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Our next speaker is, uh and, uh, is from the standpoint of your vascular, uh, where she's a pa in the Paris Brain Institute. And she's now neurobiologist in the European, uh, dialogue and a half and disease. And she's particularly interested in, uh, studying the notes of, uh, formation. And they're all in Europe. Glare crosstalk, uh, and and I hand it over to you. Um, hopefully you can hear us. Hi. I hope you can hear me. Yes, very good. So I'm trying to show my screen right now. Uh, thank you very much for the introduction and to the organizer for inviting me to present at the stock today. I hope you can see the slides. Yes. Okay. Um, so, uh, I am indeed a member of the liver. It's Keystone often at ICM, which is a group mixing clinicians and neurobiologist. I am neurobiologist myself, and we are basically interested in, uh, remyelination mechanism in the central nervous system, uh, in particular in regard to multiple sclerosis. So I am on the basic science, uh, side of the team, and today I will present you some recent data we generated on the New Deal. Interactions at the notes of health and disease. Uh, so I will be very brief on the first part of the introduction because I think done already very nicely introduced many points. But as you are all aware of, uh, myelin is essential as it can ensure a fast saltatory conduction along axons. And this is possible, actually, because the insulated mile in segments can alternate with these small child domains called the notes of rosy. And these are very highly enriched in sodium channels and potassium channels, and these proteins will allow to regenerate action potential and interactional conviction. So, um, basically, as done mentioned, um uh, in, uh, multiple sclerosis DEMYELINATION will lead to the disorganization of structure and, uh, will lead to functional deficits, which can be reversible, uh, in particular, if the accents are preserved and remyelination can a cure. Uh, remyelination is not only important to restore the conduction, but it also has been shown to actually have, uh, neuro protective role. And, uh, it is why we are interested in understanding remyelination mechanism because it could be important to target it properly, uh, to ensure the the art of the progression of the disease. Um, so regarding the notes of remitting multiple sclerosis, They actually have been described as an early target. Uh, the disease even a priority. Uh, demyelination or cures. So, indeed, was that what had been shown both in, uh, s, uh, mouse models as well as in the M s patient issues is that prior to the medication in normal appearing white matter, you have a concomitant disruption of some paranoia area, which are actually the area surrounding the notes where there is a strong extradural junction. Uh, and this goes together with microbial activation. So it very early stages of the disease in the tissue, uh, furthermore, later on, it was shown that there could be some focal actual damage at the notes, even for a demyelination, and that there was also at the notes, some recruitment of macrophages and so far more with time, when the national cures, you will have a loss of the restriction, uh, of the needle proteins they will diffuse along the accent. And this will participate to the conduction blocks or reduction. Very interestingly for us, actually, when we look in repair, uh, something, uh was observed, which was not the classical vision, because the classical vision was that when you mail in eight or you remain in eight the clusters, uh, corresponding to future. Not all structures will appear together with smiling. However, in early remyelinating plaques, uh, Como was a previous member of our team so that you can see some of, uh, these clusters appearing as isolated clusters early on in remyelination. And the numbers of the structures suggested that they were not just, uh, notes, which not had not issues, but that they would actually be re clustered structures. Uh, interestingly, this was also observed in various models, including some words from the breast team in retinal ganglial cell cultures and in optic nerve tissue. But it was also observed in various mothers were using in the team. So we have developed in collaboration with Natalie. So, uh, some people couple cultures where you can see a long gap Biologic neurons, specifically some of his cluster appearing prior to imagination. And in this model we have shown that actually, they correlate with an increased conduction velocity along axons bearing them, and that they can be contacted by pre marinating oligodendrocyte and they could participate in guiding my lung deposition in session at the vicinity. We also observe this, uh, structures along trucking DSL axons in organicity cultures of cerebellum license both during pollination. But also, if we let the cultures mile in eight and we induce demyelination in these cultures, we also see the structures. So there are rather common feature not of all neurons, but they appear in various types of neurons. Uh, and they may play a role, uh, functionally, uh, at the level of the neuron. So over very important players in remyelination our neuronal activity, which has been described to modulate various stages of remyelination process but also, uh, microbial cells. So microbial