Resolving the Role of Brain Lymphatic Endothelial Cells in Sleep Dependent Brain Clearance
Lead Research Organisation:
University College London
Department Name: Cell and Developmental Biology
Abstract
The brain is the most metabolically active organ in the body, which generates a lot of waste that must be removed. However, scientists still do not fully understand how the brain clears waste. In other parts of the body, excess fluid and metabolic waste are cleared in part by a network of vessels called lymphatics, but the brain lacks lymphatic vessels. Instead, the brain is thought to undergo a process called glymphatic clearance, in which fluid inside the brain is cleared of waste by exchanging with the cerebrospinal fluid (CSF) that bathes the outside of the brain. This washing process is thought to fluctuate across the 24-hour day, predominately occurring during sleep. The exact drainage routes for waste to leave the brain are still debated, but one idea is that waste then drains into lymphatic vessels that reside in the outer layer of the meninges (the layered tissues surrounding the brain).
Recently, another potential player in brain clearance was discovered, a cell type called brain lymphatic endothelial cells (BLECs) that reside in the inner part of the meninges. These cells are not only well positioned to participate in brain clearance, as they are surrounded by CSF, but they also have a very strong capacity to rapidly internalize substances that are injected into the brain. However, whether BLEC function fluctuates across the 24-hour cycle and whether BLECs are required for the clearance of material that builds up in the brain during waking is unknown. In this project, we will first observe how BLECs change shape and function across the day and night, and then we will test if disruption of BLEC function impacts behaviours like sleep. Since the build-up of toxic molecules in the brain has been implicated in neurodegenerative disease, this work will have important implications for healthy aging.
To observe BLECs across the day-night cycle, we will take advantage of the larval zebrafish, where BLECs were first described. Zebrafish make an excellent model for studying the link between BLEC function and sleep because the zebrafish larvae are optically transparent. This allows for the direct, non-invasive observation of BLECs, which are easily visible on top of the brain by using genetics to put fluorescent proteins specifically into these cells. The superficial location of BLECs also make them accessible to ablation with a laser, which allows us to test what happens when BLECs are no longer present. Finally, zebrafish larvae also have daily sleep/wake cycles that are largely regulated in a manner similar to humans, allowing us to examine how BLECs change over the 24-hour rhythm as well as during sleep deprivation or in response to sleep-altering drugs.
First, we will use microscopes to watch the zebrafish BLECs across the 24-hour day and measure how their size, position, shape, and connectivity changes. Inside each cell, BLECs form many large, round inclusions as they internalize material; we will also measure how these inclusions change over time. To test whether daily internal rhythms or sleep/wake states change BLEC form and function, we will also see how BLECs respond to constant light, sleep deprivation, or sleep-altering drugs. Next, we will inject dye into the zebrafish brain and watch BLECs internalize the dye. By measuring the rate of uptake (which rapidly occurs even within minutes) at different times of day or sleep/wake states, we will gain a better understanding of when BLECs are most active. Finally, we will ablate the BLECs with lasers or alter their uptake capacity by using genetics to eliminate key genes involved in BLEC function. Then we will observe zebrafish sleep using cameras to track their behaviour and see if altering BLECs leads to a change in behaviour.
At the end of this project, we will have a new understanding of how BLECs change either form and function across the 24-hour day and how BLECs' ability to clear toxic by-products impacts brain function and behaviour.
Recently, another potential player in brain clearance was discovered, a cell type called brain lymphatic endothelial cells (BLECs) that reside in the inner part of the meninges. These cells are not only well positioned to participate in brain clearance, as they are surrounded by CSF, but they also have a very strong capacity to rapidly internalize substances that are injected into the brain. However, whether BLEC function fluctuates across the 24-hour cycle and whether BLECs are required for the clearance of material that builds up in the brain during waking is unknown. In this project, we will first observe how BLECs change shape and function across the day and night, and then we will test if disruption of BLEC function impacts behaviours like sleep. Since the build-up of toxic molecules in the brain has been implicated in neurodegenerative disease, this work will have important implications for healthy aging.
