Scientist Stories: Peter McGuire, How do Mitochondria contribute to Health and Disease

Hello everyone, my name is Dr. Peter Maguire, and I'm the head of the Metabolism, Infection, and Immunity section at the National Human Genome Research Institute. I'm a physician scientist, and today I'm going to talk to you about how do mitochondria contribute to health and disease. So first of all, mitochondria play a very important role in metabolism, which we're going to talk about, but I think first it's necessary to define what is metabolism. So metabolism represents a balance between catabolic pathways and anabolic pathways. So catabolic pathways are essentially the pathways in which we take in food, and that food is actually broken down into a number of useful constituents for the body. One of which is energy, one of which is cellular building blocks, things like amino acids and glucose and things like that, and then the other one is heat. Now the energy and the building blocks can actually be used via the anabolic pathways to make macromolecules, and those macromolecules, of course, are important for building cells and tissues and organs. So metabolism, as I said, really represents a balance between these two things which are going on at all times. Metabolism is very complicated. It occurs in all different parts of the cell and involves many different pathways, and some people may get a headache by looking at this screen here. It almost looks like a subway map, but it involves essentially many different intersecting pathways, and I think one of the main take-home messages of this is not so much to memorize all the details and everything, but to look at the interconnectivity of all the different pathways. So up here you have various pathways dealing with carbon metabolism. Down here is something called the citric acid cycle and the urea cycle, and this is where proteins can come in, as well as here on the side, which are fatty acids. And all these feed into this essentially subway map of metabolism, which is marked by this main backbone of glycolysis, eventually feeding into the TCA cycle, and then eventually making its way down to mitochondrial metabolism or oxidative phosphorylation, which we're going to talk about, which is labeled here. They have it off to the left. I tend to like to have oxidative metabolism here down at the bottom because all of this is really a funnel. It serves to basically lead to energy production in the mitochondria. So as I mentioned before, metabolism takes place in all different areas of the cell, including the nucleus. There is metabolism and RNA processing, as well as in the cytosol. You can have things like glycolysis in various vacuoles inside the cell, and of course, most importantly, because that's what I like, is the mitochondrion. Okay, so the thing about mitochondria is they're actually quite multifaceted. You, you know, in a lot of the things that we learn, we think of them more in terms of energy and kind of the energy-producing organelles of the cell, or the powerhouse of the cell. But they have a lot of other roles, and I put up here battery, factory, armory, signal station, and assassin. So with regards to metabolism, that's kind of the battery and factory part. So not only do they store energy for use or make energy, but they also produce a lot of different things which are required by the cell. So oxidative phosphorylation, of course, for energy production, which is fed by the TCA cycle. The TCA cycle, of course, is important for not only leading into oxphosphatase but producing a lot of byproducts. The TCA cycle has stuff that comes in and goes out in terms of building parts for the cell. Beta oxidation of fatty acids, that's something else that occurs in the mitochondria. That's the breakdown of fat, which is a really high-energy containing molecule. Mitochondria are important in terms of building heme. So heme, which is a moiety which is involved in hemoglobin, it's an iron and porphyrin molecule which is important for hemoglobin and trapping oxygen. So that can be made in the mitochondria. Cardiolipin, which is important for mitochondrial membranes, and then steroids, of course. Course. Another function besides this kind of battery and factory is that the mitochondria can serve as an armory. And the reason I say armory is because it's involved in calcium buffering. So calcium is actually quite a very reactive cation actually in cells and can activate certain aspects of cellular processes that actually can lead to the destruction of the cell. So it's important to keep this calcium actually sequestered away, and the mitochondrion serves a really important role in doing that and essentially protecting the rest of the cell. So something that's been emerging over the past couple of years is that mitochondrion or mitochondria can serve as a signaling station. So they are involved in many different aspects of signaling within the cell, and that's via the production of reactive oxygen species using mitochondrial DNA, but also metabolites that can actually escape or are made in mitochondria, can escape the mitochondria and affect other processes. For instance, DNA transcription, or transcription to RNA, involves an opening of the DNA, and that involves essentially modification of histones, which you may have heard in some of the other courses. To modify histones, those are metabolites. Those are— it's acetyl-CoA, which comes from, as you can see here, at the end of glycolysis in the mitochondria, and that can actually escape the mitochondria and go on to alter histones and then alter gene expression. So metabolites that are made in the mitochondrion can alter gene expression. So there's a crosstalk that goes on between those two organelles. Mitochondria are also involved in stress responses. So the stress response is a highly coordinated series of gene transcription programs and alterations in cellular metabolism that help the cell adjust to stresses. Also, it's been discovered recently that mitochondria are involved in the antiviral response, so important for cells cells and their ability to fight off viruses. So basically viruses take over the machinery of the cell, and the mitochondria and various aspects of it are important in shutting down that machinery and preventing viral replication. And then the last part, of course, is apoptosis, or programmed cell death. And this is the assassin part. So release of cytochrome c from the mitochondria. And cytochrome c is actually part of the respiratory chain. The release of that actually can lead to a programmed cell death. Okay, so besides science, mitochondria have actually appeared in popular culture in a number of different ways. There's a really good book by Bryan Sykes called The Seven Daughters of Eve, which refers to tracing mitochondrial ancestry through— tracing ancestry through mitochondrial DNA. It also has appeared in