Mitochondrial Transfer, Obesity & Immune-Metabolism Interactions | Jon Brestoff | Episode 261

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Happy to be here. Do you want to start off by just telling everyone a little bit about who you are and what your lab studies? Yeah, my name is John Brestoff. I'm an associate professor of pathology and immunology and director of the Initiative for Immunometabolism at WashiU School of Medicine in St. Louis.

My laboratory primarily studies uh immunometabolism, which is how the immune system is regulated by metabolic pathways and how metabolic pathways regulate the um how immune cells regulate metabolic uh systems themselves. But we do a lot of work on mitochondria and the transfer of mitochondria between cells. And that has really kind of um been the main focus of my lab over the last couple of years.

I wanna start off talking about mitochondria a little bit. So we'll talk about the mitochondrial transfer stuff you've worked on, which is really interesting. But I wanna give a I wanna give people who who aren't too familiar with mitochondria a sense for how they move and what their life cycle is within cells.

So we don't need I don't think we need to start all the way at the beginning and go over what mitochondria are. I've covered that topic a number of times on the podcast. So we can keep it pretty beef brief when it comes to the summary of what mitochondria do as organelles. But inside of a cell, can you give people a sense for how many mitochondria there might be and how they sort of move around and what their life cycle looks like? Yeah. Um well

So my the number of mitochondria per cell can vary dramatically depending on the cell type. It can range from uh, you know, just a hundred mitochondria per cell to five thousand depending on the cell type. Uh, they have different morphologies and sometimes they are formed through these fused tubular networks and sometimes they're more punctate structures, the more spherical structures.

Um typically as cells divide, they pass their mitochondria onto the daughter cells. And those that is called vertical inheritance of mitochondria. Um, that can be either symmetric or asymmetric, where the mitochondria are uh potentially the older or newer mitochondria are retained by one daughter cell and the others are moved to the other cell. Um But often it's symmetric, which means that they're basically randomly distributed between the two different cell types.

Um in the last twenty years or so we started to understand that many cell types actually have the ability to transfer mitochondria to each other in the absence of cell division. So in this process, one cell type would eject mitochondria and deliver them to a di potentially developmentally unrelated cell type.

Um, those cells then take up the mitochondria and process them through a couple of different pathways. Mm-hmm. So so mitochondria can move around within cells. There can be many of them. I would assume that basically the number of mitochondria per cell scales with. the energy demands of that cell. So I'm guessing, you know, muscles have a lot and less energet energetically demanding cells have fewer mitochondria. Yeah, it is true. Um

That tends to be the case. However, immune cells themselves are quite energetically demanding, but they um they have uh sort of they're often pre programmed to rely upon glycolysis and glucose metabolism. So I would say that the demand for oxygen cons oxygen consumption dependent ATP production

really strongly correlates with mitochondrial biomass in a cell. Interesting. Interesting. Yeah, and we can get into that a little bit more later, but I I would imagine that a lot of these immune cells have to get into environments or microenvironments where there's not a lot of oxygen. So they need a way to work in the absence of it. Correct.

Interesting. So you can have uh some cell in the body, it divides, it will basically distribute its mitochondria between the two daughter cells. That can happen in a symmetric fashion, where basically it's fifty percent go to Daughter cell one, fifty percent go to daughter cell two. And it's gonna be an even split, roughly it's gonna be uh a random, you know, choosing of which which ones go which.

to which daughter cell, but there can be asymmetries there, it sounds like, depend you know, under special circumstances or just by chance. Maybe one daughter cell gets the better or the newer mitochondria, maybe another one gets the older ones, maybe Um, more of them are just in one daughter cell and fewer of them in another one and so forth.

Right. Yeah, that's all correct. Um one of the best examples of asymmetrics um vertical inheritance of mitochondria is in the context of stem cell differentiation. the stem cells tend to hang on to the newer mitochondria and dil and pass the older mitochondria on to the daughter cell that then uh further differentiates and becomes like the next progenitor cell type in the d in the development of a lineage. Mm. Mitochondrial transfer is

H this actually happens. So a mitochondria can go from one cell to another. Uh i does it only happen in the sense of like vertical transmission where it's it's a cell dividing into two daughter cells, or can cells also, you know. Send send mitochondria to another cell like I would send you a package in the mail or something.

The key distinction actually about mitochondria transfer is that it it occurs in the absence of cell division. Um so in this case, uh one cell uh the donor cell would actually eject mitochondria, they can package them up into extracellular vesicles or release them as free mitochondria. Um, or they can even form these transient cellular connections with the acceptor cell.

The acceptor cell then will import these transferred mitochondria and those mitochondria are then processed through a couple of different pathways. So there isn't uh the vertical inheritance of mitochondria is uh very distinct from mitochondria transfer and

I would also put forward the idea that there are really distinct um numbers of mitochondria that are exchanged in these ways. So vertical inheritance of mitochondria all of the mitochondria in the in the parent cell is are going to be segregated into the two daughter cells. Um and that process, those mitochondria never leave the cytoplasm.

