Secreted astrocyte proteins regulating rhythmic behavior

总结 在哺乳动物和昆虫中，脑星形胶质细胞与神经元合作调节睡眠和昼夜节律 行为（参见[1-3]）。为了鉴定调节节律行为的果蝇星形胶质细胞基因，我们 进行了星形胶质细胞特异性转录组表达分析，以确定在星形胶质细胞中调控的mRNA。 昼夜节律的方式我们发现，超过250种星形胶质细胞mRNA在核糖体结合中表现出昼夜节律变化， 表明这些转录本的有节奏的翻译（You等人，提交）。许多这样的“循环”基因 编码Bomanin（Bom）、Turandot（Tot）的小分泌型星形胶质细胞蛋白（类精氨酸或抗菌） 或已知由机械或免疫应激诱导的生长阻断肽（Gbp）家族。一 这些基因中的许多受保守的Toll/免疫缺陷（IMD）信号通路调节， 我们的研究表明，这些通路的组分也在成体星形胶质细胞中大量循环。类细胞因子 分泌因子在病理性免疫反应中调节睡眠，但也有证据表明， 正常调节节律行为。假设这些循环细胞因子和抗菌基因是压力- 诱导，我们假设它们的功能是促进睡眠，以应对与睡眠相关的压力。 清醒事实上，我们未发表的结果表明，星形胶质细胞缺乏BomBc 2- a Toll调节， 一种编码类尼古丁蛋白质的循环基因--导致睡眠量和质量下降。相关 重要的是，已经表明，小鼠Toll-4或Toll-2受体的缺陷分别改变了小鼠的免疫功能。 对睡眠丧失的反应[4]或小鼠昼夜节律活动的巩固[5]。本申请提出2个相关 目的是检验节律性激活星形胶质细胞信号通路和分泌蛋白质的假设， 有助于调节昼夜节律行为或睡眠可塑性。目标1将定义成人的细胞类型 果蝇大脑需要循环分泌蛋白或Toll/IMD信号成分来进行正常的节律行为 （昼夜节律和睡眠）。由于这些分泌的蛋白质是由机械或免疫应激诱导的，因此这一目标将 还测试了新的假设，即它们是由睡眠剥夺引起的，以Toll/IMD调节的方式， 在睡眠平衡中的作用目的2将确定分泌蛋白的体内表达模式 和由循环mRNA编码的Toll/IMD信号传导组分。它还将询问他们是否骑自行车 丰富的特定细胞类型的成年苍蝇的大脑。这一目标还将采用CRISPR方法来确定 神经元和神经胶质生物钟对星形胶质细胞蛋白质循环调节的贡献。 总之，这些目标将确定昼夜行为或睡眠可塑性所需的星形胶质细胞因子 并定义调节其循环表达的细胞内信号传导途径。

Summary In both mammals and insects, brain astrocytes cooperate with neurons to modulate sleep and circadian behavior (reviewed in [1-3]). To identify Drosophila astrocyte genes that modulate rhythmic behaviors, we conducted an astrocyte-specific transcriptome-wide expression analysis to define mRNAs that are regulated in a circadian manner. We show that >250 astrocyte mRNAs exhibit circadian changes in ribosome association, suggesting rhythmic translation of these transcripts (You et al., submitted). A number of these “cycling” genes encode small secreted astrocyte proteins (cytokine-like or antibacterial) of the Bomanin (Bom), Turandot (Tot) or Growth Blocking Peptide (Gbp) families that are known to be induced by mechanical or immune stress. A number of these genes are regulated by the conserved Toll/Immune Deficiency (IMD) signaling pathways and our studies show that components of these pathways also cycle in abundance in adult astrocytes. Cytokine-like secreted factors modulate sleep during pathological immune responses, but there is also evidence that they normally regulate rhythmic behavior. Given that these cycling cytokine and antibacterial genes are stress- induced, we hypothesize that they function to promote sleep in response to the stress associated with wakefulness. Indeed, our unpublished results show that an astrocyte deficit for BomBc2 – a Toll-regulated, cycling gene that encodes a cytokine-like protein – results in decreased sleep amount and quality. Of related significance, it has been shown that deficits for the mouse Toll-4 or Toll-2 receptors, respectively, alter the response to sleep loss [4] or consolidation of mouse circadian activity [5]. This application proposes 2 related aims to test the hypothesis that rhythmically activated astrocyte signaling pathways and secreted proteins contribute to the regulation of circadian behavior or sleep plasticity. Aim 1 will define the cell types of the adult fly brain that require cycling secreted proteins or Toll/IMD signaling components for normal rhythmic behaviors (circadian and sleep). As these secreted proteins are induced by mechanical or immune stress, this aim will also test the novel hypothesis that they are induced by sleep deprivation, in a Toll/IMD-regulated manner, to function in sleep homeostasis. Aim 2 will determine the in vivo expression patterns for the secreted proteins and Toll/IMD signaling components encoded by cycling mRNAs. It will also ask whether they cycle in abundance in specific cell types of the adult fly brain. This aim will also employ CRISPR methods to determine the contribution(s) of neuronal and glial circadian clocks to the regulation of cycling astrocyte proteins. Altogether, these aims will identify astrocyte factors that are required for circadian behavior or sleep plasticity and define intracellular signaling pathways regulating their cycling expression.