Structural and molecular basis of choline uptake into the brain by FLVCR2
By Nitin Vuppalapu
Choline is an essential nutrient for our brain, meaning our bodies can’t make it and so must obtain it from the food we eat. This molecule is an important factor in the production of acetylcholine, a neurotransmitter released by our neurons that controls memory and muscle control, and for making phosphatidylcholine, which is the most abundant lipid in all of our cell membrane. How does it get from our gut to our brain? It gets into our blood vessels and then travels up to the brain, but there it has to cross the blood-brain barrier (BBB). It is well known that the endothelial cells that line blood vessels in the brain are special. They don’t allow many substances to enter the brain, and this is thought to protect our brain and all its delicate neuronal pathways. These properties are what we call the BBB. But some molecules, including nutrients like choline, need to be transported across the BBB and into the brain by specialized transporter proteins, because the brain can’t make them itself. The identity of the BBB transporter that allows choline to enter the brain has stumped researchers for many years. Two potential choline transporters based on previous research include FLVCR1 and ChT. However, neither of these are found in BBB endothelial cells. In this study, researchers reveal that FLVCR2 – a protein highly related to FLVCR1 (sharing 55% amino acid sequence identity) – is a choline transporter expressed in BBB endothelial cells (cells that make up the lining of your blood vessels).
To determine FLVCR2’s location within the cell, the authors looked in tissue with a technique called immunofluorescence labeling (a method that fluorescently labels specific biological targets within a tissue sample). Using this method, scientists found that FLVCR2 is present exclusively in endothelial cells of all brain vascular segments. They found that FLVCR2 is expressed in the plasma membrane of these cells. This plasma membrane is a lipid bilayer that separates the endothelial cell from the outside space. For brain endothelial cells, one side of the cell faces the blood (the luminal membrane) while the other side faces the brain (the abluminal membrane) one side of the membrane cell content from its surroundings. They found that FLVCR2 is expressed on both the luminal and abluminal membranes of the brain endothelial cells, but with more expression on the side that faces the blood – an ideal location for importing choline from the circulation into the brain.
To verify that FLVCR2 actually transport choline across the BBB, the research group measured the uptake of radiolabeled choline in the brains of FLVCR2-conditional knockout mice (mice that don’t have the protein) and compared it to a control group that had the protein. Results showed that the control group had significantly more absorption of choline into the brain than the control group, indicating that FLVCR2 indeed plays a role in choline transport. To understand the protein’s mechanisms and structure, they used cryogenic electron microscopy (cryo-EM) which involves freezing the protein in vitreous ice and bombarding it with electrons to produce millions of 2D images of the protein from different angles that can then be used to reconstruct a 3D structure of the protein with a very high amount of detail. These structures enable the researchers to see that Aromatic amino acids provide a binding site for the positively charged choline molecule to bind in and be stabilized through cation-pi interactions. This binding pocket looked as though it might be able to bind other similar molecules, and so the researchers tested this, and discovered that other molecules that look similar to choline can also bind here. A possible goal of the scientific community could be to attain a better understanding of FLVCR2 and verify whether the protein is responsible for the uptake of other molecules into the brain. This area of study would guide the development of new treatments for neurological disorders that can cross the BBB via an FLVCR2-mediated mechanism, and reach their site of action in the brain.
It may seem ordinary for research like this to be performed, but in reality, this study is extremely precise and truly astonishing once put into perspective. Thanks to the CRISPR-Cas9 discovery, scientists can alter the language of life itself; the idea that we now have the ability to change the genetic makeup of an organism is astonishing to grasp and its revelation has expanded all fields of research. This technology enabled the researchers in this case to eliminate the protein from brain vascular cells in mice and discover what the protein was doing. The analysis of the protein structure uses cryo-EM which allows us to find the exact protein structure down to the exact amino acid and allows us to understand how the protein binds choline and allows it to be transported into the brain. With this, we can analyze the various intermolecular interactions within the protein which would help us determine its properties. Although it may seem like we have found a simple mechanism for the uptake of a molecule in the brain, through further research this discovery can become a stepping-stone for developing future therapies for brain diseases and help future generations to come.
