Dr. Patrick: Omega-3 and its brain transport may turn out to be an incredibly important target intervention for dementia and the preservation of brain capillary health. People with high levels of omega-3 DHA are almost 50% less likely to develop Alzheimer's disease compared to people with low levels. But why?

Brain transport of DHA via the MFSD2A transporter is crucial for maintaining the blood-brain barrier. Furthermore, where we lose pericytes, which prevent brain barrier leaks just so happens to overlap with the loss of these transporters of omega-3.

Dr. Montagne: No, I cannot agree more, I think there is more and more studies coming up nowadays on omega-3. And you mentioned Mfsd2a, which is one of the markers we are studying carefully because it's specific to the smallest blood vessels in the brain, so the capillaries, and that's where most of the pericytes are. And there is a recent study that shows that as we age and with dementia, Mfsd2a, so the receptor for omega-3, it's reduced on the blood vessels. And there is also a link that where there is a reduction of Mfsd2a on blood vessels, that's where we see pericytes loss. So, we can almost connect what we were seeing earlier. Of course, these are just a few studies and it has to be confirmed.

But apparently, there's more inflammation of the blood vessels, to go back to the first question to link it, which, as we age also, Mfsd2a transporters and other transporters, not only this one but this one in particular, is decreased at the capillary bed. Which have an impact apparently on the pericyte function because we can see that this hotspot of Mfsd2a loss are also hotspots of pericyte loss, which means that where we have the leakiness of the barrier. So, yes, I cannot agree more, I think we have to study a bit more DHA omega-3 and how this impacts blood-brain barrier function because that could be some preventive interventions and things like that. Even like targetable with drugs, but yeah, I cannot agree more.

And just to...we can talk a bit more about this, but just to go back at the beginning of your question, you were mentioning exercise and I think exercise is very important. Even me admit...you know, I'm not too old, I'm not even 40 years old, and I try to practice every week. Knowing all the recent data that we have, we have to do some exercise and aerobic exercise to make sure that the vessels...we have tiny blood vessels in the brain, so, if you don't exercise, if you don't make sure that your heart is pumping at a high rate regularly during the week, those tiny vessels that are even smaller than your hair in terms of diameter, they will start to collapse.

And you're going to have...and remember, the capillaries, it represents 90% of your brain vasculature, all these tiny blood vessels. So, if you start not...if you don't do enough exercise, you will start chronically to have some vessels that will basically constrict and collapse and disappear. Meaning that the surrounding neurons that are here, they need oxygen and nutrients and everything from these vessels. If these vessels disappear, you're going to lose neurons, right? So, exercise is the number one thing. You have to do that, not only for the brain, obviously, for the heart it's true, for the eyes, obviously.

If you don't exercise, you tend to lose eye vision quicker than a normal person as you age. But very important for the brain, you're going to turn...you know, if you're prone to go to cognitive decline because let's say you have a major genetic risk, but if on top of that, you don't exercise, you're going to accelerate as we know from studies. So, I think exercise right now, it's highly funded, there's a lot of work going on, and that's one of the biggest things we have to put to the world, it's like, "Okay, we need to exercise no matter what." If you want to stay healthy in terms of brain function, yeah, no other choice.

Dr. Patrick: I could not agree more. When you're talking about not exercising and how the small vessels lining the brain, they start to get...you know, basically, go away. Is that small vessel disease, or is that something different?

Dr. Montagne: So, we have to be careful, yeah, it's not like a person...if you don't exercise, you're going to get small vessel disease. I mean, it's a bit more complex and I think that's a word I didn't say at the beginning. But this dementia, Alzheimer's disease is one but the second major form of dementia is cerebral small vessel disease. And I forgot what I wanted to say. What was the question again?

Dr. Patrick: Oh, yeah, I was asking about, I mean, what is small vessel disease?

Dr. Montagne: Yeah, okay. Yes, so I wanted to just mention, I don't want people to freak out. Of course, it's a multifactorial disease. You know, it's not like...if you have a genetic risk factor on top of that, you can kind of counterbalance that by doing more exercise and eating better and stuff like that but we can discuss about this. But small vessel disease, it's very different from Alzheimer's disease, although there's a lot of commonalities in terms of brain imaging. If you look at two brains, Alzheimer's and small vessel disease, you're going to see a lot of similarities, meaning that you're going to see what we call microbleeds.

