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uh, welcome, everybody. Um, it's going to be a very exciting day. Half a day. Um, so the first speaker who will be given a big chunk of time to give us a perspective of his work is Dr Don McGahn. He's a senior clinical lecture at the University of Edinburgh, is a clinical and basic science research researcher, and focus is on multiple sclerosis and energetic six, particularly mitochondria, as well as assessment of fatigue ability in people with a mess at the end ruling clinic. So don't, uh, platform is yours. Thank you. Thank you very much. Memory and thank you. All the organizers for inviting me as well. It's great to be back in Belfast, I think maybe six or seven years ago, Michael Hutchinson and sat next to Alan Thompson as well who's a very memorable meeting. That was, um So what I'd like to do is maybe share with you, Um, one of the sort of the novel mechanisms that we've discovered for neural protection in in M s. Um, you know, it's one of those kind of moments where you've been sort of working for about 10 years with, you know, starting off with the small grants going to the Ms Society is getting sort of innovative awards and challenge awards and then bigger project grants. And when you actually put the pieces together, you come up with a simple idea and then you think, Why didn't I think about this just 10 years ago? You know, it's just so straightforward. So So when I when I sort of mentioned that 10 years after to my director in Edinburgh, he said, Well, you should go to Edinburgh Innovations and ask for a pattern. So So that so So we're in that process. So So hopefully I'll take you through the journey and and where we are at now in terms of this sort of neural protective mechanism And then how we perhaps going to go forward? Um, and you can see at the bottom of this slide it says armed. So that's basically stands for axonal response of mitochondria to demyelination. Okay, um, so let me sort of get into this. So if you start off, you know, looking at the clinical course of M s, the clinicians will be very familiar with this one. Um, of course, you know, we're familiar with the different phenotypes, right? The relapsing remitting forms and the progressive forms the primary secondary. Um, but to a large extent, these are really sort of a clinically driven, um, classes sub classifications. But pathaphysiology ically, you know that that there's a lot more in common between these different phenotypes than differences. So here, you would see, um, here in the Y axis is neurological, disability or impairment. And, you know, people start off healthy, and then you get a relapse. So you get impairment and then relapse recover. And sometimes these relapses don't fully recover. So that leaves you with a fixed deficit and then more relapses. You have more fixed deficits that you accumulate, which are sort of irreversible at, but so far anyway. And then it hits a point where you get this gradual worsening of neurological function, the progressive stage that could be superimposed with some relapses. But eventually the relapses burn out, and there's little arrow arrows here indicate degree of MRI activity. So it's very much a very active inflammatory disease early on, and then that's sort of dies down while the brain atrophy here in the dotted line carries on. Okay, um, so this is This is a video When I was doing a fellowship at the Cleveland Clinic from Bruce. Strap is very kindly agreed for me to share it. Um, and hopefully this. So this is a patient over about 14 years, and you'll see how how these patients can have been changing, um, with multiple sclerosis. So the first thing you could see on the right hand side, there's been a huge amount of atrophy over the 14 years. Um, and you see how the ventricles are getting larger brain tissue is getting smaller. Okay, so, um and and and And it's quite a profound, uh, extent of atrophy that, you see, So I'm just going to play that again. So the atrophy is you know, I think it's fairly obvious. And then if you look at what goes on in the white Matter, you'll see you know, there's a lesion. Burden has increased over time, which is what happens to the amount of demyelination if you like. But you also see some lesions come and go. Okay, So try that again. Um, you see, you're doing the right hemisphere. You see some lesions, uh, appearing and disappearing. Okay. Is that is that? Yeah. Okay, Um, so the treatments that we have the disease modifying treatment is actually very good at can be good at getting rid of those new spots that are appearing. So by the time patients diagnosed, you know, some of the damage is already done. So they've had a subclinical phase. This lesion load could be very variable, so there's already areas of established demyelination and inflammation is going on. Um, and clearly the progression, as we all know, carries on independent of really the immune therapies. Even the license immune