A story about how α-synuclein changes the comings and goings of dopamine
A summary of Threlfell et al 2021 by senior author Katherine Brimblecombe
Please note that underlined words are fully described in the accompanying glossary.
The big picture
In the brain there are lots and lots of cells. These cells need to talk to each other to send messages from the brain to the body. Brain cells talk to each other by the first cell releasing a chemical (* in the pictures below )that can fit into special “locks” on the outside of cells. This special lock is called a “receptor”. To make sure the message is just the right volume, the right amount of the chemical must be released and then it needs to be taken away, so the receptor doesn’t keep sending the same message. Some cells recycle their chemicals by carrying them from the outside (where it can talk to other cells) back inside so it can be re-packaged ready to send the next message. Cells can fine-tune the messages by changing the amount of chemical released and how long it stays around for. Here we are explaining our new findings that shows how α-synuclein changes how dopamine is recycled by the dopamine-cells. We think these changes to how much dopamine is around and how long it stays around for could be important for our understanding of Parkinson’s.
Step 1: the chemical is released. Step 2: The chemical unlocks the receptor to send its message. Step 3: the chemical messenger gets taken away. Step 4: the chemical is recycled so it is ready to send another message. The “volume” of the message can be adjusted by changing the amount released (1) or the amount taken away (3). The message shouldn’t be too loud (B) or too quiet (C,D) but just right (A). Our new data shows how α-synuclein can make the message too quiet (D).
A bit of background
The main reason people with Parkinson’s have problems moving is because a group of brain cells die. The brain cells that die are supposed to release a chemical called “dopamine”. The reason the dopamine-cells die in people with Parkinson’s seems to have a lot of causes, and the amount a single reason makes a person get Parkinson’s still isn’t known. But the substance “α-synuclein” often seems to be a factor. We are still trying to understand what α-synuclein normally does in a cell to help it work, and why in Parkinson’s it stops helping and starts hurting the dopamine-cells. We have previously found that less dopamine is released from mice that have a lot of α-synuclein. We know that the amount of dopamine available to signal to other brain cells depends on how much is released and how quickly it is taken away. Dopamine is removed by a specific carrier called the “dopamine-transporter (DAT)”. The DAT sits on the outside of dopamine cells and carries dopamine that was released by the cell, back inside it. We also think that the DAT is important for controlling how much dopamine is released by the cell, but that dopamine isn’t directly carried from the cell to the outside by the DAT.
Some scientists have found that α-synuclein and DAT are often found close to each other and that if you change the amount or type of α-synuclein, the ability of DAT to carry dopamine also changes. Another thing that changes how DAT carries dopamine is cholesterol. Cholesterol is a substance that is very important for brain cells. Cholesterol is a key part of “myelin” which is the electrical insulation of the brain. Cholesterol is also found in cell “membranes”. Membranes are the outer layer of cells (like a skin), it is in the membrane where the DAT sits to carry dopamine from outside the cell back into it.
What we tried to find out
Is there less dopamine released from mice that have a lot of α-synuclein (1) because DAT carries it back into the dopamine-cell faster? (2) or does α-synuclein make the DAT better at dialling down the amount of dopamine to be released? (3) and is cholesterol the reason α-synuclein changes what DAT does?
The tools we used
We compared mice that make a lot of (normal human) α-synuclein and mice that make no (human or mouse) α-synuclein. “Fast-scan cyclic voltammetry (FCV)” is our main (favourite!) method. It lets us measure the amount of dopamine released and how long it stays around for. We can compare the amount of dopamine released between areas of the brain and between animals. We use an electric pulse to tell the dopamine cells to release dopamine in the same way that they do in our brains. We use drugs that we know stop certain bits of cell-machinery from working so we can see what their job is. “Molecular biology” is a group of techniques that we use to measure the amount of an important substance (e.g. α-synuclein) or which substances are near each-other. “ToF-SIMS” lets us measure the amount of cholesterol inside brain cells. We make lots of measurements in the same brain and from different animals to get an “average” measurement that we can compare between the mice with lots of α-synuclein and those with none. We use both male and female animals.
What we found
1) Is there less dopamine released from mice that have a lot of α-synuclein because DAT carries it back into the dopamine-cell faster?
