Mitochondrial Medicine in the Pandemic Era by Douglas C Wallace

Good afternoon, one and all present here. We welcome you all to the Distinguished Lecture Series. We are honored to have Professor Doug Wallace with us today.

So, I was watching this video on YouTube before coming here to relate better with the, uh, lecture and the talk. Earth, the two million years old. It came one of the most momentous events is about to play out. In the, what had happened in the primordial struggle for survival, a tiny bacterium is engulfed by a single-celled organism. Neither organism is harmed. Instead, the bacterium integrates into its host, turning oxygen into energy in exchange of protection from the outside world. Symbiosis. We are the result of that today. The Invaders would be live on in each of us in the form of mitochondria, the powerhouse in our cells.

Are today's guest of honor, Professor Doug Wallace of Children's Hospital of Philadelphia, spent decades studying them, uncovering mitochondrial mysteries, and shedding light on the origins of humankind. We welcome you all, Professor Doug Wallace. May I now invite our honorable Vice Chancellor, Professor BJ Rao, the Dean, School of Life Sciences, Professor Shiva Kumar, our guest of honor, Professor Wallace, on the stage.

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We would like to welcome the dignitaries with the bouquet presentation now.

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Thank you so much. Now, I would like to request Dean's, School of Life Sciences, Professor Shiva Kumar, to come and address the gathering.

A very good afternoon to one and all. Honorable Vice Chancellor, distinguished guest and speaker of the day, Doug Wallace, who has been very kind enough to visit us in this week and also giving us a distinguished lecture. He has been to the School of Life Sciences earlier, but I am sure, sir, you must have seen a lot of improvement in terms of the academic strengths and the building infrastructure facilities which have improved over the period of years from your last visit. And we have in the School of Life Sciences, very distinguished faculty, numbering more than 60, working in different areas of research. And we have also ongoing teaching programs in different subjects at Masters level. And we recruit PhDs through an entrance exam across the country. And all our students who are graduating from here are finding very good placements outside, either for jobs or for industry. Some of them are also starting up with setting up startup industries.

Which University of Hyderabad and School of Life Sciences is the host for. We have a bio-nest on the top floor. I don't know if you had a chance to visit it. So that harbors as many as 50 different startup companies which are doing very fairly well. And with the recent NEP 2020 in place and the IOE status to the University of Hyderabad, we are growing in strength academically, both to infrastructure. And today happens to be a very auspicious day. One is your excellent talk. The second one, our VC is going to lay the foundation stone for an interdisciplinary Research Unit building which is going to come up on the University of Hyderabad campus, which I foresee as a very huge investment in terms of infrastructure. And because of the IOE status, we are able to get this possible. Without wasting much time, I would now, we look forward for the talk by the distinguished guest.

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I would request Professor BJ Rao to come on stage and address the gathering.

It's a very large, beautiful university. It's a multi-disciplinary university. We are also an institution of eminence in the country, which is a rare honor that we deserved and we got it. And also, welcome to Hyderabad city, which is a happening city. We have a large number of very, very prominent institutes in this city. So, my, my suggestion is, keep visiting us as often as possible.

Now, Doug Wallace, of course, doesn't need much introduction because he has been a pioneer in mitochondrial biology in many, many ways. But one thing I want to, uh, sort of mention here that when we started learning biology, we were taught that there is what is called central dogma, DNA to RNA to protein. But as we learn more and more biology, we realize that that central dogma actually plays a much smaller role than the overall biology. Because ultimately, the central dogma has to be instructed, has to be instructed to do what, what it has to do, right? Central dogma is just a program that must be told, do this, don't do that. Who does that? Organelles such as mitochondria, ribosomes, membranes. So, it is actually not just the central dogma, it is instruction going backwards to the nucleus that does a lot of biology, which is where I think you, you come in as a pioneer. Mitochondria being what it is, the instruction center for the cells to do what it should do based on the energy levels. And more importantly, what it should not do. So, I think you, you start the biology in some sense. So, therefore, I think it's always a pleasure to hear you. This slide is already very loaded, but there is a lot in this. I am sure you will tell us a lot of things about mitochondrial regulation and perhaps even take us to the population level of mitochondrial events. So, we are all looking forward to your talk, Doug. And keep visiting us. Keep us much more energetic. Thank you.

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The talk is about energy, so we would be energetic, sir. Uh, I would now, I would like to request Professor Naresh Sepuri to come on stage and address the gathering.

Okay, actually, I want to introduce the speaker. So, good afternoon to all. And on behalf of the Department of Biochemistry, School of Life Sciences, University of Hyderabad, it's my great honor and privilege to invite you and to introduce you as a speaker in the Distinguished Lecture Series. Professor Douglas A. Wallace is a pioneer in the field of human mitochondrial genetics. Professor Wallace is the director, currently director of the Center for Mitochondrial and Epigenomic Medicine and Children's Hospital of Philadelphia, USA. And he also holds the Michael and Charles Perne Endowment Chair in Pediatric Mitochondrial Medicine and Metabolic Diseases at Children's Hospital. So, the synonym of synonym of mitochondria is Doug Wallace. More than 35 years ago, Wallace and his colleagues founded the field of human mitochondrial genetics. They contributed to several fundamental discoveries in the mitochondrial area. So, one of them, landmark finding of his being that mitochondria is inherited exclusively from the mother. Second, pioneering contribution is a genetic alteration in the mitochondrial DNA can lead to several mitochondrial, several metabolic and metabolic disorders and degenerative diseases. He proposed the use of mitochondrial DNA as a molecular marker of diseases. Not only a geneticist, but also an evolutionary biologist, he revolutionized the field of mitochondrial biology. So, briefly, his education. He has a BS in Genetics and Developmental Biology from Cornell University. He obtained MPhil and PhD from Yale University in the area of Human Genetics. And he continued postdoctoral studies for some time at Yale University. And then he moved as an Assistant Professor to Stanford University. And he became a Professor at Emory University in the area of Biochemistry and Pediatrics. And then again, he moved to the University of California from 1996 to 2002 as the Chairperson and Senior Editor of the Mitochondrial DNA Local Specific Database for the Human Genome Organization. In 2002, he assumed Professor of Molecular Energetics at the University of California, Irvine, and founded the founded the Center for Molecular and Mitochondrial Medicine and Genetics. In 2010, he moved to his current position at the Children's Hospital of Philadelphia. And he has several awards. So, I could mention only a few. So, in the distance, one is the Fellow of the National Academy of Sciences. And is a Lifetime Achievement Award in Genetics and Benjamin Franklin Life Sciences Medal in 2017. And Dr. Paul Johnson Award for Biomedical Research in 2017. And besides several medals, he contributed immensely to the field of mitochondrial biology and genetics and several top-tier publications from his group. We are extremely, sir, we are extremely fortunate to have you and represent your work to our fraternity. And also, your presence is highly inspirational to the faculty and as well as students. And your interaction with our students and neighboring institutes' faculty is highly helpful and inspirational. And thank you for accepting our invitation and coming to Hyderabad. And I had an opportunity to work with you for three months last year. And I would say he is truly inspirational and a gentleman and a great human being. And he is kind enough to have some kind of collaborations with my lab in the University of Hyderabad. Thank you, sir, for you're kind enough to give some kind of collaboration to establish at the University of Hyderabad. I hope this collaboration extends to not only our institute and neighboring institutes and we make Hyderabad as a center for mitochondrial biology along with your center. Thank you for accepting our invitation.

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Well, thank you very much for all those kind words and for inviting me here and for, uh, all the distinguished people that have chosen to come. I'm greatly honored. I really want to thank my friend Naresh for, uh, having me and for being willing to collaborate with us on our Gates Foundation Grant on the role of mitochondria in SARS-CoV-2. So, we're going to start with the wonderful introduction that the young lady provided. She gave a very nice introductory lecture to mitochondria, which is, in fact, a symbiote living inside cells. So, each cell, then, you see, you are actually composed of a hundred trillion individual individuals which are called cells. And inside each of those cells, there's a colony of bacteria called the mitochondria. So, there's, in the order of 10 to the 17th bacteria sitting in your chair. Those are not in your gut microbiome, they're actually in your cells. And they are, in fact, central to your, everything that you do, because they provide 90% of all the energy that you use in your life. And so, today, we're going to talk about why energy is important in medicine and in human health, disease, and evolution.

So, the mitochondria really was a symbiote, uh, started about two and a half billion years ago. There was a nuclear component which was a kind of archaeobacterium. And that archaeobacterium had evolved the ability to interact with bacteria close to its cell membrane and ultimately engulf them. And one of, one of the things that engulfed was this oxidative bacterium which can convert carbohydrates and fats into energy by a process of oxidation and phosphorylation. Now, because these two organisms, uh, were initially co-equal, they came together. Each of them had its own information storage and retrieval system, DNA to RNA to protein. And amazingly enough, even today, two billion years later, they've retained this same system, two parallel systems for information. Now, originally, the original bacterium, the mitochondria, had a genome as big and complex as its host organism. But bacteria and are very able to exchange genetic information. And so, as it turned out, most of the genetic information in the bacterial genome was transferred to the archeobacterial genome, ultimately to give rise to what we now call the nucleus.

