Comparative and functional analysis of brain lymphatic endothelial cells

点击翻译按钮获取中文摘要

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.