Analysis of Mammalian Circadian Mechanism with Cell-permeant Clock Proteins

描述（由申请人提供）：人类和大多数其他生物表现出由内源性生化振荡器控制的昼夜节律，该振荡器调节睡眠，心血管功能和新陈代谢的时间。这些“生物钟”对人体生理学很重要。例如，精神病学和医学研究表明，昼夜节律性与某些形式的抑郁症、“时差反应”、药物耐受性/有效性、记忆力、失眠和其他睡眠障碍有关。因此，了解生物钟的生化机制可能会导致对与睡眠、心理健康和药理学相关的疾病的诊断和治疗有用的程序。昼夜节律钟的显著特性——24小时时间常数、高精度、温度补偿——目前还无法用生物化学方法来解释。目前的昼夜节律转录和翻译反馈模型认为，节律时钟蛋白丰度对时钟功能至关重要。在模型系统中，有可能通过在特定阶段诱导时钟蛋白合成来重置昼夜节律的阶段。这种方法很难在哺乳动物系统中实现。目前的项目将通过使用新方法通过肽介导的跨细胞膜转导将蛋白质直接引入细胞，来测试有关哺乳动物节律钟蛋白丰度意义的假设。这项技术将使我们能够调节细胞、组织切片和完整动物体内时钟蛋白的细胞内浓度。本项目适合NIH探索性/发展性资助（R21）计划，因为它满足以下所有主要标准：(1)创新研究方向，(2)探索实质性领域的新方法，(3)开发新技术和方法。

DESCRIPTION (provided by applicant): Humans and most other organisms manifest circadian (daily) rhythms that are controlled by an endogenous biochemical oscillator that regulates the timing of sleep, cardiovascular functions, and metabolism. These "biological clocks" are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, insomnia, and other sleep disorders. Therefore, understanding the biochemical mechanism of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders that are relevant to sleep, mental health, and pharmacology. The salient properties of circadian clocks-24 hour time constant, high precision, temperature compensation-are presently impossible to explain biochemically. The current Transcription and Translation Feedback Model for circadian rhythms posits that rhythmic clock protein abundance is critical for clock function. In model systems, it has been possible to reset the phase of circadian rhythms by induction of clock protein synthesis at specific phases. This approach has been difficult to accomplish in mammalian systems. The current project will test hypotheses concerning the significance of rhythmic clock protein abundance in mammals by using new methods to introduce proteins directly into cells by peptide-mediated transduction across cell membranes. This technology will allow us to modulate the intracellular concentration of clock proteins in cells, tissue slices, and intact animals. This project is appropriate for the NIH Exploratory/Developmental Grant (R21) Program because it fulfills all of the following primary criteria: (1) innovative research directions, (2) exploration of approaches that are new to a substantive area, and (3) development of new technologies and methods. PUBLIC HEALTH RELEVANCE: Rigorous testing of the current model for circadian oscillators is crucial for our understanding of these biological clocks that control so much of human physiology. These studies will yield results of theoretical importance, but recent evidence also implicates sleep disorders, obesity, cardiovascular disease, and cancer when normal circadian clock function is disrupted. Therefore, these studies also have the potential for designing treatments for sleep disorders, jet lag, insomnia, cardiovascular disease, and other clock-related disorders.