Molecular dynamics of circadian timing in a mouse model of human sleep disorder

我们已经知道一段时间了，生物钟调节着人类和其他动物的睡眠和觉醒的节律行为。这些每日节律是内源性的，因为它们在恒定条件下自由运行，并且不需要与外部因素如光和暗同步，因此被称为昼夜节律（“一天左右”）。身体的主要节律发生器位于大脑的下丘脑内，并被称为视交叉上核（或SCN）。SCN具有在实验室条件下培养时将继续振荡的独特性质。调节生物钟的基因已经被克隆出来，我们知道调节时间的一个关键特征是这些基因的蛋白质产物如何在细胞周围实时循环。这在一定程度上受到一类称为激酶的酶的调节，激酶将磷酸键添加到蛋白质（磷酸化），从而影响其活性。生物钟最著名的突变之一是激酶（CK1e），20多年前在仓鼠中发现，导致昼夜节律周期缩短。这被称为tau突变，因为昼夜节律生物学家使用术语tau来表示周期。已知相同或相似激酶系统的突变会导致人类睡眠障碍。我们在小鼠中重新制造了这种突变，并表明它加速行为活动周期的程度与仓鼠相似。我们现在提出的工作旨在研究这种激酶突变如何加速大脑和外周生物钟的生物钟。我们早期使用仓鼠的研究表明，由于核心时钟蛋白的细胞核中的加速周转，生物钟可能会在一天中的特定时间通过相位的突然变化而加速。这在机械方面相当于齿轮箱缺少几个关键齿轮，导致它在每次旋转时跳到一个新的位置。我们的目标是通过使用新的转基因动物实时研究蛋白质运动来测试小鼠中的这一想法，我们的目标是创建其中关键时钟蛋白质用荧光标记物标记的转基因动物。这些类型的研究只能在小鼠中进行，因为这些是唯一可以进行这种遗传修饰的动物。我们将使用这些动物来确定激酶如何作用于其靶蛋白，通过研究蛋白质的哪些区域（结构域）被这种激酶磷酸化。通过将我们的时钟蛋白标记小鼠与tau突变动物杂交，我们将能够确定tau如何加速周期以及它作用于哪些蛋白质。这一点很重要，因为从长远来看，描述如何实现这一点可能会导致开发影响人类睡眠和觉醒周期的新药。通过遗传修饰技术，已经从小鼠基因组中删除了一些参与昼夜节律产生的核心基因。这些所谓的基因敲除小鼠仍然是有节奏的，因为其他残留的时钟元素足以驱动行为。我们将我们的tau突变小鼠与这些敲除小鼠杂交，并确定哪些敲除小鼠表现出轮跑活动周期的缩短。这将告诉我们tau蛋白是否可以在缺乏特定基因的情况下发挥作用，以及它可以在多大程度上缩短周期。通过这种方法，我们的目标是确定哪些时钟基因蛋白可能是tau调节行为活动周期的靶点。最后，我们的目标是利用SCN和其他组织在培养中继续振荡的事实。我们将使用产生光的时钟基因的专门报告物（荧光素酶报告物）监测培养组织的活性。通过使用专门的光子记录设备监测光水平，我们将能够研究生物钟如何调节组织中的时间，以及它们对可以重新设置时钟的刺激的反应。

We have known for some time that daily clocks regulate rhythmical behaviour of sleep and wake in man and other animals. These daily rhythms are endogenous as they free-run in constant conditions, and do not require synchronization to external factors such as light and dark, and are therefore termed circadian ('around a day'). The major rhythm generator of the body resides within the hypothalamus of the brain, and is termed the suprachiasmatic nucleus (or SCN). The SCN has the unique properties that it will continue to oscillate when cultured in laboratory conditions. Genes regulating the circadian clock have been cloned and we know that a key feature regulating timing is how the protein products of these genes cycle in real-time around the cell. This is regulated in part by a class of enzymes called kinases, which add phosphate bonds to the protein (phosphorylation) thereby affecting its activity. One of the best known mutations of the circadian clock is a kinase (CK1e) and was discovered in hamsters over 20 years ago, causing a shortening of circadian period. This was termed the tau mutation, since the term tau is used by circadian biologists to denote period. Mutations in the same or similar kinase systems are known to induce sleep disorders in man. We have re-made this mutation in mice and shown that it accelerates behavioural activity cycles to a similar extent as hamsters. Our proposed work now aims to study how this kinase mutation accelerates the circadian clock, both in the brain and in peripheral body clocks as well. Our earlier research using hamsters has shown that the circadian clock may be accelerated by an abrupt change in phase at a specific time of day, due to accelerated turnover in the nucleus of the cell of core clock proteins. This is equivalent in mechanical terms to a gear box missing a few key cogs, causing it to jump to a new position at each rotation. We aim to test this idea in the mouse by studying protein movements in real-time using new transgenic animals which we aim to create in which key clock proteins are tagged with a fluorescent marker. These types of studies can only be addressed in mice, as these are the only animals in which it is possible to make such genetic modifications. We will use these animals to define how the kinase acts on its target proteins by studying which areas (domains) of the protein are phosphorylated by this kinase. By crossing our clock protein-tagged mice to the tau mutant animals, we will be able to define how tau accelerates period and on which proteins it acts. This is important as a description of how this is achieved could in the longer term lead to the development of novel drugs impacting on sleep and wake cycles in man. Some of core genes involved in the generation of circadian rhythms have been deleted from the genome of mice by genetic modification techniques. These so-called knock-out mice are still rhythmic as other residual clock elements are sufficient to drive behaviour. We will cross our tau mutant mice into these knock out mice and define which of the knock-outs exhibits shortening of wheel-running activity cycles. This will tell us whether tau can act in the absence of a specific gene and also the extent to which it can shorten period. By this means we aim to define which clock gene proteins are likely targets for regulation of behavioural activity cycles by tau. Finally, we aim to capitalize from the fact that the SCN and other tissues continue to oscillate in culture. We will monitor activity of cultured tissues using specialized reporters of clock genes which generate light (luciferase reporters). By monitoring light levels with specialized photon recording equipment, we will be able to examine how the circadian clock regulates timing in tissues, and their responses to stimuli which can re-set the clock.