Dr. Patrick: Going back to the APOE4 because it is such a big genetic risk factor for late-onset Alzheimer's disease and as you mentioned, at least about a quarter of the population has one allele. I know you had done some research, we talked about how blood-brain barrier seems to break down, and at least you're able to measure it, you know, in earlier or in cognitively normal individuals. But you also mentioned in one of your papers, there was a protein, cyclophilin A-matrix metalloproteinase, as a protein that seems to be really involved in...you know, it's an inflammatory protein, but how does it affect the blood-brain barrier?

Dr. Montagne: Yeah, so in the same paper, we were mentioning earlier that people carrying at least one allele of APOE4, they tend to have more leakage in the medial temporal lobe that we can see with MRI and biomarkers. But, of course, it's a great descriptive study but we went deeper to try to understand how this is happening. And what we found is the people carrying the APOE4 allele...so APOE, you have to know that the major source of APOE are astrocytes. So, when astrocytes make APOE, so if you have APOE3, it makes APOE3, if you have four, it makes APOE4, we know that there is a different affinity to one of the APOE receptors that is called LRP1 on pericytes.

So, astrocytes and pericytes are close to each other, they are here to maintain the vasculature as well as neural function and neurovascular coupling. But if you...so the APOE4 allele is...I don't want to say anything wrong, but there's a different affinity to the LRP1 receptor. So, when you have APOE3 basically, you have less chance to bind to LRP1, which will less likely to induce a cascade within the pericyte that involves NF-kappa B, it's a bit complicated, cyclophilin A, which then will lead to expression of MMP9 from pericytes and endothelial cells. So, basically, if you have the APOE4 gene, you will induce much more of that cascade than people having APOE3 because it doesn't bind to the LRP1 on pericytes, so it doesn't trigger the cascade of expressing cyclophilin A, which will release MMP9. And MMP9 is the matrix metalloproteinase 9 that is basically doing two things that you don't want.

Number one is disrupting the tight junctions between the endothelial cells. So, all the endothelial cells are tightly close to each other and there's tight junctions in between, so these MMP9 will eat up those tight junctions. So, if you think about this, you're going to get some gaps and that's what we call the breakdown of the barrier, so you can start to see leakage. And the other thing, there is a basement membrane...we haven't talked about this, but there is a basement membrane wrapping around the vessels and wrapping around the pericyte, and MMP9 is also eating up this basement membrane. So, two things that will basically break down the integrity of the blood-brain barrier. And interestingly, MMP9, it's good to go back a bit a few years back.

It's a very nice marker when it comes to blood-brain barrier leakage. We know after a stroke, when you have a blood clot in your brain, within the next few hours, you have a breakdown of the blood-brain barrier and big ischemic lesions and things like that. And we know that this breakdown is correlated with high levels of MMP9, there's a perfect correlation of this breakdown. But very interestingly, when you have an ischemic stroke, you have a second breakdown a few days later. So, it's what we call biphasic breakdown of the barrier a few hours and a few days and same thing for MMP9, two peaks of expression. So, it's a very nice marker that goes very well with the blood-brain barrier property.

So, we found this in APOE4 and both MMP9 and cyclophilin A levels were elevated in the cerebrospinal fluid of people carrying the APOE4 gene. So, we were talking about three to fourfold, if I remember correctly, compared to people carrying the APOE3 gene. So, that's a major thing. And we also used pluripotent stem cells, but we also used human-derived pericytes, so iPSC-derived pericytes in culture. And when you look at them, these are just cultures of human pericytes, human endothelial cells, and different cell types, and we did that for donors from APOE4 and donors from APOE3.

And just at baseline, we were able to also see that the pericytes, just at baseline, looking at them, they produce much more cyclophilin A and MMP9 than the pericytes that do have APOE3. So, these are the evidence that we put and I think what goes very well with that is we published a follow-up study in mice, where we decided to target cyclophilin A, to block cyclophilin A, to see whether if we have mice that do have APOE4, if we block cyclophilin A, can we restore vascular function? And ultimately, the goal is to restore neuronal function and cognition, right? So, we did that. We used the humanized APOE4 mice, so these are mice that have the APOE4 gene of the human.

So, we have also the mice that do have the APOE3. We follow them as they age, we do MRIs on them, we look at their blood-brain barrier function, how the blood flow and everything, and we see exactly what we see in humans is they have reduced blood flow in the brain, they have a leakier blood-brain barrier, they have some behavioral problems, also in terms of cognition, what we call novel object recognition, novel object location, those memory issues. So, these mice are quite, you know, doing what we were expecting to. And we gave every day for one month to the APOE4 mice, we gave an inhibitor of cyclophilin A that is called Debio-025. So, that's a drug that is available currently in clinical trial for hepatitis C.

So, we gave it to the mouse every day for one month and we check the same thing. We did MRI, we looked at brain tissue analysis, we did behavior. And as we were expecting, just by doing that, we were able...again, it's not a full recovery, but we were able to partially and significantly restore vascular function. So, just by blocking cyclophilin A, we were able to restore the tight junctions, we were able to restore the pericyte coverage of the vasculature, and ultimately, those mice had less neuronal damage and less cognitive problems. So, it's a good paper that says if you target the vessels, it can have a big impact on neuronal function and cognition. So, that's kind of the whole story into two papers regarding the APOE4, yeah.

Dr. Patrick: That drug, is it...a couple of questions regarding it. So, is it the same as the Alisporivir, I think, is what they...?

Dr. Montagne: Yes.

Dr. Patrick: It is?

Dr. Montagne: Yes.