Star Wars, of course, which is very important. Back in 1999, when the second set of Star Wars movies came out, the second trilogy, they referred to something called midichlorians, which was something that lives in symbiosis with the host and was important for something in the movie which they described as the Force, for all those Star Wars aficionados. So appearing in popular culture, and in which case you could actually measure the level of midichlorians within an individual and tell how strong they were in terms of being a Force user in the Star Wars world. So that was all based on mitochondria. Mitochondria do live in symbiosis symbiosis with us as a host. And eventually, we'll talk about in a few minutes over evolution how that actually developed. Okay, so how many mitochondria actually are there in cells? Well, it really depends on the energy requirements of that cell. So for instance, white blood cells have a relatively small amount, anywhere between, you know, 30 mitochondria to about 100 mitochondria. Where then you go to a much larger organ system or organ such as the liver, the an individual liver cell can actually contain between 1,000 and 2,000 mitochondria. And that's because the energy requirements of that cell way surpass what a white blood cell actually needs. So where do mitochondria actually come from? This is what I was referring before, this idea of symbiosis with the host. And this is the endosymbiotic hypothesis. So this was Lynn Margulis back in the 1960s and '70s, I believe, where she proposed a hypothesis where 2 organisms developed a symbiotic relationship. So an ancient bacterium, which is essentially what mitochondria are, and there's a lot of evidence for that, actually developed a symbiotic relationship with another host bacterium where they were actually incorporated into that bacterium, and the 2 of them had a beneficial relationship with regards to the incorporation of that ancient bacterium. And over time, that evolved into a situation where the mitochondrion actually gave up a lot of its functions to the rest of the cell, but was still required for certain aspects of function within that host cell. So the reason that mitochondria actually developed, or they helped develop this symbiotic relationship, one of the hypotheses is that it has to do with the rise of oxygen in the atmosphere. So roughly about 2.2 billion years ago, oxygen actually started to rise within the atmosphere on Earth. And unfortunately, you know, that oxygen is actually— oxygen is actually quite toxic. And this is a nice book here by Nick Lane, which is called Oxygen: The Molecule That Made the World. And it's during that time that this symbiotic relationship was developed. And part of it was probably because those ancient organisms needed some protection against essentially oxygen, and oxidative phosphorylation was able to do that, basically take what can be a toxic molecule and turn it into something useful. So mitochondria come in all shapes and sizes depending on the different cell types. So they can be elongated, as you see here on the left. They can be more circular. And inside you have what are called cristae, or kind of these infoldings of the membrane or invaginations of the membrane. Here you can see a rather elongated mitochondria. Mitochondria can also fuse with each other, and we'll talk about that in a little bit, and what are the benefits of that. But also you can see that the cristae can also change their structure as well, where in some instances they can be quite thin, and in other instances they can be quite thick, and that has implications which we'll talk about in terms of cellular bioenergetics. So the anatomy of the mitochondrion, this is kind of a, you know, idealized model of what an individual mitochondrion looks like. So you have the outer membrane here, And then inside you have the mitochondrial matrix. And the cristae, as I mentioned, are these invaginations of the membrane which form kind of walls inside the mitochondrion. In the matrix you can have things like granules as well as mitochondrial DNA. And if you'll notice here, what's important about this picture is that there's not just one circular loop of mitochondrial DNA, there are actually multiple. And these, just to remind you, that mitochondria's bacterial ancestors also have DNA which is in a circle. So they did retain at least some of the aspects of their bacterial ancestry. Now in the matrix are where a lot of biochemical reactions occur. And here on the cristae, you can kind of see these dots that are occurring. Those are the ATP synthase molecules, which we'll talk about in a minute. Which are the energy-producing part of the mitochondria. So this is actually what, um, essentially oxidative phosphorylation looks like. So within those invaginations, or the cristae, of, um, the mitochondria, this is where oxidative phosphorylation takes place. And oxidative phosphorylation, of course, is the important process for the production of ATP. So, um, during, um, metabolism, what happens is you have— so via, you know, glycolysis and the TCA cycle and fatty acid oxidation, you have the production of what are called reducing equivalents. So reducing equivalents are NADH or FADH2. And what happens is NADH passes its electrons. So NADH produced by all these different biochemical processes within the cell, or metabolic processes in the cell, pass their electrons to complex I of the respiratory chain. And the respiratory chain, or oxidative phosphorylation, is made up of 5 different components: complex I, II, III, IV, and V. So NADH passes electrons to complex I, which binds to iron-sulfur clusters, and that of course gets shuttled to complex III. So it doesn't go 1, 2, 3, it goes 1 to 3 or 2 to 3. So there's— it's actually not in series. So from complex II, actually it's FADH2 where you're getting your reducing equivalents. So I and II wind up passing their electrons to complex III, and that's via coenzyme Q, and then from complex III to complex IV by cytochrome c. Now this process of passing electrons actually causes, in each, complexes I, III, and IV, causes a conformational change in this protein complex, which allows it then to pump protons from the matrix into the intermembrane space. And when you do that, you build up essentially potential energy, and that potential energy can be leveraged. And that potential energy is actually leveraged by complex V, or the ATP synthase. So the buildup of electrons in the— excuse me, the buildup of protons in the intermembrane space are then go through this channel in complex V, or ATP synthase. And by going through this channel, ATP is synthesized by the turning of this. This is actually known as the smallest molecular motor. So as a result, 3 ATP are produced per NADH, so via complex I where it initially enters, and 2 ATP via FADH2, which is through complex II. So this essentially is how