Comparative and functional analysis of brain lymphatic endothelial cells

脑淋巴内皮细胞的比较和功能分析

• 批准号: BB/T001844/1
• 负责人: Jason Rihel
• 金额: $ 69.08万
• 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: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT001844%2F1
• 关键词: Comparative; functional; analysis; brain; lymphatic

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.

大脑是一种充满活力的器官，在使用过程中会产生大量的代谢废物。尽管取消有毒的副产品的重要性很重要，这些副产品的积聚是导致神经退行性疾病的主要因素，但大脑如何完成这种去除的理解很少。在人体的其他部位，专门的淋巴管保持液体的平衡，并协助清除组织和器官的不良副产品。从历史上看，大脑被认为没有专用的淋巴系统。近年来，已经开发了几种用于脑液平衡和废物清除的机制，新发现的脑膜淋巴管可能在此过程中起关键作用。系统的工作原理恰好存在争议。但是，脑膜淋巴管可能会导致脑液体稳态，并对健康的衰老产生影响。我们最近在覆盖斑马鱼的脑（脑膜）的组织中发现了另一种类型的淋巴样细胞，我们将其命名为脑淋巴内皮细胞或BLEC。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，能够从大脑的液体中吸收大分子。由于BLEC居住在一个非常适合参与大脑清除的解剖位置，并迅速将各种注入大脑的分子内化，因此我们建议BLEC对于去除在大脑活动期间产生的废物产物很重要。在该项目中，我们将首先确定是否在小鼠和人类销售中发现了BLEC，然后将在斑马鱼中进行一系列功能研究，以检查BLEC对BLEC的反应以及对BLEC的存在的响应，进而研究小鼠和人类脑膜的存在，我们可以通过其特征性的分子构成和超构件特征来区分其他细胞。我们工作的初步数据（请参阅支持案例）表明，BLEC确实在小鼠和人类中保守。然后，我们将转向斑马鱼进行一系列实验，以测试BLEC和大脑活动之间的关系。斑马鱼是研究BLEC的绝佳模型，因为它们在幼虫阶段是光学透明的，它允许对大脑进行直接的，无创的观察，包括BLEC，我们已经在斑马鱼中标记了具有遗传编码的荧光蛋白。斑马鱼幼虫也每天都有睡眠/唤醒周期，使我们能够检查BLEC在24小时的节奏中以及睡眠剥夺期间如何变化。首先，我们将观察BLEC在大脑中使用大脑的大分子，使用直接注入大脑的荧光染料，以直接注入大脑的荧光染料，以可将大脑注入蓝色的内在化。我们将特别测试睡眠期间BLEC的清除率是否增加，这是为小鼠脑清除率提出的想法。我们还将观察BLEC如何应对神经元活性的变化，例如，在癫痫发作的极端活性或长时间睡眠剥夺之后，视觉刺激响应于视觉刺激。最后，我们将消除BLEC，并询问长期活性后大脑的恢复是否受到干扰，例如导致剥夺后较长的反弹睡眠或癫痫发作后的恢复阶段增加。在该项目的结束时，我们将确定BLEC是否存在于小鼠和人类销售中。我们还将获得对BLEC可能功能的新见解，包括它们作为清除活性大脑有毒副产品的支持系统的推定作用。如果BLEC对于大脑清除至关重要，则有一天，它们可能是对抗衰老疾病的合适治疗靶标，例如，通过增强其自然脑清除功能。

项目成果

Loss of slc39a14 causes simultaneous manganese hypersensitivity and deficiency in zebrafish.

• DOI: 10.1242/dmm.044594
• 发表时间: 2022-06-01
• 期刊: Disease models & mechanisms
• 影响因子: 4.3

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.

• DOI: 10.7554/elife.59683
• 发表时间: 2021-01-08
• 期刊: eLife
• 影响因子: 7.7
• 作者: Kroll F;Powell GT;Ghosh M;Gestri G;Antinucci P;Hearn TJ;Tunbak H;Lim S;Dennis HW;Fernandez JM;Whitmore D;Dreosti E;Wilson SW;Hoffman EJ;Rihel J
• 通讯作者: Rihel J

Loss of slc39a14 causes simultaneous manganese hypersensitivity and deficiency in zebrafish

slc39a14 缺失导致斑马鱼同时出现锰过敏和缺乏

• DOI: 10.5167/uzh-218619
• 发表时间: 2022
• 作者: Tuschl, Karin

The zebrafish as a novel model for the in vivo study of Toxoplasma gondii replication and interaction with macrophages

• DOI: 10.1242/dmm.043091
• 发表时间: 2020-07-01
• 期刊: DISEASE MODELS & MECHANISMS
• 影响因子: 4.3
• 作者: Yoshida, Nagisa;Domart, Marie-Charlotte;Mostowy, Serge
• 通讯作者: Mostowy, Serge