First we checked that we still find that there is less dopamine released from mice that have a lot of α-synuclein, and found that we do see the same thing again J (picture 1a) (replicating your findings is an important step in science, it might feel like a waste of resources but in reality a single study doesn’t “prove” anything, we need to build a picture with lots of pieces of evidence to know how true a finding is.)
Next we measured how quickly the dopamine signal goes away in mice that have lots of α-synuclein and those with none (Picture 1b) and found that the dopamine signals from mice with lots of α-synuclein go away faster. This was a good start, but we wanted to use an extra measurement to make sure we weren’t seeing a difference because the two types of mice start with different amounts of dopamine. Imagine comparing who was faster in a race Usain Bolt or Mo Farah? E.g. It would depend on how far they running, and whether they had a rolling or a standing start (probably many other factors too!), so we need lots of different “races” to see how true our findings are.
So we compared how long the dopamine signals lasted when a little bit of dopamine is released when we use a little electric pulse; or a lot of dopamine is released when we use a big electrical pulse and drugs that dial up dopamine release (picture 1c). For these comparisons we make sure we match the measurements, so they have the same amount of dopamine to start with. We found that when we give a normal stimulation or a big stimulation (to make lots of dopamine come out), the signals go away faster in the mice that have a lot of α-synuclein than in those with none.
As a final check we used a special kind of graph (called a “Michaelis-Menten plot”)that compares a snap-shot of the fastest point of dopamine being carried away by the DAT at different starting dopamine amounts between the two types of mice (Picture 1d). This special graph told us that there might be more DATs in the mice with lots of α-synuclein, but that the DATs they have, don’t carry dopamine quite as quickly [For those interested: Lots of α-synuclein mice have a higher “Vmax” and a higher “Km” a nice overview is on YouTube about what Vmax and Km mean. The measurements we have made show that dopamine is carried away by DAT faster in mice with lots of α-synuclein, and this special graph gave us a clue how it might do it (to be continued…).
Picture 1: A) These wavelike lines are called “Dopamine transients”. They show the amount of dopamine we are measuring before, during and after we use an electric pulse to make the mouse brain release dopamine. The black arrow is when we give an electric pulse. You can see a flat baseline (red dotted line) before we give a pulse that goes up quickly and then slowly goes back to the baseline in an arc. We compare the highest points of these lines to see how much dopamine is released and the steepness of the curved line going back to baseline to see how quickly the dopamine is taken away. We compare these two measurements in mice with no α-synuclein (none, dotted lines) and the mice with lots of α-synuclein (Lots, solid lines). We measure the amount of dopamine lots of times in the same animal and then again in different animals to get an “average”. The shaded bit around the lines shows “the error”. The graph with lots of little circles shows the peak amount of dopamine for each measurement we take. We try to show our measurements in lots of different ways, especially at the start so it’s clear (especially to other scientists) how we got our measurements. The *** show that the difference in dopamine amounts we measure are “statistically significant”.
B) We can measure how steep the curved line going back to the baseline is using an “exponential decay curve”. When the curve is very steep and the line goes back to baseline very quickly you have a big k number, when the curve is shallow, and it takes a long time to go back to baseline you have a small k number. In the mice that have lots of α-synuclein the curves are steeper and go back to baseline faster than in the animals that have no α-synuclein.
C) instead of looking at the “k” for each time we measure dopamine we lined up all our dopamine measurements, so the highest points matched and then measured how steep the curve to baseline is. We did this for measurements when we gave a normal stimulation and when we gave a big stimulation to make lots of dopamine come out. Both times the curve is steeper for mice with lots of α-synuclein. The red box shows how we’ve zoomed in on curved part of the dopamine transient. D) This is a complicated graph that basically shows a snapshot of the steepest part curved line going back to baseline and the amount of dopamine it started with. We can then ask the computer to draw a line that looks like a “Michaelis-Menten plot” so it can tell us what numbers for “Vmax” and “Km” work for the measurements we tell the computer. Not one of our measurements are perfect, so we look at the numbers in lots of different ways to check they are all telling us more-or-less the same thing.
2) Is there less dopamine released from mice that have a lot of α-synuclein because α-synuclein makes the DAT better at dialling down the amount of dopamine to be released?