we extract energy from our food and make ATP for energy. And essentially what mitochondria are are a battery, right? So what do batteries do? Batteries store potential energy for future use. And in this particular case, the storage of potential energy happens in the pumping of protons into the intermembrane space. So one of the ways that, um, the respiratory chain or oxidative phosphorylation can kind of leverage or, or develop a conformation which is ideal for energy production is something called supercomplex formation. So, as you know, in the picture that I showed you, the idealized picture of the respiratory chain, each individual complex was actually separated in physical space, but it turns out that they actually are not separated. They, they form something called a supercomplex. So in this picture, you can see components of complex IV and complex III and complex I all come together to form something called the respirosome. So in other words, all these individual protein components, and they're made up of multiple proteins, are in close approximation. So those electrons can be shuttled actually quite easily, and it actually increases the efficiency of oxidative phosphorylation by this close approximation. Of these components of the respiratory chain. And part of this involves actually remodeling of the cristae. So the cristae, as I said before, which are invaginations of essentially a membrane here inside to the mitochondrial matrix, within those invaginations and those cristae are the components of the respiratory chain, which are denoted by these colors here. By the— by what happened— what happens actually is the cristae actually tighten, or they become closer together. So multiple components of the respiratory chain are brought into close approximation of each other, and this then actually increases the efficiency of oxidative phosphorylation and the ability to produce ATP. When you actually cause a deletion in a gene called OPA1, which is important for this crystae formation or this tightening of these cristae, you get rather these loose association of cristae. And as a result, what happens is you don't get this respirosome formation, which I showed before, which is this close approximation of these components of oxidative phosphorylation, and therefore mitochondrial respiration actually goes down. So this is a process— this tightening of cristae, bringing together, close approximation of these respirosomes— actually increases respiratory efficiency or efficiency of oxidative phosphorylation. Okay, so ATP. So ATP is the result of oxidative phosphorylation, and many people in the past, and it probably still appears in textbooks, that ATP is a high-energy, you know, high-energy-containing molecule, and it's because of these, you know, high-energy phosphate bonds. Turns out that's actually not true. If you look at the physiochemistry of it, these phosphate bonds on ATP are actually not high energy containing, right? So ATP contains an adenine base and a 5-carbon sugar known as ribose and these high energy phosphate bonds, which really are not high energy phosphate bonds. The point is that ATP can actually be used in the cell to do work. So one of the ways that can happen is, for instance, let's take membrane proteins. So membrane proteins often allow solutes to cross between the outside of the cell and the inside of the cell, and to do that they often require a conformational change. Now what happens is ATP can be leveraged where you can actually pluck off a phosphate group from ATP and put it on this membrane protein, and as a result that membrane protein will change its shape or its conformation, and the reason for this is because while these are not high-energy phosphate bonds, they are actually very negatively charged. So when you put a phosphate group onto this membrane protein, you actually can have, because of this highly negative charge, a change in the conformation of this protein because the negative charge pulls positive charges towards it, and in doing so then you cause a change in the conformation of that protein, and as a result you can pass various solutes across the membrane. It's also used— so that's, that's for membrane proteins. It can also be used for transport of various organelles inside the cell itself. So there are tracks, microtubules inside, and actin filaments inside cells, and organelles are not static. They move around within the cell, and part of that involves essentially the use of ATP. So phosphate groups can actually be taken from ATP and put onto these motor proteins, which carry things like vesicles, or even mitochondria can be carried this way. And as a result, it causes conformational changes in this motor protein, which actually causes it to walk, you know, going on and coming off of this motor protein. You're putting on a negative charge, you're taking it off. At one point, you're pulling positive charges towards that phosphate group, it comes off, and then you release. So you can imagine this kind of walking of this protein due to this change of phosphate groups being put on and taken off of this motor protein. So this is really the way ATP works. It allows the cell to do actual work. Okay, so the mitochondria itself, we saw kind of some idealized pictures of it, and I did describe the matrix and the cristae, etc. But also I want to point out the anatomy of mitochondrial DNA. So within the matrix here, you can, you contain numerous copies from you know, anywhere from hundreds to thousands of copies depending on the cell of mitochondrial DNA. And mitochondrial DNA took its cue from its bacterial ancestors where it actually is circular, and it contains 37 different genes. And here we're showing some of the components. It contains ribosomal RNA genes, 2 of these, and then it contains about 13 different components of the respiratory chain which are shown here. The other components of the respiratory chain are actually also encoded in the nucleus. So the respiratory chain takes its gene products not only from mitochondrial DNA, but also from nuclear DNA. And then also within here, which are not pictured, and a lot of them are on the inner loop of mitochondrial DNA, are various transfer RNAs, which are also involved for mitochondrial protein synthesis. So within mitochondrial DNA, there are actually ancient mutations which have developed, and these are what form mitochondrial haplogroups. So over time, you know, a long time ago, as we rose out of— as human beings, as we rose out of Africa and migrated to different areas of the world, different mutations developed in mitochondrial DNA over that time. And what these different mutations can serve as is essentially markers of ancestral origins. And the reason this is important is because an important property of mitochondrial DNA is that it is actually passed through the maternal line. So mothers are the only ones who are able