Transcriptome analysis of glia responding to injury

描述（申请人提供）：胶质细胞是人脑中最丰富的细胞，在中枢神经系统的功能和健康中起着关键作用。神经胶质细胞调节突触信号，包膜轴突投射，重要的是，通过充当防止神经元损伤的第一道防线来保护大脑。成人大脑中包含了一系列不同的神经胶质亚型，但人们对不同类型的神经胶质的独特遗传谱知之甚少，这些遗传谱使它们能够发挥重要而多样的功能。此外，确定神经胶质细胞的转录谱是如何在神经损伤的反应中改变的，已经提出了一系列独特的挑战，因为从大脑中分离神经胶质细胞进行转录分析的过程本身对细胞来说是高度紧张的。最近的研究表明，成年黑腹果蝇的大脑中含有各种各样的神经胶质亚型，这些亚型与脊椎动物的神经胶质亚型惊人地相似。此外，急性神经损伤在果蝇中诱导的神经胶质免疫反应与在哺乳动物神经胶质中触发的反应高度相似，包括必需的神经胶质免疫基因的上调。本项目将利用胶质细胞的这些进化保守特征，结合体内RNA标记和高通量深度测序等领域的前沿进展，生成完整成人大脑损伤前后果蝇胶质细胞的综合转录组。我们将使用新的基因

DESCRIPTION (provided by applicant): Glia are the most abundant cells in the human brain and they play key roles in CNS function and health. Glial cells regulate synaptic signaling, ensheath axonal projections and, importantly, protect the brain by serving as the first line of defense against neuronal damage. The adult brain contains a striking array of diverse glial subtypes, but little is known about the unique genetic profiles of distinct classes of glia that alow them to carry out their important and varied functions. Moreover, determining how the transcriptional profile of glial cells are altered in response to neural injury has presented a unique set of challenges, since the process of isolating glia from the brain for transcriptional analysis is, in and of itself, highly stressful to the cells. Recent work has shown that the adult Drosophila melanogaster brain contains a variety of glial subtypes that are strikingly similar to those described in vertebrates. In addition, acute neural injury induces glial immune responses in flies that are highly reminiscent of those triggered in mammalian glia, including upregulation of essential glial immune genes. This project will take advantage of these evolutionarily conserved features of glia and integrate cutting-edge advances in the fields of in vivo RNA labeling and high throughput deep sequencing to generate a comprehensive transcriptome of Drosophila glial cells in the intact adult brain before and after injury. We will use novel genetic drivers that are expressed in discrete glial subtypes in the adult fly brain to genetically "label" RNA in each class of glia in vivo and then biochemically isolate the labeled RNA to sequence glial subtype transcriptomes by RNA-seq. Using a well-established axotomy assay, we will perform these experiments in uninjured and injured flies to compare the transcriptional profiles of glia before and after acute axon injury. Finally, we will validate the expression of glial genes identified by RNA-seq and begin to characterize the functional role of the newly discovered immune genes that are acutely regulated in glia responding to axotomy. This work (a) will provide critical mechanistic insight into the function of diverse glial subtypes in the adult brain (b) offers a unique opportunity to investigate how gene expression is altered in glia responding to neurodegeneration in the intact CNS and (c) will generate a valuable genetic toolkit for the scientific community to investigate many unexplored aspects of glial cell biology.