But in the context of the mitochondria transfer, there are only a few mitochondria that are passed between cells. It's a relatively small proportion of the donor cell's mitochondria that are delivered to the acceptor cell. Um, and in those cases, mm in most of those cases, the mitochondria are um actually ejected from the

from the donor cell type. So they're actually released by that cell for capture by the acceptor cell. Okay. So from a terminology standpoint, it's inheritance. A cell divides into two or transfer, you know, one one shift its mitochondria to another one one way or another. In terms of transfer, I want to talk about how and why this happens and and you can pick the particular examples and stuff that are that are the best to illustrate this.

But in terms of the how this happens, it sounds like there's at least a couple ways that this happens. One, they can be packaged and sent off in a vesicle, probably s you know, at least somewhat akin to a neurotransmitter release in a neuron or something like that. You know, they're packaged in a vesicle just like other things are packaged in a cell and then released. Um let's talk about that one first. Um is it just like

You know, normal vesicle release or are these special big vesicles that can hold a big organelle like this? What does the structure look like here? Well it's not so clear actually. Um because in large part because the the precise molecular mechanisms are not worked out for the how mitochondria are released. Um, we know that the the vesicular structures are much larger than the vesicles that you would find in the context of an axon.

Synapse where neurotransmitters are released. Those are very small vesicles, and you we know a lot about the mechanisms of membrane fusion there to facilitate release of the neurotransmitter. But for mitochondria we're talking about cargo that's up to a micron in diameter. Um the typical um machinery like clatherin coated pits are sterically hinder hindered and cannot accommodate cargo that's that large. So um

Th this is really an open question about exactly how the mitochondria are released, but it likely does involve a membrane fusion event with a vesicle. And that's how free mitochondria are released. Um when mitochondria are released in inside of an extracellular vesicle, so the released component is a mitochondrion with another membrane around it. Um that uh that process occurs through the multivascicular body.

Um so basically lots of different vesicles fuse together and form this multi vesicular body, they um they then can either be move to the lysosome or in the context of energy depletion, this the the donor cell will actually fuse the multivascicular body with the plasma membrane and release the contents into the extracellular space. And that process we know a little bit about now, that is dependent on this protein called RAB seven.

Um and when it's in its um energy replete state, it favors the lysosome uh targeting to the lysosome and when it's energy depleted, it favors ejection. And then the third mechanism of transfer is the formation of transient cellular connections. The most A well studied example and the most frequently observed one is through the formation of tunneling nanotubes. Which is when one cell type sends a projection to another cell type, and they form this sort of like tubular highway structure that.

um allows for there's basically like a microtubule and actin based um filament system that allows for the mitochondria to be passaged from one cell type to another using the Miro. uh transport system. So literally i the cytoskeleton of one cell is used to create a tube or a tunnel and a mitochondrion can literally go through it.

Wow. Yep. So yeah. So this is this is um this is an active process, it seems. It's not um it's not like somehow mitochondria get into something like a vesicle and kind of slip out of the cell. It this this is a very regulated process, it seems. Like the cells are trying to do it, so to speak. I think so. Uh um I mean I there's a lot of interest in trying to figure out under what context mitochondria transfer is regulated. I do think that it is likely highly regulated.

Um but we just don't know enough about it yet. It's just such a new biology. Yeah, yeah. So what are some of so thinking about why this does happen, what are some of the contexts in which this has been observed? Um w what types of cells are re transferring transferring their mitochondria and and what does that actually look like?

I guess let me start by saying that this process has been observed in a lot of different organisms. Um it's been found in yeast, plants, mollusks, fish. Uh And uh human cells. So we know that this process is occurring in evolutionarily distinct organisms. In vivo. So this is all in intact animals.

That's all in intact animals except in in humans where we don't have the capacity to actually detect mitochondria transfer currently because the methods are really based off of using fluorescent reporter proteins and um So the best evidence, the most extensive evidence for mitochondria transfer is in my is in mice.

Um, we study this in the context of the adipose tissue. We have made mice that have a fluorescent reporter protein that tags the mitochondria in specifically in adipocytes or fat cells. And what we found is that adipocytes are frequently transferring their mitochondria to other cell types in the adipose tissue. The most frequent acceptor cell type is the macrophage. Um it seems like in the healthy state, the macrophages are degrading the mitochondria that are released from the adipocyte.

And the idea is that this may be a qual a mitochondria quality control mechanism that helps the adipocyte. Because some of those mitochondria are gonna be so badly damaged that they are just there's no point in recycling them, you know. And macrophages like a professional incinerator. Right. So Um so but the point is so this is happening all the time. It's frequently observed that these adibicytes, these fat cells, are getting rid of their mitochondria. They're giving them two macrophages.