The DAT is a carrier that moves dopamine from the outside of the cell back inside the dopamine cell. For it to be good at its job at clearing dopamine from outside it needs to be linked to how much dopamine is released by the cell. Other scientists have shown that the DAT stops too much dopamine from being released. We can see how much the DAT dials down the amount of dopamine to be released by using drugs that stop the DAT from working. We used three different drugs that stop the DAT from working: cocaine, GBR12935 and nomifensine. All three of these drugs made the amount of dopamine released go up, showing that these anti-DAT drugs help dopamine be released, which means that normally DAT dials down how much dopamine is released. For all the anti-DAT drugs, dopamine release goes up even more in the animals that have lots of α-synuclein than the mice that have no α-synuclein (Picture 2a). This tells us that α-synuclein makes the DAT better at dialling down the amount of dopamine to be released.
To check that it makes sense that α-synuclein can change the way DAT works we asked our colleagues in the OPDC to see if α-synuclein and DAT are often close together. They used a technique called “proximal ligation assay (PLA)” and found that they are (picture 2b).
Next we wanted to test the clue that the special Michaelis-Menten plot gave us: that there are more DATs in the mice that have lots of α-synuclein. Our colleagues in the OPDC used molecular biology to let us count the amount of DATs. Using brain slices that are very thin and labelled with green fluorescent tags called “immunohistochemistry”. We find that there are more DATs in the mice that have lots of α-synuclein (Picture 2c). To make extra sure, we asked our colleagues in Pharmacology to count the number of DATs that can “see cocaine” using “radio-ligand binding”. They did this on very thin slices of brain and again we found that mice with lots of α-synuclein have more DATs than those with no α-synuclein (Picture 2d).
Picture 2: A) ) The dopamine transients show how much dopamine is released before (black lines) and after cocaine (blue lines) We used cocaine to stop the DAT from working. You can see that cocaine makes the highest part of the line go up a lot higher than they did before and the curved line going back to baseline (red dotted line) is a lot shallower than before, showing it’s taking a lot longer for the dopamine to be taken away. Cocaine did this for both types of mice, but it did it more in the mice with lots of α-synuclein than the mice with none. Cocaine, GBR 12935 and nomifensine all stop the DAT from working and for all these drugs dopamine release goes up more in the mice with lots of α-synuclein than mice with no α-synuclein.
B) “PLA” is a way of telling if two things are very close together. Every red dot shows when one DAT and one α-synuclein are very near to each other. The blue blobs are cells.
C) The green dots are labels that stick only to DATs. The more green the more DATs there are.
D) cocaine is made radioactive so we can see when it sticks to the DATs. The dark bits show where cocaine is. The more cocaine sticks, the more DATs there are.
3) Is cholesterol the reason α-synuclein changes what DAT does?
For this part of the study we needed to play a game of “dot-to-dot” using what other scientists have already found to try to work out what explains why α-synuclein changes how DAT works.
i) Other scientists have found that cholesterol changes how DAT works.
ii) Other scientists have found that α-synuclein changes how much cholesterol there is and where it goes
So this made us think that maybe α-synuclein changes cholesterol which then changes how DAT works. So first we had to see if giving brains more cholesterol looked the same as having more α-synuclein.
First we put brain slices in a bath of cholesterol before measuring the dopamine release. We found that less dopamine is released from brains that have had a cholesterol bath (like α-synuclein) (picture 3a); dopamine signals go away faster in brains that have had a cholesterol bath (like α-synuclein) (picture 3b); and cocaine (that stops DAT from dialling down dopamine release) makes the amount of dopamine that is released go up more in brains that have had a cholesterol bath (like α-synuclein) (picture 3c).
Picture 3: A) The dopamine transients show that the cholesterol line doesn’t go as high, showing less dopamine is released. B) The curved lines going back to baseline (red dotted line) show that the cholesterol curve is steeper, so dopamine is taken up faster than the brains that haven’t had a cholesterol bat. C) cocaine makes the dopamine transient go up more in brains that have had a cholesterol bath. D) There is less cholesterol measured in brains from mice with lots of α-synuclein than those with none.
For the next experiment we asked our colleagues in Gothenburg to measure the amount of cholesterol in the brains of mice with lots of α-synuclein and those with none. They told us that there was more cholesterol in the brains of the mice with no α-synuclein than the mice with lots of α-synuclein (Picture 3d). This was a bit of a surprise to us. We thought that because the measurements from the cholesterol bath looked like the measurements from the lots of α-synuclein animals, that it meant that the α-synuclein animals had lots of cholesterol. After checking (and double checking) that nothing was mis-labelled we had another think and did some more reading.