cells are the resident immune cell of the central nervous system, and it has been shown that they can participate in all these stages by leading to remyelination going from OPC recruitment to maturation differentiation, uh, into, uh, oligodendrocyte and remyelination. What has been described as when it is the work of L. B and polio is that, uh, if you maintain the microbial cells in the print inflammatory state, uh, it actually will alter other remyelination process. So Michael J. A. L cells are known to directly interact with neurons. It has been described previously that they can interact with the Zometa and ritzy area of the neurons as well as the exponential segments. And this is the work from the Yasmin team. And, uh, we wondered whether actually they could contact the notes of Rosy because, uh, these are the only domain which are actually accessible to the microbial cell to contact directly the neuron in particular in the white matter and the composition. The organization of these domains is very close to the organization of the exponential segment from a molecular biology. So this was actually what led us to study this, uh, this question And this is the work of two previous PhD students in the team as well as, uh, the work of, uh, Stephanie go with, uh, engineer in the in the And so what we first looked at was the existence of microbial interaction, microbial contacts on other structures in various parts of mouse brain taking normal marinated breads and spinal code. And what we see is actually, uh, in all the parts of the brain and spinal cord, we looked at a microbial cell here in green can contact the dose of radiation red, so this is, uh, more clearly shown here on this Free the reconstruction. We confirmed by electron microscopy that this contact was a direct contact on the model. Excellent. Now and that micro gel processes could also contact the first paranoid, the loops, uh, neighboring the notes. Um, so we we then wanted to, uh, assess whether this was also occurring in human tissue. And so for that we look first at control. Human was Martin tissue, and we observed similarly that microglia tend to contact the nodules structures surrounding them in my eliminated, uh, central nervous system area. So we then wondered office interaction could evolve following the imagination. So for that, we first turned to, uh, demyelinating model, uh, using focal injection of listless sitting in the dorsal column of the mouse spinal cord. And we looked at, uh, correlation of tissue at the peak of the imagination because in the core of the lesion at that time, point out know, uh, other structures remaining. So we just looked in the surrounding tissue where we still had some of the, uh, structures. And then we need we looked sorry in, uh, remyelinating lesions and what you can securely on this diagram here is that there is a clear enforcement of, uh, the percentage of note, uh, contacted during remyelination compared to control, condition and the relation of tissue at the peak of the imagination. And interestingly, for us, it was actually independent of the number of not all structures or of the density of microbial cells. So we then assessed the stability. Uh, this interaction in time, uh, by developing some life imaging of mouse dorsal, spinal cord in control animals and following the imagination. So here's what you can see are images taken from a three hour movie where you have the microglia in green and the notes in red, and you can see that a long time the microbial will change shape a lot, but that it will still contact the notes. And that's something you will securely here on the corresponding video. So we felt quantified this, uh, looking at the hour, different condition and what we saw is that at the peak of demyelination in the barrel additional tissue, the interaction is actually less stable between microglia and other structures, but that in this model the interaction is restored when remyelination proceeds. So because the time uh, we were looking at We're quite long. We thought that actually, there must be a signal in mediating this interaction. And, uh, we had, uh, multiple ideas regarding that. We first tested very classical signaling pathways such as fragile can and 80 p, which are known to be implicated in, uh, in your, uh, mitochondria microbial. Uh, interaction. But, uh, they were actually not implicated in this, uh, interaction in myelinating tissue. So we then wondered whether actually neural activity itself could be implicated. And so to assess, uh, that question we went to the Xgeva, uh, organ. It'd be culture, uh, cerebella slices model. And, uh, we first treated, uh, the slices, uh, with a t t x tetrodotoxin, which will inhibit, uh, neural activity or abdomen, which will, specifically specifically history activate, uh, poking yourself neuron. And what you can see from this study is actually, the amount of interaction between microglia and note varies according to the amount of neuronal activity. So this suggests that actually, uh, you know, activity modulates this interaction. So then we wondered which, uh, molecular mechanism could be underlying, uh, cross talk. And we thought that potassium, which is released, uh, when action potentials are fired at the notes. Could be maybe a mediator of these signalling. Uh, indeed, it is known that microbial