And because that's the cell type they're giving them to, the hypothesis is. Maybe there's so many damaged mitochondria in there. Maybe they can't be cleaned up by the adipocite or not fast enough or something like this. There must be a reason why it's handing it off to the macrophage. And a good guess is that because it's a macrophage, it's sort of built to do this type of thing.

That's right. That's right. And in that case, the mitochondria tend to be degraded. Um interestingly, in the context of a high fat diet. the the dietary long chain fatty acids in the diet actually suppress the ability for the macrovages to take up those mitochondria. And that leads to the diversion of the of the adipocyte derived mitochondria that are that have been released. into the blood. So instead of being taken up by the macrophage, they get into the circulation

And we and others have shown that they go to other organs. So these mitochondria are actually being transported from the adipocyte to cells in the liver and the heart and other organs. So um so so it sounds like the implication here is If you've got a high fat diet

And we'll dig into exactly what that means in a minute. But basically these adipocytes are getting rid of their mitochondria in in the presence of certain dietary fats, instead of those mitochondria being shipped off to the macrophages where they're just going to be incinerated basically. They don't get into the macrophage. They're just sort of floating around in the bloodstream and they just get to other organs and then go inside of cells there.

That's right. Right. Exactly. Yeah. And they yeah, they it we thought maybe they would get taken up by other cell types in the in the adipose tissue because there is an interstitial space. Mm-hmm. And there are other cell types present there, but that's not the case. Um and presumably presumably these are dysfunctional or problematic mitochondria in some way. And so now the worry would be if you're getting taken up by other cells, they might be now having a problem.

Well yeah, so the actually the literature suggests that The mitochondria may be damaged, but what the what that does in small doses actually is it tickles the antioxidant uh pathways in the acceptor cell types. Um and it's a it creates a little bit of oxidative stress that allows the cell to basically boost its antioxidant capacity so that it's better conditioned to deal with a more severe an uh oxidative stress load.

And this is a concept uh sometimes referred to as mitohoresis. It's uh you know, a little a little touch of Oxidative stress is actually beneficial. Right. So a little bit of a stressor induces a beneficial effect because it's inducing sort of anti-stress or in this case antioxidant pathways. that will be net beneficial, but then at some point you can have too much of that stress and the antioxidant capabilities can't keep up with it. So this could be good at at a sufficiently low dose.

Exactly. And so we think it may actually be a way for cells to communicate with each other on an organ inter organ level. Um, you know, the adipocyte has a really unique point of view on the metabolic status of an animal system. They get to see all of the lipid biology that occurs in a way that no other cell type does. Um And I I just think that it's it's intriguing that perhaps the adipocyte is telling other cell types like cardiomyocytes in the heart w about the

about the metabolic status of the adipocyte using its mitochondria. Yeah. Yeah. So I guess yeah, there's there's multiple I I'm I'm guessing we don't know all the details here, but I can immediately sort of reinterpret the first observation in a in a potentially different way now. So if these adipocytes are just sort of giving off mitochondria at some baseline rate, maybe that can change under different conditions.

You know, one hypothesis is, oh, they're getting rid of mitochondria that are a problem. And that's why they go to macrophages to get rid of them. And that's basically what's happening there at baseline. Another way to think about this, I suppose, would be that they're just giving them off at a constant background rate to advertise the metabolic status that they have, whatever that may be.

And in the case where certain lipids inhibit their destruction by macrophages and their transfer to macrophages, that could mean You know, that could mean say uh there's now a metabolic problem in the adipocyte, and it does not want it destroyed by a macrophage because it wants other cells to know what's going on so they can react and adapt to that or something of of that nature. Exactly. Exactly. Yes.

And so talk a little bit more about the diet, the dietary fat composition that induced this um lack of transfer to macrophages. Yeah. So um what we figured out was that it's actually the dietary long chain fatty acids that are found in um lard. So this the high fat diet that we typically use is lard based. What what are those specific fatty acids? So the main fatty acids in lard are um palmitate, uh palmitic acid, steric acid, oleic acid, and linoleic acid. These are

uh saturated and monounsaturated and polyunsaturated fatty acids. Um they have a longer carbon length. And um what we figured out is that if they are in the diet specifically, they inhibit the ability for macrophages to take up mitochondria. Um they do that by Uh decreasing the expression of enzymes that are involved in the synthesis of the attachment factor for mitochondria.

A c a few years ago we did a um a large genome-wide CRISPR screen to figure out what that pathway is. And there uh it's a call type of molecule called heparansulfate. Um so the enzymatic machinery that's used to make those heparan sulfates is downregulated when s macrophages are exposed to uh dietary long chain fatty acids. Mm-hmm. And so um, did you guys actually measure the fatty acid composition of this diet yourself directly?

Uh no. We didn't measure it directly ourselves. Uh it's the composition is based off of um manufacturer provided details about fatty acid composition and the known source of the lipids. Got it. Got it. Um okay, so you give this high fat diet to the animals. They presumably put on weight. Um, their dipsites change and among other things. the mitochondria stop getting transferred to the macrophages and start getting transferred to uh other organs ultimately.

