Craig Thompson: A New Kind of Mitochondria

Transcript

Speaker 1 (00:12):

I am Daniel Levine, and this is MIT Cast, A MIT World podcast. Mitochondria have long been thought of as being uniform organelles throughout the cells in our body. In November, though a study in the journal Nature changed our fundamental understanding of mitochondria. Researchers reported that in response to stress, subpopulations of mitochondria are created with unique functions. Today we’re joined by Craig Thompson, a member of the Cancer Biology and Genetics Program at the Sloan Kettering Institute at Memorial Sloan Kettering Cancer Center and senior author of the Nature Paper to discuss those findings and their implications. Craig, thanks for joining us.

Speaker 2 (01:05):

Daniel, it’s a real pleasure to be here and I look forward to our interview.

Speaker 1 (01:09):

Well, we’re going to talk about a new paper you co-authored in Nature that fundamentally expands our understanding of mitochondria, the significance of your findings and how we might be able to bring greater coherence to mitochondria as a distinct field of study. Let’s start with mitochondria themselves, though most people think of them as having a role in regards to producing energy to power cellular functions, although they do a lot more. What role do mitochondria play in cell growth and proliferation?

Speaker 2 (01:44):

Well, that’s a great question, Daniel. I think we all encountered mitochondria sometime when we took our first biology class and we were told they were the powerhouses of the cell. And that’s because ATP provides so much of the energy for cells to maintain their order and function. And mitochondria were discovered a hundred years ago as being the major source of ATP in most of our tissues as we go about our adult life. But those scientists who worked on this tremendous story of how mitochondria took the food that we take in and burn to CO2 and water inside the mitochondria through the TCA cycle or tricarboxylic acid cycle that generates CO2 and water as a byproduct, but it also captures electrons that can be donated to the mitochondrial membranes that transports those electrons just like an electrical wire.

(02:42):

The electrical system is used to first pump hydrogen outside the mitochondria and then at the end of this process assimilate that potential in the formation of ATP by a complex that looks just like a propeller, believe it or not, that drives the formation, from ADP and inorganic phosphate, of ATP that provides us the energy, the source of energy for all things we do. That was such an exciting discovery and led to the Nobel Prize in 1978 and was considered the last great achievement of biochemistry. But those scientists were actually after the discovery of how we got food every day and turned it into ATP effectively so that all the tissues of our body can make enough energy for each of the organs to do their job, for muscles to contract, for our brain to think, for our liver to digest things, for the heart to beat.

(03:38):

They weren’t really concerned with the two problems that our laboratory works on. Our laboratory works on the normal process, wound repair. When you cut yourself or damage yourself, you’ve got to build new cells, you’ve got to grow back the skin that’s broken. You’ve got to grow back the blood vessels that are damaged. You’ve got to grow back the underlying connective tissue. That requires not just ATP but building blocks for the ability to build new proteins, new nucleic acids, new lipids. Those are the three major constituents of cells. And through their buildup allows cells to make more copies of themselves by dividing, and that’s anabolic, as we say, it builds mass. So you can’t afford to take all the food that those cells are getting and burn it to CO2 and water and extract the electrons. You have to put them together in the various building blocks that make life and cells possible.

(04:33):

And so we asked the question, why does a cell burn everything to CO2 and water? If it’s under stress, how does it reserve some of the building blocks to actually synthesize things? We discovered the most important amino acid for building structural integrity during wound repair, the amino acid proline, which is special for building the connective tissue that provides bone or cartilage, tendons, as well as what’s called the extracellular matrix. We discovered that proline was uniquely dependent on the mitochondria for synthesis.

(05:17):

We next asked how does the mitochondria balance the essential role it plays in proline synthesis versus its essential role in making enough ATP so cells could just live to fight another day. And we started just examining these two processes during times when cells are exposed to lower nutrients than they would normally get if the blood vessels are intact. That’s simple to model in tissue culture. You can take away the nutrients as if the blood supply has been disrupted or as if a cancer cells growth outstrip the vasculature. For example, we can mimic the levels of nutrients that are present in a tumor mass that we and others had measured over time. And we discovered much to our surprise, even though these processes should be competitive, most cells, most healthy cells doing wound repair and most cancer cells that are growing are able to carry on both simultaneously. And that breaks the laws of thermodynamics. You simply can’t burn more to CO2 and water and use more to build and not get into conflict if those processes occur in the same place. So we wondered how does that actually happen? And that led to the study we’re talking about.

Speaker 1 (06:43):

In the paper, what you report is that in states of stress when more energy is needed, mitochondria create distinct subpopulations. Can you explain the differences between these subpopulations?

