Understanding suppression of transcytosis in formation of the blood-brain barrier (BBB) and how Calcrl/Ramp2 signalling limits BBB permeability

The aim of this research is to understand how the leakiness, or permeability, of blood vessels in the brain are controlled so that we can identify new ways to treat diseases where these vessels become leaky. Blood vessels transport fluids and cells around our body. To do this effectively, the ability of substances, including liquids and cells, to pass through blood vessels, or permeability, must be tightly controlled to prevent leakage. Blood vessels within the brain are unique because they are the least leaky of all vessels within the body and so form a barrier between the circulating blood and the brain known as the blood-brain barrier (BBB). The BBB protects the brain from substances in the blood which could damage it, or could cause brain infection, while allowing important nutrients to reach the brain. In many diseases such as diabetes and stroke, increased blood vessel permeability within the brain causes damage or build-up of fluid that induces swelling. This can lead to organ failure and even death. Blood vessel permeability is controlled by signals within the body, which make vessels more, or less leaky by altering how tightly blood vessel cells stick to each other, or by controlling how easily substances can pass through blood vessel walls.

For over 100 years, BBB leakiness was thought to be mainly controlled by the ability of blood vessel cells to stick tightly to each other, reducing substances passing from the blood into the brain. More recently, it has become clear that to form the BBB, blood vessel cells within the brain actively use signals which stop substances passing through vessel cell walls into the brain. In animal models of stroke, transport of substances through vessel cell walls is increased and is an early sign of barrier breakdown. This arises before stickiness of blood vessel cells deteriorates and bleeding into the brain occurs. The signals which reduce vessel leakiness are not well understood and there are no treatments to effectively reduce this in humans.

We use zebrafish to understand how blood vessel leakiness is controlled since we can label blood vessels fluorescently. This allows us to observe vessel leakiness in a living organism. Importantly, zebrafish and humans share many of the signals and mechanisms which control blood vessel leakiness. We have made zebrafish that have leaky blood vessels within the brain. Using these zebrafish we have identified a new signal (Calcrlb/Ramp2a) which reduces vessel leakiness by making it harder for substances to pass through blood vessel walls into the brain. In humans, differences in Ramp2 and Calcrl are linked with increased risk of stroke. We want to understand precisely how Ramp2a/Calcrlb prevent vessels becoming leaky because this may allow us to identify new ways of treating vessel leakiness in people with diseases such as stroke, diabetes, or vascular dementia.

Using our zebrafish with reduced Calcrlb/Ramp2a signals (these signals normally prevent blood vessels from becoming leaky), we will anaesthetise zebrafish embryos, inject small amounts of fluorescent dye into their blood vessels, or fluorescently label their blood vessels, and measure the amount of leakiness using our lightsheet microscope. The dyes we use are very large and cannot leak from normal blood vessels. We will also treat our zebrafish Calcrlb/Ramp2a mutant embryos with drugs that reduce the ability of substances to pass through blood vessel walls and measure how leaky the blood vessels are afterwards. We can also label the places where blood vessel cells are joined together in our Ramp2a/Calcrlb embryos and by using a very high magnification electron microscope, see if these areas are abnormal when the blood vessels become leaky. We will also identify which signals are increased and reduced in brain blood vessels in our Calcrlb/Ramp2a mutants. These experiments will tell us exactly how Calcrlb/Ramp2a signals prevent brain blood vessels from becoming leaky.

The blood-brain barrier (BBB) is a highly selectively permeable network of blood vessels within the brain. The BBB is essential for central nervous system (CNS) homeostasis by controlling molecular flux between the blood and the brain and protecting it from pathogens and toxins. Selective permeability of the BBB is controlled by the presence of endothelial tight junctions and supportive non-endothelial mural cells which limit permeability. Unusually low levels of vesicle trafficking at the BBB recently led to identification of genetic programs which establish a functional barrier by active suppression of transendothelial permeability, or transcytosis. This indicates that CNS endothelial cells (ECs) possess a developmental programme which actively suppresses transcytosis during BBB formation. We have only limited knowledge of the basic biology of BBB transcytosis, and the real-time dynamics of BBB transcytosis are unknown.

We have identified a genetic program in zebrafish ECs which limits transcytosis in the BBB via the Calcrl/Ramp2 G-Protein Coupled Receptor (GPCR) complex. Zebrafish calcrl and ramp2 mutant embryos display extravasation of high molecular weight dyes from the BBB but exhibit normal blood and lymphatic vessel formation at these stages. We hypothesise that signalling via the Calcrl/Ramp2 GPCR receptor complex promotes BBB function by suppressing transcytosis in ECs. However, the mechanism by which Calcrl/Ramp2 suppresses transcytosis is unknown.

We propose to test this hypothesis by determining contribution of mural cells, transcellular and paracellular permeability to Calcrl/Ramp2-mediated development of BBB permeability in zebrafish embryos. We will determine mode of extracellular vesicle release suppressed by Calcrl/Ramp2 signalling and identify the transcellular transport pathways downstream of Calcrl/Ramp2 which limit BBB transcytosis. These studies will identify precisely how Calcrl/Ramp2 signalling controls barrier function within the BBB