Right. Yes. Um, okay, so and so what comes next in this story? Did you guys were you guys able to look at like what they do in those other organs or get closer to answering the question of like, okay, what is what exactly is being communicated and why when this happens?

Well, I think this is one of the biggest questions in the field at the moment is uh what is that information that is communicated and how does it impact the function of different acceptor cell types? I think one of the best clues comes from Phil Sherer and Claire Crew.

Um they're uh they basically demonstrated that the adipocyte derived mitochondria that are released in extracellular vesicles, that those mitochondria, when they're taken up by the heart, uh induce this antioxidant response pathway in cardiomyocytes. and uh confer protection against ischemia reperfusion injury after a heart attack. So um their data strongly suggests that there is a cardioprotective effect of these mitochondria. Um

But that's the only example that's been reported. And I think there's a lot of a lot of exciting avenues for new research in other organ systems. Um are there any other major context in which we've observed this type of mitochondrial transfer in vivo, other tissue or organ systems. And can you maybe just give us like a survey of of where this has been seen?

Sure. Uh it's been observed in uh as far as I know, every m most organ systems. Um sure uh surely everyone that we've looked at uh and that haven't published yet. Um, there's some very nice literature in the bone. Um there mitochondria transfer has been shown to occur in the brain, in the heart, in the uh immune system. in the adipose tissue, bone. Those are the best examples. Okay, so it seems to be a pretty widespread phenomenon.

Mm-hmm. Yes. And so how do we how do you start thinking what are some of the ideas that come to mind here for you and other researchers when you start noticing this is happening? it maybe have this sort of ormesis-like effect where it can induce beneficial uh antioxidant on antioxidant effect in in donor cells. Um are there Is there potential here that like we could start doing mitochondrial transfer on purpose? You can start thinking about maybe therapeutic applications where you're taking

healthy mitochondria from a healthy tissue and a healthy person, transferring them into sounds like perhaps you could just transfer them into the blood and they could just kind of go to different places. Um have people done those sorts of experiments in animals and and thought about it in a therapeutic context like that? Yes, they have. Um there is a technique called mitochondria transplantation.

which is defined as a procedure in which mitochondria are isolated from a biological uh source and then administered to an animal with therapeutic intent. Um, that procedure has been used in a number of animal studies um in lots of different disease states and has shown promising results in terms of therapeutic efficacy. Um and there have been several clinical trials that have been uh published that demonstrate that this procedure is uh likely safe in patients.

Um and that it uh induces uh improves outcomes in ischemia reperfusion injury types of scenarios. Yeah. So it fits with what what you shared a few moments ago. And just to be concrete about this, are we literally talking about what what what is the exact procedure here? How do you isolate mitochondria? And are you just literally injecting like a syringe into the bloodstream? Yeah. Quite literally isolating mitochondria through a variety of different methods that uh that have been published.

Uh and then injecting the mitochondria that have been isolated into various um organ systems. It um it can be done in the intravenous system for into the blood. Uh we've done intraperitoneal injections. You can do it with intracranial injections. Yeah. injections directly into tissue parenchyma. Yeah. Yeah. There are different ways of doing the transplant. Hm.

And so um you mentioned earlier that, you know, obviously macropages can, at least under certain circumstances, take up mitochondria from cells. Mitochondria, you know, long, long, long time ago used to be um other organisms, uh bacteria What is the general relationship between mitochondria and our and our own immune systems? Um, is there an immune reaction that's generated to them or is that largely absent now because they've been integrated into our cells for so long?

Yeah, we've been worried about that actually for quite a while because especially because there are several major inflammatory components that have been described in mitochondria. Um one of them is actually the double s uh circular double stranded DNA. Um

genome, the mitochondrial genome itself, that is can be pro inflammatory. Got it. So they're in other words, their their DNA is basically the structure of bacteria DNA. And one would think that our immune system would naturally see that and because it can see other bacteria DNA and and it does react to it.

Correct. And then another great example are these formulated uh N methionine peptides. Um, these are basically the first amino acid in the proteins that are synthesized in mitochondrial from mitochondrial DNA. uh have a formal group attached to the first uh methionine and that is only found in bacteria and in mitochondria genome encoded proteins.

Um and uh those formulated uh methionine residues are very pro-inflammatory. They bind to these receptors called formal peptide receptors or FPRs one, two, and three, and they induce a very potent immune response. So we've been concerned about could these transplanted mitochondria actually activate the immune system. Um And so we've tried to interrogate that in some of our studies. Uh we've characterized the numbers and frequencies of basically every immune cell population and multiple tissues.

uh after a single or multiple transplant kind of model. and looked at inflammatory gene expression as well and found no effects of the mitochondria transplant on the composition of the immune system or the inflammatory, major inflammatory pathways. So The other piece here is that a number of studies have now shown that these if you do mitochondria transfer into T cells, it actually causes them to differentiate towards a regulatory T cell phenotype.

