Resolving the Role of Brain Lymphatic Endothelial Cells in Sleep Dependent Brain Clearance

大脑是人体新陈代谢最活跃的器官，会产生大量必须清除的废物。然而，科学家们仍然没有完全了解大脑是如何清除废物的。在身体的其他部位，多余的液体和代谢废物部分通过称为淋巴管的血管网络来清除，但大脑缺乏淋巴管。相反，大脑被认为经历了一个被称为液泡清除的过程，在这个过程中，大脑内的液体通过与沐浴在大脑外部的脑脊液(CSF)交换来清除废物。这种洗涤过程被认为在一天24小时内波动，主要发生在睡眠期间。废物离开大脑的确切途径仍然存在争议，但有一种想法是，废物然后流入位于脑膜外层(大脑周围的层状组织)的淋巴管。最近，另一种潜在的大脑清除机制被发现，一种名为脑淋巴管内皮细胞(BLECs)的细胞类型位于脑膜内部。由于这些细胞被脑脊液包围，它们不仅处于有利的位置，可以参与大脑的清除，而且它们还具有非常强大的能力，可以迅速内化注射到大脑中的物质。然而，BLEC的功能是否在24小时周期内波动，以及BLEC是否需要BLEC来清除清醒时大脑中积累的物质，目前尚不清楚。在这个项目中，我们将首先观察BLEC是如何在白天和夜晚改变形态和功能的，然后我们将测试BLEC功能的中断是否会影响睡眠等行为。由于大脑中有毒分子的积聚与神经退行性疾病有关，这项工作将对健康衰老具有重要意义。为了在昼夜周期中观察BLECs，我们将利用首次描述BLECs的斑马鱼幼体。斑马鱼是研究BLEC功能和睡眠之间联系的一个很好的模型，因为斑马鱼的幼体是光学透明的。这使得可以直接、非侵入性地观察BLEC，通过利用遗传学将荧光蛋白特异性地放入这些细胞中，可以很容易地在大脑顶部看到这些细胞。BLEC的表面位置也使它们可以用激光消融，这使我们能够测试当BLEC不再存在时会发生什么。最后，斑马鱼幼虫也有每天的睡眠/醒来周期，这在很大程度上是以类似人类的方式调节的，这使我们能够研究BLEC在24小时节律以及在睡眠剥夺期间或对改变睡眠的药物的反应中是如何变化的。首先，我们将使用显微镜观察斑马鱼BLEC在24小时内的变化，并测量它们的大小、位置、形状和连接性的变化。在每个细胞内部，BLEC在内化材料时形成许多大的、圆形的夹杂物；我们还将测量这些夹杂物随时间的变化。为了测试日常内部节律或睡眠/清醒状态是否会改变BLEC的形态和功能，我们还将观察BLEC对持续光照、睡眠剥夺或改变睡眠的药物的反应。接下来，我们将把染料注入斑马鱼的大脑，并观察BLEC将染料内化。通过测量一天中不同时间或睡眠/清醒状态的摄取率(即使在几分钟内也会迅速发生)，我们将更好地了解BLEC何时最活跃。最后，我们将用激光消融BLEC，或通过遗传学消除参与BLEC功能的关键基因来改变它们的摄取能力。然后，我们将使用摄像头跟踪斑马鱼的行为，观察斑马鱼的睡眠情况，看看改变BLEC是否会导致行为的改变。在本项目结束时，我们将对BLEC如何在24小时内改变形态和功能以及BLEC清除有毒副产物的能力如何影响大脑功能和行为有一个新的理解。

The brain is the most metabolically active organ in the body, which generates a lot of waste that must be removed. However, scientists still do not fully understand how the brain clears waste. In other parts of the body, excess fluid and metabolic waste are cleared in part by a network of vessels called lymphatics, but the brain lacks lymphatic vessels. Instead, the brain is thought to undergo a process called glymphatic clearance, in which fluid inside the brain is cleared of waste by exchanging with the cerebrospinal fluid (CSF) that bathes the outside of the brain. This washing process is thought to fluctuate across the 24-hour day, predominately occurring during sleep. The exact drainage routes for waste to leave the brain are still debated, but one idea is that waste then drains into lymphatic vessels that reside in the outer layer of the meninges (the layered tissues surrounding the brain).Recently, another potential player in brain clearance was discovered, a cell type called brain lymphatic endothelial cells (BLECs) that reside in the inner part of the meninges. These cells are not only well positioned to participate in brain clearance, as they are surrounded by CSF, but they also have a very strong capacity to rapidly internalize substances that are injected into the brain. However, whether BLEC function fluctuates across the 24-hour cycle and whether BLECs are required for the clearance of material that builds up in the brain during waking is unknown. In this project, we will first observe how BLECs change shape and function across the day and night, and then we will test if disruption of BLEC function impacts behaviours like sleep. Since the build-up of toxic molecules in the brain has been implicated in neurodegenerative disease, this work will have important implications for healthy aging.To observe BLECs across the day-night cycle, we will take advantage of the larval zebrafish, where BLECs were first described. Zebrafish make an excellent model for studying the link between BLEC function and sleep because the zebrafish larvae are optically transparent. This allows for the direct, non-invasive observation of BLECs, which are easily visible on top of the brain by using genetics to put fluorescent proteins specifically into these cells. The superficial location of BLECs also make them accessible to ablation with a laser, which allows us to test what happens when BLECs are no longer present. Finally, zebrafish larvae also have daily sleep/wake cycles that are largely regulated in a manner similar to humans, allowing us to examine how BLECs change over the 24-hour rhythm as well as during sleep deprivation or in response to sleep-altering drugs.First, we will use microscopes to watch the zebrafish BLECs across the 24-hour day and measure how their size, position, shape, and connectivity changes. Inside each cell, BLECs form many large, round inclusions as they internalize material; we will also measure how these inclusions change over time. To test whether daily internal rhythms or sleep/wake states change BLEC form and function, we will also see how BLECs respond to constant light, sleep deprivation, or sleep-altering drugs. Next, we will inject dye into the zebrafish brain and watch BLECs internalize the dye. By measuring the rate of uptake (which rapidly occurs even within minutes) at different times of day or sleep/wake states, we will gain a better understanding of when BLECs are most active. Finally, we will ablate the BLECs with lasers or alter their uptake capacity by using genetics to eliminate key genes involved in BLEC function. Then we will observe zebrafish sleep using cameras to track their behaviour and see if altering BLECs leads to a change in behaviour. At the end of this project, we will have a new understanding of how BLECs change either form and function across the 24-hour day and how BLECs' ability to clear toxic by-products impacts brain function and behaviour.