Speaker 2 (06:59):

Sure. As I said, we could mimic various conditions to put cells under stress. One is to put them under stress by making cells more dependent on ATP by increasing their need for ATP. The other way was to increase their need for having proline. We simply asked how do they sustain their ability to make proline when they’re absolutely dependent on the mitochondria for oxidative phosphorylation. We discovered under both conditions, if we put them on increased ATP demand or increased proline demand, the cells divided into two distinct populations, one which became a more classic mitochondria.

(08:03):

The inner membrane is invaginated and has a highly ordered electron transport structure that allows the subpopulation to efficiently make ATP. A smaller population of them started to look different. They still had double membranes, but instead of having the inner membrane complexes make ATP, they had a series of filaments in the middle and those filaments stacked on each other and this subpopulation became efficient machines to make two essential building blocks, ornithine and proline. You could tell them apart just by their morphology. People were surprised that we could actually take pictures of them and show them as entirely distinct. And they’re dynamic. They move independently and under a wide variety of stresses, both anabolic and catabolic needs that the mitochondria supports for a cell could be satisfied by putting each job with a specific distinct subset.

Speaker 1 (09:17):

Is this link to P5CS?

Speaker 2 (09:21):

Yes. So the reason we focused on P5CS is that it is an enzyme that turns glutamate, which normally supports the TCA cycle, into a precursor critical to maintaining the TCA cycle. Completely by serendipity, we had discovered that P5CS was the limiting enzyme in converting glutamate to proline.

(10:20):

One of the problems associated with cells producing ATP by oxidative phosphorylation is that it causes stress on the cell. Excess electron transport produces hydrogen peroxide and superoxide radicals, and they have to be handled with repair enzymes that are dependent on NADPH. And we knocked out the enzyme required to make mitochondrial NADPH called NADK2. It was discovered because it was a homolog of a plant enzyme. Mitochondria without NADK2 were no longer able to make NADPH, but much to our surprise, they didn’t get into stress, redox stress. Instead, they lost their ability to make proline. That said that most of the energy that’s devoted to the synthesis comes from NADPH and most of the degradation energy goes to another molecule called NADH. And that fuel drives one or the other of these processes. And that’s why we started concentrating on P5CS. Some computational methods also suggested P5CS was the linchpin between these two processes, ATP production and precursor synthesis. The full name of P5CS is pyrroline-5-carboxylate synthase and nobody can remember that name. So, they just call it P5CS. But it’s a critical enzyme to decide what mitochondria do with glutamate, which is the breakdown product of the most common amino acid in our blood: glutamine. You use it to maintain the TCA cycle or to build proline and ornithine.

Speaker 1 (11:46):

So how does the P5CS enzyme determine, or what role does it have in the creation of these subpopulations of mitochondria and balance the need for energy production with the need for creating cellular building blocks?

Speaker 2 (12:03):

It was pretty clear if mitochondria were going to do more with a limited amount of substrate available, they had to get more efficient at what they were doing. One thing that had been reported in plants and in insects was that having enough proline was essential. Plants need proline because it provides structural integrity and rigidity to proteins. A plant obviously needs to stand up to maintain its integrity. So there’s a lot of proline in the structural proteins the plants need to maintain their tissue integrity. And it was known that when they are stressed by lack of food, that P5CS forms a filament. When we saw these mitochondria where the reductive subpopulation had a filament, we were able to demonstrate that P5CS was not only forming filaments, those filaments were bundling together. And those bundles together basically acted like a big raft that collected other reductive enzymes in what’s now called in the common vocabulary of science, a condensate, and that allowed mitochondria to separate this complex from the expanding inner membrane invagination required to undertake efficient ATP production.

Speaker 1 (13:34):

So mitochondria, to create these subpopulations, they can join together or they can divide, this is known as either fission or fusion. Can you explain how that process works?

Speaker 2 (13:48):

There are two things about mitochondria that you learn when you take an honors biology class. And that is mitochondria require a specific molecule of DNA inside them to maintain their ability to synthesize their electron transport chain. The mitochondria in an average cell have 500 to a 1000 copies of this DNA. that constantly supports the need for the replacement of proteins damaged by ETC activity. And so scientists had come up with a realization that cells have to get rid of the damaged ETC components and replace them with new components all the time. To maintain all the mitochondria in a cell, mitochondria are constantly reshuffling their contents.