T cells often will produce these pro inflammatory cytokines and they help to orchestrate the immune response. But regulatory T cells express a cytokine called interleukin ten. And that is the sort of master suppressor of the immune response. It is the break that the immune system uses to keep itself in Kind of so that is induced with mitochondria transfer. Right. So so immune system, there's lots of different cell types.

Some of them are basically pro-inflammatory cells, some of them are anti-inflammatory cells. There's always some kind of dance that happens with how many of each type you have and when they're working and and how they're turning on and turning off all these different immune responses. You're saying that in some cases at least mitochondria can go into a regulatory T cell and basically cause it to become the more inflammation resolving type of T cell. Right.

Right. Got it. That's there's uh there's some literature showing that that is the case. It likely is um sort of both at the same time. There's probably a little bit of an induction of an immune response and an also an induction of an anti inflammatory response and that they are likely offsetting. That's my prediction. Yeah.

And so per I mean, I'm I'm just speculating here. So perhaps, you know, we've had mitochondria in our cells for so long that things have evolved to a place where despite the fact that these things have many of the feet or at least some of the features of other pathogens. They are bacteria-like. Somehow they're we're we're kind of adapted to them and they're not inducing a strong pathogen-like response from the immune system. They're actually inducing, you know, a very different kind of response.

Yes. Uh I think that that's likely the reason why they're not why they're so well tolerated and why they don't induce an inflammatory response is that We evolved with mitochondria transfer as part of our physiology. And this is just a normal process that occurs. So our immune system is trained, I think, to um to interact with cell free mitochondria and to interpret what the what what those mitochondria are and to respond accordingly. Do we know much about

What happens to the mitochondria in between cells? So if they're ejected from one cell before they go into another cell, can they survive in the extracellular space and hang out? What what do they do in between? Uh what does that even look like? Yeah, they the mitochondria are definitely stable in the extracellular space for at least a a relatively short period of time, like our scale, likely longer than that.

Um they are found in the circulation. There some studies suggest that they range like the average would be like one point five million cell free mitochondria per mil of blood. So there are a lot of them. In fact, there are mo there are as many circulating mitochondria in the blood as there are immune cells. Um, but they're probably taken up very quickly once they're in circulation. I don't think they likely circulate for very long. Um and uh

Yeah, but this is the the kinetics of this have not really been worked out yet. And this is an open question in the field. Mm-hmm. Um, I want to talk a little bit more about

obesity and just the immune system generally. Um, this might be a good area for for you to comment on just uh in terms of like where the field's at. One of the questions I've become interested in in is Okay, if you're an obese individual, you have more fat. But my understanding is oftentimes the fat tissue becomes more inflammatory over time.

Um, to what extent is that true? Is the fat tissue in obesity actually inflammatory? And what can you tell us about like the nature of that inflammation? Where is it coming from? Why would fat tissue be inflammatory? Great questions. Um yes, so in the context of obesity, there is uh in an inflammatory process that occurs in the adipose tissue. Um

The it's thought that the inciting incident are dying adipocytes. There are some adipocytes that become uh so large that they undergo a cell death pathway that is cas base dependent. Um and that leads to the release of all of the adipocytes cargo in or c components into the interstitial space, including a huge amount of lipids. Many of those lipids are pro-inflammatory themselves. And what happens is macrophages get recruited into the adipose tissue.

to basically wall off this dying adipocyte and engulf all of that material. And it looks like a crown sitting on a head. So it got it's been termed the crown like structure. Um these macrophages are uh start to induce expression of many different pro inflammatory cytokines and it leads to a vicious cycle. where there's the in initiation of an inflammatory response to clear out these adipocytes potentiates more inflammation and it just keeps going.

And there are a number of studies that have shown over the last thirty years that that d that the monoc that the uh degree of inflammation in the adipose tissue is closely related to the risk of developing metabolic complications like type two diabetes.

Interesting. And so um are there any are there any indications that for obesity or other things, but you know, just metabolic dysfunction in general, that Either within an individual you can transfer healthy mitochondria from other cells into dysfunction metabolically dysfunctional cells. Or even in between individuals in a more therapeutic setting, you could do the same thing and and reverse things like obesity or diabetes or conditions like this.

Yeah, there have been actually uh four studies that have been published doing mitochondria transplants in the context of obesity. None of them come from my lab, but um They do support this notion that if you do uh mitochondria transplants, that that does decrease the severity of obesity and it reduces the degree of inflammation in the adipose tissue. So so basically you're taking mitochondria from a non obese individual.

injecting it into an obese individual, the mitochondria then get to cells somewhere, probably multiple places in the body, and the obesity phenotype uh goes down at least. Right. And these are studies that have been done in rodents, not Right Right, right. And do we know do we know more detail there? Like where exactly are they going, which cell types really matter for that and all all that?