So, um, the, the question then is, why study the mitochondria? Um, so, what's interesting about Western medicine, which even here in the Indian subcontinent is the dominant, uh, philosophical paradigm, is that it's oriented toward anatomy. So, why is it oriented toward anatomy? It goes back to a man named Vesalius that lived half a millennium ago and, and worked in Italy, a country in Europe. And he was the first to really formally define the structure of the human body. And that was a very big innovation because at that, before that, there was really no structure to put medicine into. And so, that was so important that from then on, all people that wanted to be doctors came and they studied different parts of the anatomy. So, then we have this very elaborate description of the human body, all the way down to capillaries and different parts of the body. And every medical student that studies medicine, the first thing they're asked to do is go to the, uh, anatomical lab and spend a year dissecting a human body and memorizing a lot of Latin and Greek terms for all the different parts of the body. And then that is very important because that sets the stage for their thinking about human medicine. And so, this anatomical idea of medicine creates a set of sub-disciplines that are based on anatomy. So, once you become a physician, you then specialize. You either become an ophthalmologist to study the eye, or a neurologist to study the brain, or a cardiologist to study the heart, or nephrologists to study the kidney. And all that is fine, except for it leads to an unstated corollary. And that corollary is, if I have a symptom, let's say a headache, then the assumption is that I should go see the neurologist because there's something wrong with my head. Or if I have a nervous breakdown, I go to the psychiatrist, and the psychiatrist thinks that by talking to me, he can make me feel better because I have something wrong with my head. So, the problem is that many of these approaches have not been very successful in treating and understanding the common diseases. So, common diseases like diabetes, Alzheimer's disease, Parkinson's disease, cardiovascular disease, on and on and on. So, that leads to the question then, why isn't this anatomical approach to medicine being more successful at helping us understand disease? And, um, I thought a lot about that coming from a physics background as an undergraduate in college, where Newton said 500 years ago that mass doesn't change or move without energy. And it occurred to me then when I went to Yale, as we just heard, in 1970, after I got out of service, that it seemed very bizarre that we were worrying about human anatomy when, in fact, humans were the most animated thing in my environment. And I said, Newton would say that those people must have a lot of energy flow. So, therefore, I said, why aren't people studying human energy?

So, that leads to the idea that maybe we could understand the common diseases not by studying anatomy, but by studying energy. And so, basically, I came up with this very simple formula that life is not just about anatomy, but the energy that animates anatomy, and then information necessary for anatomy, and information necessary for energy. And therefore, it seemed biological thing, since human genetics was just developing, why not study the genetics of energy?

So, why do we think that the common diseases might be due to energy problems? Well, if you think about the diseases that are most common, they affect the brain, the heart, the kidney, liver, and the endocrine systems. Well, what's characteristic of those, as opposed to say, your skin or your foot? Those are the organs that have the highest energy demand. So, if the organs that are commonly affected have the highest energy demand, then maybe the problem of those organs is related to energy. So, what creates the energy? 90% of our energy comes from this bacterium. So, that led to the realization that maybe, in fact, what we're overlooking is the energy-generating organelle. So, that led to the hypothesis that maybe the common diseases were not going to be solved by studying anatomy, but we're going to be solved by studying energy. So, that seemed like such an obvious and simple answer. Why hasn't anybody been doing that? Well, it turns out that the way people think about genetics, based on studies that were done more in the early 20th century, based on concepts from a guy named Gregor Mendel. And he said that genetic information is transmitted, didn't know the word genetics, that was transmitted from parents because each parent had two copies of a particular gene, and then one went into each gamete, and then on fertilization, two came back together. That is genetics is diploid. And even when you're trained as a medical geneticist, like I am, one of the first things you're required to memorize is a formula: H squared plus E squared equals P squared, or, uh, genetics plus environment equals phenotype. And if you want to understand genetics, what you do is you study identical twins because identical twins will have identical genes, and they will then have represent H squared. And then everything else is E squared. But the problem is that identical twins can be quite different. And the question was, was that all environment? So, that led to the question that, well, maybe there's a different aspect to genetics than just Mendelian genetics. So, the idea then is that that might, in fact, be mitochondrial genetics. But remember that mitochondria is a separate life form. So, it isn't like one gene, one polypeptide, one disease, as Beadle and Tatum said. But in fact, it's that whole network of different biochemical pathways and different genetics. And that was then very complicated and very difficult to elaborate. So, the idea then was that maybe these bacterial microorganisms were having some effect on energetics, and that was affecting the energetic organisms, and that would then affect disease.

So, how do we address that? We had to understand the genetics of this organism. And it turns out, after 50 years of working on this subject, that the mitochondrion is assembled from literally over a thousand genes scattered across the chromosomes in the nucleus, but also in this very unique piece of DNA that Mendel didn't even know about, which is, in fact, as we heard, inherited exclusively from the mother. And that is the mitochondrial DNA. So, what's good about the mitochondrial DNA? It is the wiring diagram of the power plant. So, all the most interesting energy genes are in a non-Mendelian genome, and therefore were totally overlooked and put not in H squared, but in E squared, and therefore that made it very difficult to understand.

Okay, so we're going to then follow that up with the questions: How does this process, oxidative phosphorylation, to make energy? Um, how is that, uh, process genetically encoded with all these genes? Uh, what is the role of mitochondrial dysfunction in rare and common diseases? How is the dispersed mitochondrial genetics coordinately regulated? How do we have all these genes work together? And how does an environmental challenge, in this case, SARS-CoV-2, because now, uh, University of Hyderabad and our center now has a collaborative grant on that, how would changes in an environmental challenge such as a viral infection affect mitochondrial function? And does that give us any insight on how we might treat?

Okay, so getting back to then this idea, we now have two systems: the nuclear cytoplasmic system, where the nuclear genomes are in the nucleus, nuclear genes are in the nucleus, that makes sense. Uh, they're replicated, transcribed into RNA, translated onto proteins, and the thousand-plus genes that are in the nucleus but go to the mitochondria are selectively imported into the mitochondria where they are used to assemble the structure. But now we have this circular mitochondrial DNA, which is left over from the original bacterial genome. And it now has been reduced to 16,569 base pairs. And those base pairs code for 13 polypeptides that are central in making energy. So, it's literally like building a power plant here for the university. You would have the blueprint for the power plant with the Chancellor's office, but the actual wiring diagram inside the power plant would be in the power plant, because every power plant has a different wiring diagram, and you can't just make a generalized wiring diagram, you have to have a specific one. And that's, in fact, exactly how our mitochondria are organized. So, the mitochondrial DNA then codes for the critical genes necessary to generate the energy, the wiring diagram of the power plant. And they are transcribed into transfer RNAs, ribosomal RNAs, and messenger RNAs, of which there are 13: seven of the 45 proteins of this enzyme, which we'll call Complex I; one of the 11 proteins of Complex III; three of the 13 proteins of Complex IV; and ATP A6 and eight are the two subunits of Complex V.

So, what are these complexes? These complexes are the most important enzymes necessary to make energy. And they are arrayed along the mitochondrial inner membrane. This is a very simplified diagram of the mitochondria: the outer membrane, intermembrane space, inner membrane, and the matrix. Now, uh, what we do in making energy is we burn hydrogen from our foods with the oxygen that we breathe to make water. So, let's just take, as an example, we could use either carbohydrates or fats, but we'll use carbohydrates as an example. We take glucose through glycolysis to make pyruvate. Pyruvate can be reduced to give you lactate, or it can get an amino group to give you alanine. And then, or pyruvate can go through pyruvate carrier and then pyruvate dehydrogenase to make acetyl-CoA, which drives the tricarboxylic acid cycle. Now, one might wonder why it's important for all undergraduates and graduate students to memorize the tricarboxylic acid cycle. And as I tell my medical students, it's not to anesthetize medical students, but it's very good at doing that, which, that was supposed to be funny. Laugh, ha! But in fact, what its real job is to strip the hydrogens off off the hydrocarbons and put it on a carrier, NAD. So, we're going to now take the hydrogens off the hydrocarbon and put them on the reduced form, NADH, which has two electrons to then be burned. And then the NADH is burned by Complex I, NADH dehydrogenase. The electrons flow through Complex I to CoQ to Complex III to cytochrome C to Complex IV, and then reduce an atom of oxygen to a molecule of water. And as you can see, these enzymes are along the inner membrane. So, as the electrons flow through these complexes, they're used to pump positive charges from the mitochondrial matrix through these complexes into the intermembrane space to create a capacitor that's acid and positive on the outside, and alkaline and negative on the inside. So, sitting in your chair right now are 10 to the 17th capacitors, each of which has a membrane potential of potential of 0.2 volts. So, the total amount of potential energy sitting in a chair is the equivalent of a lightning bolt. So, the difference between being alive and dead is whether you, in fact, are breathing or not. And if you're breathing, you're maintaining that membrane potential, and now you have the potential energy to do everything you want to do. But when you stop breathing, the membrane potential collapses, and now you do not have any potential energy, and then you become a cadaver. And so, as I tell my medical students, it's okay to spend their whole first year memorizing the detail of a cadaver, but if the person they come to see in the doctor's office leaves in the state of the cadaver, there was nothing wrong with its anatomy, it was just dead. And so, the important thing is to remember the difference between being alive and dead, and that is mitochondrial function. So, why haven't Western medicine studied body control function? Because in 1970, nobody even thought about it.