So, we were talking about blood-brain barrier breakdown. So, if there is a significant breakdown of the vessels, you can start having red blood cells going to your brain that are detectable using MRI, so we can see those microbleeds. That's one feature. Also, small vessel disease compared to Alzheimer's disease does tend to have small strokes, what we call lacunes. So, they have small spots or small lesions that we can detect in the brain, which chronically and over time will...you will have a deterioration of your cognitive functions. The first thing that is very important is white matter hyperintensities, so these are white matter disease. It's a hot topic, a hot research area because it overlaps. As we age, we have white matter disease. Normal aging, we do have some.

Alzheimer's disease, there is quite a lot of white matter disease. Small vessel disease, there is a lot also. And blood-brain barrier breakdown, obviously. In Alzheimer's and small vessel disease, these are key features of these two diseases especially to explain the white matter hyperintensities. This white matter disease are from a vascular source, so it's still ongoing research but we know that this area do have leaky vessels, possibly the pericyte detach, there is leakage of blood toxins into the brain which will damage the myelin sheaths and all the axons and the white matter fibers that makes your cognition running properly. So, these are the features of small vessel disease. And small vessel disease is also a common cause of stroke.

And so, there's many...small vessel disease, we have a cohort here in the UK that we are following, the leader is Joanna Wardlaw, so I invite people also if you want to learn more about small vessel disease, she is probably the world's expert in small vessel disease. So, that's a disease that is independent of amyloid, so that's a big difference, obviously, from Alzheimer's disease. But they do have...the microvessels do have some problems, and I can tell some of them which we've talked a little bit. The fact that the endothelium gets pro-inflamed, so that's something that is very present in small vessel disease.

And to give you an example, I told you about the cell adhesion molecules that are expressed at the luminal side of the microvessels of the brain. When they get shedded, they are released into the blood and we can measure them. And we know that people that do have small vessel disease, they have very high levels of soluble form of the cell adhesion molecules that we can detect in biofluids. So, that's already a feature, what we call endothelial dysfunction. So, the endothelial of the blood vessels are getting very inflamed and they don't do a proper job, which imply also, and there's some evidence, that the pericytes surrounding them are also dysfunctional very quickly in this disease.

And we can measure one...I don't know if you mentioned that today, but the soluble form of, a very complicated name, PDGF receptor beta, so it's platelet-derived growth factor receptor beta, that's a receptor on the pericytes. And that receptors, the same thing, it can be shed and released into the plasma and into the CSF and that can be measured. So, basically, we know in small vessel disease, they have high level of pericyte dysfunction and high level of pro-inflammation of the vasculature. And those things, we can also...what we do nowadays is we try to relate those biofluid biomarkers with the neuroimaging features that we see in people to try to correlate whether if you have a high level of pericyte problems, do you have more white matter disease and things like that, so we try to relate what we see in the blood and CSF to what we see in the brain.

And obviously, we look at cognition, whether there is some predictive value or whether there is some correlation or association between these biomarkers and cognitive problems. But now, we are fairly confident that targeting somehow the blood vessels, the microvasculature in small vessel disease is the way to go. We have to fix these blood vessels very quickly to make sure that these people don't go into small lacunes, microbleeds, and other things that will make them deteriorate very quickly in terms of cognition. And the last thing maybe I forgot to mention is this…is the sporadic form of small vessel disease, but there is also the genetic form. Same with Alzheimer's, we haven't talked about this, but there is the sporadic form, the most common form, and the genetic form. So, in terms of small vessel disease, there are two diseases that are quite known now and quite studied. One is called CADASIL, the other one is called CARASIL.

And interestingly, these two diseases involve pericytes and vascular smooth muscle cells, so these are the two cells of the brain that are basically wrapping around the vessels. And it involves aggregates...I mean, for CADASIL, it's Notch-3 proteins that aggregate in pericyte and vascular smooth muscle cells which makes vessels dysfunctional very quickly and those people develop white matter disease at the age of 30-40 years old and they go towards cognitive decline very rapidly. It's very severe. And CARASIL, it's about the same thing, it's HTRA1 protein that is involved, also a genetic form that has exactly the same features: white matter disease, microbleeds, lacunes, and blood-brain barrier issues. And we can measure that, we have all the tools now to measure those things very early before people really go to cognitive problems. Which means, again, we have to target those vessels to make sure that it functions properly.

Dr. Patrick: How common is the small vessel disease and do any...how common is late-onset small vessel disease, and how do any of those people actually that have small vessel disease end up getting Alzheimer's disease?

Dr. Montagne: So, it's very common because AD and SVD, so Alzheimer's disease and small vessel disease are two major forms of dementia. So, it's very common, more common than we think. The second question was about...sorry.

