Non-circadian Role for Clock Genes in Sleep Homeostasis

描述（由申请人提供）：睡眠的稳态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是基于遗传和分子研究。在这个提议中，我们认为，在SCN中被确定为昼夜节律起搏器的关键成分的几个基因在睡眠稳态中的大脑皮层（也许还有其他大脑区域）中也有单独的非昼夜节律作用。我们发现，缺乏昼夜节律“时钟”基因隐花色素（cry 1，2-/-）的小鼠不仅缺乏昼夜节律，而且表现出高睡眠压力的所有皮层电特征。这种意想不到的表型与转录调节因子period（per）1和per 2的脑表达升高相关，period（per）1和per 2在转录上受到转录抑制。睡眠剥夺后的野生型小鼠大脑皮层中的per 1，2 mRNA水平也有所升高。在这里，我们建议在mRNA和蛋白质水平上确认并进一步表征睡眠-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SCN外相互作用的时钟基因的转录网络是睡眠稳态的基础这一假设至关重要。为了将稳态与昼夜睡眠调节方面分开，将进行SCN损伤。我们预计，在没有SCN提供的昼夜节律输出的情况下，每一个表达中的皮质振荡将由睡眠和觉醒驱动，就像在完整的动物中一样。我们将通过检查缺乏cry 1、cry 2、per 1、per 2、clock和npas 2的小鼠的睡眠调节来直接操纵这种时钟基因网络。将记录缺乏cry 1或cry 2的小鼠，以评估仅一个cry基因的失活是否可以复制cry 1，2-/-睡眠表型。CLOCK和NPAS 2是per 1，2，cry 1，2和其他基因的正转录调节因子，并且应该提供与cry 1，2-/-表型的重要对比。考虑到这些基因家族中的部分冗余，我们希望对同时缺乏per 1、2或npas 2、clock的小鼠进行比较，尤其能提供信息。对睡眠稳态的分子基质的进一步理解可能有助于各种各样的急性或慢性睡眠问题的个体。

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.