No, we don't know that actually yet. Um it's actually proven to be quite difficult to track the mitochondria since they've been transplanted. Yeah. Um we've done this type of work in the context of uh an inherited mitochondrial disease called Lee syndrome. Um and we have uh We we have basically used molecular approaches using um

uh mitochondria that come from a strain of mice that has a different mt DNA genome that we can detect using uh PCR. And uh when we do the mitochondria transplants in those mice, actually the compartments that seem to retain the most mitochondria that we've transplanted are actually em enriched in the immune system. So bone marrow, blood, spleen, peritoneal cavity. So it looks t to us like maybe the immune system is preferentially taking up these mitochondria.

But it's not so clear because um the signal detection is dependent on the mitochondrial biomass of the acceptor cells and immune cells have tend to have the fewest mitochondria compared to things like the hepatocytes and cardiomyocytes. So it can just be a limited detection problem. Um

But they are yeah, this is a this is an important question. Uh what are the tissues that preferentially get the mitochondria after a transplant and what cell types within those tissues? Right. And it so so it sounds like part of where this sub discipline is at right now is there's a lot of these

interesting and big questions that are that are fairly obvious. Uh next question is to answer, but there's probably technological limitations that that, you know, we don't quite have the techniques yet that allow us to track the mitochondria everywhere and do things with enough precision to actually answer some of these things yet.

Yeah, I think that's a fair characterization. Um there are lots of good studies now that are very rigorous showing important roles for mitochondria transfer in physiology and also um in cancer uh progression and cancer pathogenesis. And also a lot of phenomenological studies demonstrating therapeutic benefits of mitochondria transplantation for a lot of different disease states.

But the mechanisms of the mitochondria transplantation are poorly understood. Um I think there's only one, in my opinion, there's only one unifying hypothesis that that um that connects them all, and it's really not been fully established yet. But that hypothesis is that the mitochondria, when they're taken up by the endothelial cells in the vasculature, that it induces an angiogenic response that allows for very rapid angiogenesis. And that may actually be the explanation for

a lot of the f uh phenomenological findings about how mitochondrial transplantation works, not just for ischemia reperfusion injury, but possibly also for diseases like obesity. Yeah. Yeah. Interesting. Um So backing up a little bit, so you you kind of in general in your lab, you study interactions between immunity and and metabolism. So the immune system and just the the metabolism of cells and what they're doing.

Um, why why did you h why and how did you get into this area? Um, I think it's probably a very broad area, but when I when I think about my own education, immunity and metabolism, the f the thing that I remember learning about first was something like type one diabetes. where you think about the immune system misfires in some way and it kills a cell, such as a pancreatic cell, and that gives you a metabolic problem. But it's basically an autoimmune misfiring of some kind.

Um, but that's you know, that's far from where the interactions end when it comes to the immune system and and metabolism. There there's just it sounds like there's constant crosstalk there. And, you know, what we what we choose to call immune cells and what we choose to call metabolism, there there's fuzzy boundaries there. There's interactions between these systems, um, even under normal, non-pathological conditions.

True. Um, in fact I would argue that the immune system and metabolic organs have really co evolved their functions with each other. Um There is there's really good evidence of that, in my opinion. Uh, fever is actually a complete change in our metabolic status. Where even something as important as core body temperature it it gets elevated and, you know, resting metabolic rate goes up and that is entirely driven by the immune system.

Um, and many of those metabolic pathways I I believe are hardwired into our physiology for other organ systems, including the adipose tissue.

Um, but not just fat, also the liver and the heart. I mean, there's really close interactions between the immune system and the metab and the major metabolic organs. Yeah. Yeah. Um So the way I got into this was um I had I've always been really interested in obesity and type two diabetes and uh did my PhD on the immune system and its role in uh regulating obesity pathogenesis.

Um, and it occurred to me when I was a resident, I read this paper from Eric Ballard's laboratory in Quebec. Um, he published a paper in blood in 2014. demonstrating that when platelets get activated, they release all of their mitochondria and that that that they are get uh they stimulate neutrophils and that it leads to a local inflammatory response. Um, he also showed that there are a lot of mitochondria in the blood and

um that those are likely platelet derived. And it occurred to me after I read that paper that maybe this type of biology is also occurring in the fat in the context of obesity. when these dying adipocytes release all of their c contents into the interstitial space, one of those com major components are going to be mitochondria.

Um and in a dying adipocyte that's um those are likely going to be damaged mitochondria. So I thought that the that the biology of mitochondria transfer and the fat would likely be very pro inflammatory. and might be contributing to the formation of the crown like structure that I described before and the subsequent inflammation that occurs in obesity.

