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。在这一提议中，我们认为几个基因被确定为SCN中昼夜节律起搏器的关键组成部分，在大脑皮层（可能还有其他大脑区域）的睡眠稳态中也有一个单独的、非昼夜节律的作用。我们发现，缺乏昼夜节律“时钟”基因隐色素（cry1,2-/-）的小鼠不仅缺乏昼夜节律，而且还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周期（per） 1和per2的大脑表达升高有关，这两个周期被cry1,2转录抑制。野生型小鼠在睡眠剥夺后，大脑皮层的per1,2 mRNA水平也有所升高。在这里，我们提出在mRNA和蛋白质水平上确认和进一步表征睡眠-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验证SCN外相互作用的时钟基因的转录网络是睡眠稳态器的基础这一假设至关重要。为了分离体内平衡和昼夜睡眠调节方面，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，皮层的per表达振荡将由睡眠和清醒驱动，就像在完整的动物中一样。我们将通过检查缺乏cry1、cry2、per1、per2、clock和npas2的小鼠的睡眠调节，直接操纵生物钟基因网络。缺失cry1或cry2的小鼠将被记录下来，以评估是否只有一个哭泣基因失活可以复制cry1,2-/-睡眠表型。CLOCK和NPAS2是per1,2, cry1,2和其他基因的正转录调节因子，并且应该提供与cry1,2-/-表型的重要对比。考虑到这些基因家族的部分冗余，我们期望对同时缺乏per1、2或npas2时钟的小鼠进行比较，尤其具有信息性。对睡眠内稳态的分子基础的更好理解可能对各种患有急性或慢性睡眠问题的个体有所帮助。

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.