therapies really reduce the rate of progression. It doesn't stop the progression. It doesn't improve neurological function. It just makes the progression less fast, if you like. Um, Okay, so let me just the next one, Um, in terms of the pathology, um, before sort of the late nineties axon degeneration wasn't really recognized as one of the major features of M s. It was, you know, Bruce Trap. And you, Peri, um, in 97 98 published these papers, um, indicating that actually, that's quite a profound axon degeneration. Um, it isn't just about inflammation and demyelination and and we begin to appreciate these different types of lesions that people characterized as active, active or chronic, active and in active lesions. And that's just different extent of inflammation and demargination. So they all got demargination. So this is the top right? Here is myelin, so you can see these lesions and then the inflammatory component is variable. So in the acute lesion, the whole demyelinating areas got inflammation. The chronic active lesion is like a ring of fire. Okay, the edges are quite active, like the acute lesion. But in the middle, the myelin is already been burnt, right, and some of the accents have disappeared. And eventually this lesion will turn out where the inflammation dies down and at least with naked axons, uh, without violence. So when you look at on the left hand side, you see here in green, that's an axon. Some of the myelin has been stripped off, and this vaccine has been transacted. Where and you know, inflammatory demyelinating response has actually damaged the axon irreversibly, so it causes terminal avoid or the axon bulb. Um, when you look at the extent of these, when you look at an acute lesion, you see lots of these bulbs right. So here you can see that where the inflammation is. This is demyelination. And you said, you know, it's quite quite abundant. Um, damage to the Axiron. So these are sort of irreversible, if you like in in the central nervous system. And And you could imagine a patient getting a new lesion. Acutely inflammatory lesions like you saw on the MRI scan, demyelination inflammation and lots of these action, all transactions. And this could potentially be one of the reasons why the recovery is not complete. Okay, So if somebody you know makes a 70% recovery, that could be because they've lost too many accidents, they can't quite compensate, right? So sort of the remyelination therapies and so on would only really work for those remaining accents. Okay, So the the therapeutic strategies really so far for protecting these accents been to target inflammation, which is which is great because that's what causes the damage in the first place. So what I'm going to show to you is a neuron centric view that how the neuron actually also needs to be targeted besides information as long as the demyelination is ongoing in this disease. So So you know after after those 10 years of work, this is This is a very simple concept that I'm going to sort of show you. Um, so So if you look at unmyelinated fiber's, probably the optic track is probably the best one where you have the unmyelinated segment in the lamb in a crib, Rosa. And then the axons are pollinated in the optic nerve. You see the difference here. So you see lots of mitochondria in the UN martinated fibers or the segments, and then the monitor fibres. You see less, and you can understand that because energetically pollination makes it much more efficient. So you need less ATP. Mitochondria don't hang around. If they're not needed, disappear, you get less of them. So during development pollination, you see that? And then when you demand unit, uh, the Axiron, all of all of a sudden it kind of goes back to is if you like the unmyelinated level the iron panel. Although there's some differences in the subtypes redistribute, uh, and the energetic changes, and they actually requires similar kind of energy to the unmyelinated ones. Uh, and you see these terminal, uh, ovoid of the axon being damaged in this process. Um, but those accidents that survive actually in the demand is that when you look at them, they have they have kind of gone back to these sort of unmyelinated levels. Okay, Somehow, when they lose the myelin, the absence of manage some of the absence of managed reset, too. They're sort of a pre myelinated levels, if you know what I mean. So it's that type of time period, which is really vulnerable. So here you see, myelination reduces the axonal content in the accident. When you demyelinate, it needs to go back to the level sometimes higher. And it's this period where while while it resetting, where the accent seems to be very vulnerable. And if you can make this much more efficient, you can then protect at least the proportion of these accents. Okay, so it's it's kind of a neuronal mechanism. So here's the optic nerve that I showed you you can see here. This is the unmyelinated segment. Here's the myelinating segment. If