Molecular and anatomical basis of sleep regulation by SLEEPLESS

描述（由申请人提供）：睡眠是一个必要的，进化保守的过程，如果不满足，会导致人类病理。睡眠的重要性被其严密的体内平衡控制所强调：睡眠驱动随着清醒时间的增加而增加，随着睡眠时间的增加而消散。然而，睡眠调控的分子机制和控制睡眠稳态的神经回路在很大程度上是未知的。果蝇（Drosophila melanogaster）已被证明在识别与行为、人类健康和疾病有关的基因方面很有用，它也成为研究睡眠的一个有价值的遗传模型系统。通过前向基因筛选，我们发现了一种新的失眠基因（sss），它是睡眠剥夺后基线和稳态恢复睡眠所必需的。在sss突变体中，我们发现睡眠调节K通道Shaker （Sh）的水平降低，导致sss与睡眠驱动降低膜兴奋性的假设。最近我们还表明，除了Sh电流的振幅和动力学外，sss还可以调节Sh通道的定位。与SSS对Sh的直接调控一致，我们已经证明，通过通道和SSS之间形成稳定的复合物，Sh的表达在转录后被促进。sss受RNA编辑机制的控制，编辑后的sss在促进睡眠方面的效果不如不可编辑的sss。因此，我们假设RNA编辑控制SSS与Sh相互作用的能力，从而改变通道的活性和亚细胞运输。SSS- sh相互作用的结构基础特别有趣：SSS是一个大家族的创始成员之一，这个大家族的蛋白质类似于神经毒素，通常作用于离子通道，这提高了这个家族的其他成员可能调节兴奋性和睡眠的可能性。本提案的重点是确定sss调节睡眠的分子基础，特别是关于Sh，并描述所涉及的神经回路。具体目的是：1)确定sss调节Sh的机制，2)确定sss的rna编辑在调节睡眠和Sh电流中的作用，以及3)确定sss在大脑中调节睡眠的位置。总的来说，这些研究将提高我们对睡眠需求的分子基础的理解，以及它如何导致大脑电活动的重大变化。这些发现也可能有助于确定干预睡眠障碍和与一般神经元兴奋性调节不当有关的障碍的新目标。

DESCRIPTION (provided by applicant): Sleep is an essential, evolutionarily conserved process which, if unfulfilled, contributes to human pathology. The importance of sleep is underscored by its tight homeostatic control: sleep drive increases with time spent awake and dissipates with time spent asleep. However, the molecular mechanisms underlying regulation of sleep and the neural circuitry that controls sleep homeostasis are largely unknown. The fruit fly, Drosophila melanogaster, which has proven useful for identifying genes involved in behavior, human health and disease, has also emerged as a valuable genetic model system for studying sleep. Using a forward genetic screen, we identified the novel gene sleepless (sss) that is required for both baseline and homeostatic recovery sleep following sleep deprivation. In sss mutants, we found that levels of the sleep-regulating K channel, Shaker (Sh), are reduced, leading to the hypothesis that sss couples sleep drive to lowered membrane excitability. More recently we have also shown that sss can regulate the localization of Sh channels in addition to both amplitude and kinetics of Sh currents. Consistent with direct regulation of Sh by SSS, we have demonstrated that Sh expression is promoted post-transcriptionally via the formation of a stable complex between channel and SSS. sss is under the control of RNA editing machinery, and edited sss is less effective than uneditable sss at promoting sleep. We thus hypothesize that RNA editing controls the ability of SSS to interact with Sh, thereby altering activity and subcellular trafficking of the channel. The structural basis for SSS-Sh interactions is particularly intriguing: SSS is one of the founding members of a large family of relatively uncharacterized proteins that resemble neurotoxins, which often act on ion channels, raising the possibility that other members of this family may regulate excitability and sleep. The focus of this proposal is to determine the molecular basis of sleep regulation by sss, particularly with regard to Sh, and to describe the neural circuitry involved. The specific aims are to: 1) determine mechanisms by which sss regulates Sh, 2) determine the role of RNA-editing of sss in modulation of sleep and Sh currents, and 3) determine where in the brain sss acts to regulate sleep. Collectively these studies will improve our understanding of the molecular basis of sleep need and how it leads to major changes in electrical activity in the brain. Such findings may also help identify new targets for intervening both in disorders of sleep and in disorders related to misregulation of neuronal excitability in general.