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

视觉系统神经胶质细胞神经递质回收域的蛋白质组学

• 批准号: 8539640
• 负责人: Helmut J Kramer
• 金额: $ 18.86万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-05 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8539640
• 关键词: Adopted; Astrocytes; Axon; Biochemistry; Biological Models; Biology; Biotin; Biotinylation; Blood Vessels; Cells; Characteristics; Chemicals; Complex; Cultured Cells; Detection; Development; Drosophila genus; Endothelial Cells; Enzymes; Evaluation; Feedback; Generations; Genetic; Glutamates; Glutamine; Goals; Hand; Histamine; Homeostasis; Horseradish Peroxidase; In Vitro; Knowledge; Label; Mass Spectrum Analysis; Mediating; Methods; Modeling; Myelin; Nerve Endings; Nervous system structure; Neuroglia; Neurotransmitters; Oligodendroglia; Phenols; Phenotype; Photoreceptors; Proteins; Proteome; Proteomics; Recycling; Role; Staging; Streptavidin; Structure; System; Testing; Transgenes; Visual system structure; Work; base; candidate validation; capitate bone; foot; forging; improved; in vivo; method development; neurotransmission; novel; research study; tool

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.

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专门任务。两个最突出的例子是少突胶质细胞在髓磷脂片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设计了许多独特的细胞区室，包括沿着血管的内皮细胞上的髓磷脂片或“星形胶质细胞脚”。尽管这些和其他独特的神经胶质区室很重要，但由于我们对其组成部分的了解不完全，对它们的形成和功能的理解仍然有限。由于难以确定这种复杂的神经胶质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，因此阻碍了这一目标的实现。 在这里，我们建议开发一种新型蛋白质组学工具，用于对体内此类神经胶质区室进行系统分析。该方法基于不同区室中存在的蛋白质的局部限制性生物素标记以及随后通过质谱法进行的分离和鉴定。作为原理验证模型，我们重点关注头状投射，即神经胶质细胞深度内陷到果蝇视觉系统中感光器的神经末梢。我们最近证明了这种神经胶质区室在神经递质回收中的直接作用。使用该模型系统，我们将在目标 1 中优化体内该区室中存在的蛋白质的生物素标记，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来说，严格验证已识别的候选蛋白质非常重要，因此我们的目标 2 将重点系统地测试已识别的蛋白质在神经递质循环和头状投射形成中的作用。对候选者的分析以及对其在头状投射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，以了解不同神经胶质区室的组成和功能。