Dr. Patrick: If people that had...you said they're two separate diseases, but I was wondering if people that do develop small vessel disease end up sometimes getting also Alzheimer's, like, if there's any...?

Dr. Montagne: Okay. So, yes, that makes the research more complicated, right? Yes. To give you an example, we have the UK Biobank here where we have brain tissue samples where we can really look at the features of these different things, Alzheimer's and small vessel disease. And a good proportion of small vessel disease-diagnosed patients, they do have some sort of CAA that we've talked about earlier, cerebral amyloid angiopathy, and some of them have amyloid plaques. So, that's the complexity of dementia, there is no pure Alzheimer's disease, there is no pure SVD, there's always all the neurological comorbidities that are coming into play, and so it's difficult to disentangle things.

But at least when people are alive, there is good imaging tools and biomarkers that we can measure in blood and CSF that tells us who is what. But some people have both and that's true. And I couldn't say...I'm not an expert on that, I couldn't say the ratio of how many can have both. Some people have SVD and Lewy body, some people also have some Parkinsonism traits. So, yeah, unfortunately, it's very common that we can have two forms of dementia kind of simultaneously. But, yes, I hope that answers your question.

Dr. Patrick: It does. Circling back to something you mentioned sort of briefly when you were talking about blood-brain barrier not functioning well, you mentioned glucose metabolism and you decrease...you know, it's important for the...the blood-brain barrier being intact is also very important for glucose transport. Reduced brain glucose metabolism can be measured, you know, up to decades, even before any Alzheimer's disease symptoms or dementia symptoms occur. Hence, you hear the term type 3 diabetes. What effect does the breakdown of the blood-brain barrier have on brain glucose metabolism?

Dr. Montagne: That's another great question and ongoing studies are tackling this because it's not that clear and quite controversial. Just to tell you a bit more, so the main transporter of glucose at the blood-brain barrier to make sure glucose comes to the brain is GLUT1. So, that's a receptor, GLUT1, that has been found to be reduced in Alzheimer's disease. So, people have looked at, again, postmortem brain tissue bank and looking at the microvasculature and bigger vessels, and they found that Alzheimer's patients, they have much less GLUT1 at the blood-brain barrier, which means that there's less potential for the flowing glucose in the blood to penetrate the brain.

So, that's one thing, and we can measure that with several techniques, but one of them is FGD/PET. So, it's Positron Emission Tomography, and we can measure what we call FDG, which is a marker of what people say neuronal activity and things like that. So, we know that when we inject that tracer, it's a fluoro-deoxy-glucose tracer, into a patient. If you have Alzheimer's and if you don't, you will have much less FDG signal if you have Alzheimer's compared to a control. But what is interesting and there's a bit of controversy, I don't want to tell too much about this, but people are explaining this data and just saying that there's less neuronal activity and that's all. But what they don't take into account is these people have less GLUT1, so there's less probability that the radioligand will go, if there's less GLUT1 receptor, into the brain.

So, I think the FDG-PET signal has to be carefully analyzed in the sense that it could be a neuronal activity, but it also could be indirectly a marker of vascular problems because of what I've just said. But again, there's experts in the PET field that will tell you the reason why it is not and vice versa. It's very technical, and I don't have that expertise. But again, we know there's less GLUT1 independently of the reduction of the vascular network in Alzheimer's disease. So, I think the root cause is playing a big role here and it's currently investigated. I don't know much more than that, sorry.

Dr. Patrick: Well, I mean, I think it's very interesting and it certainly doesn't seem like we have, you know, the consensus, you know, on the answer to that question. I'll just mention, I remember reading a couple of studies years ago, animal studies, where omega-3 deficiency caused a reduction in GLUT1 transporters in the brain. Again, of course, omega-3 deficiency also breaks down the blood-brain barrier, you know, so it's kind of what's first, you know? Like, is the reduced glucose getting into the brain affecting the blood-brain barrier? Is the blood-brain barrier affecting the glucose transporters? Or both? You know, I mean, it seems tricky to sort of figure out but...

Dr. Montagne: That's the chicken and egg question all the time. Same thing with...I mean, I'm not an expert on omega-3 but it's basically the same thing when it comes to pericyte loss, endothelial activation, like pro-inflammation, breakdown of the barrier, loss of blood flow, what is happening first? And it's always difficult to address those questions because in human, most of the clinical studies are cross-sectional, you look at one time point. Now, I think there's more and more studies that follow individuals longitudinally with scanning, with looking at biomarkers, looking at neuro-psych testings. So, we're going to get more into this very, very soon, there's big centers working to do that. But for the omega-3, yeah, I'm not surprised you said that omega-3 deficiency leads to a reduction of GLUT1. Is that what you said?