And it turns out that that it is actually completely incorrect. Um mitochondria transfer is actually most robust in a healthy state. And if you induce if you enforce an obese phenotype with a high fat diet, especially one that's made from lard, um, which is an animal derived fat, that tends to inhibit mitochondria uptake by macrophages instead of promoting it. Yeah. Um Um an yes, animal drive. One thing I wanna so this is something I I thought about recently too and and

wrote about and stuff. So lar so the classic high fat diet in the literature forever basically has been this high lard diet, which is, I believe, sixty percent of energy from fat. And lard is pig fat and pigs are animals. But pigs are not ruminant animals like cows. So they actually have the fat composition of whatever it is they're eating, basically. And um what they mostly eat today is grain-based diets high in polyunsaturated fats. And in fact,

The classic US I just learned this um not that long ago. The The database from which the fatty acid profile is drawn that is reported or at least cited, hopefully, in a lot of the high fat literature over the last few decades. Um, actually doesn't match what is actually in the lard today. Um, there's actually, you know, somewhere between 10 and 20% more polyunsaturated fatty acids. in the lard high fat diet than most studies in history seem to uh realize.

Oh god, thank you for sharing. I didn't know that actually and uh behooves us to do some measurements then on the fat the the high fat diets that we source. Yeah. Yeah. No, I think it could be interesting because basically the

the discrepancy between what was reported in that database and what was measured independently later on was essentially um much higher in linoleic acid, but then lower in, you know, oleic acid, palminic acid and and a few others. Um so it it could be, it could be interesting to look at. Yeah, yeah, for sure. We have looked at all of those fatty acids indiv individually for their impact on mitochondria transfer inhibition. Um

And they it does seem to interestingly enough correlate with the number of carbons that are present in the fatty acid. So the basically the longer the fatty acid, the less mitochondrial transfer. Correct. Correct. Yep. And we've looked at fatty acids ranging from medium chain all the way to long ch various long chain fatty acids.

the double adding a double bond slightly in um reduces the inhibit inhibitory effect, but it's still there. It's really the number of carbons is the dominant feature. So the idea would be that the more The more the diet contains these longer chain fatty acids, the less mitochondrial transfer there would be. And therefore, if the purpose, say, of mitochondrial transfer is to communicate for one cell type to communicate metabolic status to another to enable some kind of adaptive change.

that would happen less in the in a context of a diet with longer chain fatty acids. Right. There would be more with with more longer chain fatty acids, there'd be more inhibition of mitochondria transfer to at least the macrophages. Yeah, at least the macrophages. Right. Interesting. Um, so what other things are you guys working on in the lab today, whether whether or not it has to do with mitochondrial transfer?

Well, the uh one of the major topics is actually trying to translate mitochondria transplantation as a new therapeutic to treat inherited mitochondrial diseases. Uh We've been doing a lot of work on this disease called Lee syndrome. This is a absolutely devastating disease. Um it occurs in one to two thousand to one to thirty thousand individuals, depending on the population that you're looking at. And

Um, these kids initially will develop normally. They'll achieve their early developmental milestones. And then often around six to twelve months of age, they present with with regression of achieved developmental milestones. So they will um actually that's the major initial presentation. They have failure to thrive, they lose muscle tone, they develop paralysis, uh, and a very prominent encephalopathy.

And that brain dysfunction ultimately kills ninety percent of these patients by the age of four. They have no therapeutic options. They are offered supportive care and a range of different clinical trials. And, you know, families are desperate. I mean, these patients are these poor kids are have have really have no hope because of

the lack of available therapeutics. And what maybe we could use mitochondria transplantation to treat that disease. Yeah. So what's actually wrong with their mitochondria? So the uh while there are over a hundred twenty five different mutations that have been described that cause Lee syndrome.

But what they share in common is that they all disrupt the function of the of the electron transport chain or oxidate phosphorylation in some way. Yeah. So their cells are basically just not able to generate energy well. Correct. Correct. And so it is remarkable that it's even survivable um for a short period of time. But they uh Yeah, it's a really difficult disease. And we thought maybe we could plug in healthy mitochondria and that that might actually reduce disease severity.

So we did some mitochondria transplants initially in these mice, trying to look at the uh impact on the mitochondrial metabolism of macrophages. Um and what we found was when we looked at the um flux of basically a sort of an assay called the seahorse assay that helps us kind of interpret the metabolic capacity of mitochondrial metabolism in a cell.

Uh, what we found was that in healthy wild type cells, that the mitochondria transplant did almost nothing to the aerobic respiration, this small effect, but it was detectable but not super prominent. But then the knockout cells, the from the Lee syndrome mice that lack this gene called NDUFS4, that's required for complex one to assemble in the mitochondrial um electron transport chain. Um those mice get Lee syndrome and they have the macrophages have really low mitochondrial metabolism.

