Genetic Analysis of Glia-to-Neuron Communication

描述（由申请人提供）：尽管有“胶质传递”研究1,2，以及在哺乳动物模型中星形胶质细胞可以调节神经元兴奋性和行为3,4的迹象，但关于成人大脑中胶质细胞与神经元通讯的重要性仍存在争议。例如，一些人质疑神经胶质Ca2+依赖机制在神经元调节中的重要性5-7。此外，尽管某些研究表明胶质递质释放与水疱性胞吐有关，但对其机制的了解仍然有限。在这个应用中，我们提出了无偏的分子遗传学方法来鉴定胶质细胞和神经元因子对胶质细胞到神经元的通讯至关重要。我们之前的研究表明，消除果蝇胶质特异性因子Ebony基因抑制多巴胺转运蛋白（DAT）突变体8的多活性表型，表明胶质-神经元通讯在调节活性水平中的作用。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从生理上调节运动活动和昼夜节律。我们的研究也证明了Ca2+依赖机制和果蝇星形细胞样胶质细胞在运动活动调节中的关键作用9。在这个R21应用程序中，我们建议研究鉴定细胞信号机制和细胞内通路，这些通路对成年动物的胶质细胞到神经元的通讯很重要。果蝇是这类研究的一个很好的模型，因为成人大脑的胶质细胞类别已经被很好地描述了10-12，其中一类与哺乳动物星形胶质细胞具有发育、形态和分子相似性11,13,14。我们的研究将利用多种细胞类型特异性和可逆的条件扰动方法来识别介导神经元和行为的神经胶质调节的新颖，保守的因子和细胞内通路。目的1将利用我们最近开发的果蝇菌株和翻译分析方法来识别由胶质信号调节的神经元因子和分子途径。目的2将使用行为遗传策略来定义神经胶质信号机制，这对神经元调节是重要的。这一建议代表了神经蛋白组学变化的第一个广泛的研究，这些变化是神经胶质信号传导的结果。它也代表了介导成人神经元调节的神经胶质机制的第一个遗传分析。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到神经元信号的分子途径，并为研究奠定基础

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. PUBLIC HEALTH RELEVANCE: Glial cells constitute 90% of the cells of the adult brain and participate in processes ranging from neuronal support to the regulation of synaptic transmission and complex behaviors. In this application, we propose studies to examine the regulation of neuronal functions by glial cells. The proposal has significant relevance for normal brain function as well as the many neurological and psychiatric diseases that are associated with glial cell dysfunction.