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

解决脑淋巴内皮细胞在睡眠依赖性脑清除中的作用

• 批准号: BB/Y001206/1
• 负责人: Jason Rihel
• 金额: $ 88.15万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY001206%2F1
• 关键词: Resolving; Role; Brain; Lymphatic; Endothelial

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.

大脑是体内最活跃的器官，它会产生许多必须清除的废物。但是，科学家仍然不完全了解大脑如何清除浪费。在身体的其他部位，多余的液体和代谢废物部分被称为淋巴管的血管网络清除，但大脑缺乏淋巴管。取而代之的是，人们认为大脑会经历一个称为淋巴间隙的过程，其中通过与沐浴大脑外部的脑脊液（CSF）交换来清除大脑内部的液体。人们认为，在整个24小时的一天中，这种洗涤过程主要发生在睡眠期间。仍然存在争议的废物排水路线，但一个想法是，废物随后流入淋巴管中，该血管位于脑膜外层（大脑周围的分层组织）中，发现了大脑清除中的另一个潜在潜在的参与者，一种称为脑淋巴结型内皮细胞（Blecs）的细胞类型，是构成内部一部分的一部分，是层次的一部分。这些细胞不仅可以很好地参与大脑清除率，因为它们被CSF包围，而且它们也具有很强的能力，可以快速地将注射到大脑的物质内化。但是，BLEC功能是否在整个24小时周期中波动，以及是否需要BLEC才能清除在唤醒过程中在大脑中积累的材料的清除率尚不清楚。在这个项目中，我们将首先观察BLEC如何在白天和晚上改变形状和功能，然后我们将测试BLEC功能的破坏是否会影响睡眠等行为。由于大脑中有毒分子的积累已经与神经退行性疾病有关，因此这项工作将对健康的衰老具有重要意义。要在整个昼夜周期中观察BLEC，我们将利用幼虫斑马鱼的优势，首先描述了BLEC。斑马鱼是研究BLEC功能与睡眠之间联系的绝佳模型，因为斑马鱼幼虫在光学上是透明的。这允许对BLEC进行直接的，无创的观察，通过使用遗传学将荧光蛋白专门放入这些细胞中，在大脑顶部很容易看到。 BLEC的表面位置也使它们可以通过激光消融，这使我们能够测试BLEC不再存在时发生的情况。最后，斑马鱼幼虫还具有每天的睡眠/唤醒周期，这些幼虫在很大程度上受到与人类相似的方式进行调节，使我们能够检查BLEC在24小时的节奏中以及在睡眠剥夺期间以及在改变睡眠药物的情况下如何变化。在每个细胞内，BLEC会在内部化材料内化时形成许多大型圆形包含物。我们还将衡量这些夹杂物如何随着时间而变化。为了测试每天的内部节奏或睡眠/唤醒状态是否会改变BLEC形式和功能，我们还将看到BLEC对恒定的光，睡眠剥夺或改变睡眠的药物的反应。接下来，我们将染料注入斑马鱼大脑，观看BLEC将染料内化。通过在一天中的不同时间或睡眠/唤醒状态下测量摄取速率（即使在几分钟之内迅速发生），我们将对BLEC何时最活跃的何时获得更好的了解。最后，我们将使用激光消除BLEC或通过使用遗传学消除涉及BLEC功能的关键基因来改变其吸收能力。然后，我们将使用摄像头观察斑马鱼睡眠来跟踪其行为，并查看改变BLEC是否导致行为的改变。在该项目结束时，我们将对BLEC在整个24小时内如何改变形式和功能有了新的了解，以及BLEC清除有毒副产品的能力如何影响大脑功能和行为。