And if we give a single dose of mitochondria the day before, um, we completely rescue the aerobic respiration capacities of those cells. So that told us that basically

Healthy cells seem to preferentially rely on their own mitochondria. And if they see exogenous mitochondria from a transplant, they tend to ignore them. But metabolically compromised cells, which um whether it's genetic uh or pharmacologic metabolic catastrophe that occurs, they those cells can recognize their metabolic status and it seems like they can plug into, tap into these transplanted mitochondria much more effectively. Hmm. Yeah, so one one way or the another cells might have ways to

Intelligently sort of pick and choose from the mitochondria available to them. Yes, yes. And I suspect that Yeah. I suspect that it's sort of like the equivalent of like um like a generator. So like, you know, your you know, a home many homes are powered off the grid. And if you lose grid power, one potential option to get by with essential functions is uh plugging in a generator.

And I suspect that mitochondria transfer is a similar or analogous to that, where it's not sufficient to fully power Uh, the cell, but it may give enough juice to facilitate the most essential functions in order to overcome a transient metabolic insult. Interesting. Um so so what else are you working on today um that you're particularly excited about um that you might tell us uh in terms of what's coming up?

Yeah. So um, well, we started based off of those studies that I just described, we started treating mice uh with Lee syndrome with mitochondria every week. And we found that those that it actually reduced the severity of neurologic dysfunction in those mice and it also extended their lifespan. So we published those studies at the end of last year, and now we're doing work with patient derived fibroblasts.

where we're trying to screen a bunch of different uh patient derived cell lines fr with different mutations that each cause Lee syndrome. And we're trying to optimize mitochondria transplantation to support uh to support these kids and hopefully facilitate a compassionate use clinical trial. Yeah. I have a I have a question about the syndrome and how it gets started. So They are inheriting mitochondrial mutations that give them an impaired electron transport chain in their mitochondria.

So if you they get their mitochondria from their mom, how does this work in terms of the inheritance? So the the mom is perfectly healthy, but how s how do the kids inherit these mitochondria with these mitochondrial DNA mutations? Yeah. Oh sorry, we froze. Sorry, froze for a second. Did you catch that? I I caught the question. Um yeah, so um many of the genes that are that cause Lee syndrome are nuclear genome encoded and are autosomal recessive.

I see. Got it. Got it. Two mutations from one from the father, one from the mother. So this is actually important to tell people. So I think what you're getting at here is So although many of the components of the electron transport chain and those proteins and things in mitochondria are in the mitochondrial genome, that's not true for all of them. Some of them are in the nuclear genome. In fact, most of them are actually in the nuclear genome. Okay. Uh yeah. So um

Right. So if there is an MT DNA encoded mutation, that does follow a different inheritance pattern that's maternal. Um But uh yeah, so there often it's autosomal recessive. There are multiple inheritance patterns. Interesting. Um, is there anything that you want to reiterate or any final thoughts you want to leave people with about this topic of mitochondrial transfer or just sort of immune metabolism interactions generally?

Well, um, I think what's so exciting about the biology of mitochondria transfer is that I think that it links back to the evolutionary origins of life, uh eukaryotic life at least. Um, I I think that if my hypothesis is that this biology helped to support the evolution of eukaryotic life. Um, and I think it's very hardwired into our physiology. Um

It's very early days and there are a lot of people who are very excited about the idea of mitochondria transplantation, but I really d think that they're uh I would caution against the hype around it. Um, it's not a panacea. It is a

There it I think there are risks to the procedure and it really requires the right type of scenario to use mitochondria transplantation. Yeah. You know, I I didn't actually think about it this way. This all this mitochondrial transfer stuff that does really speak to the the origins of eukaryotic cells potentially, because the classic endosymbiotic theory that I've always had in my head since I was, you know, a teenager.

Is somehow, someway, way back when the mitochondria all got into some cells and then they just kept getting inherited after that. And What you're basically saying is it's it's actually very common for mitochondria to go in between cells to and from, come and go. And so um it may not have been such a um a rare singular event. This type of thing, transfer of these basically miniature cells between larger cells may have been common the whole time.

Yeah. And I actually view mitotransfer as being a a a relic of that evolutionary history. Um, uh preserved one because I think it's beneficial to there's an advantage to doing it. Um but just imagine this, like, you know, in the earliest prim primordial soup, you know, you've got uh the last universal common ancestor and it acquires these um intracellular bacteria that ultimately became mitochondria.

those bacteria had the capacity to get into and get out of cells in the first place. So that biology is kind of already right. um part of that evolutionary history. And it's thought that those the most of the genes in the that um alpha proteobacterial species were cut and pasted into the nuclear genomes of the eukaryotic cells. uh conferring control over the mitochondria to the um winning endosymbio is symbiotic. So um

You know, like you could imagine that this uh this biology of mitochondria transfer is really actually still being controlled by those same genes. It's just that the cell now uses it for physiologic purposes more so than anything else. Right. Interesting. Well, uh, Professor Brestov, thank you very much for your time and I look forward to talking to you again in the future. Thanks so much for having me. It was a fun discussion, Nick. Have a great day.

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