(15:02):

They’re constantly fusing and dividing. There’s machinery that joins the membranes together so mitochondria become fused and then also a separate set of machinery that comes from the cytosol side that pinches the centers of mitochondria to separate them in two.  By constantly fusing and then fissioning, you could do two things. You could make all the mitochondria homogenous, they’re being replenished with DNA and other structural components into all the same. So they were efficient and equivalent for ATP production and all the damage components collect away from the DNA and are remodeled by fission away from the rest of the mitochondria. There’s a beautiful set of papers that showed that that actually occurs and are removed from the body by a process known as autophagy. So, everybody celebrated a few years ago that success to understand how you constantly repair mitochondria and replenish them through reshuffling so that everything’s homogeneous. That’s a great success and all true. And so when cells are really well fed and they have extra nutrients, that’s exactly what we see. But you put them under nutrient stress and now they need to increase their specialized functions. Mitochondria are just as important evolutionary for their synthesis role as they are for their ATP role.

Speaker 1 (16:28):

How was this discovery made?

Speaker 2 (16:31):

So the discovery was made by simply asking what happens when cells are challenged, they don’t have enough proline or they don’t have enough ATP. And then looking at what happens to the structure of mitochondria do over time, and we were helped by the fact that we could observe P5CS redistribute from the entire mitochondrial network into just a small subpopulation of mitochondria over an eight hour period. And if we give back proline, all of it comes back and redistributes everywhere.

Speaker 1 (17:12):

My sense is that mitochondria were previously thought of as being a uniform organelle.

Speaker 2 (17:18):

Right.

Speaker 1 (17:19):

How does this change our understanding of mitochondria or cellular metabolism?

Speaker 2 (17:26):

I think a lot about this, and this is where we hope some of the work will go. It gets back at the evolutionary origins of mitochondria and what we have used them for throughout the eukaryotic kingdom. So all eukaryotic cells have mitochondria. Many unicellular eukaryotics can do without mitochondrial ATP synthesis, but they still require mitochondria to make things. However, as multicellular animals, we require mitochondria to produce enough ATP to survive, so we depend on delivering oxygen and efficient ATP production. For mitochondria to act as powerhouses, they use oxygen. Animals have a vasculature to deliver oxygen to your central organs through a vascular system to maintain mitochondrial ATP synthesis. Mitochondria are powerhouses that efficiently burn nutrients to make sufficient ATP to survive. And that led to a misunderstanding for those of us who focus on mammals. The synthetic functions required by single cell organisms are also just as important to animals. We’re back to thinking about that in new ways.

Speaker 1 (19:02):

What’s known about the role these subpopulations play in say, cancer or metabolic diseases?

Speaker 2 (19:10):

It’s a really good question. So, I’ll give you two examples that we’ve talked about and then we and others are going to work on. If you look in vivo, one of the cancers that’s the most aggressive and literally among one or the most lethal or second most lethal cancer is pancreatic cancer. Almost the same number of people are diagnosed with pancreatic cancer every year as die of pancreatic cancer because it’s lethal usually within a year or so of diagnosis. And so the question became why is it so lethal? It’s because these cells are constantly dividing and invading. When people study them and measure the nutrients in their environment, the nutrients available are below those a normal cell requires to grow and divide. When we looked at biopsies of human patients biopsies, de-identified human biopsies of pancreatic cancer.

(20:09):

The tumor cells that were growing all had mitochondrial subpopulations. They knew to adapt. They needed these two subpopulations, we discovered. The second is another population of cells that are key to both cancer but also to a variety of diseases. In the immune system, most of our immune cells are just sitting around as quiescent B and T lymphocytes. When active, B cell make antibodies and T cells make the cytokines and do cellular killing. Most of the time we want them just resting in our lymph nodes, but when we get exposed to a pathogen, we need T cells to come and recognize the infected cells and kill them. And so we asked, what’s the metabolism of a T cell? At rest, they’re living off of very basal metabolism and resting T cells were known for years to depend on oxidative phosphorylation to survive because they don’t have glucose transporters on their surface. They all have mitochondria that are divided into subpopulations. Normally they only have around 20 mitochondria, but they’re divided into the two subpopulations already. Resting T-cell mitochondria were said to not have classical mitochondria, and that’s because half of them were doing something else.

Speaker 1 (21:33):

Does this do anything to change our understanding of aging?

Speaker 2 (21:38):

So it’s very funny, you should ask. So one of the challenges that the reviewers made for the paper and that we took to ourselves was to really say, is this process critical? Is this process of filaments coming together, of bundling together to make this efficient condensate of proteins necessary? It’s in the subpopulation that we call the reductive subpopulation. Is it really required? Well, oddly enough, about 10 years ago it was discovered that a disease of premature aging called cutis laxa with progeroid features. Patients look like they have progeria, they have this abnormal face. Their skin gets all wrinkly and disordered.