self expressed some potassium channels in particular frequent, Uh, and these are implicated in the, uh, prosthesis, the potassium homeostasis of microbial cells. So to check whether potassium could actually be the mediator of this interaction, Uh, we used a pharmacological approach again to inhibit specifically the nodules, uh, external potassium channels, or to inhibit Szechwan on the microbial side. Uh, and so what we observed using this approach is is again if we're in a bit potassium signaling we have a reduction in the interaction. So this suggests that potassium is a mediator of this interaction which keeps microglia at the notes of roughly. So, uh, we basically wanted to take advantage of this to try to alter the interaction inr imagination and see what was happening to the tissue. And, uh, we first checked that actually, this, uh, potassium signaling was still the mediator in, uh remyelination. And it was the case. And we then looked at microglia and imagination following the activation of micro panels interaction in remyelinating slices. And so what we observed was. Actually, When we inhibit this interaction, we have a reduction of the pro regenerative microbial cells. So here, as soon as in the i g F one market, and we have concomitantly an increase in the expression of print inflammatory markers, uh, such as a nose. Uh, furthermore, what we saw is that this was actually correlating with a reduction in the remyelination rate in the slices. So, uh, to confirm this in vivo, we used, uh, approach, which allowed us to deliver the in veto, uh, which will alter the microbial note interaction. Uh, in vivo on the spinal cord, which had been previously imagine ated and using that approaches, we reproduce what we had observed the Xgeva. Uh, so we have a reduced expression of, uh, g f one micro liter, and we have a delay or, uh, an alteration of the imagination with, uh, an increase in the non REM eliminated area in the treated mice compared to control. Um, so, uh, reciprocally. We then wondered whether by actually activating neuronal activity, we could, uh, induce. We could facilitate. Uh, the switch towards, uh, pro generative micro. Really helpful. And so for that we develop, uh, various approaches. Today I will show you only the dreads. So basically, we, uh we went back to our exit for model. And what we did was to express this receptor which combined to it's ligand and close up, uh, enables to increase the firing rates of packages cells, uh, in the slices. So this is the work of, uh, another PhD student in the family who's name is Clement. Pero. And so what you observed is that when it does that it has, uh, an increase of, uh, the nodules structure contacted, uh, in slices treated with, you know, compared to control slices And when looking in remyelinating slices, what is so is actually, we could indeed promote uh, the pro regenerative state of microbial, uh, using this increase of neuronal activity and this a further lead to a higher remyelination rate in the slices. So in conclusion of this spot, what I should do is, uh, that, uh, microglia contact the notes of the Xgeva. So and, uh, that, uh, it is a preferential interaction, which is rain fast in repair, that it is modulated by normal activity and in particular by, uh, artistic signals, uh, released at the note and that it can participate in the modulation of, uh, microbial phenotypic state and, uh, as a result, participate probably in a modulation of the imagination. So something I have to mention here is that actually, we looked at this using, uh, models which are not very inflammatory, and in particular, there are weak recruitment of circulating immune cells in this system. So, following this works, we were keen on addressing all the inflammatory issues that are observed in, uh, mess could actually, uh, modulate this interaction between microbial and notes of the And so for that, uh, the students in the team, that's the key part is developed an inflammatory model of M s to assess this question. So, uh, basically, she uses the A A model. And again, we have a peak, uh, in the disease, which is absurd through scoring of the animals and then with time, uh, animals enter in a remission phase is where they have usually, uh, recovery. A partial recovery, uh, with the lowering of their, uh, score. So the first thing as she looked at was, as we had done for the LPC model, uh, the percentage of not contacted uh, at the peak of the disease and in the remission phase is compared to control condition. And so she saw something relatively, uh, similar to what we have seen in LPC with an increase. A significant increase of the contact in the remission phase is but contrary to lp see where we had a relative, uh, virginity between the animals here. We had the impression that actually we had some animals we would have, uh, not very much increased amount of interaction, whereas some were, uh, comparable to what we have seen. Uh, LPC model. And so what facility did as a first approach that, uh, these are very preliminary data is that she tried to correlate this with, uh, something functional regarding the animals. And actually, what she saw is that the percentage of not contacted during remission correlates with the recovery, Uh, the clinical recovery of the animals. So we are trying to investigate that point now, to understand