you look at the mitochondrial activity in this case, cytochrome see oxidase or complex for activity, see a lot more, uh, in the unmyelinated segment. Similarly, if you do e m. And look at the mitochondrial density. You see a lot more in the unmyelinated segment, so these are healthy, healthy, uh, optic nerves, Uh, and you can see how how more energy is required. So if you if you if you get the difference in iron channels So now 1.6 for an example following. Demyelination persistently let sodium in and you need to get rid of that sodium, using sodium potassium ATPase, which, of course, requires ATP hence greater reliance of the demyelinating plaques on on on ATP. Okay, so this is the respiratory change you probably remember from your sort of levels of the school school days. It's got five complexes. The electron transport change from complex, complex four and that's cytochrome. See, oxygen is this is the oxygen binds to okay, and the proton pump to make the electrochemical gradient. And 80% is a complex five, actually, then generates the ATP using the electrochemical gradient. Um, now. So when we started off, what we wanted to do was based on that hypothesis, that when you lose my balance, you need more energy. We were expecting to see more mitochondria in the demyelinating plaques. And so this is a whole bunch of autopsy from MDS cases, and actually, this is what we saw. So if you if you look at the complex for activity and you localized to the axons and in the demyelinating lesions the ones that I showed you early on and you see there's a huge spread, of course, you see a significant increase in the mitochondrial activity compared to, uh, control tissue without m s or normal appearing white matter from, uh, those who have had m s. Um, interestingly, if you look at the accents that were damaged so the ones that's with the terminal avoid or ones that accumulated, uh, things like a pee pee or synaptophysin. We did not see that sort of increased activity, Right? So suggesting that this phenomenon is is sort of a compensated process that I was alluding to early on. But when we reported this, the literature was actually suggesting that this is a pathogenic process, right? So we were saying, having more mitochondria, more activity is actually bad news, because eventually these guys will produce more toxins and the Axiron will eventually degenerate. So there's a possibility of this happening. Um maybe we can mention that later on. But certainly the acute scenario Our prediction at that time was this is actually helpful. Helpful process. So So that's a mess. Tissue. And then if you and and looked at the experimental models demyelination. Uh, this is a collaboration with Robin Franklin. Looking at TV, Enbrel might induce lesions. We saw the same thing here. You see, men interactions demand interactions with lots more mitochondria. And interestingly, remyelination didn't bring it back down to the myelinated levels. Now, you know, we only pursued this for I think, uh, something like 84 days following demargination. So in the long term, whether it goes back to the myelinated levels or not, we're not too sure. But at least the remyelination itself didn't really reverse it. Uh, within that time frame. Um, since since that we've actually looked at several other models and I'll show you later on, and that increased mitochondria is a very robust, very consistent finding, right? It doesn't matter how you do, moderate. So then we started to dissect This is a good thing or a bad thing. So with Bruce trap what we we We use the the center filing now. Mice. So essentially, center filing is a axon specific mitochondrial docking protein. When you knock that out, the mitochondria don't stop, and they just keep moving. So when you then demyelinate these mice, they can't make that response right. They can't sort of stop and fuse and increase the mitochondria volume. They just jump up and down instead. Um, and you can you can see here. Basically, when you when you knock out the mitochondrial content increase, you see a lot more terminal avoids in these mice compared to, uh, the wild types. Okay, So, um, so the next thing what we wanted to do, uh, was to see what happens if you actually boost that response. Right? So we know if you prevent that response is bad. Boosting it is that Is that any good? So to do that, we took several different approaches. We took micro flutic chambers. We use cerebella slices in vivo and here with the cerebellum slices we did with the Pecos Spritzer. We basically spread in, uh, mitochondria. Targeted photo convertible. D I m e s too. So here you see a park in the sell. You see its action going along and you could photo convert segment of interest. So here we just did the proximal part, And you see the cell body to the to your left. Um, And then you can do time lapse imaging of that segment and see the mitochondrial movement, both antegrade from the cell body and retrograde from the distal end