To observe BLECs across the day-night cycle, we will take advantage of the larval zebrafish, where BLECs were first described. Zebrafish make an excellent model for studying the link between BLEC function and sleep because the zebrafish larvae are optically transparent. This allows for the direct, non-invasive observation of BLECs, which are easily visible on top of the brain by using genetics to put fluorescent proteins specifically into these cells. The superficial location of BLECs also make them accessible to ablation with a laser, which allows us to test what happens when BLECs are no longer present. Finally, zebrafish larvae also have daily sleep/wake cycles that are largely regulated in a manner similar to humans, allowing us to examine how BLECs change over the 24-hour rhythm as well as during sleep deprivation or in response to sleep-altering drugs.
First, we will use microscopes to watch the zebrafish BLECs across the 24-hour day and measure how their size, position, shape, and connectivity changes. Inside each cell, BLECs form many large, round inclusions as they internalize material; we will also measure how these inclusions change over time. To test whether daily internal rhythms or sleep/wake states change BLEC form and function, we will also see how BLECs respond to constant light, sleep deprivation, or sleep-altering drugs. Next, we will inject dye into the zebrafish brain and watch BLECs internalize the dye. By measuring the rate of uptake (which rapidly occurs even within minutes) at different times of day or sleep/wake states, we will gain a better understanding of when BLECs are most active. Finally, we will ablate the BLECs with lasers or alter their uptake capacity by using genetics to eliminate key genes involved in BLEC function. Then we will observe zebrafish sleep using cameras to track their behaviour and see if altering BLECs leads to a change in behaviour.
At the end of this project, we will have a new understanding of how BLECs change either form and function across the 24-hour day and how BLECs' ability to clear toxic by-products impacts brain function and behaviour.
Technical Summary
The brain is the most energetic organ, yet how the brain clears waste is poorly understood. One hypothesis posits that sleep-dependent exchange between the brain's interstitial fluid and cerebrospinal fluid (CSF) is an important clearance mechanism, but the routes and mechanisms involved in this clearance remain uncertain. Recently, brain lymphatic endothelial cells (BLECs, also called muLECs or FGPs) were described to reside in the meninges of zebrafish and other vertebrates, where they are capable of rapidly internalizing macromolecules from the CSF. We will now test the hypothesis that BLECs participate in either circadian or sleep-dependent clearance of macromolecules from the brain. We further hypothesize that BLEC function is important for removal of macromolecular waste that accumulates in the brain during wakefulness and that failure to remove this material will impact sleep and other behaviours.
Our research programme will first take advantage of the optical transparency of zebrafish larvae to observe how BLEC morphology, connectivity, and endosome generation change across the day-night cycle, under "free-running" conditions, during sleep deprivation, and in response to sleep-altering drugs. We will also use a brain-ventricle dye injection method to observe BLEC uptake rates at different times of day, lighting conditions, and sleep/wake manipulations. These experiments will allow us to dissect the contribution of internal circadian rhythms, direct effects of light, and the impact of sleep/wake internal states to BLEC form and function. Then, we will manipulate BLECs using genetics and laser ablations followed by multi-day videotracking to determine the impact of BLEC function on sleep/wake behavior. Together these experiments will link circadian rhythms or sleep/wake state directly to BLEC function and will determine whether and how alterations in BLEC activity impact the brain and behavior.
Our research programme will first take advantage of the optical transparency of zebrafish larvae to observe how BLEC morphology, connectivity, and endosome generation change across the day-night cycle, under "free-running" conditions, during sleep deprivation, and in response to sleep-altering drugs. We will also use a brain-ventricle dye injection method to observe BLEC uptake rates at different times of day, lighting conditions, and sleep/wake manipulations. These experiments will allow us to dissect the contribution of internal circadian rhythms, direct effects of light, and the impact of sleep/wake internal states to BLEC form and function. Then, we will manipulate BLECs using genetics and laser ablations followed by multi-day videotracking to determine the impact of BLEC function on sleep/wake behavior. Together these experiments will link circadian rhythms or sleep/wake state directly to BLEC function and will determine whether and how alterations in BLEC activity impact the brain and behavior.
