Non-circadian Role for Clock Genes in Sleep Homeostasis

描述(申请人提供)：睡眠的动态平衡调节机制在很大程度上是未知的，尽管基因表达的作用似乎基于遗传和分子研究。在这项建议中，我们认为在SCN中被识别为昼夜节律起搏器的关键成分的几个基因在大脑皮层(也许还有其他大脑区域)中也具有独立的、非昼夜节律的作用，以维持睡眠稳态。我们发现，缺乏昼夜节律基因的小鼠，不仅缺乏昼夜节律，还表现出高睡眠压力的所有大脑皮层特征。这种意想不到的表型与大脑转录调节期(PER)L和PER2的表达增加有关，这两个基因在转录上受到CRY1，2的抑制。睡眠剥夺后的野生型小鼠大脑皮质中PER1，2mRNA的水平也特别高。在这里，我们建议从mRNA和蛋白质水平上证实并进一步描述睡眠-觉醒与这些分子变化之间的关系。确定这种关系和这些变化的神经解剖学特异性对于检验这样一个假设至关重要：SCN外相互作用的时钟基因转录网络是睡眠稳态的基础。为了将动态平衡从昼夜睡眠调节方面分离出来，将进行SCN损伤。我们预计，在缺乏SCN提供的昼夜节律输出的情况下，PER表达的皮层振荡将由睡眠和清醒驱动，就像在完整的动物中一样。我们将通过检测缺乏Cry1、Cry2、PER1、PER2、Clock和npas2的小鼠的睡眠调节来直接操纵这个时钟基因网络。缺乏Cry1或Cry2的小鼠将被记录下来，以评估只有一个CrY基因的失活是否可以复制Cry1，2-睡眠表型。Clock和NPAS2是per1，2，Cry1，2和其他基因的正转录调控因子，应该是与Cry1，2-/-表型形成重要对比的基因。考虑到这些基因家族中的部分冗余，我们预计对缺乏per1，2，或两个npas2时钟的小鼠的比较将特别有意义。更好地理解睡眠稳态的分子底物可能有助于各种患有急性或慢性睡眠问题的人。

DESCRIPTION (provided by applicant): The mechanisms of the homeostatic regulation of sleep are largely unknown, although a role for gene expression seems likely based on genetic and molecular studies. In this proposal we suggest that several of the genes identified as critical components of the circadian pacemaker in the SCN also have a separate, non-circadian role in the cerebral cortex (and perhaps other brain regions) in sleep homeostasis. We found that mice lacking the circadian 'clock'-genes cryptochrome (cry1,2-/-), not only lack circadian rhythms but also exhibit all the electrocortical hallmarks of high sleep pressure. This unexpected phenotype was associated with elevated brain expression of the transcriptional regulators period (per)l, and per2, which are transcriptionally inhibited by CRY1,2. Wild-type mice following sleep deprivation also have elevated levels of per1,2 mRNA in the cortex specifically. Here we propose to confirm and further characterize the relationship between sleep-wake and these molecular changes at both mRNA and protein levels. Determining this relationship and the neuro-anatomical specificity of these changes is critical to test the hypothesis that a transcriptional network of interacting clock genes outside the SCN underlies the sleep homeostat. To separate homeostatic from circadian sleep regulatory aspects, SCN lesions will be performed. We expect that in the absence of circadian output provided by the SCN, the cortical oscillation in per expression will be driven by sleep and wake, as in intact animals. We will directly manipulate this clock-gene network by examining sleep regulation in mice lacking cry1, cry2, per1, per2, clock, and npas2. Mice lacking either cry1 or cry2 will be recorded to assess whether inactivation of only one cry gene can replicate the cry1,2-/-sleep phenotype. CLOCK and NPAS2 are positive transcriptional regulators of per1,2, cry1,2, and other genes, and should provide an important contrast to the cry1,2-/- phenotype. Given the partial redundancy in these gene families, we expect comparisons of mice lacking both per1,2, or both npas2,clock, to be especially informative. An improved understanding of the molecular substrate of sleep homeostasis could be helpful to a wide variety of individuals with acute or chronic sleep problems.