to give mitochondrial DNA, um, to their children. So by looking at these markers that accumulated over, um, a long time during our history as human beings, you can actually trace back your ancestry using mitochondrial DNA. And initially, you can see here, this is a map of the world. We rose out of Africa anywhere around 200,000 years ago, and during that time, as we migrated across the different continents, we developed different changes in our mitochondrial DNA, which then served as markers. And those markers, when you put them together, together can form something called a haplogroup, or kind of a fingerprint of your origins. So you can see here the initial ancestral haplogroups in Africa are L0, L1, L2, L3, but as we migrated and different changes occurred, they developed different haplogroups, such as ones for Northern Europe— or sorry, this is actually Southern Europe, Northern Europe, as well as into Asia, as well as into the New World. And you can actually, via these changes, these haplogroups, it— what it really tells us is something about the migration of human populations during, you know, these past 200,000 years. And all of these different companies that look at your ancestry, right, use mitochondrial markers essentially, and haplogroups, cannot— to determine kind of where your roots are. And you've probably seen this on— actually, this is an old slide because I don't know if this commercial is even on television anymore, but this is an individual who thought he was initially German because of his family history, and then because of ancestry.com, he found out that he was actually Scottish. So, and that was based on essentially, you know, the use of mitochondrial haplogroups to see where his origins are. Okay, so that's some background just on some of the basic biology of mitochondria, but most importantly, because I am, I am a physician, I'm a scientist, I take care of kids who have mitochondrial disease, so I'm going to tell you a little bit about what happens when mitochondria fail. So mitochondrial— so, so I want to draw two distinctions. There's mitochondrial disease, which is due to inherited mutations in genes involved in mitochondrial function, and then there's mitochondrial dysfunction. So since mitochondria play such an important role in energy production and really are a, a real hub for the cell, mitochondrial dysfunction actually occurs in a lot of different diseases. So patients with mitochondrial disease do have mitochondrial dysfunction, right? But patients with all these other diseases, which are shown here, can have mitochondrial dysfunction, but they don't necessarily have mitochondrial disease, right? So mitochondrial dysfunction contributes to all different, you know, pretty much almost any disease you can think of has been demonstrated to have mitochondrial dysfunction, including cancer. But here you see things like Alzheimer's and Huntington's and Parkinson's and autism, etc. So mitochondrial dysfunction, you know, as I said, being a hub for energy production, really plays a key role in a lot of different diseases. What I'm going to talk about is mitochondrial disease. So I thought it'd be fun to talk about a clinical case just to kind of give you an example, introduce you to what mitochondrial disease actually looks like. So this is a clinical case of a 3-year-old male who was adopted with both of his siblings. The biologic mother was noted to have substance abuse. So during his development, for those of you who have kids, you probably remember your kids developed certain motor milestones as they were growing up, such as, you know, walking talking and babbling and mama, dada, stuff like that. So he had difficulty meeting his motor milestones and such as, you know, sitting up or crawling, etc. And then eventually actually lost his motor milestones. Now, the failure to meet motor milestones initially was attributed to, well, you know, there's a history there of biologic mother with substance abuse, so therefore that could, that could account for his failure to meet motor milestones. But the loss of motor milestones that he actually was able to achieve actually says something else, that that is of concern. And has to be looked into, and that was one of the triggers to have him looked into. The other thing too is when he did have a febrile illness, he actually experienced further neurologic decline basically and eventually wound up on a ventilator with tracheostomy. So, some of the things that would tip you off— the failure to meet milestones was clouded a little bit by this family history of the biological mother with substance abuse, but some of the things that would tip you off are loss of milestones that had already been achieved, as well as this kind of stressful precipitating event where there's further neurologic decline. That, to us clinicians, speaks to an inborn error of metabolism, and specifically, you know, in this particular case, mitochondrial disease. So this is actually his MRI, and what happened during his febrile illness is he actually developed what we call metabolic stroke, or we're not really using that term as much anymore, but what it means is it's neuronal death and it's not due to vascular issues, it's due to an energy issue. So here we're looking at his MRI, and just to give you an idea to orient you with MRIs, you have to imagine that you're looking up through the feet of an individual in slices. So now the slice they're at now is at the level of, of the brain essentially, or the lower part of the brain. And here you can see the eyes just for some orientation. This This is the nose. So we're looking up. You know, we've already passed the jaw, so you won't see the jaw at all. And what you see here is the cerebellum. And in here you can see that there are certain areas that appear kind of whitish. And then also this is part of the brainstem, certain areas which are whitish here. And if we move over to this section here, this is looking straight onto the individual and you're doing slices going from the nose back. And you can see here's the brain here, and here's his brainstem, and you can see this kind of swollen light-colored areas of brainstem here. This is from the side, swollen areas of brainstem here. This is the cerebellum, which I oriented to you over here, or oriented you to over here. So anyway, he has lesions that developed essentially in the cerebellum and in his brainstem, and as a result, the brainstem is important for controlling breathing or the drive of breathing. So as a result, he wound up with a tracheostomy. So in other words, a tube in his throat to help him breathe and wound up on a ventilator, which he still kind of requires at night. He has respiratory weakness because he doesn't have that drive. And this is what resulted from his illness that he had. So this illness brought this on, this very stressful event where he had nerve