Okay, so here we are. We have a membrane potential. We're going to use that to make chemical energy. And we convert that chemical energy by this enzyme, ATP synthase. And the protons flow through the ATP synthase to condense ADP and phosphate to make ATP. Then that exchanges across the adenine nucleotide translocator out through a sieve-like protein. And the ATP then is hydrolyzed to do work. So, you're using oxidation to make a membrane potential, and phosphorylation to convert it into chemical energy, hence the process, oxidative phosphorylation.

Now, it turns out everybody in this room has a slightly different efficiency of making ATP from the food you eat. Let's say that you're very efficient at making ATP. So, you generate your, your hydrogen, you burn the hydrogen, and you get the maximum amount of membrane potential. Then you use that to make ATP. So, you get the maximum amount of ATP for the minimum amount of calories you burn. Now, a calorie is a unit of heat. So, every calorie you burn increases your body temperature. So, the mitochondria is regulating your body temperature by burning hydrogen. Okay, so if you're very efficient at pumping protons out and converting them to ATP, as we say, tightly coupled, then you will burn the minimum amount of calories for the maximum amount of energy. That's great. But what if you're less efficient at pumping protons out and converting them to ATP? Well, now you still need the same amount of ATP, but now you have to burn more calories for the ATP. But now you're going to generate more heat. And so, the mitochondria regulates your body temperature by this coupling efficiency.

The mitochondria also is a furnace, and it may smoke. And the smoke are called oxygen radicals. And most of the oxygen radicals come from the mitochondria. And the way that happens is electrons from the early stages of this electron transport chain can be donated directly to O2 to give you an unpaired electron, superoxide anion. And that wants another electron because you always want pairs of antiparallel electrons in your orbitals. And so, this one will grab up electrons from any other protein, nucleic acid, or lipid. So, we have an enzyme, manganese SOD, that takes two of these to make hydrogen peroxide, which is more stable. But if it gets another electron from a transition metal, it will give you hydroxyl radical, again with an unpaired electron, and that's the most potent oxidizing agent in your body. So, these are the reactive oxygen radicals. We have an enzyme, glutathione peroxidase, that could take hydrogen peroxide to water using reducing equivalents from an enzyme, nicotinamide nucleoside transhydrogenase. Don't you love that? Anyway, that, though, however, is a rate-limiting step. So, hydrogen peroxide builds up in the mitochondrial matrix.

The mitochondria also regulates calcium by the uptake of calcium using the membrane potential by the calcium uniporter. It has a self-destruct system, which most people now think is the ATP synthase. And that then measures the membrane potential, oxidative stress, the amount of calcium. And when they get out of balance, it goes from a closing door, closed door, to an open channel that short circuits the membrane potential. Fluids flow into the mitochondrial matrix, the inner membrane swells, two proteins, Bax and Bak form a mega channel, they release these stored proteins, they go out, and they then activate apoptosis. That is, they degrade all the proteins in the cell. Why would you want to do that? Because this is a perfectly good bacterium. And if you release the bacterium in your bloodstream, you're going to get a lot of inflammation. So, you just burn it up with, um, caspase digestion before it ever gets out. So, but mitochondria then has a very unique genetics. Um, the mitochondrial DNA itself codes for 13 proteins. The nucleus codes for a thousand or more proteins. And at least 150 nuclear and mitochondrial proteins work together to make oxidative phosphorylation. The mitochondrial, mitochondrion has its own DNA, which are in these nucleoids. And those then code for these 13 polypeptides. The oxidative phosphorylation system generates most of the energy, regulates redox balance, generates reactive oxygen species, which are both signaling and toxic, regulates calcium, regulates apoptosis. And as you'll see, the intermediates generated by the TCA cycle turn out to be the substrates to regulate the epigenome. And that's how the mitochondria coordinates nuclear gene expression to get along with its subsidiary bacterium.

Okay, so, um, what about this genetics? Well, the mitochondrial DNA is, as I said, only 16,569 base pairs. But that is, uh, an understatement of its importance because it's absolutely packed with information. There are no three prime or five prime non-translated regions, no introns. Every, almost every nucleotide is used. So, here is the mitochondrial DNA circle. Uh, this is the regulatory region, codes for a small and large ribosomal RNA, 22 transfer RNAs that punctuate all the genes.

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ND1, 2, 3, 4L, 4, 5, and 6 are the seven subunits of Complex I. Cytochrome B is the one subunit of Complex III. CO1, 2, and 3 are the three subunits of Complex IV. And ATP A6 and 8 are the two subunits of Complex V. So, that is then the wiring diagram of the power plant.

Now, this mitochondrial DNA is highly sequentially variable. As I said, almost everybody here has a different, um, different coupling efficiency. And that variation is mostly due to variation in the mitochondrial DNA. So, therefore, you have a problem if, uh, you have a person who has tightly coupled mitochondria, and if perhaps she married a person with loosely coupled mitochondria, and we allowed recombination, then you might get a loosely coupled Complex I with tightly coupled Complex III and IV. And the loosely coupled Complex I would short circuit. So, that's a big problem. You can't do that. All the enzymes, all four enzymes, one, three, four, and five, have to co-evolve to use the same membrane potential in the same way. So, that really, uh, was disturbing to Mother Nature. And she thought and thought and said, what's the problem? And the problem is clearly men. Now, if you'd ask any woman, she could have told you that right away. So, anyway, the problem being men, then the answer is to get rid of men. So, the solution from Mother Nature was to have the mitochondrial DNA transmitted from her daughters to all of her children and her daughters' children. But males' mitochondrial DNA enter the egg, are seen as foreign, and are selectively destroyed. So, poor men have been thrown out for three billion years. So, nothing has changed.

So, anyway, each cell then has hundreds of mitochondria. They're constantly replicating inside your cells, and they're accumulating mutations. So, when you get to be an old guy like me, you have pretty much blasted your mitochondrial DNA. Don't tell my wife. Anyways, the point is that you can now have a mixture of mutant and normal mitochondrial DNA. So, we call that heteroplasmy. If the cell divides down the middle, then both would have some mutant and some normal. But if the cell divided this way, then this cell would have only normal, and this would have twice as many mutants. So, as a heteroplasmic cell, say an oocyte with a million mitochondrial DNAs, as it divides into individual germ cells with only a few mitochondrial DNA, it then can have segregate its different genotypes. And so, you could have some cells that have predominantly good mitochondrial DNA and high energy output, and others with high numbers of mutant mitochondrial DNA. And every damaged mitochondrial DNA takes out a wiring diagram and takes that power plant offline. So, the more mutant mitochondrial DNAs you have, that means more mutant mitochondrial DNA should have, yeah, then the less energy. So, as the energy declines, then from the mitochondrial DNAs, uh, it ultimately falls below the minimum for that organ. And that then gives you a phenotype, the equivalent of a metropolitan brownout.

So, when my mother was 75 years old, she said, well, the problem, because see, I'm 75 years old, is I just don't have the energy I used to. And I said, aha, I think that's the answer. I better study mitochondria. Now, now that I'm 76 years old, I think she really underestimated the problem. But anyway, this was a situation we call that the bioenergetic threshold.

Now, why is that interesting? Because the brain is 2% of your body weight, but uses 20% of all your mitochondrial energy. So, a 5% reduction in mitochondrial function is going to have a very big effect on your brain. Okay, so the mitochondria is inherited uniparentally. This isn't as fun. This is the first 50 years, anyway. The mitochondrial DNA is, uh, inherited along the maternal lineage. So, it's accumulating sequential mutations. So, the number of nucleotide differences between any two of you is the time since you shared a common mother. So, we could sequence everybody in the room, and we could reconstruct all your maternal history. So, what we did is then we went around two different indigenous populations around the world, sequenced our mitochondrial DNAs, linked them up genetically, and then considered where they are located. Allowed us to figure out how women migrated around the world. And what we found is that mitochondrial Eve, as it's called, was originally in sub-Saharan Africa. The ancient mitochondrial DNA we call L0, L1, and L2 are two different pygmy lineages. This is 90,000 years old. This is about 30,000 years old. Then L3 is the out-of-Africa people in sub-Saharan Africa. And then L3 gave rise to two mitochondrial DNAs, M and N. Only these mitochondrial DNAs left Africa and colonized the rest of the world. M moved along the tropics all the way down to Australia, and then much later accumulated mutations and went into Central Asia. Whereas N moved directly into Europe, and there it generated lineages which we gave letters to, H, J, T, etc. And then much later moved laterally along the temperate zone into Asia. So, Europe has primarily lineages from N, and Asia has both M and N. And then only one M, two M's, and one N became enriched in Chukotka across the Bering land bridge and gave the first migration to Paleo Indians. And subsequent migrations occurred.