(22:39):

Most of those people, if they’re classic progeria, they have a defect in a structural protein that’s rich in proline called laminin. But they also, have defects in connective tissue. The big surprise was a subpopulation of these patients have mutations in P5CS but they are still enzymatically active. They can still make the enzyme product, but the mutant proteins can’t form filaments. And if they can’t form filaments, they’re incapable of maintaining their extracellular matrix and their skin becomes lax just like in progeria. This suggests mitochondrial subpopulations contribute to maintain our connective tissue.

(23:45):

We think that a medical insight the work will help explain are so-called fibrotic diseases. One of the largest set of diseases that remain idiopathic, we don’t know what causes them, are idiopathic fibrosis of the lung and idiopathic fibrosis in the liver which are fatal. And that’s a dysregulation of how much of these extracellular matrix proteins you make. Collagen is the critical protein component, and proline is a critical rate limiting step of that synthesis. And so we think that regulation and the coordination between the mitochondria and the cytosol may play a set point for why some people have extra scar tissue and why people don’t repair well enough.

Speaker 1 (24:31):

What do you see as the potential to apply this research in a clinical setting?

Speaker 2 (24:38):

So I would say right now we are still just trying to understand when and why cells use this. So this is what’s always disappointment to a disease community. For people that have mitochondrial diseases, how are we going to use this going forward? I would say the fact that there are subpopulations may help with diagnostic studies before it’s going to help with therapy. Oddly enough there are already some possible examples of why kids with electron transport chain disorders, which is one form of mitochondrial disease that’s common throughout the world and why adults that have so-called mitochondrial diseases where their mitochondrial DNA or their mitochondria become unfunctional is that they have empirically tried various remedies to help make them better. One thing found for the kids with electron transport change discovered actually in the early nineties was that those kids during development had a difficulty in making enough amino acids to fuel protein synthesis.

(25:47):

In that case, the amino acid, it looked like they had a deficiency in, might have been aspartate. Aspartate is critically important for the ability of cells to divide. Interestingly, most of our aspartate is probably made in our mitochondria. A couple of classic papers years ago discovered that electron transport chain activity is required for normal aspartate metabolism. And so people started to add more amino acids to the diets of patients that have electron transport chain deficiencies. But which ones are most helpful and why remain unclear. And so every group you go to, talks about amino acid supplements.

(26:51):

And so one of our follow-up studies of these mitochondrial subpopulations is trying to understand why certain amino acids, such as aspartate and asparagine, may help patients with mitochondrial diseases. And there are several other amino acids that are dependent on mitochondria for their synthesis in vivo, things like arginine that are critical for maintaining their immune response and other things. So we do think an understanding of the synthetic power of mitochondria is going to fuel more rational ways to provide supplements for people with both mitochondrial disease and ETC defects. But that’s down the road. We’ve not proven that, it’s all things that have been proposed which now are being worked on.

Speaker 1 (27:58):

Does the research suggest potential new therapeutic targets through either the subpopulations of mitochondria or perhaps P5CS?

Speaker 2 (28:09):

I do think in cancer that there’s a likelihood that we’ll find things like P5CS as a potential target. I don’t think that’s going to be true for the mitochondrial diseases, but it has renewed attention to how important mitochondria are for synthetic function and to maintaining the ability to maintain the right mix of building blocks to sustain cellular integrity. It’s not just all about the ATP, which has been so much of the focus.

Speaker 1 (28:41):

This seems to be so fundamental to understanding mitochondria and to think mitochondria were first identified in the 1850s. It took another a hundred years to develop a theory of ATP production. Where are we in terms of efforts to understand mitochondria?

Speaker 2 (29:05):

I think it had never really been seriously considered that mitochondria are an evolutionarily adapted organism that lives symbiotically with us and that we’ve captured it not just for the ability to make ATP, but to have a variety of synthetic processes that help us with our cellular, tissue, and organismal integrity, and that those processes are dynamic. We thought about it purely as a powerhouse that provided the ATP all the time. It’s really much more like a Swiss army knife. There are many things that mitochondria provide cells and we’ve missed the opportunity to bolster some of the things that people with mitochondrial diseases might benefit from by not understanding those other roles that mitochondria play in synthesis or in the ability to form enzymes that do unique jobs. Hemoglobin, for example, is made by mitochondria. The heme group for hemoglobin is made by mitochondrial synthesis. That’s recently been discovered in important new insights why do people with mitochondrial disease feel so tired all the time? They can’t make heme groups. And so you say, well, it’s not their blood that’s abnormal. It’s their muscles. Well, muscles need heme groups to hold oxygen and to carry out other activities as well.

