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

我们早就知道，人类和其他动物的生物钟调节着睡眠和觉醒的节律行为。这些日常节律是内源性的，因为它们在恒定的条件下自由运行，不需要与光和暗等外部因素同步，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，被称为视交叉上核（SCN）。SCN具有独特的性质，当在实验室条件下培养时，它将继续振荡。调节生物钟的基因已经被克隆，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内实时循环。这在一定程度上是由一类被称为激酶的酶调节的，这些酶在蛋白质上添加磷酸键（磷酸化），从而影响其活性。最著名的生物钟突变之一是一种激酶（CK1e），它是20多年前在仓鼠中发现的，导致昼夜节律周期缩短。这被称为tau突变，因为术语tau被昼夜节律生物学家用来表示周期。已知相同或相似激酶系统的突变可引起人类睡眠障碍。我们在老鼠身上重新制造了这种突变，并表明它加速了行为活动周期，其程度与仓鼠相似。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。我们早期对仓鼠的研究表明，由于核心时钟蛋白的细胞核更新加快，昼夜节律时钟可能会在一天中的特定时间发生突变。在机械方面，这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。我们的目标是在老鼠身上测试这个想法，通过使用新的转基因动物实时研究蛋白质运动，我们的目标是创造出关键时钟蛋白被荧光标记的动物。这些类型的研究只能在老鼠身上进行，因为这些是唯一有可能进行这种基因改造的动物。我们将用这些动物来确定激酶是如何作用于它的靶蛋白的，通过研究蛋白质的哪些区域（结构域）被这种激酶磷酸化。通过将时钟蛋白标记的小鼠与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。这很重要，因为从长远来看，描述如何实现这一目标可能会导致影响人类睡眠和觉醒周期的新药的开发。一些参与昼夜节律产生的核心基因已经通过基因改造技术从小鼠基因组中删除。这些所谓的“基因敲除老鼠”仍然有节奏感，因为其他残留的生物钟元素足以驱动它们的行为。我们将把我们的tau突变小鼠与这些敲除小鼠杂交，并确定哪些敲除小鼠会缩短滚轮跑步的活动周期。这将告诉我们tau是否能在缺乏特定基因的情况下发挥作用，以及它能在多大程度上缩短周期。通过这种方式，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目标。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（荧光素酶报告）来监测培养组织的活性。通过用专门的光子记录设备监测光照水平，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生物钟的刺激的反应。

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.