So, why would then only two mitochondrial DNAs leave Africa? Why is their high regionality of mitochondrial DNA in even today's world? And the answer is that these mitochondrial DNAs are adapted to different environments. So, what's happening here in Africa? If you want to survive and you're a woman, you need to run away from lions. Well, how to run away from lions? You need a lot of ATP, and you do it's very hot, so you don't want to be hot. So, you have to have tightly coupled mitochondria, minimize heat, maximize ATP, run away from lions, great. Okay. But up here, the lions rose to death. That wasn't a problem for you. The problem is now you're going to freeze to death. So, how do you deal with that? You get mutations in your mitochondrial DNA that make them less efficient. But now you eat more calories, that is a high-fat diet, and that then compensates to make more ATP. But in the meantime, you've made more heat, and that then allowed you to survive in the winter. So, these mitochondrial mutations that allowed our ancestors to adapt to different environments. And then finally, the mitochondrial DNA accumulates somatic mutations over time, and those are the aging clock. And I will get back to that in just a minute.

So, let's look at then the mitochondrial DNA. There are three important classical disease-causing mutations. Mutations occur on the maternal lineage that give deleterious mutations. Mutation ancient polymorphisms that allow people to adapt to different environments, but now in different environments are incompatible and give disease. And the accumulation of age-related mutations. So, there is in our mitochondrial sequence database, Mitomap, now 50,000 mitochondrial DNA sequences, about 20,000 polymorphisms, and about 1,000 pathogenic mitochondrial DNA mutations. So, our first mutation was in 1988 where it was reported. And since then, a thousand, uh, pathogenic mutations have been found. These are just examples. This mutation of the tRNA leucine gene at nucleotide position 3243, that one can be heteroplasmic, and it can give you phenotypes from diabetes all the way up to lethal childhood disease. And we'll get back to that mutation. In this position here, at 11778, if you inherit that from your mother, you, if that is homoplasmic, pure mutant, but you'll wait until midlife and then you'll lose vision in one eye and then the other. Leber's hereditary optic neuropathy. Mutation in ATP A16 at 8993, at about 70% mutant will give you, uh, problems with your retina. 85% mutant gives problems with your brainstem and your cerebellum. And then at 90%, kills you as an infant with Leigh syndrome. So, variable heteroplasmy. So, there are literally hundreds of these pathogenic mutations. But there are also ancient polymorphisms. So, that variant arose here in Africa early on, and it represents three-quarters of all sub-Saharan African lineages. And then this variant is found in half of Europeans. And variants A, B, C, and D arose here at Asia, across the Bering land bridge, and colonized the Americas. And these are variants that mark lineages that were adaptive to different environments.

So, lastly, we accumulate these age-related mutations. And so, what we did is we took an enzyme called catalase, that takes hydrogen peroxide to water. And remember, the mitochondria is accumulating hydrogen peroxide. So, that then is making reactive oxygen species that are damaging the mitochondria. So, we reasoned if we put catalase in the mitochondria of a mouse, using a mitochondrial targeting peptide, we would get rid of the hydrogen peroxide and maybe then decrease the aging clock. And so, what we found is we could decrease the mitochondrial DNA mutation rate by half, and we could increase the lifespan of both male and female animals by over 20%. So, that is the aging clock.

Okay, enough of that. So, this is the picture I showed you before. If we then get rid of anatomy, put that on the side, and put energy in the middle of medicine, then we can see that all, all of the common diseases have the same pathophysiological mechanism: energy deficiency. So, we have oxidative phosphorylation and have energy deficiency, increased ROS, redox potential alterations, and calcium. And if you have a chronic energy defect, you'll screw up mitochondrial DNA replication, you accumulate mitochondrial DNA damage, and that will erode the energy till you get to be my age, and then you'll be very, very tired, and that's aging. Or it can also augment a minor inherited defect. And that's why you can have diseases that have a delayed onset and progress. Of course, because you have to wait till the somatic mutations cross that expression threshold. But if you have just partial mutations or defects in oxidative phosphorylation, they can occur by either nuclear mutations, changes in gene expression from the epigenome, ancient adaptive polymorphisms, or recent deleterious mutations, changes in your diet, whether you exercise or not, whether you smoke and then poison your mitochondria. And if you get a partial reduction in oxidative phosphorylation, the first thing that's got to be affected is your brain, because a 5% reduction in energy is very bad for your brain. So, that's going to affect the brain, the heart, the muscle, and the renal system. All these organs that are commonly affected in the common disease. If you inhibit oxidative phosphorylation, you accumulate carbohydrates and fats, and that's diabetes and obesity. If you, in fact, damage mitochondrial DNA, you can, in energy, you cannot undergo apoptosis. You release all of those mitochondrial antigens in the bloodstream, and that then activates the inflammasome and the cGAS-STING pathway for interferon. And that's why all of these diseases have an inflammatory component. And then finally, you could have changes in energy metabolism in cancer, and also environmental challenges such as viruses that affect energy.

So, anyway, guess what? That was background. Okay, so let's talk about something a little bit more interesting. So, here's a family. This individual has a mutation that changes this proline to leucine at codon 25 in the ND1 gene, nucleotide position 14,600. This woman had 50% mutant in her blood, and she had optic atrophy and cerebellar ataxia. Her sister had only 5% in her blood, was perfectly normal. So, she had three different consorts, and every one of her children was 100% mutant, and they all died. So, this was a heteroplasmic mutation that segregated to homoplasmy in our oocytes and killed off all of her children. Ah, I'm falling apart. Okay, I hope this still works. So, anyway, this doesn't work. Yeah. Conserved changes, highly conserved proline. So, maybe the hello magic put it there, guy. Okay, so anyway, uh, we wanted to see if this mutation really caused the disease. So, uh, for those of you that study microbiology, you know that you have to fulfill Koch's postulates. Okay, Koch was a guy who, uh, probably died of some terrible disease, but anyway, he had this idea that how would I prove that bacteria cause a disease? So, he said, what I'll do is I'll isolate the bacteria, and then I'll feed it to my neighbor, and if my neighbor dies, then the bacteria caused a disease. It makes sense. The only problem is that his neighbor shot him before she died. But anyway, the point is that we could then determine if this mutation in this bacterium, which is inside cells, caused the disease by a dis- following Koch's postulate. But we couldn't very easily feed the bacteria to the mouse. So, what we did is we took mouse cells, oops, sorry, we took mouse cells and we treated them with a mutagen, and then we screened a whole bunch of different mouse cells for ones that had defects in mitochondrial function. Then we sequenced all the mitochondrial DNAs, and lo and behold, found a single cell line with the ND6 mutation that changed Proline 25 to Leucine. Good, first step. So, then we took the mouse cell line, we removed its nucleus, took its cytoplasmic fragment, we made a female embryonic germ cell line, we then removed its mitochondria with this drug, fused in the sick mitochondria to make what's called a transmitochondrial cybrid. It's a hybrid, and then we put that into a blastocyst, and it then went into the foster mother and gave chimeric animals from both the black cyst and the agouti animals, and then bred the females for those that picked up the agouti in the germ line and thus the mitochondrial mutants. Okay, so that, that worked after 20 years. And so, here we have then the bowels that turned green because I wasn't very happy. But anyway, the point is it had about a 60% reduction in respiratory Complex I because it's a Complex I mutant, that makes sense. And that would then cause a defect in oxidation of NADH to NAD. And so, by using fluorescent lifetime imaging microscopy to look at NADH levels, you can see that in the hippocampus of a normal mouse, we have almost all NAD. But in the mutant mouse, we have huge amounts of NADH. So, that makes sense. The extra NADH is going to reduce then the electron transport chain, and that then is going to create a lot of electrons to make ROS. And these animals that have very high oxidative stress, and that causes their neurons to swell up and to ultimately fail and give neurological symptoms.

