Comparative and functional analysis of brain lymphatic endothelial cells

脑淋巴内皮细胞的比较和功能分析

基本信息

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

项目摘要

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.

大脑是一个能量密集型器官，在使用过程中会产生大量代谢废物。尽管清除有毒副产物很重要，这些副产物的积累是神经退行性疾病的主要促成因素，但人们对大脑如何实现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平衡并协助清除组织和器官中不需要的副产物。从历史上看，人们认为大脑缺乏专门的淋巴系统。近年来，已经开发出几种脑液平衡和废物清除机制，新发现的脑膜淋巴管可能在这一过程中发挥关键作用。该系统到底如何运作仍存在争议。然而，脑膜淋巴管可能有助于脑液稳态并对健康衰老产生影响。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淋巴样细胞，我们将其命名为脑淋巴内皮细胞（BLEC）。与形成管状血管的传统淋巴组织不同，BLEC 形成一组松散连接的细胞，能够从脑液中吸收大分子。由于 BLEC 位于非常适合参与大脑清除并快速内化注入大脑的各种分子的解剖位置，因此我们认为 BLEC 对于清除大脑活动期间产生的废物非常重要。在这个项目中，我们将首先确定小鼠和人类脑膜中是否存在 BLEC，然后在斑马鱼中进行一系列功能研究，以检查 BLEC 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BLEC 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 在小鼠中确实保守，也可能在人类中保守。然后我们将转向斑马鱼进行一系列实验来测试 BLEC 与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，这使得可以直接、非侵入性地观察大脑，包括 BLEC，我们在斑马鱼中用基因编码的荧光蛋白标记了 BLEC。斑马鱼幼虫也有每日睡眠/觉醒周期，这使我们能够检查 BLEC 在 24 小时节律以及睡眠剥夺期间如何变化。首先，我们将使用荧光染料观察 BLEC 从大脑中摄取大分子的速率，直接注射到大脑中以可视化 BLEC 的内化。我们将专门测试 BLEC 的清除率是否在睡眠期间增加，这是针对小鼠大脑清除提出的想法。我们还将观察 BLEC 如何响应神经元活动的变化，例如，对视觉刺激、癫痫发作的极端活动期间或长期睡眠剥夺后的反应。最后，我们将消融 BLEC，并询问长时间活动后大脑的恢复是否受到干扰，例如导致睡眠剥夺后恢复时间更长，或癫痫发作后恢复阶段增加。在该项目结束时，我们将已确定 BLEC 是否存在于小鼠和人类脑膜中。我们还将对 BLEC 的可能功能获得新的见解，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 BLEC 对大脑清除至关重要，那么有一天它们可能会成为对抗衰老疾病的合适治疗靶点，例如通过增强其自然的大脑清除功能。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
作者：
通讯作者：

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