Dr. Patrick: Okay. How safe is that? I mean, is that, you know, something...I mean, first of all, I mean, if you have an APOE4 allele, you know, could you take that prophylactically? Would you have to take it, like, your whole life? You know, like, that would be interesting.

Dr. Montagne: Yeah, I mean, these are good questions. I don't know if I'm the expert to address these questions, but Alisporivir, is it safe? So, first of all, I wouldn't say it's safe. So, there had been a few clinical trials, and unfortunately, some of them have been stopped and I think it was for...designed for hepatitis C. It's also currently used...by the way, it has been used a year ago for COVID-19 in France for the lung, people having, like, a lot of lung inflammation, things like that, I think, but again I haven't checked recently. But I know they stopped the trial for one person that had some cardiac issues after the treatment. So, I cannot say it's safe or it's not safe.

What I think is we need to design the trial and give it to the right people, I think, rather than giving like this. I think you have to go through criterias before being able to give it. I don't know if targeting cyclophilin A is the best approach, at least we know it partially worked in animals, but it might be...another possibility might be to target MMP9. So, these are close to each other. This has been also studied quite a lot in the context of stroke. But as you know, there are thousands of compounds that failed in stroke for many reasons, but I don't think anyone has tried targeting MMP9 to look at more subtle blood-brain barrier leakiness and see whether we can kind of seal the barrier.

Yes, I'm not...yeah, so basically, that's the whole purpose of what we're doing right now is we are...we know there's a few targets that might not be the safest or maybe not the best target, but we have a list of other targets that we are studying, which will hopefully do the same thing. We want to restore pericyte function, we want to restore endothelial function, and make sure the blood-brain barrier is, you know, not leaky, pushing the flow as you need to for your brain. And make sure...also something we haven't really talked about, the transporters at the blood-brain barrier, if you start having a leaky barrier, you're going to have some transport issues.

So, it comes back to glucose, oxygen, but also amyloid. If you have a leaky barrier and if you have your vessels that are not functional, there's a few receptors, RAGE, LRP1, there's a few others, if they don't work properly, you're going to...you won't be able to waste...I mean, to clear your waste from your brain while you sleep and things like that. So, you will have a tendency to accumulate more amyloid oligomers and possibly develop plaques. And so, everything is interconnected. But again, yeah, I think fixing the blood vessels...whether cyclophilin A is the right target, I don't know, but I think it's promising. We just have to see who will benefit from getting the drug.

Dr. Patrick: So, if you're having a dysfunctional blood-brain barrier, your glymphatic system isn't going to be working properly during sleep. Is that correct?

Dr. Montagne: Say that again.

Dr. Patrick: So, you know, if you're basically having blood-brain barrier breakdown, everything you've been describing, the glymphatic system, which is when you sleep, it squirts the cerebrospinal fluid into the brain to clear out the amyloid plaques. Is that also impaired somewhat?

Dr. Montagne: So, there is active research in this area, obviously, but that's fair to say. Yes, of course, if you have...because, you know, at the arterial level, you have the perivascular space, which is enlarged when you sleep, which has been shown. And yeah, of course, if you have a disruption of these vessels, obviously, the perivascular space will be...the properties and the cells being around, the perivascular macrophages, and everything will be highly disturbed. Yes, it's fair to say. It's just the glymphatic system, it's very controversial nowadays, as you may know.

We don't really know whether...I mean, the fact that we clear things from the arterial level and then it gets basic convective flow that drains the interstitial fluid back to the veins, that's kind of the theory of the system. But I think it requires a bit more work to prove that this is truly happening, in my opinion. But there's also another system, there's the IPAD, so that's the intramural periarterial drainage. So, there's basically either the glymphatic and/or the IPAD system to clear out toxins out of the brain. Again, I'm not the expert on that but it's fair to say that, of course, if you have vascular dysfunction, the clearance of toxins, not only amyloid but alpha-synuclein or other things, will be highly disturbed, yes.

Dr. Patrick: In my opinion, that sort of puts, of course, the vascular function and blood-brain barrier integrity upstream of amyloid in a way because having that dysfunction first will then lead to more amyloid accumulation if you're not able to clear it out, right? What do you think of...so there's been a lot of failed attempts to target amyloid beta plaques, soluble amyloid, although recently there's the Lecanemab, which targets the protofibrils, so it's still like a soluble form of amyloid, but there's some positive results with that drug. Do you have any thoughts about it? Do you think...?

Dr. Montagne: Yeah, I think...I like to be optimistic and I like to be positive. So, yeah, I think it's good that we have drugs not only, you know, for research, but for people also, for people and for the families affected, it's good that we have a drug that can remove, to some extent, the plaques and there's evidence of that. What is a bit more controversial is the impact on cognition. So, there is some, you know, improvement but it's not what you would hope for, which goes back to, "Okay, targeting amyloid oligomers is probably not the way to go and it's probably not sufficient or it might be too late also." So, it goes back to our important research, if you tackle things and target things much more earlier than that, you will have a better chance. Yeah, so I don't have much to say.

I think we'll see it more as a cocktail of treatments if you give something that will make your blood vessels working as they should work in terms of pericyte function, clearance, endothelial function. For us, the drug like an anti-amyloid of any form, I would bet that you would have more chances to have a bigger impact, bigger positive impact, very positive impact on cognition. But again, it has to be done very early. If your brain is full of plaques, it's probably too late already. So, that's the importance of the research we do right now. But I see that as a cocktail maybe, improving the blood vessels because we know we need those blood vessels to be functional to clear out amyloid. Yeah, so the combination would be probably a viable solution in the near future, maybe.