right in that segment. So we did that with and without demyelination to see if there is a difference. Okay, so these are a bunch of money donated axons in that initial secretary left is the cell body. So we're looking at the proximal segment of the Axon. The photo conversion isn't 100%. So you still see some residue or green in there, but you'll see some green mitochondria without read that are sitting along. Okay, so you see some, some retrograde, some antegrade. So these are over about 20 minutes. Time lapse. Yeah. And then we added lysolecithin to these slices and and looked at the managed vaccines. Okay. I mean, it was it was like striking. So when Simon I was doing a PhD with me at the time when he came back and showed me these. I mean, it's quite tricky to get this right because he had to make sure you get the Pecos Spritz. All right? The neurons have to survive. So he did a lot of work to try and collect these a movie. Thought it was just sort of incredible. Um, Okay, so then he did a bit of analysis. Uh, make sure that he deserved to get his PhD. You know, you had that wasn't good enough. So he had to do a bit more. Um, so basically, what you find is you see the number of much contrary increases with the imagination. Um, the total area increases the size, you know, So on. Uh, So there's significant changes if you look at these chemo graphs here. So basically, those videos put into, uh, sort of, uh, over over time, uh, images here. So you see, in myelinated axon some of the mitochondria moving that way and that greatly, this one's moving retrograde. And when you demanded you see a ton of these mitochondria moving right, so clearly the cell body is responding to demomination really quickly. So within 16 hours of myelin being damaged, you see these massive response, and presumably that's that's how the mitochondria content in the action goes up rather than mitochondria being generated or synthesized in the in the action percent. So So really, this is quite exciting, because when you look at the neuropathology in M s, you see a massive axon loss towards the end in autopsy. 70 80% is a massive, uh, synapse loss. Similarly, 70 80%. But if you look at the cell bodies, that's usually about 15 2025% except in these B cell follicle type cases, which is quite aggressive, and in the superficial layers, you see quite extensive neuronal loss. But it's what sort of distinguish is M s in some ways from the other classical neurodegenerative disorders is the cell bodies are surviving, you know, relatively speaking. But the neurons been disconnected, right? The accents have gone, the synapses have gone. And therapeutically, if you're going to target a neuron, that really gives you an opportunity. Because if you can target cell body, whatever the process of that going on, hopefully you could prevent the axons and the synapses in M s. Um, so to try and sort of figure out what we could do with this response we wanted to sort of first look at the relationship between those terminal avoid formation and the time course of the response. Right. Um, And here what you could say you can see is if you look at the the time course of the multi cultural increase and it peaks. So in this model, here is about five days, and here and and here it's seventies. But by by the time the axonal responses peaked, you've already had the maximum axon damage, right? So the extent of, uh, avoid formation peaks within a day here and within five days. So the so the mitochondria response peak actually lags. So So what that was telling us is maybe, actually, yeah, it is a natural sort of homeostatic response, but it's not good enough. It's not fast enough to That's why some of these accents are are dying. So we wanted to find a way of enhancing that, Um, so we use several different methods to enhance the mitochondria response. So we use P. D. C one alpha said to be the master regulator of mitochondria biogenesis. It does lots of other things too. So we basically produce more mitochondria in the cell body hoping that more will be transported and that that will speed up the demyelination response. We always expressed mirror mirror one which is involved in trafficking mitochondria. Uh, and also you get a get a drug screen and picked up a drug that's been licensed for use in diabetes called pioglitazone to see Oops. Uh, what happens? So maybe there's some videos here, Uh, and you can see what these different manipulations do. Um, so basically, these three methods revved up the antegrade transport of the mitochondria. Right. So that's a way of really enhancing the mitochondria response. So here, this is all contraindicated. You could see the numbers speed, size and occupancy the impact of that, uh, of manipulations. And this is an example from the micro flutic chambers so you can put the drug so we put the neurons here, the axons grow, and then we have a core culture system in the other chamber, so you can demand on it putting lysolecithin