So, we have two different mice that we've been working with: the ND6 mouse with Proline 25 Leucine, and an ND5 mutation that's a Serine codon 204 to Proline that we're going to talk about. So, we're going to compare these two mice. And what's interesting from the work of, uh, my colleague, uh, Ray, uh, Ryan Morrow, we can isolate mitochondria from the cerebral cortex of the two mice, and we can compare the respiration. And what's interesting is this is the normal mouse, this is the ND5 mouse, and this is the ND6 mouse. And you can see that they have very specific but subtle differences in energetics. So, then we can take these mice, and and my colleagues Kate and, uh, Jess can then do behavioral studies on these mice. This is called a radial arm maze. You put the mouse in this closed area, and then it will venture out into the more scary area. And you can measure how many times it goes out in the scary area, the idea about how anxious it is. And if you look at the results they got, this is the control mouse with the highest mitochondrial function. This is the ND5 mouse with the next highest level. And this is the ND6 mouse with the next highest level. There is a direct proportionality between behavior and mitochondrial cortical defect. And that's why I believe that all the neuropsychiatric disorders are about mitochondrial dysfunction. You can also ask, this is a study on depression. And not to go into great details, but you could see that this animal has lower depression, this one has slightly more, this has slightly more. And then if you feed them lithium, which is a classic treatment for depression, then you normalize, and they don't have depression anymore. So, these mice then have neuropsychiatric symptoms that are completely due to a point mutation in the mitochondrial DNA.

So, then we can ask, is there a behavioral problem? So, we can look at our ND6 mouse, put it into a cage where this is a novel object and a novel mouse. And most normal mice will tend to visit with the novel males. But these mice, this is the, the normal animal, this is the mutant animal, would tend less to look at the novel males, but actually much prefers looking at the novel object. So, it has autism. And this mouse is also hyper-compulsive, and it has obsessive-compulsive disorder. So, a single point mutation in the mitochondrial DNA, resulting in a 10 to 20% reduction in mitochondrial respiration, generates all the neuropsychiatric disorders. Right, okay, that's the point.

Alrighty. So, then why would it have this problem? The one of the most obvious things that makes reactive oxygen species. So, we can measure that by using ethidium bromide, which diffuses through the body of a mouse. And our colleague Bob March put a fluoride-18, which you can hardly see, which is a PET probe. Now, when ethidium bromide sees superoxide anion, it gets a positive charge. So, then you can put in the ethidium bromide, let it bind, get oxidized, and then that's not oxidized, wash out, and then do PET analysis. So, this is a normal mouse. And then if we treat it with a bacterial antigen, we get inflammation. So, we see more signal. But if we look at the ND6 mouse, it already has the inflammation of a mouse with the bacterial antigen. But we add a lipopolysaccharide, and we get a cytokine storm. So, why do we get a cytokine storm in the ND6 mouse? Because to get cytokine production, you need to get T cells. So, you have T glycolytic cells, T effector cells that make the cytokines and ROS. But they turn out to be glycolytic, that is, they have low oxygen consumption. But the T regulatory cells that keep these cells in check, they turn out to be oxidative. And so, the problem is that the ND6 mutation causes the oxidative cells to die off. So, you lose the negative regulation, and you then get the inflammatory system. So, that then is a very important aspect to where the cytokine.

The storm comes from, and we'll get back to that. So then the question is, well, we've shown that a mouse can get autism, but do humans get autism due to mitochondrial defects? So what we did is we looked at these mitochondrial haplogroups and we asked, in a population of autistic families and children, do people with certain mitochondrial haplogroups have an increased risk of autism? And the answer is that a number of these low incident frequency European lineages have odds ratios of two, which is higher than any nuclear gene ever found.

And if, if you look at gender, autism is much more common in males and females, about four-fold more common in males. And you can see that these mitochondrial lineages are half as important as gender. And if you look, add up all this, this is 55 percent of the mitochondrial disease in Europeans. Therefore, Mighty counter then has a huge risk factor for autism. But these people don't necessarily have autism. So why not? Because they have to have a second hit. And the second hit is due to somatic mutation.

So this is the work of Zen Longu and former postdoc of ours, Mountain Barton Picard and Ichin Wang. And they then look for somatic mutations in siblings of families where one child had autism and the other did not. And in every case, the child with autism also had higher somatic mutations. So they inherited the risk factor with their haplogroup and then they acquired somatic mutations to cross over to the expression threshold.

Okay, so why would then there be this interaction between the mitochondria and the nucleus? So what we did is we looked at this family. The, uh, in the early 1980s, this individual had lactic acidosis, growth retardation. These are all of her children with lactic acidosis, growth retardation, progressive dementia, stroke-like episodes, hypertrophic cardiomyopathy, cardiac conduction defects, the oxidative fibers degenerating, the glycolytic fibers being fine. And all these young men and women died in their late teens, early 20s of heart failure and neurological disease. So these people had a mutation in the tyrosinase gene at 3243 at about 70 percent mutant. So they have what's called the MELAS syndrome, which is lethal. But if they had only 10 to 30 percent mutant, they would have either type 1 or type 2 diabetes or autism. And if they had 100 percent mutant, they would die of Leigh syndrome. So why would the same mutation if totally different clinical phenotypes? So that, that was a puzzle.

One of the, the chemical phenotypes, as I said, at a low percentage heteroplasmy is diabetes. So this is a pedigree we studied in Asia where we have the maternal transmission of diabetes. But this was a surprise because we knew there was a 3243 mutation. But when we quantified the number of mutation, Almighty Cardiology instead of being 20 to 30 percent, it was 88. So how come these people didn't have Leigh syndrome?

So we then did a haplogroup study of Asians for the risk of diabetes, hypertension, and obesity. And these are European lineages that we've given names to. And what it turned out is this lineage N9a has an odds ratio of less than one. That is, its protective. And when we looked at the background of this particular family, they were all N9a3. So even though they had the high mutation level, they had a protective background. And that, that compromised the severe effect and gave them diabetes. So we have an interaction then of nuclear, of mitochondrial new mutations and background defects.

So then we asked the question, well, how could you have different phenotypes when you have the same genotype? So what we did is we made cell lines with different percentages of the mutant mitochondrial DNA but the same nucleus, so-called cybrids. Three, two, four, three mutant. Then we did RNA-seq on those individuals and we did principal component analysis of the RNAs. And this was the transcriptional profile where you had normal mitochondrial DNAs. This is the profile when you have 20 to 30 percent mutant mitochondrial DNAs. These are the proton profiles when you have 50 to 90 mutant mitochondrial DNAs. This is 100 percent mitochondrial disease. And this is a cell with no mitochondrial DNA at all. So what was amazing is that the percentage of mutant created transcriptional profiles that exactly correspond to the stepwise differences in phenotype. So it implied then that the quantitative mitochondrial genotype was having a quantized nuclear effect that gave rise to the phenotype.

So how could that be? Well, it turns out it changes different transcription factors that then create changes in the nuclear gene expression. So why would that be? Well, if you have a mutation in the mitochondrial DNA, it's going to affect the respiratory chain. And that's going to affect the oxidation of NAD, NADH to NAD. And that then is going to affect the flux of pyruvate through acetyl-CoA and oxaloacetate to give you citrate. And citrate then drives, as you all memorize, the TCA cycle. Now, if you have very reduced NADH, then these pathways require oxidative reduction of NAD to NADH. But if you already have reduced NADH, you're going to stall the electron transport chain.

So what's going to happen? Citrate is going to go out through the citrate carrier. And then ATP citrate lyase is going to cleave it into oxaloacetate and acetyl-CoA. And acetyl-CoA that is going to be the acetylation of all the acetylation groups of the histone acetyltransferases. And mitochondria makes all the single carbon molecules go through S-adenosylmethionine. And that does all the methylation of the histones and the DNA. And then alpha-ketoglutarate is the critical component for all the dioxygenases for, what, alpha for the histones and for the DNA. That's the Jumonji and the TET methyltransferases. And so then finally, succinate as the actual inhibitor of these systems. So the point of this was all of the regulation of the epigenome was due to substrates that came directly from the mitochondria, which were regulated by the mitochondrial DNA genotype. And so you can see as a percentage of mitochondrial DNAs increases, alpha, in this case, alpha-ketoglutarate starts low, goes high, and then down. And the histone methylation starts high, goes down, and goes up, the opposite effect.

So after all of this work, if we correlated all the substrates with all the histone modifications against all the mitochondria genotypes, we come up with 150 discrete histone modifications that regulate the interaction between the mitochondria and the nucleus. So basically then, the mitochondria is the integrator of nuclear and mitochondria gene expression and the environment. And if you perturb the system, you send out these small molecules that then regulate the epigenome and regulate nuclear gene expression to hopefully maintain homeostasis and normal health. But if they cannot, you get disease.

Okay, so what then is an example? Isn't this fun? Okay, wake up everybody. I'm still here. I think. Okay. All right. So last example, what is the role of the environment? How are we going to study that? We have to have an environmental challenge. So we chose something specific that is SARS-CoV-2 because a number of us were a bit concerned about it. They threw us out of our labs, that's why. Anyway. So what is SARS-CoV-2? It is a single-stranded RNA virus of 30 kilodaltons. It codes for polyproteins called ORF3, orphan ORF2, and then a series of open reading frames, including some structural proteins, the, uh, envelope protein, the membrane protein, the core protein, and the spike protein. So if you take a cultured cell and you infect it with SARS-CoV-2, it takes the normal looking mitochondria with their imaginative cristae and converts it into a mitochondria that's epignotic with the cristae open, very definitely sick. So what's going on?