Speaker 1 (30:28):

It seems there have been these types of leaps in our understanding of mitochondria, but as a field of research, it remains fairly fragmented. Why is that?

Speaker 2 (30:39):

Well, the sad reality of this is that most kids who are born with defects in the electron transport chain have a great deal of difficulty making it through development to puberty. They can have a huge range of developmental abnormalities because mitochondria are central to all the things we do. There’s no stable population of people that reminded us we need to do better because these kids largely have motor and neurologic symptoms that keep them out of the public eye. And so while we have better care for them, but we haven’t had that breakthrough where kids are having a normal life. And then the same thing for adults. The symptoms of mitochondrial disease are weakness and fatigue, and they get so dismissed when people talk about it that it’s underdiagnosed and underappreciated for its importance, and we don’t really understand yet why mitochondrial DNA acquires a mutation and then the mutated DNA starts to win out over the normal mitochondria. The constant remixing to make mitochondrial homogeneous was supposed to eliminate damaged mitochondrial DNA. And it doesn’t do that. Probably that’s because the DNA is itself part of this reshuffling of subpopulations. We are working on that part of the problem right now.

Speaker 1 (32:04):

Do you see the potential for say, the field of mitochondrial biology and medicine to emerge as a unique field similar to what’s happened with genetics?

Speaker 2 (32:16):

I don’t think that is going to be. I mean, all of us have genetics because we are born with the DNA that we inherit from mom and dad. But much the way how you use that information is a combination of genetics and this exploding field of epigenetics. We have argued that half of epigenetics is mitochondrial metabolism. And so we are going to be spending a lot of time trying to convince people that the root to understanding how cells execute our genetics goes through the mitochondria in a much more fundamental way than had been appreciated before 10 years ago.

Speaker 1 (32:53):

We’re at the start of a new administration. There appears to be bringing some uncertainty across the biomedical research landscape right now. How are researchers dealing with this, and is there a need to think about new funding sources in any way?

Speaker 2 (33:12):

I do think the new administration is going to look to chase success, and one way they measure success is people donating to causes and things that matter to them. Oddly, the health issue that everyone faces is wound repair. The need to understand the diversity of mitochondria is, as I just started this conversation with, was for wound repair and things like burns and those kinds of wounds where you have to replace a tissue. And I do think that has never gained traction in the public. Everybody thinks their cut’s going to heal, yet we see diabetics lose their foot from a cut that doesn’t heal. We see all kinds of issues around burns and the lesions and the scarring that’s left from a recovering burn. Injury is debilitating. Tissue regeneration has been folded into the aging movement. If we could understand the regenerative role of mitochondria, mitochondria could be critical to understanding aging as a health issue, the healthy aspects of repair that allow us to regenerate and maintain function for as long as we can during our life. So we have healthy aging. Being part of the focus on healthy aging and avoiding the defects of mitochondria is a way forward. And that’s partly the way those of us interested in Mito World have come together around getting the public to understand healthy aging starts with mitochondrial health.

Speaker 1 (34:49):

Well, what can be done to bring more coherence to the study of mitochondria as a field of biology and medicine?

Speaker 2 (34:57):

There’s a big transition in education right now, when you and I went to school, every course you took had a textbook. No kid in high school and college today wants to lug around their textbook. They’re reading articles, they’re reading things on the internet. They’re part of social media. And we have not changed the perception of the central role of the mitochondria in the integrity of us as an organism in 50 years anyway since 1978. And we need to, as a group of individuals who care about those afflicted with diseases of the mitochondria, as well as those of us interested in how cells work, we have got to raise the importance of a mitochondria to the importance of other organelles such as the nucleus.

Speaker 1 (36:00):

Craig Thompson, a member of the Cancer Biology and Genetics Program at Sloan Kettering Institution at the Memorial Sloan Kettering Cancer Center and senior author of the Nature Paper, which appeared in the November 6th issue. Craig, thanks for your time today.

Speaker 2 (36:16):

Thank you very much for having me, and I look forward to keeping you up to date on our work as we go forward. Thank you very much for the interview.

Speaker 1 (36:25):

To stay up on the latest news and research for the world of mitochondria, be sure to check in with MIT World regularly. For MIT Cast, I’m Daniel Levine. Thanks for watching.