what could be the mechanism behind, uh, and see if we can actually, uh, promote the recovery. Uh, in the in this animal, playing on this interaction a second point facility looked at was as we had done before the, uh, stability of the interaction, Uh, a long time using the similar individual approach using time lapse imaging. And so, uh, as you may remember, when we had looked in the LPC model, we had seen that, uh, at the peak of demyelination in the population of tissue, we had the reduction of the stability of the interaction. Uh, in this mice and what we saw here in is very similar. However, contrary to the LPC model in the remission phase is we didn't see any recovery in the stability of the interaction, uh, suggesting that actually, uh, this, uh, interaction is not totally back to normal and that something is preventing. Uh, it's the return to it's stability. So what she felt or looked at was the final type of microbacteria in this mice and what she saw is actually compared to NPC. Uh, she has a much more pro inflammatory, uh, status of the microbial cell. And however, when we look at this population, uh, and their ability to contact the notes we see as observed in LP see that the, uh you know, cells are slightly less contacting and the idea of fun are very prone to, uh, contact the notes. So, in summary of this part, what I showed you is that when we increase neuronal activity in your imagination, we have, uh, sorry. We have, uh, a promotion of the interaction between, uh, microbial cells and, uh, and notes that this will participate in increasing the degenerative phenotype in this tissue. And furthermore, uh, this project narrative cell have a affinity for the notes, and this may actually participate in a sort of virtues feedback loop, helping, uh, to promote the, uh, the pro generative state of microglia and repair processes. However, when we are looking at inflammatory, uh, situation, what we observe is a lower amount of, uh, degenerative cells and increase in the pro inflammatory cell, which have a tendency to contact less, uh, the notes. And maybe we are then, uh, participating in a vicious circle this time where we prevent actually, partially the switch, uh, this micromelia towards the pro generative state. So the last part I wanted to show you because it's, uh, still very preliminary. But I think it's interesting to to mention the context of this master class is that we are in parallel looking at various, uh, M s tissue to assess the existence and the characteristics of microglia nodes, interaction, uh, in various m s, uh, context. And so what I can tell you is in all the context we looked at as long as we have, um, some of those structures there is a certain amount of interaction between microbial cells and, uh, notes of refills. So this is something which is maintained, uh, in the in this context and what we quantified already is looking at, uh, anesthetic microbial cells, so expressing P to a 12 hour receptor. Uh, what we can see is that, uh, we have a reduction of the interaction in, uh, normal appearing white matter of Ms patients compared to control, but that we have a restoration of the interaction in shadow place. So remyelinating And so this suggests to us that actually, it's quite similar to what we see in our animal models, and it encourages us to pursue the study of the mechanism in our models to better understand it and how it could be part of the repair processes. So, in conclusion, uh, I showed you that microglia can contact notes of, uh, human tissue in particular. In normal appearing white matter and various lesion types of food, a type of interaction could vary, and it's output. Uh, and when looking at the anesthetic microglia, we saw this engagement from the notes in normal appearing White Matter and the restoration of the contact in shadow place And finally, in a very interesting point for us is that the extent of microbial contact at notes during remission correlates with clinical recovery. And, uh, we are now investigating this, uh, heterogeneity in our minds to see, uh, this variable amount of contact could actually participate Maybe in the different the viability of recovery in these animals. And so to conclude, I wanted to leave you with this. Take a message that we think in the team that the note of it could be a kind of neurological communication. Uh, so it has been described previously that astrocytes, uh, contact nodes, and they are comparing little astrocytes. And, uh, some potential function of this interaction has been recently described in relation with the regulation of, uh, the note of of the size and myelinating tissue. It has also been described that actually, uh, obviously can also contact the structure in a certain condition, and I showed you today that micro liters are also present there. What we did was to assess whether actually one given note could be contacted by multiple global cell type, and it is indeed the case. So the notes of rosacea seems to be a place where, uh, there could be a cross between you're on Onglyza and also maybe between, uh, themselves and which could be important, uh, to synchronize the behavior in health and in repair. Indeed, uh, we have observed that both microglia and Premarin ating oligodendrocyte can contact early little structures prior to imagination or imagination. And, uh, we