Q&A with Dr. Dibyanti Mukherjee and Dr. Rosemary Cater (authors of the article)
Q: You show that FLVCR2 is expressed on the luminal side of endothelial cells. How do you think choline gets from the other side to other cells of the brain?
Dr. Mukherjee:
We do not know exactly how choline gets out of endothelial cells to the other side of the brain. Flvcr2 is expressed on both sides of endothelila cells (luminal more and abluminal less). It is possible that flvcr2 functions as both exporter and importer of choline. Or it might be possible that there are some other exporters of choline present in brain ECs, but they have not yet been identified. So, our next goal after this paper was to find out how choline gets exported from the endothelial cell and into the brain parenchyma and if Flvcr2 plays a role in this process.
Dr. Cater:
While our data demonstrate the FLVCR2 is mostly expressed on the luminal membrane, it is also expressed on the abluminal membrane – see Figure 1.
So, the hypothesis can go one of two ways:
FLVCR2 can also export choline from endothelial cells across the abluminal membrane into the brain parenchyma
Or, there is another choline transporter expressed on the abluminal membrane.
There is data out there from other researchers demonstrating that FLVCR2 can also act as an exporter so this would be in support of the first hypothesis here. i.e., the transporter just works with the concentration gradients – to always allow choline to flow down its concentration gradient. So while this may be the case, it is complicated by the proton dependence we observe, which to be honest with you I do not know the explanation for – the proton dependency we observe is a bit confusing because there is no substantial proton gradient across the plasma membrane and there are no charged residues in the central cavity of FLVCR2 to carry the protons – so while our data do strongly demonstrate a proton dependency, the molecular mechanism by which this occurs is still a puzzle to me! Perhaps it is not a tight coupling, more research needs to be done to understand this, but there is a chance that FLVCR2 on the abluminal membrane also exports choline out of the endothelial cell into the brain parenchyma…. However, in saying this… what would drive this transport to ONLY be across the abluminal membrane, if FLVCR2 is simply a uniporter then what would be encouraging choline to be exported via FLVCR2 on the abluminal membrane rather than the luminal membrane and just back out into the blood? As you can tell, this is something I think about a lot and a fantastic question that still requires further investigation. As for the second hypothesis – there may be other choline transporters expressed on the abluminal membrane, but this has not been thoroughly investigated yet.
Q: What do you know about the role of this gene in the human brain?
Dr. Mukherjee:
Flvcr2 plays a very important role in brain angiogenesis during development. Angiogenesis occurs through tip/stalk selection. Tip cells are non-dividing and are present at the beginning of the blood vessels where they form sprouts helps in penetrating the tissue while stalk cells are dividing cells, and they are at the back of tip cells forming the lumen of blood vessels. We have found that during development if we delete Flvcr2 from endothelial cells then the endothelial cells show abnormal angiogenesis meaning they will not form the tip cells but rather form more stalk cells. This indicates that Flvcr2 helps in tip/stalk selection and therefore promotes angiogenesis in brain development.
Dr. Cater:
As for the role of this transporter in human brain function, we find this something very exciting. We know that the gene is involved in blood vessel development in the brain, indeed mutations in this transporter cause severe cerebrovascular malformations and fetal death/newborn death – without blood vessels in the brain, the brain can simply not function properly as it can’t get all the nutrients and oxygen it needs to grow. These mutations also cause hydrocephalus.
The substrate of the transporter (choline) is absolutely critical for cell development and growth. Our cells (including all cells in the brain and BBB endothelial cells) need this choline to make phosphatidylcholine which is the most abundant lipid in the cell membrane – so as you can imagine, without choline we would simply not be able to make cell membranes, and so you wouldn’t be able to make the cells in our brain or the endothelial cells that line blood vessels in the brain – so it’s really important for brain development and growth – this fits in with what we see in the humans with mutations in the transporter – we can’t grow blood vessels in the brain without this transporter and the choline it supplies. This is also very important for myelination which is very rich in phosphatidylcholine. Of course, this choline is also used to make the important neurotransmitter acetylcholine. Phosphatidylcholine is also implicated in Alzheimer’s disease – and so there could be a link here.
Link to the article here: Article - Structural and molecular basis of choline uptake into the brain by FLVCR2
Figure legend: Choline uptake in the brain by FLVCR2 in endothelial cells.