Comparative and functional analysis of brain lymphatic endothelial cells

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.

The mechanisms by which the energetically intensive brain maintains tissue homeostasis remains a fundamental mystery. We propose to test the hypothesis that a newly discovered brain lymphatic endothelial cell (BLEC) is involved in solute clearance from the brain during periods of high neuronal activity. Understanding how this novel lymphatic-related cell population participates in brain clearance will provide insight into how dysfunctions in this system may contribute to diseases of ageing.

Our programme of study will firstly carry out molecular, immunohistochemical and ultrastructural studies to determine whether BLECs are present in mammalian, including human, meninges. This work will build on our preliminary data and firmly establish the evolutionary conservation of BLECs. To gain functional insight into BLECs, we will use the experimentally tractable, optically transparent zebrafish larvae, in which BLECs can be non-invasively 4D imaged during behavior using confocal and 2-photon microscopy. To manipulate neuronal activity, we will use both natural visual stimuli and 24-hour sleep-wake cycles, as well as pathological states, including sleep deprivation and seizure. Following behavioral manipulation, we will use 4D imaging to assess morphological changes and combine cell sorting and RNA sequencing to determine molecular changes in BLECs during behavior. We will also functionally assess macromolecule uptake by BLECs using dye injection and photobleaching techniques. Finally, to assess whether BLEC function is required for normal behavior, we will either laser or genetically ablate BLECs and quantify changes in the recovery of larval zebrafish locomotor behavior following sleep deprivation or seizure.

Who might benefit from this research?

We have identified numerous beneficiaries of our research, including direct collaborators, academic, industry, charity, and public stakeholders.

Academic beneficiaries include the research communities involved in studying sleep, lymphatics, neuroscience, neuroanatomy, immunology, healthy ageing, and neurodegenerative disease.

Although our work is basic research, industry stakeholders may benefit in the longer term. Industrical beneficiaries may include those who work on healthy ageing, especially if our research clearly implicates BLECs in the clearance of toxic by-products from the brain.

Charity stakeholders include the Alzheimer's Research UK, with whom we have an existing relationship, as well as other charities involved in diseases of ageing.

The public is the largest stakeholder and ultimately is the source of funding for this research. This group includes concerned patients and their families who are personally invested in gathering more information regarding neurodegenerative disease, the general public who are interested in healthy ageing, schoolchildren who are engaging in scientific experiments which develop into careers, and countless others. Public interest is growing, especially with the increase in the aging population within Britain in the last decade.

How might they benefit from this research?

These groups may benefit from our work on many levels. Our direct collaborators will benefit from up-to-date insights into the molecular and functional characteristics of BLECs and their roles, which will impact and inform their own experiments. We will cultivate a strongly collaborative atmosphere, keeping everyone in regular contact through email, skype, and telephone, and have also budgeted lab visits to physically share techniques and ideas in real time.

The wider academic community will benefit from a greater insight into how BLECs, the lymphatic system, and the brain interact to impact brain function and development. Please see "academic beneficiaries" as well as the "Pathways to Impact" sections for more details.

Our work is predominantly basic research, but, long-term we envision that brain clearance (in which BLECs may have a role) could become a potential target for drug discovery, with pharmaceutical and clinical industry stakeholders. If so, these groups will directly benefit from the public availability of our datasets on the molecular characteristics of BLECs, which may assist in the design of drug discovery pipelines. They will also benefit from the experimental validation of these cells as a therapeutic target worth investigating.

Charities involved in neurodegenerative disease will benefit from this work by gaining new insights into a previously unknown process that may be involved in disease.

We will continue to work with the ARUK to keep donors, patients, doctors, and academics who partner with these charities informed of our work through lab visits, talks, and other communication.

The public will benefit from a greater fluency in scientific concepts and an appreciation for how basic research may impact health in a translational manner. We will continue to engage the public through lay talks, interviews, websites and blog posts, and school visits.