Comparative and functional analysis of brain lymphatic endothelial cells

大脑是一个能量密集的器官，在使用过程中会产生大量代谢废物。尽管清除有毒副产物的重要性，其积累是神经退行性疾病的主要贡献者，但大脑如何完成这种清除却知之甚少。在身体的其他部位，专门的淋巴管维持液体的平衡，并帮助清除组织和器官中不需要的副产品。从历史上看，大脑被认为是缺乏专门的淋巴系统。近年来，脑液平衡和废物清除的几种机制已经被开发出来，新发现的脑膜淋巴管可能在这一过程中发挥关键作用。该系统的确切工作原理仍然存在争议;然而，脑膜炎可能有助于脑液稳态并对健康老龄化产生影响。我们最近在覆盖斑马鱼大脑（脑膜）的组织中发现了另一种类型的神经胶质样细胞，我们将其命名为脑淋巴内皮细胞（BLECs）。与形成管状血管的传统淋巴组织不同，BLEC形成了一组松散连接的细胞，能够从脑液中摄取大分子。由于BLECs位于一个非常适合参与脑清除的解剖位置，并迅速内化注入大脑的各种分子，我们认为BLECs对于清除大脑活动过程中产生的废物很重要。在这个项目中，我们将首先确定BLECs是否存在于小鼠和人类的脑膜中，然后将在斑马鱼中进行一系列功能研究，以检查BLECs如何响应并反过来影响大脑活动。我们将检查小鼠和人类脑膜中BLECs的存在，我们可以通过其特有的分子组成和超微结构特征将其与其他细胞区分开来。我们工作的初步数据（参见支持案例）表明BLEC确实在小鼠中保守，可能在人类中也是如此。然后我们将转向斑马鱼进行一系列实验来测试BLECs和大脑活动之间的关系。斑马鱼是研究BLECs的一个很好的模型，因为它们在幼虫阶段是光学透明的，这允许直接，非侵入性地观察大脑，包括BLECs，我们在斑马鱼中用遗传编码的荧光蛋白标记了BLECs。斑马鱼幼体也有每天的睡眠/觉醒周期，这使我们能够研究BLECs在24小时节律以及睡眠剥夺期间的变化。首先，我们将观察BLECs从大脑中摄取大分子的速率，使用直接注射到大脑中的荧光染料来可视化BLECs的内化。我们将专门测试BLECs的清除率是否在睡眠期间增加，这是一个针对小鼠大脑清除的想法。我们还将观察BLECs如何响应神经元活动的变化，例如，在响应视觉刺激，在癫痫发作的极端活动期间，或在长时间睡眠剥夺之后。最后，我们将消融BLECs，并询问长时间活动后大脑的恢复是否受到干扰，例如导致剥夺后更长时间的反弹睡眠或癫痫发作后恢复期增加。在本项目结束时，我们将确定BLECs是否存在于小鼠和人类脑膜中。我们还将对BLECs的可能功能有新的认识，包括它们作为清除活跃大脑中有毒副产物的支持系统的假定作用。如果BLECs对大脑清除至关重要，那么它们有一天可能成为对抗衰老疾病的合适治疗靶点，例如通过增强其天然的大脑清除功能。

The brain is an energetically intensive organ that produces a large amount of metabolic waste during use. Despite the importance of the removal of toxic byproducts whose buildup is a major contributor to neurodegenerative disease, how the brain accomplishes this removal is poorly understood. In other parts of the body, specialized lymphatic vessels maintain the balance of fluid and to assist in the clearance of unwanted byproducts from tissues and organs. Historically, the brain was thought to be devoid of a dedicated lymphatic system. In recent years, several mechanisms for brain fluid balance and waste clearance have been developed, and newly discovered meningeal lymphatic vessels may play a key role in this process. Exactly how the system works remain controversial; however, it is probable that meningeal lymphatics contribute to brain fluid homeostasis and have an impact on healthy ageing. We have recently discovered another type of lymphatic-like cell in the tissue covering the brain (the meninges) of zebrafish, which we have named Brain Lymphatic Endothelial Cells, or BLECs. Unlike traditional lymph tissues, which form tubular vessels, BLECs form a loosely connected set of cells that are capable of taking up large molecules from the fluid of the brain. Because BLECs reside in an anatomical location well suited to participate in brain clearance and rapidly internalize a variety of molecules injected into the brain, we propose that BLECs are important for the removal of waste products created during brain activity. In this project, we will first determine whether BLECs are found in mouse and human meninges and then will perform a series of functional studies in zebrafish to examine how BLECs respond to, and in turn affect, brain activity.We will examine mouse and human meninges for the presence of BLECs, which we can distinguish from other cells by their characteristic molecular makeup and ultrastructural features. Preliminary data from our work (see Case for Support) indicates that BLECs are indeed conserved in mice, and possibly in humans. Then we will turn to the zebrafish to perform a series of experiments to test the relationship between BLECs and brain activity. Zebrafish make an excellent model for studying BLECs because they are optically transparent in larval stages, which allows for the direct, non-invasive observation of the brain, including BLECs, which we have labelled in zebrafish with genetically encoded fluorescent proteins. Zebrafish larvae also have daily sleep/wake cycles, allowing us to examine how BLECs change over the 24 hour rhythm as well as during sleep deprivation.First, we will observe the rates by which BLECs take up macromolecules from the brain, using fluorescent dyes that are directly injected into the brain to visualize internalization by BLECs. We will specifically test whether the rate of clearance by BLECs is increased during sleep, an idea that has been proposed for mouse brain clearance. We will also observe how BLECs respond to changes in neuronal activity, for example, in response to visual stimulation, during the extreme activity of seizures, or following prolonged sleep deprivation. Finally, we will ablate BLECs and ask whether the recovery of the brain following prolonged activity is disturbed, for example leading to a longer period of rebound sleep following deprivation or an increased recovery phase after a seizure.At the end of this project, we will have determined whether BLECs are present in mouse and human meninges. We will also have gained new insights into possible functions of BLECs, including their putative roles as a support system for clearing toxic byproducts from the active brain. If BLECs are critical for brain clearance, they may one day be suitable therapeutic targets for combating diseases of ageing, for example by boosting their natural brain clearing functions.