Genetic Analysis of Glia-to-Neuron Communication

描述(申请人提供)：尽管在哺乳动物模型3、4中，有研究表明星形胶质细胞可以调节神经元的兴奋性和行为，但关于成年大脑中神经胶质细胞到神经元的交流的重要性仍存在争议。例如，一些人质疑神经胶质细胞钙依赖机制在神经元调节中的重要性。此外，目前对神经胶质递质释放的调控机制仍知之甚少，尽管某些研究表明它涉及囊泡胞吐作用2。在这一应用中，我们提出了无偏见的分子遗传学方法来识别神经胶质细胞与神经元通讯所必需的神经胶质和神经元因子。我们之前的研究表明，果蝇胶质细胞特异性因子乌木的消除在遗传上抑制了多巴胺转运体(DAT)突变体8的过度活动表型，表明胶质细胞与神经元之间的通讯在活动水平的调节中发挥了作用。在最近发表的研究中，我们已经证明成年果蝇的神经胶质细胞可以生理地调节运动活动和昼夜节律。我们的研究还显示了钙依赖机制和果蝇星形胶质细胞样神经胶质细胞在运动活动调节中的关键作用9。在R21的应用中，我们提出了一些研究，以确定在成年动物中神经胶质细胞到神经元通讯的重要细胞信号机制和细胞内通路。果蝇是这类研究的优秀模型，因为成年大脑的胶质细胞类别已被很好地描述为10-12，其中一类别与哺乳动物星形胶质细胞11、13、14在发育、形态和分子上具有相似性。我们的研究将利用多种条件摄动方法，这些方法具有细胞类型特异性和可逆性，以确定新的保守因子和细胞内通路，这些因素和细胞内通路介导了神经元和行为的神经胶质调节。目的1将利用我们最近开发的果蝇品系和翻译图谱方法来识别受神经胶质信号调节的神经元因子和分子通路。AIM2将使用行为遗传策略来定义对神经元调节重要的神经胶质信号机制。这一建议代表了对神经胶质信号导致的神经元蛋白质组变化的第一次广泛研究。它也代表了对调节成年神经元的神经胶质机制的第一次遗传分析。因此，这些结果将对神经胶质生物学产生重大影响--它们将定义神经元和神经胶质细胞内介导神经胶质细胞间信号传递的分子通路，并为研究奠定基础。 在哺乳动物模型中研究保守因子，以了解它们在人类健康和神经疾病中的作用。

DESCRIPTION (provided by applicant): There is controversy about the importance of glia-to-neuron communication in the adult brain, notwithstanding studies of "gliotransmission" 1, 2 and indications that astrocytes can modulate neuronal excitability and behavior in mammalian models 3, 4. For example, some have questioned the importance of glial Ca2+-dependent mechanisms in neuronal modulation 5-7. In addition, there is still limited knowledge of the mechanisms regulating gliotransmitter release, although certain studies suggest it involves vesicular exocytosis 2. In this application, we propose unbiased molecular genetic approaches to identify glial and neuronal factors essential for glia-to-neuron communication. We previously showed that elimination of a Drosophila glial-specific factor called Ebony genetically suppressed the hyperactivity phenotype of a Dopamine Transporter (DAT) mutant 8, indicating a role for glia-to-neuron communication in the regulation of activity level. In more recent published studies, we have demonstrated that adult Drosophila glial cells can physiologically modulate locomotor activity and circadian rhythmicity9. Our studies also demonstrate a critical role for Ca2+- dependent mechanisms and Drosophila astrocyte-like glia in the regulation of locomotor activity 9. In this R21 application, we propose studies to identify cell signaling mechanisms and intracellular pathways that are important for glia-to-neuron communication in adult animals. Drosophila is an excellent model for such studies as the glial classes of the adult brain have been well characterized 10-12 and one class has developmental, morphological and molecular similarities to mammalian astrocytes 11, 13, 14. Our studies will take advantage of multiple conditional perturbation methods that are cell type-specific and reversible to identify novel, conserved factors and intracellular pathways that mediate the glial modulation of neurons and behavior. Aim 1 will utilize Drosophila strains we recently developed and translational profiling methods to identify neuronal factors and molecular pathways that are modulated by glial signaling. Aim2 will use behavioral genetic strategies to define glial signaling mechanisms that are important for neuronal modulation. This proposal represents the first broad study of neuronal proteomic changes that occur as a consequence of glial signaling. It also represents the first genetic analysis of glial mechanisms that mediate the modulation of adult neurons. As such, the results will have a major impact on glial biology - they will define molecular pathways within neurons and glial cells that mediate glia-to-neuron signaling, and set the stage for studies of conserved factors in mammalian models to understand their functions in human health and neurological disease.