cells that actually died here. And this is something called Leigh syndrome. So this is a type of mitochondrial disease, and a lot of times it is it is first brought out by an illness. And here what we're looking at is something called MR spectroscopy. So this is another version of MRI, but instead of looking at the actual tissue and its water content, whether it's swelling or not, or light-colored, this actually looks at metabolites in the brain itself. And one of the things that you look for in mitochondrial disease is lactate production. So lactate, actually, you can see there's a tracing here, kind of a squiggly tracing going along here, and each of these peaks actually means a certain type of metabolite. And these two, this double peak here, this doublet, actually indicates lactate. So this individual actually had an elevation of lactate in certain areas of his brain, which is highly suggestive of mitochondrial disease. Mitochondrial disease is really— the diagnosis is a a picture that you have to develop with many different pieces of evidence. And this is one piece of evidence that was highly suggestive of having this. So here— sorry, I know I pointed over here before. What I meant to point at is here. This is actually the lactate doublet right here. And in this particular case, it can sometimes be inverted. As you can see here, here's the doublet here. So anyway, He has a biochemical abnormality in his brain where he produces a lot of lactate, and that suggests mitochondrial disease. So mitochondrial disease is the most, um, common inborn error of metabolism. So inborn errors of metabolism are usually enzyme or cofactor deficiencies in different aspects of metabolism. Well, this one turns out to be one of the most common and actually almost is out of the rare disease category because rare diseases, I think the cutoff's about 1 in 2,500 or so. Mitochondrial disease has a prevalence about 1 in 5,000. So, you know, you, you most likely have bumped into someone on the street who has mitochondrial disease. There's a child born every 30 seconds with mitochondrial disease, and it does affect all age groups. We specifically study children, but it does affect all age groups, and it's deficiency of energy production and produces multi-systemic disease. So basically, organ systems that require a lot of energy are the ones that tend to be affected. So you can have problems with your eyes, with your heart, muscle, brain, you know, pretty much, you know, anything you can think of. A lot of organ systems do require a lot of energy. Unfortunately, the therapies are very limited and it's really poor long-term outcome. We really don't have any magic bullet at this point. The medical treatments are limited and in general infection can be devastating. And that's what we study in these patients. We study infection and mitochondria. Mitochondrial disease. And the recent COVID-19 pandemic has produced a lot of challenges with this. And I can tell you in the question and answer session some of the things that we're doing to address COVID-19 in patients with mitochondrial disease. Okay, so, you know, some of you may have heard of mitochondrial disease in the news. And this was a story that came out back in 2017 with regards to Charlie Gard. And, you know, Charlie Gard was a young child who had mitochondrial disease due to RBM2 mutations, and there were discussions about quality of life and offering what treatments to offer and how, how far to go with treatment essentially with this individual. So this is something that you may have heard of because in general, you know, mitochondrial disease is a pretty devastating disease. So the pathophysiology of mitochondrial disease, as I mentioned before, there is bioenergetic failure So, you know, the respiratory chain does not work very well, so your ability to make ATP is severely hindered. And as I mentioned, one of the signs of that is lactic acidosis. That can be lactic acidosis, so buildup of lactate in the blood. And that can be due to— the buildup of lactate in the blood actually can be traced back to the fact that because you cannot use oxidative phosphorylation to make ATP, The, uh, one of the other ways you can use to make ATP is actually burning glucose. So you can see here ATP, you can actually produce ATP less, it's a lot less and it's less efficient, but you can produce ATP via burning glucose or glycolysis. And as a result of burning glucose, you actually make a lot of lactate. And that's why these individuals have high lactates, dysfunctional mitochondria, so therefore they burn a lot of glucose. Another, another aspect of the pathophysiology of mitochondrial disease is oxidative stress. They produce these mitochondria— if they're not functional and can't, can't receive those reducing equivalents, they can produce a lot of reactive oxygen species, which can damage cells, damage cell membranes, damage DNA. Also, since the mitochondria is important for integration of the, the, the host cell stress response, response. You can have abnormal cell stress responses in tissues with mitochondrial disease. So it's important to point out, as I mentioned before, the source of genes for mitochondrial function have two locations. So one of which is in mitochondrial DNA, which I mentioned before, and we showed you those 37 different genes in that circular mitochondrial DNA. And that's one source. And remember, it's passed in the maternal lineage because because women are the only one that pass on mitochondrial DNA to their progeny. But some of the other— 37 genes is not enough to make and run a mitochondria. It takes thousands of genes. So a lot of those genes actually reside in the nuclear DNA. And that's inherited, of course, from all of our ancestors, right? You get essentially half your genes from your mother and half your genes from your father. So essentially, 3 sources of genes include mom, dad, and then mom again via mitochondrial DNA. And this is just highlighting some of the sources, um, or some of those genes and how they contribute to mitochondrial function. So here on the left, you can see the nuclear DNA, and it's kind of color-coded. The different genes here are colored in the nuclear DNA, and they contribute components to complex I. Complex I is made up of 46 different proteins. Right? So that's the blue arrow here, complex II the yellow. So the nuclear DNA actually contributes to the construction of these different protein complexes in oxidative phosphorylation, as does the mitochondrial DNA. You can see here the blue— of those 46 proteins in complex I, the mitochondrial DNA contributes a number of those, as you can see here, which by the blue marks here. So the basic take-home message is that for this to run properly, you have to have contributions from mitochondrial DNA and nuclear DNA. So therefore, mitochondrial DNA— excuse me, mitochondrial disease can be produced by