here without affecting the cell bodies. And you can put the drug here hopefully without affecting too much going into the core culture chamber, uh, and and see the impact and you by enhancing the mitochondrial response you could you could essentially, so you could demand it. The acts on here but still protect the action, unlike the untreated one by the accidents. Degenerate. Obviously it's not. It's not an all or nothing phenomenon. You know, it's not a perfect method, but you get a significant action protection in this system, and that's associated with the enhancement of the of the mitochondrial, um, response Similarly in vivo, if you do that, do that in the cerebral slices to, uh, again with the less less within, uh, in mice for coalitions. Here, you get significant symptoms, so it's a bit more bit more to do. So just just revving up the mitochondria. If you like mitochondria more like the power houses, right? So you get the power houses, but it needs to feel, uh, to get them to do the maximum job. So we we don't think with these manipulations, we are actually doing anything about the field. Um, so there there may be more, actually that we could do to protect if you could somehow provide more fuel into these accents, for example, uh, to to improve this mechanism. So we're really excited about that. And then we were We were then thinking about the disease m s, because this is not, like, sort of young animals or brain slices on mice. You know, it happens in, you know, with age, the progression the demyelination goes on. Um, And what we discovered before we started manipulating was when we looked at the cell bodies. These cell bodies had damaged mitochondria. So lots of different different groups using different methods, essentially all ending up showing my control defects. Right. So here, if you look at Bruce Traps group looked at, uh, nuclear encoded transcripts. They found significant reduction of mitochondrial respiratory transcripts. We found mitochondria, DNA deletions. Um, Jenny Broadwater. She did proteomics and found, uh, significant changes related to mitochondrial proteins. This is Jack Van Hanson. Found a decrease in PG. See one alpha and and so on. Okay, so this is actually quite interesting, because when you unlike a young mouse, when you look at, um, m s tissue, this is post mortem. Late stage. Undoubtedly, they didn't really. Some of these neurons didn't have healthy mitochondria. Right? So the question is, if you're going to ramp up mitochondria, okay, that's good. if it's a healthy neurons. But if you're ramping it up in some of these dodgy neurons, is it really bad, you know, because you're basically sending out these, uh, sort of defective mitochondria into the Axiron. So to do that, we basically do different models. So we took a genetic model and knocked out complex for activity inducible way. Um and, um, the first thing before we actually generated the mouse, what we wanted to do was to see Is there a correlation? Right, So So if you if you see a, uh an M s autopsy case that's got lots of mitochondrial changes in the neuron, do we Do we still see the axonal response? Because our prediction was, if there's a lot of mitochondria damage, we probably won't see the axonal response in the surviving axons, right? The ones that have gone died, they've gone, um, so So we can talk about which area of the nervous tissue should be looked at and decided to look at the dorsal root ganglia. This is a great idea from Bruce Strap, because the cell bodies are sort of like the peripheral nervous system. But they put a central projecting acts on which gets demyelinated. So things like the meningeal information cortical demomination all of that shouldn't really affect the DRG neuronal cell bodies. And when we looked at those mitochondria changes, you know, we we found quite extensive changes here in the dorsal root ganglion neurons both appropriate, receptive and nociceptive neurons. And I think this is about 20 cases. We characterize these looking at mitochondria, DNA, different mitochondria, subunits, um, and identified, um, multiple complex deficiencies in these cases. But in in six cases, we were able to get the DRG. It was actually quite quite tricky to collect DRG because not a lot of the brain banks don't collect also ganglia. And, you know, it's the last thing people that have to go in myself and actually pick them out. Uh, but in six cases that we managed to get, uh, spinal cord block at the root entry zone to have a demyelinating lesion right in the dorsal column. So So we know that's the first lesion that the cell this neuron has been exposed to, right? So hopefully any sort of multiple for collision shouldn't be influencing the mitochondria changes that we see in that entry zone block. And to our surprise, what we saw actually was the cases that had more damage. Mitochondria in the cell body actually had greater mitochondria in the accident. Okay, we we're predicting the opposite were