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，它们在中枢神经系统的功能和健康中发挥着关键作用。神经胶质细胞调节突触信号、鞘内轴突投射，更重要的是，通过充当抵御神经元损伤的第一道防线来保护大脑。成人大脑包含一系列惊人的不同神经胶质亚型，但人们对不同类别神经胶质细胞的独特遗传图谱知之甚少，这些基因图谱使它们能够发挥其重要而多样的功能。此外，确定神经损伤时神经胶质细胞的转录模式是如何改变的，这带来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，成年果蝇的大脑含有各种胶质亚型，这些亚型与脊椎动物中描述的那些亚型惊人地相似。此外，急性神经损伤诱导果蝇的神经胶质免疫反应，这与哺乳动物神经胶质细胞的反应非常相似，包括上调必要的神经胶质免疫基因。该项目将利用胶质细胞这些进化上保守的特征，并整合体内RNA标记和高通量深度测序领域的前沿进展，以产生完整的成年果蝇脑损伤前后胶质细胞的全面转录组。我们将使用新的基因 在成人大脑中表达的离散神经胶质亚型的驱动因素从基因上给它们贴上了标签 在体内的每一类胶质细胞中提取RNA，然后生化分离标记的RNA，通过RNA-seq对胶质亚型转录本进行测序。使用成熟的轴突切断法，我们将在未受伤和受伤的果蝇中进行这些实验，以比较急性轴突损伤前后胶质细胞的转录图谱。最后，我们将验证神经胶质基因的表达。 由RNA-seq鉴定，并开始表征新发现的免疫基因的功能作用，这些基因在神经胶质细胞对轴突切断的反应中受到尖锐的调节。这项工作(A)将为成人大脑中不同胶质亚型的功能提供关键的机制洞察力(B)提供一个独特的机会来研究在完整的中枢神经系统中神经变性反应中神经胶质细胞的基因表达是如何改变的，以及(C)将为科学界提供一个有价值的遗传工具包来研究胶质细胞生物学的许多未探索的方面。

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.