So what happened is Gordon, a very long list of authors, took each polypeptide from SARS-CoV-2, put it into a cell, and then asked what cellular proteins interacted with that polypeptide. And so what they found is over 360 interactions. And then our group methodically went through every one of those and asked what protein, what protein was interacted. We found that between 16 and 20 percent of all the interactions were with proteins that regulated the mitochondria. So as you can see here, we had proteins, viral proteins that regulate mitochondrial DNA replication, transcription, protein synthesis, modification of the RNAs, modification, uh, regulation of respiratory complexes, regulation of glutathione peroxidase, also proteins that interacted with all the nuclear regulatory systems that made the proteins for the mitochondria. So the virus was systematically turning off proteins that were involved in mitochondrial function. Why would you do that? Because if you inhibit the electron, if you inhibit the electron transport chain, what you're going to do is build electrons up in complex one. And that's going to make more hydrogen peroxide. Why is hydrogen peroxide important? Because it diffuses out of the mitochondria and then it interacts with the protein HIF-1 alpha. And HIF-1 alpha coordinately upregulates glycolysis. So why would the virus want to do that? Because you don't want to burn all your carbon to make energy in the mitochondria when what you really want is carbon to make virions. So you block oxidative phosphorylation and allow glycolysis to make lots of substrates for viral replication. And it does that by mitochondrial ROS production through HIF-1 alpha.

Okay, all right. So what would make sense? Sorry, I forgot. What would make sense? Well, the virus is initially going to make proteins that inhibit mitochondrial function. But the first thing the cell is going to do is make more mitochondria. So how does it sustain? How does a virus sustain the mitochondrial blockade? The way it does that by blocking transcription. And that's the object of what we want to talk about.

Okay, so why would we want to talk about that? The respiratory complexes are complex. Complex one is made of 54 polypeptides, seven from the mitochondria, rest from the nucleus. How is that done? So it turns out that the respiratory complex one is assembled from five different modules. Each module has structural subunits and assembly subunits. So you assemble each module and then you glue all the modules together to make the final complex. Likewise, the same is true for complex two, where you have two different modules. Complex four, where you have three separate modules. Complex five, when you have multiple modules. And then complex three is interesting. It's linear, but this is a module, this is a module, and this is a module. So it builds things in stepwise groups. So it needs both structural and assembly subunits. So if you were a virus and you wanted to block transcription of oxidative phosphorylation, you wouldn't block all 150 genes. That would be very, very tedious. But all you have to do is kill one module. So you would inhibit one module, can't assemble the complex, the respiratory chain is dead. So that's exactly what SARS-CoV-2 does.

Okay, so here are 700 nasopharyngeal swabs for which we sequence the RNA. So we have the total viral load and all the messenger RNAs that are made, right? So was that me falling apart again? No. So here we are, a nasopharyngeal, and this is the viral load. And it turned out that there were three different levels, but they were all very high. These are probably people at different stages of the initial infection. So the point is, lots of virus. Okay.

So now if you look at all the transcripts, and these are all the genes that are necessary to make each oxidative phosphorylation complex, complex one, two, three, four, and five. And if you then look at the expression aggregate of all these, you can see that at the high viral load, you've downregulated the genes for oxidative phosphorylation complex one, three, two, three, four, and five. And even in my middle viral load. And by the time you're at low viral load, you've only had some effects. And if you don't have any virus, you don't have an effect on oxidative phosphorylation. So now if you look down here, all the gray are downregulated, occasional red are upregulated. Now that was very odd. Why would you have some downregulated, some upregulated? Remember, the virus only has to hit one module. So I've now diagrammed these by the different modules. And what you can see is you look at this module, that all of these proteins, which are structural proteins, are downregulated, but the assembly proteins are upregulated. And you see that over and over again. So what's happening? The virus is blocking the trap, the structural proteins. The cell is saying, gosh, I don't like that. It upregulates assembly proteins to compensate. So you see that over and over again.

So here is a group of complex four genes. This is the structural subunits, this is the assembled subunits. And this one's particularly. SARS-CoV-2 puts the copper in the copper system necessary for cytochrome oxidase to work. Okay. And it's hugely upregulated to compensate for the downregulation. So you see this over and over again. Okay.

So then what you can say, you can look at each of these modules and ask, uh, some of the genes are structural, downregulated, but the associate genes are upregulated. So these are, uh, this one is another case where you have primarily the assembly genes are upregulated. Here you have a situation again, structural genes downregulated, assembly genes upregulated or not. So we have a system then of balance. The virus is trying to block the system. The host is compensating by upregulation. But as long as those viral genes are downregulated, it doesn't matter what the host does. And that's the viral strategy.

Okay, so now, uh, what happens if you go out and die? So now what we found is, amazingly enough, when you die, you have no longer any virus, but you're still dead. So why, why are you dead? A virus is gone. It turns out that the, once you have the virus, it can inhibit mitochondrial gene expression on certain organs. And this is the heart. And every nuclear-encoded gene of the heart is irreparably shut down. So now you're going to die because your heart's going to fail, even though you no longer have any virus. Okay. But at the same time, your mitochondrial DNA genes are saying, wait a minute, I don't like that. And they're strongly upregulated. Okay. So you have this balance, imbalance of the system. And so what you see is in the nasopharyngeal, everything's down. By the time you get to the lung, everything is up. But in the heart, the liver, and the kidney, things stay down and kill you. Okay. So again, now you're still again seeing this differential. Each of the organs where you have the structural genes and the assembly genes are going in the opposite direction because the host is trying to fight the virus.

So another aspect of this is the virus is then going to initiate inflammation. Well, one system is through the mitochondria. And we'll get into that. And that's through the MAVS pathway, mitochondrial-associated viral receptor. But I want to emphasize right now this system called the integrated stress response. Here we have a viral RNA that interacts with a kinase called PKR. And that activates the kinase to phosphorylate itself. And then it phosphorylates eIF2 alpha. And one of your colleagues is working on this with Dr. O'Perron. And once you phosphorylate eIF2 alpha, you then block this initiation factor. And it blocks cytosolic protein synthesis. That's the host's attempt to block viral translation. But what it does is it then activates the expression of these transcription factors ATF4 and ATF5. And they then affect mitochondrial supercomplex formation. And they turn on single carbon metabolism. And these cytokines FGF21 and GDF15, which are signaling systems. So we're going to come back to that. So we're going to look a lot at this system and this system. This is another antiviral system, but we won't talk about that right now. Okay.

Okay, so now what we're going to look at is what happens to these systems, the integrated stress response, HIF-1 alpha, and mTOR. So let's start with HIF-1 alpha. Well, what we see is that HIF-1 alpha is upregulated. Well, why is that good? What's that going to do? That's going to upregulate glycolysis because HIF-1 alpha drives glycolytic genes, pseudohypoxic state. And these are all the target genes that we know about. HIF-1 alpha, they're upregulated in every organ. So again, as I said, mitochondrial ROS production is up. The ROS activates HIF-1 alpha. HIF-1 alpha drives glycolysis. And that's good for the virus. So the virus is then making lots of substrate.

Where does the substrate come from? It comes from mTOR. So now you see that the virus is still trying to operate mTOR, but less effectively because the host is fighting that. But what about the integrated stress response? That's how the cell is going to fight the virus. And now you can see that all the genes of the integrated stress response are upregulated.

What about the mitochondrial DNA? Well, here we had a surprise. If we look at the people that are in autopsy, as I said, all their mitochondrial genes are upregulated because the nuclear genes are downregulated. But the people in the initial phase of viral infection had a very different pattern. They had many of these genes are downregulated, but these two genes, ND1 and ND6, were upregulated. Huh? How did that work? Because you don't see that in the autopsy tissue. Well, it turns out that the virus activates this production of a microRNA, 2392. And 2392 goes into the mitochondria and binds to the glutamate gene in the mitochondrial DNA. And mitochondrial DNA transcription is symmetrical around the two strands. So there's one promoter that goes around what we call the heavy strand and another promoter that goes around the light strand. So transcription starts through the ribosomal RNAs at ND1 and then hits the block in the tRNA. And therefore, this gene will be upregulated. But all of these other genes will be not transcribed. And that's what we see. ND1 up, all the remaining genes are down, except ND6. Why is ND6 up? Because it's on the other strand. And therefore, it's a different promoter. So the virus is actually initially killing mitochondrial DNA through this microRNA. But after the virus is gone, the microRNA is gone. And now all genes of the mitochondrial DNA are upregulated. But it's too late.