mutations in genes in mitochondrial DNA or mutations in genes in nuclear DNA that are involved in mitochondrial function.. So here, this is the mitochondrial DNA, and this is a morbidity map for, um, for the mitochondrial DNA. And what that means is this is where different mutations in mitochondrial DNA map for different diseases. So things like, um, aminoglycoside-induced deafness. So these are people, when they take a certain antibiotic, because of a certain change they have in their mitochondrial DNA, they actually can develop deafness as a result. These are a bunch of other mitochondrial diseases, as you can see here, and they are all kind of scattered in different genes in the mitochondrial DNA. So that's one source. And then of course, as I mentioned, the nuclear DNA can be another source. In the nucleus, by the way, I should mention, there are probably over 1,200 genes which are involved, which, which can potentially be mutated, which can lead to mitochondrial dysfunction, therefore mitochondrial disease. So one of the things that— so the thing about mitochondrial DNA is it has a number of special properties that make understanding its genetics complex. So two things that I'm going to mention are mitochondrial heteroplasmy and threshold effects. So the thing about mitochondrial DNA is in a single cell, right, this is your cell here, and inside are the mitochondria, which like little Tic Tacs inside here. And some of them are red and some of them are white. And the, um, the ones that are red are mitochondria that have a lot of, um, uh, mutant, uh, or a lot of mitochondrial DNA with mutations in it. So within it, within a single mitochondrion, you can have between hundreds and thousands of mitochondrial DNA, and you can have a balance between how many good copies do you have and how many bad copies do you have? In other words, how many of those mitochondrial DNA contain a mutation and how many don't? And what happens then is when cells divide, those mitochondrion are then randomly segregated. So the daughter cells can have then less of those dysfunctional mitochondria, so they may be okay, or they can develop more— not develop more, but have more passed on. And this is a random process. You can either have cells that eventually do okay because they don't have as many mutant mitochondria, dysfunctional mitochondria due to a large proportion of mutant mitochondrial DNA, or you can have cells that really don't function very well, and that can actually result in tissue dysfunction, organ dysfunction, cell death, things like that. So this idea of two different mitochondrial DNA within a single cell is called heteroplasmy. So in other words, the existence of mutant mitochondrial DNA and normal mitochondrial DNA. And when the mutant mitochondrial DNA passes a certain threshold, disease can actually develop. So heteroplasmy, as I mentioned, is the existence— this is just, just to try and illustrate it further— where it's the existence of essentially two types of mitochondrial DNA, one mutant form and one— essentially mutation in a gene, because right, there are 37 genes, one of those genes has a mutation in it, and then you have essentially a mitochondrial chromosome which does not. So here you have heteroplasmy, you have mitochondria which contain mutant mitochondrial DNA and those that don't, and we use these terms homoplasmic or heteroplasmic. Heteroplasmic is what I'm talking about, the reflection of these two different DNA populations, mitochondrial DNA populations within a single cell. Homoplasmic refers to one kind. So you can be a homoplasmic wild type, in which case you won't have disease. Depending on the varying levels of heteroplasmic, as soon as you pass a certain threshold, you may develop symptoms of mitochondrial disease. And then of course homoplasmic mutants have, you know, tend to have full-blown mitochondrial disease. Okay, so one of the important things with regards to maternal inheritance and how you can have, um, essentially, um, the expansion of these mutant mitochondrial DNAs, um, is something called, um, it has to do with the maternal inheritance. It has something called the bottleneck effect. So the primordial germ cells inside a woman, of course, go on to make eggs. And in that primordial germ cell, um, you have a large number of mitochondria, right? And some of those can be mutant and some of those can be normal. So red are your mutants and green are your normals. But what happens is you have this bottleneck effect. In other words, when you go on to make egg cells, that number goes from a very large amount to a much smaller amount, right? It's like, it's like 100 to 1,000 times less. And what happens then is that gives the opportunity for mutant mitochondria with mutant DNA to become more prevalent. So it's essentially a bottleneck. And then once that forms into a mature egg cell, you can actually then result and have more mutant mitochondria than you would have normal mitochondria. So essentially, this bottleneck, this constriction of reducing the number of mitochondria from the transition of this mother's primordial germ cell eventually to egg cells, this bottleneck actually can cause the— or lead to the expansion of mutant mitochondria. So in this particular case, it's 80% mutant, and in which case when joins with a sperm and then forms essentially the embryo, you can have a child with severe disease. 50% mutant, it depends on the mitochondrial gene, it depends on the mutation, they may or may not have disease. 20% mutant, they most likely won't have disease. So once again, there's this threshold of how many mutant mitochondria you have to have before you start to have tissue dysfunction and even death, etc., tissue death, etc., which then can bring up the many symptoms of mitochondrial disease. So here it's, it's essentially the same thing showing this threshold where you have the mutant mitochondria. Here we're showing a threshold of 70% or so, and in which case this person has 80% mutant mitochondrial DNA and they develop disease. So this is— this is the— I know this is a long-winded explanation, but it can be very complicated. So this threshold effect essentially, which can impart the symptoms and signs of mitochondrial disease. Okay, so the symptoms of mitochondrial disease. As we said before, since mitochondria are in essentially every cell of the body and energy is important for a lot of different tissue types, you can imagine that the symptoms of mitochondrial disease can be quite extensive. So here I'm just showing you on the side, um, a cartoon of the human body and all the different, um, things that can happen in mitochondrial disease, the different symptoms that they can have. They can have problems because the eyes require a lot of energy. You can have things like blindness, problems with eye movements. In the brain, of course, of course, a very high energy requiring