Dr. Patrick: Yes, correct.

Dr. Montagne: I haven't read that paper but it goes well with what we said earlier, right? There is a vicious circle, where I think it's likely that, of course, the endothelial-pericyte crosstalk should be very involved in that problem, in my opinion, and that's something I would love to study.

Dr. Patrick: I'll send you my paper, I wrote a sort of interactive review article back in...gosh, it must have been like 2018 or something, I think. And it was on the important role of Mfsd2a and omega-3 in APOE4 carriers. And I have references for a lot of the studies, like the deficiency in omega-3 causing GLUT1 transporters to go down. So, I'll send you my review, you might be interested in...of course, it's a little bit of a hypothesis sort of review, you know? So, I review the literature, but there's a lot of my sort of thoughts for people that are actually exploring the field because I'm not doing these studies, you know, so you might be interested in that.

Dr. Montagne: And it's just...oops, sorry.

Dr. Patrick: Go ahead.

Dr. Montagne: It just rings a bell, I just remember reading a couple of stories and it's very relevant to what we do in the lab, is that, yeah, omega-3...the fact that you give omega-3 to aged animals...and I told you, as you age, you have pro-inflammation, like a hyperactivation of the brain endothelium. And a couple of studies have given some omega-3 to the mice and I remember seeing a reduction of one particular protein that I really like to study and what we are currently studying is VCAM1, vascular cell adhesion molecule 1, and omega-3 was able to reduce these levels. And in the lab, we know that VCAM1 plays a major role upstream of pericyte detachment. So, again, there's a lot of things that can be connected. I think it's very interesting.

And just another word on VCAM1 is Tony Wyss-Coray in Stanford University, one of the big labs working on proteomics and all these fancy omics techniques, he looked at Alzheimer's and healthy controls, he looked at their plasma and he has looked at using proteomics, looking at different proteins which...Alzheimer's, sorry, and normal aging. And what he found...I think the most striking finding was normal aging, he found, I think, 30-plus proteins in the plasma that were elevated with normal aging that were related to the blood-brain barrier. And if you look down, I think the top five candidates were proteins that are part of the endothelial cells, obviously, but the number one that stood out as the number one protein that is elevated with normal aging was soluble VCAM1. So, everything...you know, if you put some studies together, it kind of makes sense, there is a bit more to dig. But I think it makes sense and it might go well with also what omega-3 is doing to the vasculature, so it'd be nice to dig further.

Dr. Patrick: It's very interesting that you mentioned Tony Wyss-Coray as well because if I recall correctly, he's also been involved in research where he's transplanted young plasma into, you know, older mice and it's sort of rejuvenated in the brain and it's like, "Well, are there proteins...?" I think the opposite was done as well, where old plasma was transplanted into younger mice and it sort of accelerated and I think if I recall correctly, blood-brain barrier breakdown was part of that. And so, it's like identifying those proteins that is causing this.

Dr. Montagne: I think he's doing fantastic work and yes, correct, he is looking at the proteins that we have in the young blood that will help not only brain functions or anything, like, to help not aging too fast, but also he found a few key proteins that are involved in maintaining blood-brain barrier as we age. So, I think it's very interesting. And he's using also the parabiosis model where he kind of linked, you know, aged and young mice, but I think it's a very important study.

Dr. Patrick: Yeah, and, you know, the other interesting thing, this kind of brings me back to some of your work where I don't know if...I think you mentioned this very early in our discussion about the blood-brain barrier when it becomes "leaky," this allows molecules that usually don't pass into the brain to then pass. And you had a paper that you had published a couple of years ago with...I think it was with your postdoctoral mentor, Dr. Zlokovic, about a protein called fibrinogen. Which, interestingly enough, is also...like, when I go and get my inflammatory biomarkers measured, I like to look at fibrinogen as well as high-sensitivity C-reactive protein. It's an inflammatory marker. I mean, it's involved in blood coagulation but you found this in the brain. What is the significance of that?

Dr. Montagne: Yes, yes, and it's not only two years ago, I think there's...I think the first report, it's probably 10 years ago. But the first report was on, again, brain tissue samples, very valuable tissue samples from donors, where we see that if you compare a control brain, someone cognitively normal, no issues whatsoever, and another time an Alzheimer`s brain or a small vessel disease brain. You start seeing what we call this extravascular deposition, fibrinogen is one of them. It's one protein that is supposedly in the blood, it shouldn't be in the brain at all, but we start seeing this extravascular deposition of fibrinogen, which means that to cross, you have to have some degree of breakdown of the barrier.