would be very keen on understanding the mechanism that are underlying this interaction, uh, assess what the rule could be in elephant repair and in particular, how they could be affected by neural activity and whether this could participate in the coordination of his actors towards, uh, an adequate remyelination process. So with that, I will thank all the member of the two and I particular, the students who participated in this work and our collaborators, and I thank you for your attention. Thank you very much. And thanks for being available for this talk. Uh, anyone would like to ask a question. Michelle, could you ask loudly? Yeah, we have one question submitted online. Help kick off the online test by and And you just want to really fascinating. And do you know what, Mike Rowe? They seek contact. Is it always the same type of microbacteria that makes contact? Or is the microbial genotype dependent on the state of the Axiron IV is it doesn't have to whether it's my imagination remyelination or healthy. So I'm not sure I got the question right. So I will try to answer. And if it's not that, please let me know. Um, basically, uh, as far as I understood, you ask whether there are different type of microbial contacting and whether it depends on the state of the neuron. Uh, so, uh, what I showed you is actually, uh, the all microglia seem to contact, but they do not have the same affinity. Apparently proinflammatory microglia a lesser affinity compared to prior regenerative cells to, uh, contact the notes. And also what we saw is that it's very much dependent of your renal status as it varies with normal activity and also I did not show you, but actually at the very onset Uh, e, uh, microglia totally disengaged from the note. This is a previously published work, but we have similar results, and it's actually the macrophages derived from monocyte. We tend to contact at the beginning of the disease and in e uh, with the the the evolution of the disease that micrographia, uh, will start to come back to the notes and to, uh, part of the interaction and macrophages Will, uh, then go away. And there are nearly no macrophages contacting in remission in a when microglia is entering most of the contacts. So I hope it answers your question. Yes, there is a note from even, thank you very much. And and, uh, I was wondering why I was listening to your talk. Is there some kind of relationship between these mitochondrial movement that Don was talking about and and and and and the the contacting and signaling between the microwave and the rodent number here? So I think it is an excellent question. Um, I I could not attend to all the talk. Unfortunately, because I was doing the test for the protection of the slide show. But my guess would be that directly or indirectly, yes, it is important. It is important because, uh, there there is a clear, uh, energetic part with all this, like potassium release and the functioning of all the eye on flux at the notes are depending on on micro on mitochondrial energy, I guess. Uh, So, um, uh, there could be over type of release. We're looking, uh, now because what we looked like for ATP, for example, uh, was only myelinating issues. So it's something we want to see Whether there could be synergistic. Uh, sorry. Synergy between the multiple pathways. So my guess is, yes. Uh, it is a very interesting point. Thanks, Sam. Hi. I'm very interesting question. Can you here, Can you hear? I'm, uh I can write out a bit more. Can you can? Yeah, it's okay. Really nice. Thought. I was wondering if you look at whether with aging this micro flyer nodal contact, it's under because everything has changed on the microbiome change. And I wonder whether it doesn't get and it contributes to the remyelination that you see with, uh, it's an excellent question. I I really want to look at this and early. Maybe, Uh so what is very difficult for us is to properly understand what come first, whether it is a maintenance or an activation of the proinflammatory state, which will lead to less interaction, and then it will kind of worse in the or whether it could be that actually, for some reason the interaction is lost, and then it will, uh, promote the maintenance of pro inflammatory cells. So I think it would be very interesting to look during aging, but early at the early steps of what could be considered as aging, to see if we could see, uh, an alteration of this contact or it's output early early on. Another thing I did not show today for the sake of time is that actually, uh, the interaction is increased with normal activity. But if the pattern of normal activity is abnormal, the output may differ, meaning that microbial cells are actually turning to a pro regenerative states only if we have an, uh, physiological increase. And if we have totally abnormal patterns as what observing E actually from like increasing activity doesn't lead to a pro generative cells, but rather proinflammatory. So we have to to think in terms of, uh, aging of the tissue we have to think of. So in terms of, uh, physiological status of the neuron and its ability to fight your anomaly. Thank you very much, Anne. And, uh, everyone is desperate for a coffee. So we'd like to invite you and to join us for a coffee, even if we actually, um and thank you very much again for this very stimulating talk.