organ. You can have developmental delay or seizures or dementia or neuronal death like I mentioned before that can be brought out by illness. The— they can have deafness, they can have problems with the heart and the, the pacemaker of the heart which sets the rhythm of the heart. They can have things like diabetes, they can have problems with their gastrointestinal symptoms. So really widespread and really speaks to all of these different organ systems which require a lot of energy. As I mentioned before, this can happen in the brain. This was our patient who essentially had neuronal death in his brainstem, which led to breathing problems because that's the main driver of respiration. Also in the eyes, it can lead to problems in the eyes, as we mentioned, and it can lead to blindness. This, and this essentially is looking at the back of the eye or the retina of the eye. And you can see here, there are various blood vessels coming out here, and you can see here on the periphery there are these spicules that develop, these kind of black-looking, almost like dirt, which accumulates inside the retina. And this is actually due to mitochondrial disease and eventually can lead to blindness. Blindness. Another thing that mitochondria are important for is not only for vision itself, but the movement of the eyes. So here, probably the best thing to look at is this lower cart— lower pictogram here, where this is showing essentially that the eyes actually move by the cranial nerves, and the cranial nerves come out from the brain and serve the muscles of the eye and basically give the message for the direction in which the eyes are to move. So here you can see when an individual is told to look straight ahead, you can see that first of all her eyelids are drooping a little bit, and that's cranial nerve 3 that helps keep those eyelids up. And also she has something called strabismus where her eye seems to be deviating— her left eye seems to be deviating a little bit when she's told to look straight ahead. When she is asked to look to her left, you can see that her eye actually looks very similar to the first picture, so it doesn't look like it moved very much. When she's asked to look to the right, you see that her eye moves back to center but cannot move completely to the right as can her right eye. So in other words, her conjugate gaze is disturbed. She cannot— the eyes move together, right? In which case, her left eye does not move to the same extent that her right eye does. So she'll have different problems with things like double vision and stuff like that because she doesn't have that conjugate gaze of her eye. And then the last thing here is she's asked to look up, so, and in which case she is unable to do so. And that's also because the cranial nerve that helps with looking up, as well as your eyelids, is actually has has dysfunction to it due to mitochondrial disease. And this is called progressive external ophthalmoplegia, or problems with movement of the eyes. Here I'm going to show you a quick video. Watch this. You can see this individual, he has an inability to raise his eyelids when he's asked to look up. And that is due to problems with the cranial nerve 3 due to mitochondrial disease. Okay, so mitochondrial disease, of course you can imagine, you know, muscle, skeletal muscle is also an organ system that requires a lot of energy. And having dysfunctional mitochondria, mitochondrial disease, actually can lead to what we call myopathy or mitochondrial myopathy. So you can see here, this is a stain for mitochondria within a muscle cell, and you can see that they can accumulate on the outside of a muscle fiber. These are muscle fibers here. These are called ragged red fibers, and this actually indicates— this is taken from muscle biopsy of an individual, and this actually is highly suggestive of mitochondrial disease. And you can see that because of skeletal muscle weakness, individuals can require— it can range in terms of their level of disability, but it can get to the point where some individuals have such muscle weakness that they actually require assistance with walking. So not only is muscle an important high-energy organ system, but so is the liver. So we're looking here at a normal liver, and all all the hepatocytes here. And this is a— on the right is a liver with mitochondrial hepatopathy, or mitochondrial disease affecting the liver, and the accumulation of fat. So fatty liver disease occurs in these individuals because of dysfunctional mitochondria. Okay, so as I mentioned, the diagnosis of mitochondrial disease really involves, you know, as clinicians, putting together a picture. No single thing, uh, you know, single aspect of investigations that we do defines mitochondrial disease. It's putting all the puzzle pieces together. So the first thing that's important for the diagnosis is being able to recognize it. Um, so, so recognition involves essentially, um, individuals who have a disease which affects multiple different organs or organ systems that don't seem to be related. So in other words, you know, like diabetes and deafness. It's hard to think of many pathophysiologic processes that would tie those two together. Okay, um, so recognizing is important. Um, blood testing— so we can test, essentially we can look at lactate levels in the blood, um, because as I mentioned before, patients with mitochondrial disease use a lot of glycolysis, they produce a lot of lactate, so we can look for lactate in the blood. We also can use blood studies for DNA, which I'll mention in a minute. Muscle biopsy, as I mentioned before, I showed you a muscle biopsy from a patient with ragged red fibers, which are suggestive of mitochondrial disease. We also use MRIs, which I showed you before, which you can not only look at the structure of the brain and swelling in certain areas, which can indicate neuronal death, but you can also look at metabolites, where we looked at lactate by MR spectroscopy. So MRI is very important. And then DNA studies, of course, putting this all together to really, you know, hone down on the diagnosis. You want to have DNA studies so you can sequence mitochondrial DNA, nuclear DNA, looking for deleterious changes. Or it used to be called mutations, now they're called deleterious variants. But for the lay public, mutations, you know, is still a term that's still used. So you're looking for mutations either in mitochondrial DNA or nuclear DNA, and these are genes that are involved essentially in mitochondrial function. Okay, so the treatment, as I mentioned, is really quite limited for mitochondrial disease, unfortunately. It involves giving vitamins and cofactors, and that's to try and enhance whatever metabolism or mitochondrial function you may have left. So that's what vitamins are, essentially. They help— they, they are— they are oftentimes things that are involved in improving enzyme