Mechanisms of Circadian Rhythmicity in CLOCK-Deficient Mice

描述(申请人提供)：昼夜节律存在于整个动物界的物种中。在人类中，昼夜节律的紊乱会导致基于昼夜节律的睡眠障碍、轮班工人的不适应和时差反应，并可能导致包括抑郁和季节性情感障碍在内的神经精神障碍。转录-翻译反馈环是生物钟机制的中心。已知的驱动昼夜节律转录的正性元件是CLOCK和BMAL1，这是两种基本的螺旋环状螺旋蛋白，它们可以二聚化来激活反应基因的表达。我们最近培育出了时钟基因零突变的小鼠。出乎意料的是，这些时钟缺陷的小鼠在恒定的条件下保持了行为的昼夜节律。我们的研究将表征时钟缺陷小鼠的生理和分子节律，并评估在缺乏时钟的情况下节律性的机制。在具有先前描述的Clock显性负突变的小鼠中，Clock-Delta19蛋白可能通过干扰其他关键的bHLH-PAS蛋白的活性来扰乱昼夜节律，这表明主要的昼夜转录激活因子仍有待确定。这个项目的一个主要目标将是确定这种明显的转录激活的第二种机制。我们将测试NPAS2，一种与时钟密切相关的bHLH-PAS转录因子，可以替代时钟，从而在时钟缺陷小鼠中保持节律性。我们还将确定BMAL1是否在没有时钟的情况下对节律性是必需的，期待一个发现将使基于对候选BMAL1相互作用蛋白功能重要性的评估的研究成为可能。这项拟议的研究对于理解生物钟的功能是必要的，时钟是生物钟机制的中心组成部分，因此对于理解和可能开发基于昼夜节律的睡眠和精神障碍的新治疗方法是必要的。此外，生物钟在调节生殖、新陈代谢、细胞生长和肿瘤进展方面发挥着不同的作用，因此了解昼夜节律产生的基本机制具有重要的意义。

DESCRIPTION (provided by applicant): Circadian rhythms are present in species throughout the animal kingdom. In humans, disorders of circadian timing contribute to circadian-based sleep disorders, maladjustment of shift workers and during jet lag, and may contribute to neuropsychiatric disorders including depression and seasonal affective disorder. A transcriptional-translational feedback loop is at the center of the circadian clock mechanism. The known positive elements driving circadian transcription are CLOCK and BMAL1, two basic helix loop helix proteins that dimerize to activate expression of responsive genes. We have recently generated mice with a null mutation of the Clock gene. Unexpectedly, these CLOCK-deficient mice retain circadian rhythmicity in behavior in constant conditions. Our studies will characterize physiological and molecular rhythms in CLOCK-deficient mice, and assess mechanisms of rhythmicity in the absence of CLOCK. In mice with the previously described dominant negative mutation of Clock, the CLOCK-delta19 protein likely disrupts circadian rhythmicity by interfering with the activity of other key bHLH-PAS proteins, indicating that a major circadian transcriptional activator remains to be identified. A major objective of this project will be to identify this apparent second mechanism for transcriptional activation. We will test the hypothesis that NPAS2, a bHLH-PAS transcription factor closely related to CLOCK, can substitute for CLOCK and thus maintain rhythmicity in CLOCK-deficient mice. We will also determine whether BMAL1 is necessary for rhythmicity in the absence of CLOCK, expecting a finding that will enable studies based on assessment of the functional importance of candidate BMAL1-interacting proteins. The proposed studies are necessary to understand the function of CLOCK, a central component of the circadian clock mechanism, and thus are relevant to understanding and possibly developing novel treatments for circadian-based sleep and psychiatric disorders. In addition, the circadian clock plays diverse roles in regulating reproduction, metabolism, cell growth and tumor progression, so the importance of understanding basic mechanisms of circadian rhythm generation has many implications.