So, it has been found in Alzheimer's disease. We also found that in animals that either do have Alzheimer's disease or have some sort of blood-brain barrier issues. And we know that this protein that is important for blood coagulation, as you said, and inflammation has nothing to do in the brain and it's toxic, it's just toxic. And we found that it's neurotoxic, so it's toxic to neurons. It's also toxic to oligodendrocytes, where we found that…the oligodendrocytes are basically cells that are making myelin and make sure that the white matter is intact and we can function properly. And those molecules are crucial in the brain.

And we show that when you have a leaky barrier and you have fibrinogen going in, the oligos are very sensitive to that fibrinogen. So, they take it up, so they internalize fibrinogen, and they die by what we call autophagy. So, it's almost like a suicide kind of death, which leads to white matter disease. And if we go back, white matter disease is a common feature of Alzheimer's and small vessel disease. And as I said earlier also, the source of white matter disease is likely a blood-brain barrier breakdown, fibrinogen might play a role in this formation of white matter disease. So, yes, it's a very important protein that we found.

Interestingly, in animals, we were able to reduce fibrinogen levels systemically in the blood, of course, at a level that you don't want to increase to have some coagulation problems or increases of bleedings and things like that. So, you have to reduce at a level that you don't start doing bleeding or clotting. But just by reducing the fibrinogen level in a mouse model that do have blood-brain barrier issues, we were able to demonstrate that there is less, obviously, it makes sense, less fibrinogen going into the brain, less white matter that is damaged. And also interestingly, by reducing fibrinogen, we were able to partially restore vascular function in terms of blood flow and also the integrity of the barrier.

So, fibrinogen has probably different roles, not only coagulation and inflammation but probably a bit more. But I think that's one other way, we can target toxic things in the blood to avoid doing damage to the brain but I think it's probably easier to fix...easier, I don't know, but fix the blood vessels rather than doing that. But at least it's another evidence that, okay, a leaky barrier is leading to damage to the brain and you don't want that obviously. And the last thing on fibrinogen is, I just remember now, fibrinogen can activate the brain resident immune cells that are microglial cells through CD11b, so that's a specific receptor.

So, when fibrinogen gets in, it can bind to microglia, so it will induce an overreaction or over-inflammation of the brain which will be detrimental for many things, for the functions of the cell surrounding. And that reminds me that there is a fantastic researcher, Katerina Akassoglou, at Gladstone University, UCSF area, where I think she developed an antibody that blocks the interaction between fibrinogen and microglia to avoid that overexpression of inflammation or overactivation of microglial cells because she's also a strong believer that vascular dysfunction is a very early and a major contributor to dementia. So, that's very interesting research.

Dr. Patrick: It's very interesting, again, like you said, it really shows the importance of maintaining the blood-brain barrier so that it doesn't allow, you know, things like fibrinogen, which is involved in blood coagulation to cross into the brain. And, again, as you mentioned, the inflammation, it's activating microglial cells and it's causing them...I mean, that whole process you described earlier, the inflammaging and pericyte detachment perhaps and all that. Also, as I was reading and doing some background research on some of your work and because I knew of the inflammatory role of fibrinogen, you know, I'd already been familiar with that protein, you know, separate of what happens when it gets into the brain, I was looking up omega-3 because I had remembered coming across some studies with it.

And interestingly, people...air particulate matter, so when you have like air pollution and particulate matter, when people are exposed to high amounts of it, it causes their fibrinogen to go up, right? It's, again, an inflammatory marker as well. But people that were taking in high amounts of omega-3, it blunted the increase in fibrinogen and plasma. And so, it would be very interesting to see in some of the animal models you were discussing if omega-3 could blunt, you know, the white matter dysfunction caused by fibrinogen getting into the brain. So, again, it's another prevention, easier, low-hanging fruit thing that people can do now, right? I mean, making sure they're taking in enough omega-3.

Dr. Montagne: Yes, exactly. No, I cannot agree more. We don't need to repeat what we said but, yeah, there's this kind of vicious cycle where omega-3 may play a major role in vascular functions, of course. It's not even, "may play," I think it does play a role and it might play an even more important role in people at risk for Alzheimer's, it's possible the idea of APOE4 carriers that we've talked about.