thinking, you know, they're not They're not healthy, so they probably can't move into the Axiron. And and there's going to be, you know, more actual damage and so on. So it was quite surprising to see this this this trend. So then then we thought, Well, we've got to generate a mouse. Um, so we got a, uh, DRG specific complex for knockout mouse. Uh, it's got some interesting phenotypes I can talk to if anyone's interested in the implications of DRG neuronal mitochondria changes, um, and basically revved up the market called demyelinated these mice and revved up the mitochondrial response. Because if if it's still a good thing, then we should see some protection here. If it's bad, then, uh, it would be the opposite. Um, so So here. When when you say is when you when you demyelinate these might still, uh, mount the response. Okay, like we've seen in the M s tissue. Um, So there's increasing mitochondrial content uh, and so on. Um, And when you when you manipulate when you enhance the mice using, uh, the drug pioglitazone you're showing in the bottom here, you see a significant, uh, some protection. Okay, So suggesting even even those neurons that have got damaged mitochondria revving up the mitochondrial response if they're still getting demyelinated is not a bad thing. Okay, so if you're going to use that as a therapeutic strategy, we at least hopefully we don't have to worry about that aspect. Um, Okay, So with the mouse were treated with prednisone, you can see that Increase the PG, see one alpha more mitochondria, biogenesis. So you get a bit more mechanistic view of that. How that drug works. Um, so So we're back to where we started. Basically showing you what what we've discovered. So, uh, myelination reduces mitochondria, content demolished, and he needs to reset back to his sort of a pre marinated level. But that's a slow process. Uh, good enough to save some of the axons. But if you read that up, you know, we've done it in three different ways, but I'm sure there's multiple, different ways to do it. You can protect some accents, but that's probably not the end of the story. You know, there's a lot more to do with, uh, providing the metabolites and so on to these accents. Um okay, So, um, this is this. Since that work was done, we've been just just to see how robust, uh, much control responses we've looked at all these different models. Now, I think the paper Yeah, it is impressed in and lots of different ways of demyelinating. If you look at the demand is consistently you see increasing mitochondria content. Now, some of these could be, you know, just the transport block. They just stuck. Some of it could be the natural response I talked to you about, so it's going to be a very sort of a mixed picture. Um, but if you look at the level of the complex for activity, it's a slightly different story. So, um, in the mornings, like e u c uh, more mitochondria. But they've lost their complex for activities. Right? So a bit like our complex will knock out mice. Uh, they're not working properly, but it's still, uh, from our economic zone. Is it still good to have those mitochondria, uh, than have less of them. Uh, the complex activity that the loss correlates with, basically, um, the extent of acts on damage. So So So while we kind of revved up this arm response, there's a lot more to do. That's the metabolites. And also that's preserving to try and preserve the mitochondrial activity of the mitochondria that are there to sort of Max maximize the benefit. Um, so this is just to summarize in the inflammatory environment showing how the reactive oxygen species in e for an example, uh, M s lesions can damage the mitochondria that have already responded. So a sub optimal response, which we could therapeutic target. So in summary, hopefully, I'm showing you this armed response, which is excellent response of mitochondria to demyelination bit of a mouthful because it's one of our postdocs like that because it's armed arming the axons to withstand the withstand the injury. Um, and, uh, it's a natural, spontaneous response. Um, and it fails in M s progressive, especially because mitochondrial not working properly, So it's not sort of the maximum. Um, and you could target with PBC one alpha, um, and other methods to try and protect it. So So what We How we see this is really very much as a mechanism to front load the remyelination therapies. Right? Because because remyelination is basically about trying to save the accidents that that have already survived, right? So the actions have already survived this acute injury. Are there to be re myelinating? So if he front load the remyelination therapies start, start this early, then we can save more axons for long enough, hopefully for remyelination to to do the long term neural protection. So that's the bit that the university was very excited about. And it's got a pattern for that front loading of the remission therapy with this mechanism to save more axons. And