function, etc. And then exercise, of course. Oh, sorry, I, I did want to mention a lot of those vitamins are antioxidants, right? Because patients with mitochondrial disease can produce a lot of free radicals and reactive oxygen species. So some of those antioxidants can be helpful. Exercise can be helpful in patients with mitochondrial disease, although it's still being, you know, evaluated. Most importantly, which is something I'm concerned about, is treating infections in these individuals, right? Because infections can be a life-threatening scenario for people. And you can see here on the right, this is just— this is not a patient, this is a picture I grabbed from the internet, but it really gives you an idea of the level of care that a child can require with mitochondrial disease when they get an infection. They can have, you know, they can be in the ICU with all this level of care that's required to keep them alive, including a respirator and including dialysis for kidney problems. And we use a lot of medications to treat the complications, basically. So we're kind of treating— we're kind of chasing our tails a little bit treating seizures and treating, you know, heart problems and treating liver problems, etc., you know, but not really, really getting at the issue, which is mitochondrial dysfunction. So some of the ideas that people have come up with for trying to treat mitochondrial disease are shown here, where, you know, things like mitochondrial replacement, where you try and take mitochondria from another human being and try and introduce them, trying to increase the stability of some mitochondrial tRNAs for, you know, making proteins, or even targeting pathogenic mitochondrial DNA specifically using some platforms like CRISPR, which you may have heard about in other scenarios. I'm not going to talk about it, but basically the idea is that you target the pathogenic mitochondrial DNA to try and reduce the number. So in other words, if the red are your mitochondrial DNA that have mutation in it. You can use CRISPR technology to try and reduce that number and try and reduce below the threshold, right? So you won't have those symptoms of mitochondrial disease. So these are just some of the ways that people are thinking about trying to approach the treatment of mitochondrial disease. Okay, so one of the things that has come up in the news is the idea of a 3-person baby right, where you can have, you know, a sperm donor, an egg donor, and then eventually you can also have a mitochondrial donor, and that can be done by pronuclear transfer, which I'm showing you here. So the couple essentially, if they have concerns about mitochondrial disease in their family due to mitochondrial DNA mutations, right, what you can do is you can actually remove the nucleus and implant it in a cell which has been enucleated, where the nucleus is removed, and essentially what you're doing is you are transferring the mitochondria, and therefore the mitochondrial DNA, of that donor couple to that individual, to that, excuse me, to that couple who have concerns about their own mitochondria. They may have a mutation or something like that, and in which case, then the cell hopefully will develop normally. So there is an individual in New York who is actually doing this, and, you know, the, the, um, there are— so far, you know, things have been fine, um, but however, this doesn't speak to the potential long-term effects of mitochondrial DNA because, as I mentioned, mitochondrial DNA and the mitochondria, you know, have many, many different roles, right, which I mentioned before, beyond metabolism, involved in cell death and calcium buffering, etc. And signaling, right? So mitochondria signal to the nucleus and can help control gene expression and things like that. So there is this potential issue of incompatibility between mitochondrial DNA and nuclear DNA, and it may do have to, have to do with this crosstalk. So this is a paper that came out, uh, I believe last year or the year before, which talks about that compatibility of mitochondrial changes in mitochondrial DNA and whether they are compatible with the nuclear DNA. So the idea is that they did— this was done in the UK where they looked at a large number of individuals and their progeny and looked for the passage of mitochondrial DNA, or changes in the mitochondrial DNA, on to the next generation. And what you would see is sometimes those changes were enriched and sometimes they disappeared. And what that led them to conclude is that there are certain changes in mitochondrial DNA that are not compatible with nuclear DNA. DNA. So basically, selective forces acted on heteroplasmy, or this level of, of changes in mitochondrial DNA versus— or mutations in mitochondrial DNA versus normal mitochondrial DNA. So there were selective forces in the female germline that was influenced by the nuclear background. There was this incompatibility between the nuclear DNA in the egg and the mitochondrial mitochondrial DNA, which led to selection. So that's a lot of material to take in. I'll be happy to answer some questions, but I just wanted to summarize. So first of all, mitochondrial disease itself is a multi-system disorder of energy metabolism, and since mitochondria are present in nearly all the tissues of our body, there is potential for different tissues to become dysfunctional or show symptoms of mitochondrial disease. Mitochondrial disease is the most common inborn error of metabolism, right? So of all those different, you know, enzymes and, and, uh, cofactors within all of that, uh, complicated subway map of metabolism which can go wrong, mitochondrial disease is the most common form. Um, it's mostly due— so I know I mentioned, um, nuclear mutations, uh, and mitochondrial mutations which can lead to mitochondrial disease. It turns out that mostly it's due to nuclear mutations, that mitochondrial disease due to mitochondrial DNA mutations is a smaller fraction. And that makes sense— there's only 37 genes in the mitochondrial DNA. There are thousands of nuclear genes which can lead to mitochondrial disease. This, as I mentioned, mitochondrial disease, mitochondrial dysfunction, has applicability to a lot of common diseases as well as aging. So mitochondrial dysfunction ties into, as I mentioned, things like Alzheimer's and Parkinson's Alzheimer's disease, et cetera. At this point, unfortunately, there is no satisfactory treatment. We do, we use things, you know, as I mentioned, like antioxidants and vitamins and stuff like that, but it really is, is really not overall making very significant impacts on the patient's life. So we have to look for new therapies and new therapies that are under investigation are mitochondrial replacement, or if it's mitochondrial DNA mutation, changing the frequency of mitochondrial DNA mutations. So I'd like to thank you, and I'd be happy to take any questions.