So to prove this, isn't this fun? To prove this, we have to then look at a few animal models. So here's the hamster model. And what's interesting is, would we acutely affect the hamster, we get a lot of virus in the lung because this is a lung infection. But almost no virus at any of the other organs. So now we can look at the expression of the OxPhos genes. Well, we get a surprise. This early infection is affecting most of the organs. But amazingly enough, in the cerebellum of the brain and the striatum, we see mitochondrial OxPhos is downregulated. And in the striatum, it's upregulated. But there's no virus there. How could that be? When we first saw this, we think this must be a mistake. But when you look at all the different genes, you see that the striatum is all the OxPhos genes are highly upregulated. Good. And the cerebellum, they're downregulated. But in all of the viscera, including the lung, it's still at an early stage, at their variable. And you can see this very nicely if you look at the mitochondrial ribosomal proteins, they're up in the striatum and down in the cerebellum. So what's going on? Because of the integrated stress response, cytokines are being made that are diffusible. One of which is GDF-15. And we think that that then is acting on the brain and altering mitochondrial expression. But why would that be important? Because this would cause your brain to malfunction. And we think this is the basis of what's called brain fog that you see in long COVID.

We can also look at a mouse model. So we have ND6 of mice and BALB/c mice infected with a mouse-adapted virus. And now what you see, these are the time frames of the infection. So we're already seeing that the virus is declining. Whereas it's declining slower in the BALB/c than slower in the Black6, then in the BALB/c because they're both at day four. So now you see an interesting, interesting thing. And, uh, this particular virus, where it's now, these are, by the way, lung. It's going toward the autopsy lung. And remember the autopsy lung increased mitochondrial transcription. You see the later onset virus, farther odd infection has now increased mitochondrial transcription in the lung. Whereas the earlier virus load in the Black6 is still in that intermediate stage. So this validates this idea that over time, you change the mitochondrial transcription of the interaction between the virus and the host.

So last point. So what's going on? The virus is affecting the assembly of mitochondrial DNA and nuclear DNA genes in these modules. And that creates what's called an unfolded protein effect. So here we're looking at complex four and five. And the host has tried to upregulate complex four with SCO2. But the virus has killed the structural subunits. So what's that going to do? That's going to make more ROS. Well, the ROS is then going to go out of the cell. And there it's going to activate HIF-1 alpha to drive glycolysis to make more virus. So we know about that. But the unfolded protein response is going to give you an imbalance to drive the integrated stress response. The unfolded protein response, a protease cleaves a target molecule, DDEL1, that goes out to the cytoplasm, interacts with this protein, which is another kinase in the integrated stress response. And it activates the phosphorylation of eIF2 alpha. And that then inhibits protein synthesis. But at the same time, activates the ATF4 and ATF5 and generates GDF15, which then creates the brain fog. Then finally, the oxidative stress of the ROS oxidizes the mitochondrial DNA. The mitochondrial DNA goes out, binds to the inflammasome and to cGAS. And that then generates IL-1 beta from the inflammasome and the interferons from the cGAS-STING pathway that generates the inflammation. But it also counteracts the synthesis of the virus by the innate immune system. And this system is driven by a rate-limiting step of the mitochondrial DNA replication called CMPT2. And we see that highly upregulated in all the tissues that are virally infected.

So the last point to be made then is what we have here is the virus initially uses its polypeptides to block mitochondrial function. That increases ROS production. That drives HIF-1 alpha. That makes more glycolysis to feed viral replication. The host says, bad news, I'm going to upregulate nuclear transcription to make more OxPhos. But at the same time, the virus has selected specific structural and assembly protein operons, if we can use that term, to block. And by knocking out just a few of the assembly complexes, kills the transcriptional response, keeping that respiratory down, increasing glycolysis, driving replication. The integrated stress response then tries to deal with that by shutting down translation. But it's too late because the virus is already replicated by that time. Even though there's no more virus, mitochondrial transcription of the nuclear genes has been shut down permanently. And we go on and die.

So we can see that here. So here, initially, the virus infects. Oops, sorry. The virus infects. The first thing it does is elaborates the microRNA. The microRNA goes in and blocks mitochondrial transcription. And that then increases ROS. And that activates the cGAS-STING pathway, the integrated stress response, and the inflammasome. But most of all, it activates HIF-1 alpha and makes more glycolysis to make virus. But then the host tries to make more OxPhos enzymes. But the virus then blocks transcription. By blocking transcription, it makes this defect even more concerted. Increases the integrated stress response, the inflammatory gas sting, and more viral proteins. But then ultimately, the virus is cleared because it's now already affected someone else. But now we're left with a situation where the lung is trying to compensate by making more mitochondria. But the viscera is irreversibly turned off. And that then results in what we believe is death and long COVID. So we now believe we have an integrated view based on mitochondrial function of an environmental challenge that is SARS-CoV-2.