that's, uh, cartoon to show how the armed response So So it's really a bridging mechanism. Okay, so it's bridging from my lung damage to myelin repair. So you basically have more axons there to for remyelination to take over and do the long term protection. So we just submitted a grant application to the M s society with some pilot data, uh, to see, actually, what happens because, you know, so So one of the questions was neurons have got. Some of the neurons have got dodgy mitochondria, so reading that, uh, it doesn't seem to be a bad thing. But if you're revving up mitochondria in neurons to save the keep the demyelination axons in M s, we'll be doing similar things to axons that are still chronically demyelinated, right. We'll be doing the same similar things to access that are remyelinating. So all of that will be going on at the same time, like so, if you think about these people, call about this floor expanding lesions. So that ring of fire I showed you early on where the myelin is getting damaged. And that's that's where this mechanism will protect the axons. This mechanism will won't protect the ones that have already survived, right? That's where the REMYELINATION would come in. Um, so so what are the implications for remyelination accents? What the implication of chronically demyelinating axons of boosting this response in the long term. So, uh, so we propose some strategies and and hopefully, you know, if that's good news, then we can, uh, take, uh, this type of manipulation to the clinic and and test it in in patients. Okay. Thank you. Very much Thank you for doing this is definitely not the bridge in in, uh, so I'd like to open the floor for questions. And since it's, uh, like, if anyone would like to ask questions from the audience online, please do that. And Michelle will read it out for me and just raise your hand if you have a question to don. Uh, please. Thanks. Sorry. It looks similar. Similar? I'm putting talk. Thanks dot So my question is what you're alluding to, and they're at the end when you enhance the chondral response. This is possibly not specific just for the demyelinated accents. But other healthy accent will be affected as well. Does this have any consequences? And could there be any sort of exhaustion? And eventually, if you constantly trigger them, those that do not need them? Absolutely. Does this affect on the Neurontin? They? Yeah. So there is. There is a suggestion for this based on shiver er right. So if you look at all these parameters, are sure you might conjure content The number sized dynamics in the Shiva a mouse is actually quite similar to the Democratic mouse. Of course, the shivering you can keep for a long time, right until that sounds to generate. So there's a group that's published in the Journal of Neuroscience. Um, uh, you need to be reproduced to see if it's if it's reproducible or not. Um, where they implicated my condo complex for deficiency. And they knocked out by crossing the shivering with the center file in and showed that there's less actual degeneration. The survival improves, and so on. So So the conclusion from that was this response. It may be fine in the in the short term, but in the long term, having more mitochondria, if they're damaged, it might be a bad thing. So So that could be true. So that's the purpose of that. Last night, I talked about to try and answer that, um uh, but I think I think if you if you can re myelinating these, then you could obviously got you got. You can counter any long term. Now the question is, is it what happens to the ones that you can't remember the name, you know? I mean, that's that's That's a very, very important question to answer. First of all, to find out, Is it bad? Is it Is it bad for even even remyelinating axons Having more mitochondria than needed might be a bad thing. Now, if you just, like, feed wild type mice, for example with your medicine, um, that there's been toxicity studies done and so on. And if you obviously increase the dose massively, you you damage the axons. Um, but but within within a sort of a lower dose is, uh, that doesn't seem to be any toxicity. Um, but also, I think, you know, like, healthy optic nerve, right? The lamina crabro so unmyelinated that sense somehow they they survive. So I guess it's to what extent you're going to ramp it up. You just don't want to overdo it and do it as short as possible, little as possible and then get remyelination to be the savior. Actually, uh, sort of a little bit joining two. Ativan's question. I was thinking during your talk that if we have more mitochondria, we will have more stuff to feed the mitochondria to produce ATP. So what happens to the metabolic, uh, profile at these? Uh, these damages, I mean, you you would expect that you need more juice. Where do you get that? exactly. Exactly. That's a great question. Because, you know, it's collaborative work with Julia Edgar from Glasgow and close Navy. You how the oligodendrocyte