We found out that other scientists had showed that α-synuclein changes how the cholesterol carrier works. They found that α-synuclein helps the carrier that moves cholesterol inside cells to outside cells (called the “ABCA1-transporter”). So α-synuclein makes more cholesterol outside cells and less inside cells. This made us think that maybe our cholesterol bath isn’t copying the lots of α-synuclein measurements because it puts more cholesterol inside cells, but because there is more cholesterol outside cells. To test this we used a drug that stops the cholesterol carrier from working (ABCA1-transporter inhibitor) called probucol. Probucol stopped cocaine being as good at making the amount of dopamine go up: i.e. probucol stops the DAT being as good at dialling down how much dopamine is released (Picture 4a). This is the opposite of what α-synuclein does. We wanted to use another way to check if α-synuclein in making more cholesterol outside the cell make the DAT work better. So we used a drug called nystatin that grabs and holds cholesterol outside the cell so cholesterol can’t do anything. Nystatin did the same as probucol (Picture 4b) and made cocaine less good at making dopamine go up. We also found that probucol and nystatin slowed down how quickly dopamine was taken away (Picture 4c) So stopping cholesterol being carried out or trapping cholesterol that is out, makes the DAT work less. This is the opposite of what α-synuclein does.
Picture 4: A) Probucol is a drug that stops the cholesterol carrier from working, it means less cholesterol is carried out of brain cells into the area between cells. Cocaine can’t boost dopamine release as well with probucol than it normally can. B) Nystatin sticks to cholesterol outside of cells and stops cholesterol from being able to stick to anything else. Nystatin also stops cocaine from making dopamine release go as high as it normally does. C) The steepness of the curved lines of the dopamine transients are less steep with probucol or nystatin compared to normal.
So we think that α-synuclein makes the cholesterol carrier work better. Meaning there’s more cholesterol outside the cell, which makes the DAT better at carrying dopamine back into the cell and better at dialling down dopamine release. Together we think this means that α-synuclein makes less dopamine be released and makes what is released there for a shorter time. Other scientists have shown that cholesterol makes DATs work on their own rather than pairs. When DATs work on their own there are more DATs to carry dopamine, but they are slower to carry dopamine. We would need to do more experiments to test if this is what happens here.
Other scientists are finding ways to change how DATs work. We hope with more research we might find a drug that can stop DATs from dialling down dopamine release and let it stay around longer.
Our colleagues in and out of Oxford: Within the OPDC we connect with many other scientists that are brilliant at getting lots of different types of measurements. We regularly show our data to one-another and happy to help each other. So when we have a question we can’t answer there is usually someone close by who can. We are based in the “department of physiology, anatomy and genetics”, but we find a lot of the same sorts of questions interesting as scientists working in the “department of pharmacology”. Without these links between scientists we would be trapped only using the techniques we know, or worse use a technique badly and get funky measurements.
Our colleagues across the world: We connected with our colleagues in Gothenburg after presenting early data at a conference hosted by them. Without their valuable insights we wouldn’t have been able to make the next step in our experiments.
We hope that this would make the symptoms of Parkinson’s disease less because dopamine will be allowed to signal more to other brain cells.
Who paid for it
Funded scientists: Parkinson’s UK monument grant (ST, BR, NCR, NBG) Project grant (KB) MRC studentship (NP, RA). Resources (Parkinson’s UK). Conference attendance (Guarantors of Brain)
What the reviewers said
This paper was reviewed by Prof Sara Jones and Prof Aurelio Galli. We submitted the paper to Frontiers in Cellular Neuroscience as part of a special edition on transporters, edited by Prof Annalisa Scimemi. Both the reviews were positive and gave us useful feedback. Most of the comments were asking for technical details to make it easier for other scientists to try and copy our methods and hopefully our results. We were happy to provide these details. When we got the reviewer comments we didn’t know who was reviewing our paper (a blind review). After the paper was accepted the reviewers said they were happy to have their names listed on the published paper.
The full published paper that is open for anyone to read can be found in Frontiers in Cellular Neuroscience.