隔室的重要性，它们的形成和功能的理解仍然是有限的，由于我们不完整的知识，其组成部分。实现这一目标一直受到阻碍的困难，确定蛋白质存在于这种复杂的神经胶质隔室，这不是很好地概括在体外培养的细胞。 在这里，我们提出了一种新的蛋白质组学工具的系统分析，这样的胶质细胞车厢在体内的发展。该方法是基于局部限制的生物素标记的蛋白质存在于Bisturen隔室和随后的分离和鉴定质谱。作为一个原理验证模型，我们专注于头状突起，这是神经胶质细胞深入果蝇视觉系统中光感受器的神经末梢。我们最近已经证明了这种胶质细胞在神经递质循环中的直接作用。使用这个模型系统，我们将在目标1优化生物素标记的蛋白质存在于这个区室在体内，并随后通过质谱鉴定。因为对于任何新的蛋白质组学方法来说，重要的是严格验证所鉴定的候选蛋白质，所以我们将集中于系统地测试所鉴定的蛋白质在神经递质再循环和头状突起形成中的作用。这种候选人的分析和评估他们的作用，头状突起的功能，然后将帮助我们进一步优化这种蛋白质组学方法，了解不同的胶质细胞隔室的组成和功能。

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.