metabolically supports the axon. Right. So when you lose the oligodendrocyte, you lost that metabolic support. So where does it get it? You know, the exercise. Um, so to try and, uh, remyelination may be good in terms of giving more metabolic support so that the Jews aspect is something that we we've completely ignored. And how do we have no doubt that that that would be another, uh, you know, low hanging fruit in terms of therapeutics to ramp up the so you get even more 80. So maybe we don't have to ramp up the response as much. You know, for the long term problems, if you could give it more juice. Uh, thank you. Because it is really is. Really? I think the window is narrow. It's It's a bit like, you know, formula one driving, you know, the pit stops, the rubber comes off, the rubber goes on, right. It's just saving the axon until until you do that, uh, if you can do the remyelination quickly, probably don't need this one But, you know, chances are we're never going to get to those, like, a few days where you can save the Axiron. So you just got to protect it for long enough, a quick one down here. That was a great job. I think at one point you mentioned that, you know, a small proportion of the axons to survive in Ms Lesions. But then you mentioned, except for the cortical, the topical, you know, Is it related to dysfunction? Maybe. Or, you know, is there anything more on that? Yeah, I think that's probably more complicated that I don't think it's a mitocondrial sort of a primary mitochondria story, Right? Because lots of inflammatory environment. So that's Richard Reynolds work, isn't it? Showing, um, you know, the superficial layers have less neurons anywhere, right? So if you get rid of a few, then you lost a higher percentage, you know what I mean? So, yeah, I'm not saying I'm not saying it's not relevant, but you just have to kind of look at it, But, um, but I think the cortical lesions were probably more complex than that, those inflammatory ones. But what's still striking, though, is a lot of these cases if you, you know, most papers is like 15 20% cell body loss atrophy. I mean, they don't work properly because the synapses and accents have gone. But cell bodies that somehow hanging around is the better one for you to get a picture of the country a schematic e and K. Yeah. Yeah. I mean, we've done a lot of spinal cord, right? E l P uh, pc. And so on. LPs keeps on spinal cord is completely spazzed, at least with. And I also looked at keeps on plus randomizing model that Bruce Trap has and even that the spinal cord is bad. So the only place we looked at was the corpus callosum. Yeah. Thank you. So question there and the question that and then, yeah, I mean, that could be particularly relevant if you know if if that's, like real and, you know, consistent and robust thing, um is to rescue how the neurons that have already got damaged mitochondria, Jeremy, especially if they've got to say genetic problems that we can't reverse. Introducing a bunch of healthy mitochondria might actually be quite if you can do it. Uh huh. Thanks dot great. So it's interesting because maybe rethink how I think about your protection. But I think it's very generically as good for everybody in this setting as a precursor to repair. That would wouldn't necessarily be everybody, because the older individuals to repair. So I guess it's sort of, you know, the clinical trial question. Who would you seek, given that you have limited shots in a clinical trial? Who, what population of people with Ms do you think would be the first group that you were tested? You want to really give away our plan? So we're planning to do a, you know, small phase two trial looking at that. So So, First of all, you got to have a situation where there's ongoing demomination, right? Uh, so you could pick up the new lesions, which is kind of unpredictable. You don't know who's going to get them, and you've got very effective drugs, so patients are on effective drugs, so it's quite hard to find a new lesion a time, you know, you know, if you're choosing, whereas if you look at a slow expanding lesions the chronic one that's expanding, you know, at the edges you get ongoing demyelination. So So that's the ones that were thinking about, uh, and to see, you know, look at more sort of a finer parameters of imaging like D T I and Marlin. And so on, too, Because if you if in those the ring ring, ring of fire, If if you're protecting more axons, we should be able to see more marlin from remyelination there. Right, there's more accents, more remyelinating marlin, and maybe even the d t. I. We might get a better signal. Um, so it's, you know, finding the drug, finding the right patients. So, you know, like, kind of highly selected sort of group to show whether it works. And then you could do it if it works. Uh, so we were able to squeeze you more for your secrets? Uh, well, I'd like to move on. Uh, we are running as usual. Uh, thank you very much.