So I'd just like to end by all the fantastic people that have done this work. So in our SARS-CoV-2 work, we have Joe and Gently, Tim, Alicia, Debbie. And then the population genetic studies work were done by Larry and Deanne. This is a subset of all the huge number of people that were required to get all of those samples, 700 nasopharyngeal samples, 40 autopsies, led by our colleague Afshin. Much of the molecular biology was done by Chris. And then these were individuals that provided the animal work and the population genetics. But then our studies, this were, this is the group that did the nuclear work on the histones, that that is Piotric, Kevin, and Ben. These are the folks that did the great work on autism, the mouse, Tau, Anna, Eric, Katie, Jess. And this is my long-term colleague here, sitting in the front, stand up, I said. So this is your contact with Hiatarad. And then Kirsten, Ryan, Martin, Megan, and now our newest addition, Arnold. So those are the people that make this happen. They don't let me in the lab because I break things. And so I sit there and try and generate money so they can break things. So anyway, I want to thank you all for that. And the newest grant that we have together with the University of Hyderabad is our Gates Foundation Grant to study the role of SARS-CoV-2 on mitochondrial haplogroup predisposition to the disease. So next year, we hope to tell you that part of the story. So thank you very much. Thank you. Can you ask some questions, audience please? Yeah, thank you, sir. You have referred actually the microRNA 2392. Can you really target that with antisense and contain the COVID-19 infection? That's an outstanding question. We have antagomirs that we've made to the 2392, and we have them in the lab, but we haven't yet taken SARS-CoV-2 infected cells and treated them with the antagomir. But that's, that's definitely in the works. So right now, I'm afraid I cannot answer your question. This gentleman, though, has been working on the integrated stress response system that I just talked about, and a very, very beautifully worked out that together with Naresh. I understand microRNA 200. Yes. What is its function like? So that's another one of great interest. It's, uh, it's also been of interest in the rodent model systems. We have not had the, uh, the person El Perro to work on that, but that's, that's also known. We're working a lot with Afshin at the far left here on several RNAs, 200, 451, 181, and 141 as possible ways to manipulate these systems. So I think that there's a lot more that can be done with non-coding RNAs to try and limit SARS-CoV-2 infection. But I don't have data for you. Yeah, yeah. Another thing, how specific is a microRNA 2392 on mitochondrial transcription? What about the nuclear transcription? Anything can get inhibited? Thank you very much. I, I glossed over that because I knew I was going rather long-winded. We also have looked at the seed state of 2392 across all known mitochondrial, nuclear, and cytoplasmic encoded genes, a thousand of them. We found 362 mitochondrial nuclear encoded mitochondrial protein genes that also have seed sites for 2392. Now, of course, just because you have a seed site, you don't know if it regulates translation. But it makes sense that the virus is using 2392 to block mitochondrial DNA transcription, but also nuclear encoded mitochondrial translation. And that involves OxPhos genes, ribosomal proteins, sirtuins, assembly proteins, all across the board. So the virus is using 2392 also to downregulate the translation of a large number of critical mitochondrial genes. So again, 2392 is not just about the mitochondrial DNA, it's, it's another approach by the virus to keep translation down while it inhibits transcription. So thank you very much. That's a very important point. Yes. Hi Doug. So, um, for the first part of the talk, so do we understand whether because haplotypes create haplogroups, create mitochondrial dysfunctions which can also again, in a complicated network, probably also impact polygamma activity? So do we understand about the somatic mutation rates, and whether that is related to haplogroups, and how does it affect different tissues? So what are the mutation frequencies, and how much of that is related to the replication frequencies of mtDNA across tissues? Do we know that? Yes. So, um, there's a very excellent question from your affiliated Tata Institute and one of its senior investigators. So the idea is, um, where did we get this haplogroup variation? Why is it regionally specific? And why is it so common? So it turns out that the mitochondrial DNA mutation rate is extremely high, many, many fold higher than the nuclear DNA. So you would think since the genes are absolutely critical for life, and those are the students that had listened to me earlier today, I apologize for going over this, you would think that that high mutation rate would kill all the respiratory chain and we'd all be dead. So in fact, that isn't what happened. And what happens is that the oocyte, which has a million mitochondrial DNAs, can have mutant and normal mitochondria specs in. But at fertilization, it cleaves into cells. And the inner cell mass cell only has, well, there's anywhere from an estimate of four to 100 mitochondrial DNAs. So what you've really done is you've gone in and you've just did a random sample of the number of black and white balls to put in each of the different cells. And then they go on to make the protein cells that go on to make oocytes. So what you're doing then is you're segregating out your heteroplasmy into isolated forms of mutant, normal. Now it turns out that once you have a mutant mitochondrial DNA, you increase mitochondrial ROS production, increase rate of continental ROS production, as far as we can tell, we're still working on this, increases apoptosis. And that in fact kills off the primordial germ cells that had the badass mitochondria. So now you have an intra-ovarian selection for the bad mitochondria. Now you still have the mild mutations that allow you to have diversity. So this is a way the system creates a lot of environmental adaptation diversity without a lot of genetic load. And that's really neat because that problem is a big problem in the nucleus because you always, you segregate nuclear genes independent of selection. But with the energetics, you can have energetic selection at the single cell level, which you can't have anatomical selection until you get to the whole organism. So the mitochondria then becomes a powerful way of generating a lot of environmental adaptive diversity without a lot of genetic load. And that, we believe, is the pre-evolutionary adaptive stage that then goes on to Darwinian selection of nuclear genes. And we think that that then, the mitochondria is the basis for all diversity of populations. So the point is then that why is some mitochondrial DNA is good in some areas and other, as I said, we think that because mitochondria subtly generates energetics, but the mitochondria now, and it regulates energetics, regulates ROS, regulates the epigenome, regulates the integrated stress response, it regulates the innate immunity. And suddenly now the mitochondria is regulating all of the aspects of human biology that are important to adapt to adaptation to cold, diet, to infectious agents, to all different aspects. So then each subtle change in the mitochondrial DNA gets tested in different parts of the world. And they then become our good. Those that variant that is good in that environment, subtly changing energetics, then is selected for. And it then gives rise to a whole bunch of other mitochondrial DNAs with that original adaptive mutation. And they then form a group of related haplotypes, which we call a haplogroup. So that's why different haplogroups are specific around the world. But now we have a problem of jet airplane lines and boats. And so then the people from one place decide to go to another place where that variant is not adaptive, but now is maladaptive. Now we think that that's then predisposes them to autism or diabetes or obesity or neurodegenerative disease because they're out of environmental context. So that's sort of the working hypothesis for how haplogroups might account for all these carbon diseases. Okay, so the second, yeah, fantastic. I love that answer. Anyway, I don't know about the question. The second part of the talk, Doug, um, so did you segregate these patients into diabetic and non-diabetic? How much of the baseline mitochondrial dysfunctions, uh, did you think was responsible for SARS-CoV-2 dependent remodeling of mitochondria? Excellent question. So he's now referring to the fact that it's well established that certain patients, of certain people with mildly challenged physiology, such as people that are overweight, or people that have diabetes, or old people like me, or males, which are course defective. All of these individuals, why are they more sensitive to dying and severe disease than say, the lovely women who seem to be resistant to everything? So it turns out that all of these changes subtly decrease mitochondrial function. And the idea then is that then when the virus challenges for mitochondrial dysfunction, if you already have a very robust system, as let's say you're a very healthy woman, then you can resist all that I've talked about and come through with still residual mitochondrial function to then have your organs repaired. But if you're a poor, sickly male, then you shut down everything and you have really hard time reactivating transcription and then going back into normal organ function. And then people that have diabetes already start out with a partial mitochondrial defect. So they then drop below this system and that shuts down their transcription. So that's how this model seems to fit in with these environmental effects. Now we're very interested in the Gates Grant to see if different mitochondrial haplogroups from different parts of the world, which you know, we know have subtle differences in energetics, if that might account for why some men and women are more and less sensitive to the virus, even though they seem perfectly healthy. So you have this situation where you can have a woman who seems fine, but then gets the virus and she will go ahead and die. Where another woman that's equally healthy, even maybe even the same community, but she just gets a mild infection. So why is that happening? So our working hypothesis is that they have this underlying mitochondrial variation that the virus is exploiting. We don't know that that's true. That's just the working hypothesis. Okay, thanks. Thanks for that. I'll pay you later. So I have, you talked about the nuclear. Is it possible that there could be a non-genetic way to modulate mitochondrial function? And that could be, uh, through this RNA-based mechanism? If you think that there's a way, I mean, first of all, how does the microRNA get in? I guess that's an open question. And also then, if RNA, which may be transcribed at the nuclear level, microorganisms could be a way to communicate to the mitochondria, modulate mitochondrial function? So then could you not potentially have, if there's an RNA-based transport, could you not potentially have a non-genetic way of modifying, of modulating mitochondrial activity? Absolutely. And, um, so we focused on 2392 because when you get your SARS-CoV-2 infection, that microRNA is very low normally in a normal person, goes to very high levels. So obviously the virus has a reason for increasing 2392. But this group of people in the second tier, we have a number of different, so that's called the COVID International Consortium. So we have members of COVID from India, from Africa, from all over the world trying to figure out this problem, put together as many different perspectives as possible. And one of our working groups is non-coding RNAs. So we actually have already have a paper in press that shows there are also non-coding RNAs that seem to be upregulated in viral infection. And they could very well be involved in either regulating nuclear or mitochondrial function or mitochondrial DNA function. And one of your colleagues is looking at non-coding RNAs, for instance, in making say, platelets out of megakaryocytes. So I think that there's a lot of other interesting ways that this could go, could be developed. And one of the projects that we're very much involved in in COVID is we've now taken all of this transcriptional data and now we've looked at expression of all of the immune and inflammatory genes. And we're now parsing out what are the inflammatory and immunological genes that are upregulated. The ROS pathway is one of them. The mitochondrial regulation of the innate immune is another one. We're seeing certain effects on T regulatory cells. So again, it turns out that many of these things can come back to mitochondrial ROS and to the integrated stress response. So I think that we're beginning to get a coherent notion of how we can then intervene with through bioenergetics. So thank you very much. So my colleague Prasad is our experimenter who's been working for how many working, almost eight years, right? And he is 10 years on trying to figure out how RNA gets in the mitochondria. And we think, we think, we think we have an experimental system that may be doing that, but we don't know that yet. But that's very, very hard. It's easy for the cell, but it's hard for us. So we would love to be able to put RNA in the mitochondria. If we could do a CRISPR system for the mitochondrial DNA, for instance, we could complement things, we could do all kinds of stuff. But trying to experimentally in the lab consistently put micro, put RNA into the mitochondria turns out to be very hard. Apologies, Professor Doug has a meeting, you know, followed by this meeting. So we request you for help in the break time for catch one or questions. Students were interested, please bear with us because he is very scheduled, is very tight. So we are wrapping up the question analysis. Yeah, please bear. I'd much rather have questions in the meeting, but okay. Thank you. Thank you. Thank you, audience. Oh, something else is happening. Yeah, thank you so much for all the hard work in providing us the information from autism to aging and all sorts of aspects. I'm graduating. [Music] [Applause] Thank you, everybody. You've been wonderful and very patient. Okay, are we done? Oh, we're not done. Okay, another plaque. Oh, sorry. Put this one down. Thank you. Thank you. Thank you. You can go now. Bye. Much gratitude, sir. I would like to request Professor Sharmaji to come and good evening, all. It's my pleasure to extend a word of thanks on behalf of the Department of Biochemistry at University of Hyderabad to all. It has been such an honor to be a part of this wonderful event. I would like to begin with extending my heartfelt gratitude to our esteemed guest, Professor Douglas C. Wallace, for accepting the invitation and spending time with students and faculty, discussing science, sharing views, ideas, giving new perspectives to mitochondrial biology. I also thank him for a very informative talk. Thoroughly enjoyed connecting mitochondrial perturbations to perturbations in the way the nucleus works, and to almost it boils down that many of our issues reflect in overall perturbations in fundamental metabolisms. Thank you so much, sir. Really enjoyed and a lot of connections between inflammation and processes, uh, physiological processes which decides the fate of a pathological condition. Thank you so much. Thank you, Professor Wallace, for sharing this exciting ongoing research question. Your lab surely is infectious, and I'm sure there would have been more interaction if the time had not been limited. Thanks again for stimulating all of us. We thank our Vice Chancellor, Professor BJ Rao, and Dean, School of Life Sciences, for the support and encouragement for organizing this event, and also engaging with scientific discussion outside, uh, the talk. We thank all the students, faculty, and colleagues from the university and other institutes for joining the event. And a special thank to Professor Naresh, who brought us all together here by organizing this event. We thank our different offices, our PRO office, which has also helped us in wide publicity of this distinguished lecture. And finally, it's my honor to acknowledge the contribution of all offices, administrative staff, and all those who worked hard for successful organization and seamless conduct of this event. Thanks again. [Applause] Thank you so much, Professor, uh, Wallace, and we wish, we wish to see you continue to be a trailblazer in the field. And as we said, we would like to have you again and again on the campus. Thank you very much. Much gratitude.